Departamento de Botánica
Facultad de Ciencias Naturales y Oceanográficas
Universidad de Concepción
VALOR ADAPTATIVO DE LA VÍA FOTOSINTÉTICA CAM PARA ESPECIES
CHILENAS DEL GÉNERO PUYA (BROMELIACEAE)
Tesis para optar al grado de Doctor en Ciencias Biológicas, Área de especialización
Botánica
IVÁN MARCELO QUEZADA ARRIAGADA
Profesor guía: Dr. Ernesto Gianoli M.
Profesor co-tutor: Dr. Alfredo Saldaña M.
Comisión evaluadora de tesis, para optar al grado de Doctor en Ciencias Biológicas Área Botánica “Valor adaptativo de la vía fotosintética CAM para especies chilenas del género Puya (Bromeliaceae)”
Dr. Ernesto Gianoli ______Profesor Guía
Dr. Alfredo Saldaña ______Co-tutor
Dr. Carlos M. Baeza ______
Dra. María Fernanda Pérez ______Evaluadora externa
Dra. Fabiola Cruces ______Directora (S) Programa Doctorado en Botánica
Septiembre 2013
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A Paula, Leonor y Julieta
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AGRADECIMIENTOS
El completar exitosamente una tarea de esta magnitud se debe, en gran medida, a todos quienes me brindaron su apoyo, consejo o ayuda en algún punto de este largo camino.
En primer lugar debo agradecer a Paula, mi esposa, amiga y compañera, por soportar conmigo estos 4 años y medio de esfuerzo, sacrificios y más de alguna recompensa. No solo ha sido soporte para mi espíritu durante todo este tiempo, sino que además fue la mejor compañera de terreno que pude haber encontrado. Agradezco también a mi hija mayor,
Leonor, inspirada dibujante, talentosa fotógrafa y la mejor asistente de muestreo que existe, cuya mirada de felicidad y asombro durante los largos viajes en los que me acompañó fue el mejor recordatorio de que la vida hay que disfrutarla, siempre. También, y aunque llegó al final de este largo camino, agradezco a Julieta, quien ha sido el impulso que necesitaba para darme a la tarea de concluír este trabajo. Sin ustedes nada de esto existiría, de eso no tengo dudas. Este logro también es suyo.
Por cierto, debo también agradecer a mis padres, Héctor y Ana, cuyo apoyo jamás me ha faltado, sobre todo en los momentos difíciles, que han sido muchos.
De igual forma, agradezco a mis profesores guía, Ernesto Gianoli y Alfredo Saldaña.
En ambos he encontrado excelentes guías y consejeros. Para mí ha sido un privilegio contar con su apoyo en todas las etapas de este trabajo, el que sin lugar a dudas contribuyeron a llevar a buen puerto.
A todos los compañeros y amigos que forman o han formado parte del Laboratorio de Ecología Funcional, con quienes compartí buenos y malos momentos, y de repente algunas cervezas. En especial quiero agradecer a Fernando Carrasco, gran amigo y compañero de
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terrenos y conversaciones, así como a Matías Pérez, Cristian Torres, Susana Gómez y
Cristian Salgado. Todos ellos, en algún momento del desarrollo de este trabajo, aportaron desinteresadamente con comentarios y sugerencias que no hicieron más que enriquecer el resultado final.
A los profesores del programa de Doctorado en Ciencias Biológicas, área Botánica, sin excepción, mis mayores agradecimientos.
También deseo agradecer a quienes, desde fuera de la Universidad de Concepción, entregaron un apoyo importante a este trabajo. A Gerhard Zotz, de la Universidad de
Oldenburg, quien me recibió en su grupo de trabajo durante casi 3 meses, y con quien aprendí muchísimo. Asimismo a don Patricio Novoa, del Jardín Botánico Nacional, cuyo aporte en información fue invaluable para ayudarme a localizar las poblaciones de Puya idóneas para mi trabajo.
