UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” INSTITUTO DE BIOCIÊNCIAS – RIO CLARO unesp PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS (BIOLOGIA VEGETAL) ECOFISIOLOGIA DA GERMINAÇÃO E CRESCIMENTO INICIAL DE Araucaria angustifolia EM RESPOSTA À TEMPERATURA LORENA EGÍDIO DE CASTRO Tese apresentada ao Instituto de Biociências do Campus de Rio Claro, Universidade Estadual Paulista, como parte dos requisitos para obtenção do título de Doutor em Ciências Biológicas (Biologia Vegetal). JULHO - 2017 UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” INSTITUTO DE BIOCIÊNCIAS – RIO CLARO unesp PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS (BIOLOGIA VEGETAL) ECOFISIOLOGIA DA GERMINAÇÃO E CRESCIMENTO INICIAL DE Araucaria angustifolia EM RESPOSTA À TEMPERATURA LORENA EGÍDIO DE CASTRO Tese apresentada ao Instituto de Biociências do Campus de Rio Claro, Universidade Estadual Paulista, como parte dos requisitos para obtenção do título de Doutor em Ciências Biológicas (Biologia Vegetal). JULHO - 2017 Castro, Lorena Egídio de Ecofisiologia da germinação e crescimento inicial de Araucária angustifolia em resposta à temperatura / Lorena Egídio de Castro. - Rio Claro, 2017 76 f. : il., figs., gráfs., tabs., fots. Tese (doutorado) - Universidade Estadual Paulista, Instituto de Biociências de Rio Claro Orientador: Gustavo Habermann 1. Fisiologia vegetal. 2. Pinheiro brasileiro. 3. Ecologia das plantas. I. Título. 581.1 C355e Ficha Catalográfica elaborada pela STATI - Biblioteca da UNESP Campus de Rio Claro/SP À minha mãe Conceição, pela vida e por todo amor e dedicação. Aos meus avós, pai, padrasto e tia Rosa, pelo carinho e cuidado. DEDICO “Digo: o real não está na saída nem na chegada: ele se dispõe para a gente é no meio da travessia” (Guimarães Rosa- Grande Sertão: Veredas) AGRADECIMENTOS Em primeiro lugar agradeço à Deus pelo dom da vida e pela oportunidade de viver mais essa experiência Agradeço à Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP, Instituto de Biociências da UNESP – Campus Rio Claro, pela infraestrutura e apoio técnico e pela oportunidade de realização do doutorado em Biologia Vegetal. Agradeço à CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) pela concessão da bolsa e apoio financeiro. Agradeço à banca avaliadora pela disponibilidade em avaliar este trabalho. Agradeço ao meu orientador, Prof. Dr. Gustavo Habermann, pela oportunidade dada, por todos os ensinamentos e por se fazer sempre presente. Aproveito a oportunidade para expressar minha admiração por sua conduta e profissionalismo. Ao Thales Barbosa e ao Felipe Pedrosa pela ajuda indispensável na coleta de dados em campo e laboratório, sem vocês não seria possível! Ao Anésio, funcionário do PECJ, pela valiosa ajuda na coleta de dados no parque. Aos funcionários da Unesp Sílvia e João pela importante ajuda nos experimentos. Aos meus pais, avós, padrasto, tia Rosa e Maria José, por todo apoio e incentivo dado durante toda minha vida. Às minhas amigas de longe que sempre estiveram presentes e me deram força nos momentos difíceis, em especial à Saritinha, Bruna, Amanda, Eldinha, Mayra e também ao amigo Isaac. Às minhas amigas queridas do laboratório: Anna, Giselle, Marina, Mariana, Brendinha, Otávia e também ao Eduardo pela amizade e pela agradável convivência, adoro vocês!! Às amigas Letícia, Betânia, Natália e Laurinha pela amizade desde o início do doutorado e por todo apoio que me deram. Á minha amiga querida Patrícia Tiemi pela amizade, por me ouvir e aconselhar tantas vezes, pela parceria no GG e na busca pelo autoconhecimento. Às minhas irmãs do coração Brendinha e Juliana por alegrarem a reta final do meu doutorado e por compartilharem comigo a busca pelo caminho do bem. Ao meu amado Pereirinha por todo carinho, por cuidar de mim e por me apoiar na reta final da tese. À todos os conhecidos, colegas e amigos que encontrei durante esta caminhada, com os quais tenho a certeza de ter aprendido algo. Fica aqui minha gratidão a todos!!! SUMÁRIO RESUMO ......................................................................................................................... 9 ABSTRACT .................................................................................................................... 9 INTRODUÇÃO GERAL ............................................................................................. 10 REFERÊNCIAS ........................................................................................................... 14 CAPÍTULO 1: Seeds of Araucaria angustifolia: To bury or not to bury? That’s the ecological question! ...................................................................................................... 18 Abstract ..................................................................................................................... 18 Introduction .............................................................................................................. 19 Material and Methods .............................................................................................. 22 Plant material and area description ......................................................................... 22 Field and laboratory experimental designs ............................................................. 22 Field seed and soil water content ............................................................................ 24 Germination rate ..................................................................................................... 24 Biometric data of seedling laboratory study ........................................................... 25 Data analysis ........................................................................................................... 26 Results ........................................................................................................................ 27 Discussion .................................................................................................................. 29 Acknowledgements ................................................................................................... 33 References.................................................................................................................. 34 Figures ....................................................................................................................... 37 CAPÍTULO 2: Increased growth of Araucaria angustifolia under warm conditions is unaccompanied by increased photosynthetic performance .................................. 45 Abstract ..................................................................................................................... 46 Introduction .............................................................................................................. 46 Material and methods .............................................................................................. 49 Plant material .......................................................................................................... 49 Study design and experimental conditions ............................................................. 49 Soil fertility ............................................................................................................. 50 Biometric parameters .............................................................................................. 51 Plant nutritional status ............................................................................................ 51 Leaf gas exchange .................................................................................................. 52 Data analysis ........................................................................................................... 52 Results ........................................................................................................................ 53 Discussion .................................................................................................................. 55 Acknowledgments ..................................................................................................... 60 References.................................................................................................................. 60 Tables ......................................................................................................................... 66 Figures: ...................................................................................................................... 68 Supplementary material: ......................................................................................... 74 CONSIDERAÇÕES FINAIS ......................................................................................... 76 RESUMO A Araucaria angustifolia é a espécie chave das florestas ombrófilas mistas, cuja distribuição está associada à climas frios, entretanto pouco se sabe a respeito das respostas da espécie à temperatura. O objetivo do presente trabalho foi avaliar o efeito da temperatura no desempenho de sementes, plântulas e mudas. As respostas das sementes foram avaliadas em condições de laboratório e em condições de campo, levando-se em consideração o efeito do enterrio das sementes, que na natureza é realizado por animais dispersores. Ao contrário do esperado, observou-se que as plântulas e mudas de um ano e meio de idade apresentaram melhor desempenho ecofisiológico em maiores temperaturas e amplitudes térmicas. Também foi observado que o enterrio de sementes proporcionou maiores porcentagens de germinação quando comparado com sementes que não foram enterradas, o que se deve à melhores condições de temperatura e umidade. ABSTRACT Araucaria angustifolia is the key species from mixed ombrophilous forests, whose distribution is associated with cold climates, but the responses of the species to temperature are little known. The objective of the present work was to evaluate the effect of temperature on the performance of seeds, seedlings, and young plants. The seed responses were evaluated under laboratory conditions and field conditions, considering the effect of seed burial, which in nature is carried out by dispersing animals. Contrary to expected, it was observed that seedlings and one year and a half plants presented a better ecophysiological performance at higher temperatures and thermal amplitudes. It was also observed that seed burial provided higher percentages of germination, which is due to the better conditions of temperature and humidity, when compared to seeds that were not buried. 