Finalmente, a Gloria Morales y Fabiola Gaete, secretarias del Departamento de
Botánica y del Postgrado respectivamente, mi enorme agradecimiento por todas las gestiones, algunas de última hora, que siempre llevaron a cabo con la mejor voluntad.
Esta tesis doctoral fue financiada íntegramente por CONICYT, mediante dos becas: Beca de
Doctorado en Chile (21090071) y Beca de Apoyo a Tesis Doctoral (24110160).
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INDICE (TABLA DE CONTENIDOS)
Estatus de los manuscritos 6
Resumen 7
Introducción general 10
Hipótesis 24
Objetivos 25
Capítulo I: “Crassulacean acid metabolism photosynthesis in Bromeliaceae: an evolutionary key innovation” 26
Capítulo II: “Crassulacean acid metabolism varies with latitude in Chilean bromeliads” 51
Capítulo III: “Latitudinal variation in the degree of Crassulacean acid metabolism in Puya chilensis: advantages of the use of two analytical methods” 84
Capítulo IV: “Phenotypic selection on CAM photosynthesis in Puya chilensis: Water availability as a main driver” 113
Discusión general 139
Conclusiones 150
Referencias generales 153
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ESTATUS DE LOS MANUSCRITOS
Capítulo I: “Crassulacean acid metabolism photosynthesis in Bromeliaceae: an evolutionary key innovation” Publicado en Biological Journal of the Linnean Society 104: 480-486
(2011).
Capítulo II: “Crassulacean acid metabolism varies with latitude in Chilean bromeliads” En preparación.
Capítulo III: “Latitudinal variation in the degree of Crassulacean acid metabolism in Puya chilensis: advantages of the use of two analytical methods” Enviado a Functional Plant
Biology. En segunda revision.
Capítulo IV: “Phenotypic selection on CAM photosynthesis in Puya chilensis: Water availability as a main driver” En preparación.
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RESUMEN
La distribución de una especie puede estar determinada tanto por las condiciones ambientales como por la capacidad de ésta para tolerar dichas condiciones y sus posibles cambios. Los atributos, conocidos como adaptaciones, que confieren a los organismos la capacidad de establecerse exitosamente en un hábitat determinado al optimizar su adecuación biológica pueden ser “arrastrados” hacia nuevos hábitats donde no son estrictamente necesarios. Esto puede tener efectos negativos sobre la adecuación biológica de los organismos al actuar como
“lastre”, consumiendo recursos que podrían beneficiar otros procesos dentro de la planta o interactuando de forma negativa con atributos que la podrían beneficiar. El efecto de un
“lastre” sobre el avance geográfico, o la permanencia bajo nuevas condiciones ambientales, de un grupo de organismos podría ser evaluado utilizando un grupo de distribución amplia y especializado en determinados ambientes, y que tenga una parte de sus integrantes ocupando zonas donde las condiciones sean distintas. Para esto se puede evaluar la forma en que el atributo en cuestión varía entre distintos ambientes, y el valor adaptativo que posee en aquellos hábitats donde, supuestamente, es innecesario. En el presente trabajo de tesis evaluamos el valor adaptativo de la vía fotosintética CAM en la familia Bromeliaceae y, en particular, en las especies endémicas de Chile del género Puya. Las especies endémicas de esta familia se distribuyen a lo largo de un gradiente latitudinal de temperatura y precipitaciones que comprende las zonas de clima desértico, mediterráneo y templado- lluvioso de Chile central y centro-sur. Nuestros resultados demuestran que, en primer lugar, la vía CAM fue una adapración clave para la familia Bromeliaceae, facilitando su diversificación a nivel continental. En segundo lugar, que la expresión de la vía CAM está directamente relacionada con la latitud en Chile central. Finalmente, demostramos que en
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algunas especies esta vía fotosintética se expresa diferenciadamente entre poblaciones que viven bajo distintos regímenes de humedad, y que mientras en las que viven en zonas semiáridas la vía CAM tiene efectos positivos sobre el fitness, en aquellas que viven en zonas húmedas el expresarla tiene efectos negativos sobre éste. A partir de estos resultados inferimos diversas consecuencias de la mantención de la vía CAM en zonas donde no es necesaria, y su posible efecto “lastre” para algunas bromelias chilenas que se encuentran al sur de los 35° S.