10 INTRODUÇÃO GERAL O crescimento e desenvolvimento das plantas é coordenado pelas variações ambientais. Dentre as variações ambientais, a temperatura é um dos principais sinalizadores para ajustes metabólicos durante o ciclo de vida das plantas (Franklin, 2009). Desta forma, a temperatura pode influenciar o funcionamento dos meristemas (Savvides, 2017), com consequências na emissão de folhas (Stewart et al., 2016), no crescimento das raízes (Schenker et al., 2014), nos processos hormonais que regulam o crescimento (Stavang et al., 2009) e na fotossíntese (Sage & Kubien, 2007). A reprodução das plantas também sofre influência da temperatura, visto que o início do período de floração é determinado principalmente por horas de luz e temperatura e em algumas espécies só é iniciado após exposição prolongada à baixas temperaturas, processo conhecido como vernalização (Capovilla et al., 2014). Incrementos na temperatura promovem aumento nas taxas metabólicas, e de modo geral estimulam o crescimento das plantas (Wigge, 2013). Maiores temperaturas relacionam-se principalmente a maior alongamento do caule e maior acúmulo de biomassa de caules e folhas (Atkin et al., 2005). Para algumas espécies, além de menor biomassa, longos períodos de frio resultam em folhas mais espessas e menor área foliar (Patel & Franklin, 2009; Gorsuch et al, 2010). A fotossíntese também responde de forma positiva ao aumento de temperatura até que se atinja uma temperatura ótima, acima da qual há queda nas taxas fotossintéticas (Yamori et al., 2014). A temperatura ótima está geralmente associada às temperaturas do ambiente de origem das plantas (centro de origem genético), mas este parece ser um caráter plástico, permitindo que as plantas mantenham a eficiência fotossintética em diferentes condições de crescimento (Slot & Winter, 2017). A maioria dos estudos fisiológicos sobre respostas às temperaturas são 11 realizados com espécies cultivadas (Bita & Gerart, 2013; Sánchezet al., 2014; Asseng et al., 2015) e pouco se sabe a respeito das respostas das espécies nativas e de climas subtropicais, como as coníferas, às variações de temperatura. As gimnospermas estão distribuídas em regiões de clima frio e consequentemente são adaptadas à esta condição. Alguns estudos mostram que o incremento na temperatura poderá provocar uma redução em larga escala das florestas de coníferas até o ano de 2100 (Mcdowell, 2016). Entretanto, há algumas evidências de que estas plantas também possam ser beneficiadas por incrementos na temperatura, uma vez que ainda não estão no limiar térmico, ao contrário das espécies de regiões quentes (Way & Oren, 2010). Dessa forma, são necessários mais estudos para elucidação das respostas fisiológicas e adaptações das plantas ao clima. A Araucaria angustifolia (Bertol.) Kuntze, também conhecida como pinheiro brasileiro ou pinheiro do Paraná, é uma gimnosperma nativa da América do Sul e caracterizada como espécie chave da Floresta Ombrófila Mista (Overbeck, 2007). As populações de A. angustifolia estão distribuídas de forma fragmentada em um amplo gradiente latitudinal, entre 19° 15' até 31° 30' S e 41° 30' até 54° 30' W, com relação inversa entre latitude e altitude (Ledru & Stevenson, 2012). Além disso, a espécie está sempre associada à climas subtropicais, com temperaturas médias anuais entre 18-24°C e pluviosidade entre 1500-2000 mm (Oliveira et al., 2010). Devido à ampla distribuição latitudinal, as populações de Araucaria apresentam variações nos períodos de ocorrência das etapas fenológicas. Populações do sudeste do país (estados de São Paulo e Minas Gerais) concentram a dispersão de suas sementes de março a maio (Mantovani et al., 2014). Já a dispersão de sementes das populações do sul concentram-se entre maio e julho (Kissmann & Habermann, 2014). Sementes de A. angustifolia são recalcitrantes, o que significa que são sensíveis à perda de água e 12 possuem curta longevidade (Berjak, 2008). Estas sementes são dispersas com alto teor de umidade (de 40 a 50 %) e não toleram desidratação abaixo de 35% (Gasparin, 2017). Na ausência de condições para germinação, a viabilidade dos pinhões é de aproximadamente 90 dias. Ao longo da história evolutiva das espécies há um direcionamento para a sincronização entre o período da dispersão das sementes e a janela climática adequada para germinação e crescimento das plântulas (Nathan & Muller-Landau, 2000). As sementes recalcitrantes são mais comuns em ambientes onde a dispersão de sementes ocorre na estação chuvosa e a germinação rápida para estabelecimento das plântulas representa uma vantagem ecológica para as espécies com sementes recalcitrantes (Tweddle et al., 2003). A Floresta Ombrófila Mista é uma fitofisionomia com alta pluviosidade bem distribuída ao longo do ano (Backes, 1999). A dispersão dos pinhões no final de março no sudeste do país e em maio no sul do país, coincidem ambas com períodos chuvosos, o que poderia indicar ajustes temporais na estratégia da espécie. Entretanto, há grandes diferenças entre as temperaturas encontradas nas duas regiões durante a dispersão, o que indica que apesar da estratégia geral da espécie, é provável que também ocorram adaptações locais populacionais no estabelecimento das plantas e isso exige mais estudo. Devido ao seu alto valor nutritivo, as sementes de A. angustifolia são intensamente predadas por aves e mamíferos (Solórzano-Filho, 2001; Ribeiro & Vieira, 2014). Alguns desses animais, tais como cutias (Dasyprocta sp.) e esquilos (Sciurus sp.) possuem o comportamento de enterrar sementes para consumo futuro (scatthoaders) e acabam atuando como dispersores de sementes (Vander Wall & Beck, 2012). Isso ocorre porque devido à alta oferta de recursos ou mesmo devido à morte do 13 dispersor, algumas sementes não são recuperadas e podem germinar, recrutando novas plantas. Sementes enterradas encontram condições de temperatura e umidade diferentes das condições encontradas por sementes dispersas sobre o solo, o que acarreta em diferenças nas respostas e no sucesso do estabelecimento da planta (Batlla & Benech- Arnold, 2006). Além de interferir no recrutamento de novos indivíduos, a alta predação dos pinhões e coleta para consumo humano (Silva & Reis, 2009) dificulta o estudo e entendimento do desempenho de sementes enterradas e não enterradas em condições naturais. Portanto, estudos que caracterizem o desempenho e as condições microclimáticas encontradas pelos pinhões no sub-bosque da Floresta Ombrófila Mista são de extrema importância e ajudam a entender as estratégias ecofisiológicas da espécie. Diante disso, testamos aqui a Tese geral de que a baixa temperatura pode beneficiar a germinação de sementes e o crescimento de mudas de A. angustifolia. Logo, procuramos responder às seguintes peguntas: 1) O enterrio de sementes no sub-bosque de uma Floresta Ombrófila Mista pode favorecer a germinação da espécie? 2) Considerando que as sementes desta espécie devem germinar no outono/inverno, será que suas plântulas crescem melhor sob temperaturas baixas? 3) O crescimento de A. angustifolia é prejudicado por condições de temperaturas elevadas? 14 REFERÊNCIAS ASSENG, S.; EWERT, F.; MARTRE, P.; RÖTTER, R.P.; LOBELL, D.B.; CAMMARANO, D.; REYNOLDS, M.P. Rising temperatures reduce global wheat production. Nature Climate Change, v.5, p.143-147, 2015. ATKIN, O.K.; LOVEYS, B.R.; ATKINSON, L.J.; PONS, T.L. Phenotypic plasticity and growth temperature: understanding interspecific variability. Journal of Experimental Botany, v.57, p.267-281, 2005. BACKES, A. Condicionamento Climático e distribuição geográfica de Araucaria angustifolia (Bertol.) Kuntze no Brasil - II. Pesquisa (Botânica), v.49, p.31-51, 1999. BATLLA, D.; BENECH-ARNOLD, R.L. The role of fluctuations in soil water content on the regulation of dormancy changes in buried seeds of Polygonum aviculare L. Seed Science Research, v.16, p.47-59, 2006. BERJAK, P.; PAMMENTER, N.W. From Avicennia to Zizania: seed recalcitrance in perspective. Annals of Botany, v.101, p.213-228, 2008. BITA, C. E.; GERATS, T. Plant tolerance to high temperature in a changing environment: scientific fundamentals and production of heat stress-tolerant crops. Frontiers in plant science, v.4.p.273, 2013. CAPOVILLA, G.; SCHMID, M.; POSÉ, D. Control of flowering by ambient temperature. Journal of experimental botany, v. 66, p. 59-69, 2014. CHUINE, I. Why does phenology drive species distribution? Philosophical Transactions of the Royal Society of London B: Biological Sciences, v.365, p. 3149- 3160, 2010. 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J.; DEMMIG‐ADAMS, B.; COHU, C.M.; WENZL, C.A.; MULLER, O.; ADAMS, W.W. Growth temperature impact on leaf form and function in Arabidopsis thaliana ecotypes from northern and southern Europe. Plant, cell & environment, v.39, p.1549-1558, 2016. TWEDDLE, J.C.; DICKIE, J.B.; BASKIN, C.C.; BASKIN, J.M. Ecological aspects of seed desiccation sensitivity. Journal of ecology, v. 91, p. 294-304, 2003. 17 VANDER WALL, S.B.; BECK, M.J. A comparison of frugivory and scatter-hoarding seed-dispersal syndromes. The Botanical Review, v.78, p.10-31,2012. VIEIRA, E.M.; RIBEIRO, J.F.; IOB,G.. Seed predation of Araucaria angustifolia (Araucariaceae) by small rodents in two areas with contrasting seed densities in the Brazilian Araucaria forest. Journal of Natural History, v. 45, p. 843-854, 2011 WAY, D.A.; OREN, R. Differential responses to changes in growth temperature between trees from different functional groups and biomes: a review and synthesis of data. Tree physiology, v.30, p.669-688, 2010. WIGGE, PHILIP A. Ambient temperature signalling in plants. Current opinion in plant biology, v. 16, p. 661-666, 2013. YAMORI, W.; HIKOSAKA, K.; WAY, D. A. Temperature response of photosynthesis in C3, C4, and CAM plants: temperature acclimation and temperature adaptation. Photosynthesis research, v.119, p.101-117, 2014. 18 1 Esse artigo será submetido na revista Plant Ecology. CAPÍTULO 1: Seeds of Araucaria angustifolia: To bury or not to bury? That’s the ecological question! 1 Lorena Egídio de Castro 1 , Felipe Pedrosa 2 , Patricia Tiemi de Paula Leite 1 , Thales Barbosa dos Santos 3 , Gustavo Habermann 4 * 1 Programa de Pós-Graduação em Biologia Vegetal, Universidade Estadual Paulista (Unesp), Instituto de Biociências, Departamento de Botânica, Av. 24-A, 1515, Rio Claro, SP, 13506-900, Brazil 2 Programa de Pós-Graduação em Ecologia e Biodiversidade, Universidade Estadual Paulista (Unesp), Instituto de Biociências, Departamento de Botânica, Av. 24-A, 1515, Rio Claro, SP, 13506-900, Brazil 3 Graduação em Ciências Biológicas, Universidade Estadual Paulista (Unesp), Instituto de Biociências, Departamento de Botânica, Av. 24-A, 1515, Rio Claro, SP, 13506-900, Brazil 4 Universidade Estadual Paulista (Unesp), Instituto de Biociências, Departamento de Botânica, Av. 24-A, 1515, Rio Claro, SP, 13506-900, Brazil. E-mail: ghaber@rc.unesp.br; Tel: +0055 19 3526 4210 *Corresponding author Abstract The success of a plant is initially dependent on seed dispersal, germination performance and seedling establishment. Araucaria angustifolia (Brazilian pine) exhibits wide geographical distribution in South America with seed dispersal occurring in the fall/winter and varing between regions. Dispersed by animals with burial behavior, we tested the germination performance of buried and non-buried seeds for 90 days at the beginning of its seed dispersal. We also monitored daily oscillation of temperature in the soil and in the air. When buried, almost half of the seeds had already germinated at 45 days, while less than 10% of non-buried seeds germinated until 60 days. We also simulated field soil and air temperatures inside germination chambers and found that germination performance of seeds at simulated soil temperature was higher than those at simulated air temperatures after 60 days. In the laboratory, we also measured height, number of leaves and dry matter of seedlings grown at constant 10, 20, 30 and 40°C. 19 At 10 and 40°C, seedlings did not develop properly, and after 60 days, seedlings at 30°C showed two times more leaves and two-fold higher dry matter in relation to seedlings at 20°C. Our data suggest that buried seeds may benefit from more stable temperature (and moisture) conditions. Germination at the beginning of seed dispersal may allow proper seedling establishment before lower temperatures develop in the winter. Keywords: Brazilian pine; germination rate; seed dispersal; seedling growth; temperature Introduction The distribution of plant species is closely related to climate and usually determined by temperature and rainfall (Woodward and Williams 1987). While water availability can influence plant performance and explain species distribution in the tropics (Habermann and Bressan 2011; Habermann et al. 2011), temperature can influence plant growth (Atkin et al. 2006). Distribution of plant species also involves seed dispersal, germination performance and seedling establishment. Dispersers and their relation to seed germination and seedling establishment of key species from tropical vegetation are relatively known (Beckman and Rogers 2013; Galetti et al. 2013). However, such studies for vegetation occurring in cool conditions of subtropical areas are less frequent. Occurring in subtropical areas of South America (south and southeast of Brazil and Argentina), Araucaria Forest is essentially comprised of a gymnosperm named Araucaria angustifolia Bertol. Kuntze (Brazilian pine). Currently threatened extinction, the remaining populations of A. angustifolia are found between 19° 15' to 31° 30' S and 41° 30' to 54° 30' W, with an inverse relationship between altitude and latitude (Ledru and Stevenson 2012). In addition, its area of occurrence is influenced by 18-24C of annual average temperature and 1500-2000 mm of rainfall (Oliveira 2010). 20 Consequently, such vast territory of occurrence subjects this species to distinct phenological timing, depending on the region. Usually, A. angustifolia seed dispersal takes place between the beginning of the fall until the winter. However, it has been observed that at low latitudes (São Paulo and Minas Gerais states in Brazil) the seed dispersal is anticipated (March) (Mantovani et al. 2004) in relation to high latitudes (Santa Catarina and Rio Grande do Sul Brazilian states, for example), where it happens at the beginning of the winter (June) (Kissmann and Habermann 2014). Besides being a response to the regional climate, seed dispersal occurring at different periods can also result in distinct seed germination and plant establishment strategies. Araucaria angustifolia seeds are rich in starch (Panza et al. 2002; Gasparin et al. 2017) and represent food source for the fauna in the fall/winter, when a reduced number of angiosperms produce animal-dispersed fruits (Paise and Vieira 2005). Squirrels (Sciurus spp.), agouties (Dasyprocta spp.) and the jay birds (Cyanocorax spp.) act as A. angustifolia’s seed dispersers, since they are scatter-hoarders (Vander Wall 2002). Scatter-hoarder animals bury the seeds in the soil to stock it for future feeding, creating favorable conditions for seed germination (Iob and Vieira 2008, Vieira et al. 2011). As far as we are aware, buried versus non-buried seed germination has not been tested for A. angustifolia in the field, but probably, buried seeds experience low amplitude in daily temperature in relation to seeds left on the forest soil surface. In addition, the soil may also maintain favorable moisture conditions for seed germination. Accordingly, seeds of A. angustifolia are recalcitrant and show dehydration sensitivity and reduced longevity (Corbineau et al 1997; Gasparin et al. 2017), which means that buried seeds could benefit from more stable conditions for prompt germination. However, seeds of A. angustifolia maintained in germination chambers simulating daily air temperature oscillations showed higher germination performance when compared to the constant 21 ideal temperature for germination of these seeds (20C) (Kissmann and Habermann 2014). Depending on the period of seed dispersal within the fall-winter interval, initial seedling development is also influenced by different temperature ranges. During seed dispersal of A. angustifolia in Santa Catarina state (June) the air temperature oscillates between 9.4 and 15C (Kissmann and Habermann 2014), while there is no such data for native areas of A. angustifolia occurring at low latitude. The 9.4-15C daily oscillation may not be suitable for the initial growth of this species. There are no studies determining the average growing temperature for A. angustifolia, but there is evidence suggesting that young plants of this species grows more with the increase of temperature (Castro et al. 2017), and this decreases the counterintuitive concept that this species is related with low temperature conditions. We monitored daily oscillation of temperature in the soil and in the air, and tested the germination performance of buried and non-buried seeds of A. angustifolia, at the beginning of seed dispersal season at a low latitude area for this species. In the laboratory, besides determining cardinal temperatures for seed germination of this species, we simulated field soil and air temperatures in order to isolate the temperature effect on seed germination. In addition, we assessed growth of seedlings at constant 10, 20, 30 and 40C. Due to previous studies (Kissmann and Habermann et al. 2014; Castro et al. 2017) we predict that large daily amplitude in air temperature is associated with higher germination performance of non-buried seeds in relation to buried ones, a condition which we confirmed more stable temperature throughout the day. We also hypothesized that the increase of temperature enhances the seedling growth of this species. We discuss seed germination and seedling establishment strategies as a function of the timing of seed dispersal and temperature range. 22 Material and Methods Plant material and area description Seeds of Araucaria angustifolia (Bertol.) Kuntz were collected from the soil surface at the beginning of the seed dispersal (March, 2016), at the Campos do Jordão state park (2245’S 4530’W; 8341ha), São Paulo state, Brazil. The study area was located at 1450 m of altitude in a remnant of Upper Montane Mixed Ombrophylous Forest. According to Köppen-Geiger classification, the climate of this region is characterized as Cfb (Álvares 2014), exhibiting mild summers with air temperature not higher than 22C, and with absence of dry seasons in the winter with frequent frosts. Soil and air temperatures were measured with 1400-103 and 1400-101 temperature sensors, respectively. These sensors were connected to a LI-1400 data logger (LI-COR, Lincoln, NE, USA), which collected data every 30 min throughout a 90-day field experiment. Mean, minimum and maximum daily temperatures were used to calculate average values for every 15 days in the field experiment. Daily rainfall (mm) was obtained from a weather station located within the Campos do Jordão state park, approximately 3 km from the experimental area, and the accumulated rainfall was also calculated for every 15 days. Soil water content was measured at 0, 15, 30, 45, 60, 75 and 90 days of field experiment. Field and laboratory experimental designs At the beginning of the seed dispersal we randomly selected five sites within the experimental area (approximately 10 ha). At each site we buried (10  1 cm in depth) 11 seed samples (made of nylon  bags) containing 15 seeds each (Fig. 1b), and we also left 11 samples with 15 seeds each displaced on the soil surface protected from predators with exclusion cages (non-buried seeds) (Fig. 1a). After 15, 30, 45, 60, 75 and 90 days 23 we collected one sample (buried and non-buried) from each of the five sites. Although we have used 11 samples at each of the five sites, we collected six samples (one at each evaluation date) due to predators and unexpected losses. On each evaluation date, we assessed seed water content (%), percentage of germinated, intact and deteriorated seeds. Under controlled conditions (laboratory), we performed a study using seeds and another one using seedlings. For the seed study, we first characterized the germination rate (G%) of seeds maintained in germination chambers at constant temperatures 5, 10, 15, 20, 25, 30, 35 and 40C under a photosynthetic photon flux density (PPFD) of approximately 80 mol m -2 s -1 . We also used the soil and air mean daily temperatures obtained in the field during the first 15 days of seed dispersal (Fig. 2a) in order to simulate these temperatures within germination chambers in the laboratory. Then, we assessed G% of seeds when soil and air field temperatures were simulated (Fig. 2b) into separate germination chambers in the lab that was monitored 90 days. For the seedling laboratory study, we used seedlings (3 mm of root) recently germinated at 20C because this temperature is considered to be ideal for the germination performance of A. angustifolia seeds (Kissmann and Habermann 2014), as also confirmed in the present study (Fig. 5). Six seedlings in plastic pots (500 mL; 15 cm in height and 8 cm in diameter) containing an organic substrate for forest nursery (Plantmax; Paulínia, São Paulo, Brazil) were put to grow at constant 10C, 20C, 30C and 40C under a PPFD of approximately 80 mol m -2 s -1 . The number of leaves and plant height (cm) was measured at every four days. After 60 days, plant biomass (root, shoot and leaves) (g), excluding the seed, was also assessed. 