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INTRODUCCIÓN GENERAL
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INTRODUCCIÓN
La capacidad de una especie para aprovechar los recursos disponibles, tolerando además condiciones ambientales variables, podría definir su rango de distribución geográfica y su potencial para avanzar hacia nuevos hábitats (Begon et al. 2006). Especies con tolerancia a una mayor heterogeneidad ambiental, mediada por los atributos funcionales adecuados, presentarán distribuciones más amplias y nichos potenciales mayores que aquellas cuyas respuestas son limitadas (Wiens y Donoghue 2004). Un ejemplo de ésto es el caso de las especies tolerantes a la desecación, capaces de tolerar un amplio rango de disponibilidad de agua y sobrevivir a periodos de ausencia absoluta de ésta, pudiendo expandir su rango de distribución hacia zonas con una alta variabilidad en la disponibilidad de agua (Yang et al.
2003, Alpert 2006).
Estos casos, en que grupos de organismos (especies, poblaciones) están especializados en las condiciones de un hábitat particular, están usualmente mediados por atributos que han aparecido como respuesta a éstas, conocidos como adaptaciones (Futuyma 2009, Futuyma y
Agrawal 2009). Una adaptación se define como “aquella variante fenotípica que resulta en la mayor adecuación biológica dentro de un grupo específico de variantes en un ambiente dado”
(Reeve y Sherman 1993). En las especies vegetales, el papel de las adaptaciones ha sido destacado en distintos ámbitos, como la defensa contra la herbivoría, formas de crecimiento o medios de dispersión de semillas (Farrell et al. 1991, Eriksson y Bremer 1992, Gaut et al.
1992, 1996, Rieseberg 1997, Andreasen y Baldwin 2001), atributos a los que se les atribuye valor adaptativo. Se considera que la aparición de caracteres que permiten el aprovechamiento de nuevos nichos ecológicos, en algunos casos promoviendo el aislamiento
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reproductivo, fomentaría los procesos de especiación (Coyne y Orr 2004). En este sentido, algunas adaptaciones, conocidas como “innovaciones clave”, han jugado un rol importante en la diversificación de ciertos clados, permitiéndoles explotar exitosamente nichos ecológicos previamente no utilizados, o subutilizados, lo que resultó en un aumento en su diversificación (Simpson, 1953; Heard & Hauser, 1995). Atributos identificados como innovaciones clave incluyen la forma de crecimiento anual, el hábito trepador, frutos carnosos, modificaciones en las semillas que facilitan la dispersión o los canales de resinas
(Farrell et al., 1991; Eriksson & Bremer, 1992; Ricklefs & Renner, 1994; Hodges, 1997;
Dodd et al., 1999; Andreasen & Baldwin, 2001; Smith, 2001; Gianoli, 2004).
Atributos “lastre”.
En el caso de que las condiciones ambientales cambien, o que parte de un grupo especializado de organismos avance hacia regiones con condiciones ambientales distintas de aquellas para las que se encuentra adaptado, el mantener las adaptaciones que permitieron su especialización podría implicar “arrastrar” hacia el nuevo ambiente algunos atributos que pueden dejar de ser necesarios, ya que no existiría el requerimiento para el cual eran útiles
(Armbruster 1997, Guimaraes et al. 2008, Johnson 2009).