24 Field seed and soil water content On each evaluation date (0, 15, 30, 45, 60, 75 and 90 days of experiment) five non-buried and five buried seeds from each of the five sites were collected. The samples were stored in plastic bags (ziplock) and immediately taken to the laboratory where the seed fresh mass (g) was weighted using a scale. Subsequently, the samples were oven-dried at 103C until constant mass (seed dry mass). Seed water content was calculated according to the standard procedures of the International Seed Testing Association (2011). Five fresh soil samples were collected below the leaf litter, at 10 ± 1 cm depth, at each of the five sites, and these samples were maintained in previously weighted (tare) hermetic glass pots (15 mL) and immediately taken to the laboratory, where fresh weight (g) was obtained using a scale. Dry soil mass (g) was determined by oven-drying the samples at 105C to constant mass, and the soil water content was calculated and expressed in %. Germination rate In the field, on each evaluation date (0, 15, 30, 45, 60, 75 and 90 days of experiment), seeds were classified into germinated and non-germinated seeds. Germinated seeds showed the protrusion of 1 mm of the primary root. Non-germinated seeds were classified as deteriorated or intact. Deteriorated seeds exhibited visual changes of appearance, exposing the reserves or the embryos, or even visually predated by insects and/or microorganisms. Intact seeds were considered as non-germinated and were not returned to buried or non-buried conditions. These seed classes were separated and results were expressed in percentage. 25 In the laboratory, we used the same seed lot used in the field experiment, and seed water content was 49%. Then, for characterizing the germination rate (G%), the seeds were sown inside hand-folded channels of several layers of filter paper that were wetted with distilled water; these layers of paper were placed inside transparent plastic boxes (13 × 13 × 4 cm) that were covered with their respective plastic transparent lids, and the (six) boxes, which contained 10 seeds each, were regarded as replications (Fig 1c). The folded paper channels ensured a high seed-paper/water contact because the seeds were 4 ± 0.5 cm in length and 1 ± 0.5 cm wide at the base of the seed, and this procedure was also performed by Roberto et al. (2011). The number of seeds that germinated was monitored daily and assessments continued until no seed had germinated for at least 14 days. The boxes were maintained inside germination chambers at constant 5, 10, 15, 20, 25, 30, 35 and 40C. For seeds that were put to germinate under simulated field soil and air temperatures we used the same procedures mentioned above, but evaluations were done at every 15 days (15, 30, 45, 60, 75 and 90 days of experiment). We used five larger transparent boxes to accommodate 15 seeds each, following the same evaluation dates and seed number used for samples in the field experiment. Biometric data of seedling laboratory study The number of leaves was counted, and the plant height (from root collar to the shoot apex) was obtained with a ruler (cm). At the end of the experiment (60 days), roots, shoots and leaves (excluding the seed) together for each seedling were oven-dried at 60C until constant mass (g). 26 Data analysis The field study was conducted using a randomized complete block design. Each of the five sites within the experimental area was considered one block (replication). In each block we used six samples for both buried and non-buried seeds. The laboratory study was conducted using a completely randomized design. We used six replications (boxes containing 10 seeds each) for characterizing G% of seeds under constant temperatures and also six pots with one seedling each for evaluating seedling growth at 10, 20, 30 and 40C. For seeds that were put to germinate under simulated field soil and air temperatures we used five replications (boxes containing 15 seeds each). A Student t test (α = 0.05) was performed (after checking for normal data distribution and homogeneous variance of data) between buried and non-buried seeds (field study), and also between seeds put to germinate under simulated field soil and air temperatures (laboratory study), after transforming %G into (G% + 0.5) 0.5 . For characterizing G% of seeds at constant temperatures in the lab, data were subjected to one-way analysis of variance (ANOVA), and mean results were compared by Scott Knott test (α = 0.05), also after transforming (G% + 0.5) 0.5 . In addition, a repeated measures Anova (RM-Anova) was performed to test for differences in plant height and number of leaves over time within each group of seedlings maintained for 60 days at 20C and 30C only (at 10C seedlings did not develop shoots and at 40C seeds did not germinate – see results). However, at 60 days of study we also run a student t test (α = 0.05) in order to check for differences in plant height and number of leaves between both groups of plants. For seedling dry mass we performed a Tukey test ( = 0.05). Statistical tests and graphics were performed using SigmaPlot 12.0 Software, and standard deviation (SD) is given in all figures. 27 Results In the field, at the beginning of the seed dispersal, the mean daily temperature was 15.8C in the air and 16.9C in the soil. However, the air temperature varied from 11.9 (6:30h) to 20.4C (13:30h), resulting in amplitude in daily temperature of 8.5C, while in the soil such amplitude was of 0.6C (Fig. 2a). When field air conditions were simulated in a germination chamber in the lab, the mean daily temperature was 15.9C, varying from 12C (5:00-7:00h) to 20C (11:00-15:00h) resulting in amplitude in daily temperature of 8C. When the field soil conditions were simulated in a germination chamber in the lab, the mean daily temperature was at constant 17C (Fig. 2b). After displacing the seeds in the field, the first 15 days showed no rainfall. However, between the 15 th and 90 th day it rained 211.1 mm, from which 80% (168.4 mm) occurred between the 75 th and 90 th day (Fig. 3a). The soil water content reflected the rainfall of the period. When measured at 0 days of field study, it was 59.4%, decreasing gradually to 39.1% (30 days), maintaining these values up to the 60 th day of study, and returned to 58.3% (75 days) and 56.0% (90 days) (Fig. 3a). The difference in seed water content between buried and non-buried seeds was of 0% (0 days; initial seed water content = 49%), 3% (15 days), 6% (30 days), 7% (45 days), 10% (60 days), 9% (75 days) and 5% (90 days), demonstrating that buried seeds showed an average of 7% more water content when compared to non-buried seeds (Fig. 3a). The mean air temperature slightly decreased throughout the experiment, being 15.9C (in the first 15 days) and 13.0C (from 61 until 75 days), although in the last 15 days of the experiment mean air temperature was 10C. During the 90 days, the amplitude in mean air temperature varied between 3 and 5C (Fig. 3b). The soil temperature also decreased from 16.9C (first 15 days) to 13.6C (last 15 days of the 28 study) (Fig. 3b), but the amplitude in soil mean temperature was 1C during the whole field study (data not shown). In the field, buried seeds showed increasing %G for seeds sampled at 15 (4%), 30 (31.7%), 45 (48%) and 60 days (96.7%) of experiment and, consequently, intact seeds decreased accordingly in this same period; buried seeds sampled at 75 and 90 days showed %G around 85% (Fig. 4a). In contrast, non-buried seeds maintained their %G below 7% from 30 days until 60 days, and intact seeds were also maintained at an average of 79  8% from 15 to 60 days. Non-buried seeds increased their %G to almost 80% at 75 days and subsequently decreased it to 45% at 90 days, while deteriorated non-buried seeds increased from 75 to 90 days (Fig. 4b). When constant temperatures were tested in germination chambers in the lab, the percentage of germination (%G) was maximum when seeds were at 20C, and the lowest %G was observed when seeds were at 5C and 35C. At 40C seeds did not germinate (Fig. 5). In the lab, seeds put to germinate at simulated field soil and air temperatures showed similar %G from 15 to 45 days, but from 60 until 90 days seeds germinated at simulated field soil temperature showed higher %G when compared to seeds germinated at simulated field air temperature (Fig. 6). In the lab, height was similar between seedlings grown at constant 30C and 20C (Fig. 7a). However, the number of leaves increased from 30 days until the end of the study, when plants grown at 30C showed almost two times more leaves (p = 0.038) in relation to plants grown at 20C (Fig. 7b). After 60 days under constant 30C, seedlings showed two-fold higher (p = 0.002) dry weight in relation to seedlings grown at constant 20C. Seedlings grown at 10C did not develop shoots and leaves, and seedlings at 40C did not survive (Fig. 8). 29 Discussion Our results demonstrate that high germination performance of buried seeds is associated with the soil temperature that was close to the ideal temperature for germination of A. angustifolia seeds. At the beginning of the seed dispersal (March/2016), the soil temperature was close to 17C, which is close to the constant ideal temperature for germination of these seeds (20C) (Fig. 5), as also confirmed by Kissmann and Habermann (2014). The high germination performance of buried seeds was also associated with the low amplitude in daily temperature in the soil (Fig. 2a). When we simulated daily air and soil temperatures and controlled seed water content in the lab, we also saw higher germination performance in stable (soil) temperatures compared to variable (air) temperatures (Fig 6), which showed daily amplitude of 8C. Seeds of A. angustifolia are recalcitrant (Farrant et al 1996; Corbineau et al 1997; Araldi et al 2015), contrasting with some orthodox seeds that require considerable amplitude in daily temperature to induce germination (Berjak and Pammenter 2008). In contrast with atmospheric conditions faced by non-buried seeds, when buried, the seeds conserved their water content. As recalcitrant, seeds of A. angustifolia do not tolerate dehydration below 35% (Gasparin et al 2017). In the present study, buried seeds showed water content varying from 49 to 53%, while non-buried seeds showed an average of 37% of seed water content (Fig. 3a). This indicates that for buried seeds, the soil maintained their water content 15% above the critical water content these seeds can tolerate. Seeds of Quercus ilex, a recalcitrant seed from western Mediterranean climate, show higher germination performance when buried (53.1%) and compared with non- buried seeds (21.8%) (Gomez 2004). In addition, this author evidenced that buried seeds 30 exhibited lower predation rate and resulted in higher plant emergence. We did not assess plant emergence in the field, but after 90 days, 44% of non-buried seeds were deteriorated (Fig. 4b), while only 13.7%, when buried (Fig. 4a). Contrarily to orthodox seeds that persist in seed banks, recalcitrant seeds do not show thick and hard coats or phenolic compounds in order to avoid predation and endure a whole season in seed banks waiting for better germination conditions in the next season (Bewley et al 2012). For A. angustifolia, it is known that scatter-hoarder animals provide effective seed dispersal (Iob and Vieira 2008, Vieira et al. 2011). These animals feed on A. angustifolia seeds and bury many seeds into the soil for future meals. As a masting tree species, A. angustifolia relies upon satiating scatter-hoarders, which enhances the chances that well-fed animals will not recover buried seeds (Theimer 2005). Scatter- hoarder may also be predated or die, leaving all cached seeds promptly to recruit. Thus, a possible extinction of specific dispersers could reduce tremendously the chance of natural perpetuation of Araucaria native Forests. Therefore, we could not support our first hypothesis that high amplitude in daily temperature enhances seed germination of this species. In the present study, it is likely that the seed burial at the beginning of seed dispersal season conserves seed water content and maintains the temperature around 17C, which is close to the ideal temperature for germination of A. angustifolia seeds. Notwithstanding, the sensitive point for such conclusions is the soil temperature or timing of seed dispersal. We have observed for three different years (2014, 2015 and 2016) that at Campos do Jordão state Park seed dispersal starts at the end of March/beginning of April and lasts until May. The soil temperature also decreased from 17C to 14C during the 90-day field experiment (Fig. 3b). In a previous study conducted in the south of Brazil (Kissmann and Habermann 2014), it is reported that A. angustifolia seed dispersal occurs between 31 May and June, as evidenced in natural Araucaria Forests remnants occurring in the states of Rio Grande do Sul (RS) and Santa Catarina (SC). These authors report daily temperature in the soil, measured during the seed dispersal, varying between 12.4 and 13.2C and air temperature oscillating between 9.4 and 15.0C. These temperature values are not close to the ideal temperature for germination of A. angustifolia seeds. Although tolerating and germinating at temperatures close to 10-15C, Kissmann and Habermann (2014) concluded that seeds of this species remain buried in the soil of Araucaria Forests before being exposed to critical temperature that allows germination, perhaps at the beginning of the spring. In the present study, we noted that buried seeds start germinating in up to 15 days after burial, and 60 days later 96.7% of the seeds are already germinated (Fig. 4a), while non-buried seeds seem to depend on rainfall because their %G peaked (%G = 79%) after 80% rain has occurred in the field (Fig. 3a and 4b). Taken together, this suggests that one must consider the current soil temperature (which is influenced by air temperature) and the regional factor as soil temperature may vary between regions during seed dispersal of this species. Although seed germination is critical for successful establishment of any species in any environment (Long et al. 2015), the rapid plant growth is also important. In the present study, seedlings were grown at different constant temperatures inside chambers that provided approximately 80 mol m -2 s -1 PPFD. Seedlings of this species grow in shade conditions of understory of Araucaria Forests and Upper Montane Mixed Ombrophylous Forest, as observed at Campos do Jordão state Park. Irradiance not higher than 200 mol m -2 s -1 is used to grow seedlings of A. angustifolia (Einig et al 1999). This demonstrates that the irradiance we used inside chambers are close to field conditions where A. angustifolia seedlings are found. Although plant height was similar between plants grown at 20C and 30C (Fig. 7a), after 60 days, the number of leaves 32 was higher when seedlings grew at 30C when compared to 20C (Fig. 7b). Thus, irradiance was not limiting and seedlings did not etiolate, as observed in Fig. 8. A previous study using one-yr-old A. angustifolia plants also shows that when grown under warm conditions (daily temperature oscillating between 19C and 33C) the number of leaves is higher than cool conditions (daily temperature oscillating between 17.5C and 20.8C) (Castro et al, 2017), indicating that this response must be conserved for this species. Our data also show that although seeds germinate at 10C (Fig. 5), seedlings do not develop shoots and leaves at this temperature even after 60 days (Fig. 8); and at 40C seeds do not even germinate (Fig. 5 and Fig. 8). This indicates that temperature around 20C allows the highest germination performance for this species as long as seeds are buried in the soil in order to maintain their seed water content above 35% (Gasparin et al, 2017) and to keep temperature as stable as possible. However, it seems that an early germination allows the establishment of seedlings before temperature reaches values as low as 10C as the winter approaches, as in fact occurred in the field experiment. In addition, (a “rapid”) seedling establishment could also avoid predation because during the winter A. angustifolia seed reserves represent a food source for the fauna (Paise and Vieira 2005), in detriment of established plants that are sclerophyllous (Castro et al 2017) and perhaps not attractive for animals. Transferring part of the seed reserve to the underground hypocotyl of A. angustifolia is a strategy for seedling establishment (Dillenburg et al. 2010) and this process lasts for at least 70 days (Einig et al., 1999), almost the same time period we evaluated seedling growth (Fig. 8). One must still consider the regional factor when judging A. angustifolia seedling establishment. As mentioned above, the seed dispersal season varies between regions 33 and temperature also varies accordingly. Therefore, an early germination and seedling establishment in the state of São Paulo (Campos de Jordão state Park) may not be so advantageous in the states of Rio Grande do Sul or Santa Catarina, where seed dispersal season occurs later and temperatures are usually lower (Kissmann and Habermann 2014). Thus, it is likely that for Araucaria Forests occurring at low altitudes but high latitudes in South America, seeds of this species must remain buried in the soil until being exposed to critical temperature that allows germination and seedling establishment at the beginning of the spring, as suggested by Kissmann and Habermann (2014). Considering that A. angustifolia seeds are recalcitrant, their suggestion merits further investigation. In conclusion, seeds of A. angustifolia benefit from burial due to the maintenance of seed water content above the critical moisture conditions these seeds can tolerate. When buried, stable temperature close to the ideal temperature for the germination of these seeds can also benefit the early germination of seeds of this species. The seedling establishment, however, might depend on the region Araucaria Forests occur because when the temperature reaches values as low as 10C during the winter, especially at latitudes below São Paulo state, it could impair seedling development and this merits further investigation. Acknowledgements We acknowledge the Coordination for Improvement of Graduate Personnel (Capes) for Ph.D. scholarships granted to Lorena de Castro and Patricia Leite, and the São Paulo Research Foundation for a Ph.D. scholarship granted to Felipe Pedrosa (Fapesp #2015/18381-6). G. 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Austral Ecol 35:.134-147. Paise G,Vieira EM (2005) Fruit production and spatial distribution of animal-dispersed angiosperms in a Mixed Ombrophilous Forest in State of Rio Grande do Sul, Brazil. Rev bras Bot 28:615-625. Panza V, Láinez V, Maroder H, Prego I, Maldonado S (2002) Storage reserves and cellular water in mature seeds of Araucaria angustifolia. Bot J Linn Soc 140: 273-281. Roberto GG, Coan AI, Habermann G (2011) Water content and GA3-induced embryonic cell expansion explain Euterpe edulis seed germination, rather than seed reserve mobilisation. Seed Sci Technol 39: 559-571 Theimer, TC (2005) Rodent Scatter hoarders as conditional mutualists. In: P-M Forget, Lambert JE, Hulme PE, Vander Wall SB (Eds) Seed Fate: Predation, dispersal and seedling establishment. CABI Publishing, Cambridge, MA. p. 283-297 Vander Wall SB (2002) Masting in animal-dispersed pines facilitates seed dispersal. Ecology 83: 3508-3516 Vieira EM, Ribeiro JF, Iob G (2011) Seed predation of Araucaria angustifolia (Araucariaceae) by small rodents in two areas with contrasting seed densities in the Brazilian Araucaria forest. Journal of Natural History 45: 843-854 Woodward FI, Williams BG (1987). Climate and plant distribution at global and local scales. Plant Ecol 69: 189-197. 37 Figures Fig 1 Experimental design of non-buried seeds protected by exclusion cages (a), buried seeds (b) and seeds germinated inside germination boxes in the laboratory (c) 38 Fig 2 Daily oscillation of soil and air temperatures observed in the field (a) and daily oscillation of soil and air temperatures simulated inside germination chambers in the laboratory (b) 39 Fig 3 Rainfall variation and water content of seeds (6 samples with 15 seeds) and soil (n = 5) during the field experiment (a). Mean, minimum and maximum air temperatures were used to calculate average values for every 15 days during the field experiment. Mean, minimum and maximum air temperatures and mean soil temperature during the field experiment (b). 40 Fig 4 Mean values (6 samples with 15 seeds each) of the percentage of germinated, intact and deteriorated seeds of A. angustifolia when buried (a) and not buried in the soil (b) 41 Fig 5 Mean values (6 boxes with 10 seeds each) of the percentage of geminated seeds of A. angustifolia within germination chambers at 5, 10, 15, 20, 25, 30, 35 and 40°C. Distinct letters show significant differences between temperatures by Scott Knott test (P < 0.05). Vertical bars are SD. 42 Fig 6 Mean values (6 boxes with 10 seeds each) of the percentage of geminated seeds of A. angustifolia at simulated air and soil temperatures inside germination chambers in laboratory conditions. Distinct letters show significant differences between temperatures by Student t-test (P < 0.05). The absence of letters indicates lack of significant differences between the temperatures. Vertical bars are SD 43 Fig 7 Mean values (n = 6) of plant height (a) and number of leaves (b) of A. angustifolia seedlings grown at 20°C and 30°C, during 60 days. Vertical bars are SD 44 Fig 8 Mean values (n = 6) of total biomass (roots, shoots and leaves, without seeds) of A. angustifolia seedlings grown at 10°C, 20°C, 30°C and 40°C in laboratory conditions for 60 days. Absence of letters indicates lack of significant differences between treatments and distinct letters indicate significant differences by Tukey test (P < 0.05). Vertical bars are SD Temperature (°C) 10 20 30 40 D ry w e ig h t (g ) 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 Lab d a b c 45 1 Artigo publicado no periódico Trees Structure and Function, (2017), p. 1-11. CAPÍTULO 2: Increased growth of Araucaria angustifolia under warm conditions is unaccompanied by increased photosynthetic performance 1 Lorena Egidio de Castro 1 , Camila Kissmann 2 , and Gustavo Habermann 3 * 1 Programa de Pós-Graduação em Biologia Vegetal, Univ Estadual Paulista (Unesp), Instituto de Biociências, Departamento de Botânica, Av. 24-A, 1515, Rio Claro, SP, 13506-900, Brazil 2 Univ Estadual Paulista (Unesp), Instituto de Biociências, Departamento de Botânica, Rua Prof. Dr. Antonio Celso Wagner Zanin s/n, Botucatu, SP, 18618-689, Brazil 3 Univ Estadual Paulista (Unesp), Instituto de Biociências, Departamento de Botânica, Av. 24-A, 1515, Rio Claro, SP, 13506-900, Brazil. E-mail: ghaber@rc.unesp.br; Tel: +0055 19 3526 4210 * Corresponding author Author Contributions: Conceived the idea and designed the experiment: LEC and GH Performed the experiment: LEC Analyzed data: LEC, GH and CK Provided reagents, instruments, material and analytical tools: GH Wrote the manuscript: LEC, GH and CK Key message: Although A. angustifolia occurs in regions with subtropical climate, warm conditions do not seem to impair the growth of young plants. 46 Abstract The effects of increasing temperature are worth studying even in tree species from subtropical climates. Araucaria angustifolia occurs in the south and southeast of Brazil and in Argentina and its growth and success may be associated with low temperatures. We measured growth, photosynthetic parameters and the nutritional status of this plant growing under artificial warm and cool conditions. We expected growth and photosynthetic performance to increase under cool rather than warm conditions. Under high daily temperature, plants showed increased leaf area per plant, more leaves, containing more nitrogen. However, CO2 assimilation rates at light saturation were similar in plants grown under both conditions, and photosynthetic nitrogen use efficiency was 25% higher in plants under cool conditions. This may be the first report of temperature effects on the growth of this species. Despite enhancing growth in A. angustifolia, warm conditions do not directly influence photosynthetic activities, but enhance leaf area per plant allowing increased CO2 uptake. Key words: Brazilian pine; ecophysiological responses; gas exchange; temperature. Introduction In general, plants grow more under warm conditions than under cool conditions. Plant growth (increase in plant size, leaf area and biomass), as a result of temperature, is dependent on thermal sensitivity of growth (Atkin et al. 2006), acclimation of respiration (Atkin et al. 2005), optimal temperature for photosynthesis (Berry and Bjorkman 1980) and, an interaction of the above-mentioned processes influences the carbon balance. Under warm conditions or when temperature rises, plants exhibit enhanced growth and increased photosynthesis (Hikosaka et al. 2006, Ribeiro et al. 2012), as the optimal temperature for photosynthesis increases due to changes in activation energy of enzymes involved in these processes (Hikosaka et al. 2006). However, some species are adapted to cool conditions. For example, Eucalyptus globulus shows increased photosynthetic rates under cool conditions (Costa e Silva et 47 al. 2009), as its genetic center of origin is believed to be in Tasmania, contrasting to its congeneric E. grandis, adapted to warm conditions. Gymnosperms are also adapted to temperate climates and, under cool conditions, are expected to have an advantage at some biological/ecological level (Arroyo et al. 1996). Larix decidua and Pinus mugo, two conifer species from montane provenances in Switzerland, show biomass production even at 6C (Hoch and Körner 2009), indicating that plants adapted to temperate climates maintain their growth capacity under conditions in which most plants would stop growing. These authors support that conifers do not deplete their carbon resources (photosynthetic activity), although extremely low temperatures may affect their meristematic processes (sink activity). There is much debate over the negative effects of increasing temperature on reproduction, growth and yields of plants that already are at their top limit responses to increasing temperature, especially of crop species with a C3 photosynthetic metabolism (Sage and Kubien 2007). On the other hand, studies discussing the effects of increasing temperature on the physiology of native plants adapted to cool or subtropical climates are less frequent (Saxe et al. 2001, Crawford 2008). Therefore, understanding the responses of plants from subtropical climate is important to predict whether these native populations are likely to expand or reduce the area of their natural occurrence in the case of a rise in temperature above average growing temperature for each species. Models predict that a 3 o C increase in the current temperature could reduce the occurrence of Araucaria angustifolia (Brazilian pine) in its endemic region in South America (Wrege et al. 2009). This species occurs in the south and southeast of Brazil and in Argentina (Reis et al. 2014). In Brazil, the distribution of A. angustifolia seems to exhibit an inverse relationship between latitude and altitude, so that in latitudes around 20°S it occurs at 1500-1800 m of altitude, while between 25°S and 30°S it 48 occurs at 500-900 m of altitude (Ledru and Stevenson 2012), indicating that this distribution pattern could be associated with cool and moist conditions of subtropical climate. In fact, a negative correlation between temperature and growth of male individuals of A. angustifolia, which grew more during the cold than in the warm season, has already been observed (Cattaneo et al. 2013). This indicates that cool conditions may benefit this species at some level. There are no studies determining the average growing temperature for A. angustifolia. Few ecophysiological studies have been conducted with this species, focusing on its response to contrasting irradiance (Duarte and Dillenburg 2000), soil compaction, water relations (Mósena and Dillenburg 2004, Cassana and Dillenburg 2013, Cassana et al. 2015), as well as comparisons between young and adult trees in the field (Franco et al. 2005). Studies about the effects of increasing temperature on the physiology of A. angustifolia are important, even though this species is associated with cool and moist conditions of subtropical climate. Indeed, no evidence has been collected on how A. angustifolia may react to a rise in temperature, or even to any contrasting temperatures. This lack of evidence may be due to the fact that the above-mentioned studies with this species concentrate on issues not related to identifying the best temperature for seedling production and reforestation, for example. Under this perspective, we evaluated growth, photosynthetic parameters and nutritional status of A. angustifolia seedlings grown under two contrasting artificial daily temperatures (warm and cool conditions). Given the natural occurrence and observations made in the field for this species, we expected that growth and photosynthetic performance to increase under cool rather than warm conditions. 49 Material and methods Plant material Plants of Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae) measuring 47  5.9 cm in height and with an age of 1.5 years were used. These plants were obtained from germinated seeds (‘pinhão’ as they are also called in local communities) collected at the municipality of Pilar do Sul, state of São Paulo, Brazil (2348’37’’ S, 4742’48’’ W) in April 2014. The leaves of this plant species are like a short-base (approximately 0.5 cm) isosceles triangle exhibiting 3-4 cm in length, a typical triangular-lanceolate leaf described for some gymnosperms, and its thickness averages 1 mm. Therefore, these are not needles, but leaves. A substrate made of oxisoil: organic substrate: sand (2:2:1 v:v:v) was prepared together and distributed to individual pots (10L) where the plants grew and were irrigated every other day. The pots were kept on benches in a greenhouse with semi- controlled conditions for five months before separating the groups of plants for the study. Study design and experimental conditions One group of 12 plants was maintained under warm conditions while another group of 12 plants was maintained under cool conditions for 120 days. The warm conditions were created using a plastic cover applied over a bamboo structure placed on a bench (3m x 1m x 0.80m) inside a greenhouse. This structure simulated a small greenhouse (3m in length, 1m in width and 1m in height above the bench) (Fig. 1A). The cool conditions were created using a plastic cover applied over a cubic structure made of aluminum frames placed on a bench inside the greenhouse. This cubic structure was 3m in length, 1 m in width and 1m in height, and it had a small window (0.45 x 0.32 m) where an air conditioner (Springer, Porto Alegre, Brasil) was placed to provide 50 cooler air than that inside the greenhouse (Fig. 1B). Air temperature (C) and photosynthetic photon flux density (PPFD; mol m -2 s -1 ) were measured inside both structures using a 1400-101 air temperature sensor (LI-COR, Lincoln, NE, USA) and a LI-190-SA quantum sensor (LI-COR, USA), respectively. Under each condition, these sensors were connected to a data logger (LI-1400, LI-COR, USA) that collected data every 30 min on a daily basis. Daily amplitude in air temperature was larger under warm conditions (14.5C) when compared to cool conditions (3.3C). Under warm conditions, temperature varied from 19C (3:00 - 6:00 h) to 33C (12:00 – 14:00 h), while under cool conditions it only varied from 17.5C (3:00 – 6:00 h) to 20.8C (15:00 h) (Fig. 2A). Using plants growing in both conditions, we measured plant height, number of new shoots, root-collar diameter, gains in shoot length, root-collar diameter and number of new shoots (n = 12) as well as leaf gas exchange rates at 30, 60, 90 and 120 days (n = 6). At 120 days of study, we measured root length, root volume, number of leaves per plant, total leaf area per plant, and the biomass of roots, shoots, leaves and total biomass (n = 5). At 120 days of study, we also measured the photosynthetic nitrogen use efficiency (PNUE), as well as total contents of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulphur (S) in the roots, shoots and leaves (n = 3). Soil fertility parameters of the substrate used in the pots of plants grown under both conditions were also assessed (n = 3). Soil fertility At the end of the study, soil samples were randomly collected from five pots from both warm and cool conditions. These samples were taken to the Soil Science Lab at University of São Paulo (USP, Esalq), in Piracicaba, SP, Brazil for routine soil 51 chemical (fertility) analysis (pH in CaCl2), which was performed according to van Raij et al. (2001), and the procedures are described in English by Dantas and Batalha (2011). Biometric parameters Plant height was measured with a ruler (cm), root-collar diameter with digital calipers (mm) and the number of new shoots (emitted since the beginning of the study) was counted. The gains in the shoot length, root-collar diameter and number of new shoots considered the increase in the length, diameter and number of these parameters since the start of treatment application. At the end of the study, root length was measured with a ruler (cm), and root volume assessed by water dislocation when excised root systems of plants were inserted into a graduated cylinder (mL). At 120 days of experiment, all the leaves of each of the five replicates were excised and counted and total leaf area per plant (cm 2 ) was measured with an area meter (LI-3100C, LI-COR, USA). Subsequently, leaves, shoots and roots of the whole plant of each of the five replicates were placed in paper bags and dried at 60°C (to avoid N volatilization) until constant mass. The biomasses (g) of organs were assessed with an analytical scale. Plant nutritional status After assessing the biomasses of organs, three (already dry) samples of leaves, shoots and roots were ground and digested in a nitric-percloric acids solution. Potassium concentration was determined in a flame photometer (Micronal B262, Micronal, São Paulo, Brazil). Calcium and Mg were determined by the atomic absorption spectrophotometer method. Phosphorus was quantified colorimetrically, S was determined using a turbidimetric method and N was measured by the micro-Kjeldahl method, all of which followed Sarruge and Haag (1974) and Dantas and Batalha (2011), and was performed in the routine Plant Nutrition Lab (Esalq, Usp, Piracicaba, SP, Brazil). 52 Leaf gas exchange The CO2 assimilation (A, mol m -2 s -1 ) and transpiration (E, mmol m -2 s -1 ) rates, stomatal conductance (gs, mol m -2 s -1 ), and intercellular CO2 concentration (Ci, mol mol -1 ) were assessed in fully expanded triangular-lanceolate leaves with an open portable gas exchange system (LI-6400xt, LI-COR, Lincoln, NE, USA). Water use efficiency (WUE, A/E) and intrinsic water use efficiency (IWUE, A/gs) were also calculated. Photosynthetic nitrogen use efficiency (PNUE, mol CO2 g -1 N s -1 ) was calculated by the ratio between A (transformed to mol CO2 g -1 leaf s -1 ) and leaf N content (g N per g dry leaves). One leaf per measurement was accommodated within the leaf chamber (6400-40 LCF, LI-COR) and measurements were performed between 9:00 and 11:00 h on cloudless days. The leaf area used to calculate gas exchange was the average from 15 leaves when disposed within the inner rubber ring, resulting in 0.7 cm 2 . The PPFD was provided by a red (90%) and blue (10%) LED light source (6400-02B led light source, LI-COR, USA), on top of the leaf chamber, set to provide 1200 µmol m -2 s -1 , as this value returned saturating A for A. angustifolia observed in a previous study (data not shown). CO2 concentration in the leaf chamber averaged 400 µmol mol - 1 , as provided by the 6400-01 CO2 mixer (LI-COR, USA). Vapor pressure deficit (VPD; kPa), relative humidity (RH) and air temperature inside the leaf chamber were not set artificially, but oscillated with the external environment. Under warm conditions, VPD was 3.17 ± 0.72 kPa, RH 47.8 ± 7.3% and air temperature 34.0 ± 2.2C, while under cool conditions VPD was 2.62 ± 1.53, RH was 50.3 ± 4.9 and air temperature was 26.8 ± 2.5C. Data analysis A Student t test (α = 0.05) was performed (after checking for normal data distribution and homogeneous variance of data) between plants cultivated in warm and 53 cool conditions, testing gas exchange variables (A, gs, E, Ci, WUE, IWUE) (n = 6) and non-destructive parameters (gains in shoot length, root collar diameter and number of shoots) at 30, 60, 90 and 120 days of experiment (n = 12). Notwithstanding, we also run an analysis of variance (two-way Anova) to check gas exchange data between treatments over time, especially for CO2 assimilation rates that could result in some consequences for explaining the plant biomass. For this, time was used as one of the factors (four levels – 30, 60, 90 and 120 days of experiment) and temperature as the other factor (two levels). Using five replicates for biometric parameters (number of leaves, shoot and root lengths, root volume, total leaf area per plant and biomass of organs) and three replicates for PNUE, macronutrient contents in leaves, shoots and roots, we also used a Student t test (α = 0.05) to test these variables at 120 days of experiment. Statistical tests were performed using SigmaPlot 12.0 Software, and standard deviation (SD) is given in all figures and tables. Results The results of soil fertility were similar for plants growing under warm and cool conditions (Table 1). However, plants under warm conditions showed 42% higher leaf N content when compared to those under cool conditions (Table 2). Leaf S content was also higher (+13%) in plants under warm conditions when compared to those under cool conditions (Table 2). Daily variations in PPFD under warm conditions were very close to daily variations in PPFD under cool conditions. At 12:00 h, average PPFD under warm conditions was 205 mol m -2 s -1 , while under cool conditions, it averaged 272 mol m -2 s -1 (Fig. 2B). 54 CO2 assimilation (Fig. 3A) and transpiration (Fig. 3B) rates were the same for plants under both conditions measured on every evaluating day when using the Student t test. When a two-way Anova was used to check differences between treatments over time, it was shown that time exerted effects on A, but no significant interaction (time x temperature) was observed (Table 3). Stomatal conductance (gs) and intracellular CO2 (Ci) were higher for plants grown under cool conditions at 30, 60 and 90 days. Water use efficiency (Fig. 3D) and IWUE (Fig. 3F) showed a great variability, but WUE was higher in plants grown under warm conditions when compared to those under cool conditions only at 30 days of experiment, while for IWUE, higher values for plants under warm conditions occurred at 30 and 90 days of experiment. The root length (Fig. 4B) and root volume (Fig. 4D) did not differ between plants grown under both conditions. However, the shoot length was 33% greater in plants under warm conditions when compared to those under cool conditions (Fig. 4A). The number of leaves was 40% greater in plants under warm conditions when compared to those under cool conditions (Fig. 4C), and leaf area per plant was 30% larger in plants under warm conditions (Fig. 4F). On the other hand, the biomass of roots, shoots and leaves was the same in both treatments, being only slightly higher in plants under warm conditions when the total biomass was compared between treatments (Fig. 4E). The gain in shoot length was greater in plants under warm conditions when compared to that under cool conditions at every evaluation day (Fig. 5A). The gain in root-collar diameter was the same for plants grown under both conditions (Fig. 5B), while the gain in the number of new shoots was higher for plants under warm conditions when compared to those under cool conditions only at 120 days of experiment (Fig. 5C). 55 The PNUE of plants under warm conditions was 25% lower when compared to those under cool conditions (Fig. 6). Discussion Contrary to our hypothesis, the results demonstrate that warm conditions enhance growth in A. angustifolia. Plants, in general, have an optimal as well as minimum and maximal air temperature for vegetative growth (McClung and Davis 2010, Hatfield and Prueger 2015). Although A. angustifolia occurs in high latitudes and/or high altitudes (Franco et al. 2005; Duarte et al. 2006), which reinforces that its growth and success may be associated with low temperatures, there are no studies showing the optimum temperature range for the vegetative growth of this species, making a contrast with studies of seed germination of this species. For seed germination of A. angustifolia, it has been demonstrated that the optimum temperature varies between 20C and 25C (Kissmann and Habermann 2014). Although the biomass of organs measured at the end of the study showed similar values between plants under both conditions (Fig. 4E), the shoot length (Fig. 4A) and the number of leaves (Fig. 4C) were 25% and 40% higher in plants under warm conditions than in cool conditions, respectively. Temperature rise, in general, may increase stem diameter, plant height, and shoot biomass (Way and Oren 2010). In the present study, the gain in shoot length showed a five-fold increase between 30 and 120 days of experiment for plants under warm conditions, while for plants under cool conditions such increment was only three-fold (Fig. 5A). This indicates that under warm conditions, the production of leaves and the shoot apex activity (sink activity) were considerably higher than under cool conditions. Therefore, the present study might be the first report on plant growth of A. angustifolia under contrasting temperatures. 56 In the present study, the temperature in the leaf chamber was similar to growing conditions for the warm treatment; however, the air temperature of 26.8C in the leaf chamber exceeded the temperature at growing conditions for the cool treatment (20 ± 2.8C; Fig. 2). This could have artificially increased A of plants under cool conditions. Nevertheless, carbon assimilation is maintained stable under a 17-34°C temperature range when measured in Quercus rubra, Q. falcata, Betula alleghaniensis and Populus grandidentata (Gunderson et al. 2010). For Populus deltoides, increase in temperature results in enhanced plant growth, but has no effect on A (Cerasoli et al. 2014). Therefore, an increment of 6C in the leaf chamber does not seem to influence A of plants grown in cool conditions. Thus, despite growing more under warm conditions, A was the same between plants in both treatments (Fig. 3A), which may refute our prediction that photosynthetic performance of A. angustifolia increases under cool conditions, and it deserves further investigation. The difference in mean temperature between cool and warm conditions was 6°C, but the amplitude of daily temperature was 3.3°C under cool and 14.5°C under warm conditions (Fig. 2). Some studies have shown that the amplitude of daily temperature exerts stronger influence on the vegetative growth when compared to the influence of daily mean temperature on plant growth (Bueno et al. 2012, Baoguo et al. 2014). For instance, higher vegetative growth and biomass of Populus deltoides (eastern cottonwood) is noted under a 5°C amplitude in daily temperature when compared to a constant daily temperature even when under the same daily mean temperature for both conditions (Cerasoli et al. 2014). However, P. deltoides is not native to temperate climates, and could benefit from any temperature rise, including the amplitude in daily temperature. When Larix decidua and Pinus mugo, two conifer species, are submitted to constant vs. variable 6°C and 12°C (minimal and optimum temperatures for both 57 species), these plants show relative insensitivity of growth to the presence or absence of variability in temperature (Hoch and Körner 2009), suggesting that species adapted to cool conditions are not plastic to temperature variability. There are no studies evidencing the minimal, optimum and maximal temperatures for growth of A. angustifolia, and this may be the first study contrasting temperatures for this species. Since all plants within both treatments tested here originated from one population (Pilar do Sul, SP, southeastern Brazil), other provenances of A. angustifolia should be tested under contrasting temperatures. Thus, in the present study, it is possible that A. angustifolia might have benefited from the higher amplitude in daily temperature under warm conditions in contrast to the five-fold lower amplitude in daily temperature that occurred under cool conditions, and this topic merits further investigation. Plants under both conditions showed similar A values over time (Fig. 3A), although plants under warm conditions exhibited higher total biomass (Fig. 4E). This could be due to gas exchange measured at saturating light, while growing conditions provided lower light intensity (Fig. 2B). However, A. angustifolia young plants also grown under 200 mol m -2 s -1 of PPFD exhibited light saturated (at 1000 mol m -2 s -1 of PPFD) A value of 5.4 ± 1.8 mol CO2 m -2 s -1 (Einig et al. 1999), which is similar to A values of plants grown in warm and cool conditions in the present study. Therefore, it is unlikely that gas exchange measurement was biased by different light intensities between growing and measurement conditions. These same authors demonstrated that mature fully developed leaves should be used for measuring gas exchange in this species, as A tend to drop by 50% in young leaves of A. angustifolia. In the present study we also used mature leaves to assess gas exchange data. Although plants under cool conditions maintained higher gs values until 90 days of study in relation to plants grown under warm conditions (Fig. 3E), gs did not 58 influence A of any plant group because A was similar between treatments during this period. Cool conditions were provided with an air conditioner, which also dries air. However, the relative humidity varied by only 5% in measuring conditions between treatments. In addition, VPD did not seem to influence gas exchange in measuring conditions, as no curve could be regressed between VPD and these rates (see Fig. S1 in supplementary material). For plants under cool conditions, the larger gs for the first 90 days of study did not represent any advantage, such as to increase A, although their Ci values were higher than those observed in plants under warm conditions for the same period (Fig. 3C). This did not represent any disadvantage either, as it did not allow increased E. Nevertheless, plants under warm conditions showed 30% larger total leaf area per plant (Fig. 4F). Therefore, plants grown in warm conditions can take up more carbon at similar CO2 assimilation rates as plants under cool conditions. High temperature is also associated with variations in nutrient uptake and usage by the plant. In Pseudotsuga menziesii, a native tree from temperate climates, leaf N content was lower and plants showed different amino acid groups in a high day/night temperature (30/25°C) when compared to a mild day/night temperature (20/30°C) (Baoguo et al. 2014). In the present study, plants under warm conditions showed almost 30% more N in their leaves in comparison to plants grown in cool conditions (Table 2), although both groups of plants grew on substrates with similar fertility (Table 1). This could be associated with the higher growth of plants under warm conditions because tree growth and foliar N content seem to be positively and linearly associated (Davidson et al. 2007), although these authors used legume and non-legume species in their study. However, although accumulating more N in their leaves, plants under warm conditions showed a 20% lower PNUE in relation to plants in cool conditions (Fig. 6). It seems that the uptake of more N allowed plants under warm conditions to produce more leaves 59 increasing their total leaf area per plant (Fig. 4F), which eventually led to take up more carbon at similar CO2 assimilation rates as plants under cool conditions (Fig. 3A). Leaves of A. angustifolia are very sclerophyllous. This indicates that these leaves invest less in leaf area and symplastic components and more in thickness and structural components, which requires less N and results in reduced specific leaf area (leaf area per leaf mass, SLA) (Reich et al. 1998). In fact, most of the N absorbed by adult and young plants of A. angustifolia in the field is metabolized in the roots, suggesting a minor role for N in shoots and leaves (Franco et al. 2005). Thus, more N in plants under warm conditions did not directly influence A, which was similar between treatments. In addition, it is unlikely that N was distinctly allocated to photosynthetic (chloroplasts and cytoplasm) or structural (cell walls and fibers) apparatus of A. angustifolia’s leaves of plants from any of the conditions because SLA was similar for plants grown in both conditions (see Fig. S2 in supplementary material). Therefore, warm conditions enhanced N uptake and the production of morphologically similar leaves of A. angustifolia plants. One could argue that plants under cool conditions were subjected to 25% more sunlight when compared to plants under warm conditions, specifically between 10:00h and 16:00h (Fig. 2B). However, plants under cool conditions grew less than those under warm conditions, indicating that 25% more PPFD did not make any difference to the performance of these plants. More importantly, it confirms that the temperature contrast was more important than the PPFD difference. Our results indicate that despite the fact that A. angustifolia can grow more under warm conditions this could not be explained by carbon assimilation rates, which was similar for plants growing under both conditions. Warm conditions enhanced N uptake and the production of leaves, increasing the total leaf area per plant. This 60 allowed plants grown under warm conditions to take up more carbon at similar CO2 assimilation rates as plants under cool conditions. Acknowledgments Lorena E. de Castro acknowledges the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes) for a Ph.D. scholarship. C. Kissmann acknowledges the Post-Doctoral program at São Paulo State University (Prope/Unesp, IB Rio Claro). G. Habermann acknowledges the Brazilian National Council for Scientific and Technological Development (CNPq) for a productivity fellowship (Grant 308902/2014- 9). 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