En ese escenario, algunos atributos funcionales que pueden haber servido como adaptaciones podrían convertirse en un “lastre” evolutivo. Estos atributos dificultan la diversificación o el avance de una especie dentro o hacia una nueva zona geográfica, ya sea por el costo en recursos que implica mantenerlos como por su interacción negativa con otros atributos que podrían ser beneficiosos para el organismo (Wilson 1988, Begon et al. 2006, Andrade et al. 11
2007). Un ejemplo de esto es la partenocarpia en algunas especies de Pistacia, atributo que habría surgido como una distracción para predadores de frutos, pero que reduce significativamente la eficiencia reproductiva de estas especies en lugares donde los predadores no se encuentran (Zangerl et al. 1991, Fuentes y Schupp 1998, Verdú y García-
Fayos 2001, 2002). Otro ejemplo es la producción de frutos de gran tamaño en algunas especies tropicales, los que eran consumidos por la megafauna local, la cual se encargaba de dispersar las semillas. Actualmente, con la megafauna extinta, la mayor parte de los frutos de estas especies se pierden al podrirse en el suelo luego de caer, y solo unos pocos son consumidos por fauna introducida (Guimaraes et al. 2008, Johnson 2009). La producción de estos frutos de gran tamaño implica un enorme gasto de recursos para los individuos, los cuales se ven desperdiciados al no cumplirse el objetivo que es la dispersión de sus semillas.
En este caso, una especialización cuyo fin era utilizar a la megafauna como dispersora se habría vuelto un lastre.
Surge la interrogante, entonces, sobre cuál es el valor adaptativo de determinados atributos que, si bien se cree que pueden haber sido claves para la diversificación de un grupo bajo condiciones determinadas, han sido conservados aun cuando parte de dicho grupo se encuentra en zonas donde las condiciones ambientales no los hacen necesarios. En esta tesis, consideramos evaluar el valor adaptativo de la vía fotosintética CAM, una adaptación fisiológica a la aridez. Este mecanismo involucra la apertura de los estomas durante la noche para capturar el CO2 atmosférico, el que es fijado en ácido málico mediante la enzima
Fosfoenolpiruvato Carboxilasa (PEPC) y almacenado dentro de las vacuolas celulares.
Durante el día el ácido málico es transportado fuera de las vacuolas y descarboxilado, recuperándose el CO2, el que es integrado al ciclo de Calvin, que se realiza con los estomas 12
cerrados (Ting 1985, Ehleringer y Monson 1993). Esta separación temporal de las fases fotosintéticas le permite a la planta realizar sus procesos fotosintéticos diurnos sin alteración, pero evitando la pérdida de agua por transpiración que se produciría al abrir los estomas a las horas de mayor radiación solar (Taiz y Zeiger 2002, Lüttge 2002, Larcher 2003, Heldt 2005).
A nivel de especie, la vía CAM puede ser constitutiva, es decir que el individuo siempre la utiliza, o facultativa, es decir que los individuos pueden pasar de la vía C3 a CAM o viceversa dependiendo de sus necesidades (Taiz y Zeiger 2002, Lüttge 2004, Winter et al. 2008), y se ha registrado variabilidad dentro de algunas especies en la expresión de CAM a lo largo de gradientes de humedad (Kluge et al. 1991; Scarano et al. 2002; Lüttge 2004). La fotosíntesis
CAM es el tipo de fotosíntesis más eficiente en cuanto al uso del agua (Ehleringer y Monson
1993) y es bastante frecuente en familias especializadas en hábitats xéricos, como Cactaceae,
Bromeliaceae y Orchidaceae (Ehleringer y Monson 1993, Cushman 2001, Taiz y Zeiger
2002, Lüttge 2002).
Para evaluar la posible calidad de “lastre” de la vía CAM consideramos recurrir a un grupo de especies filogenéticamente cercanas, que presente distribución amplia y que se destaque por su adaptación a la aridez, y del cual uno o más clados tengan presencia en zonas en las que la disponibilidad de agua no es limitante, aunque manteniendo el uso de dicha vía fotosintética.
Bromeliaceae.
Una familia con presencia prominente de la vía CAM, y altamente especializada a la aridez, es la familia Bromeliaceae (Smith y Downs 1974, Benzing 2000). Esta familia es de 13
distribución casi exclusivamente Neotropical, salvo por la presencia de una especie en África occidental (Smith y Downs 1974, Smith y Till 1998, Givnish et al. 2004). En América se encuentra desde el sur de los Estados Unidos hasta la zona centro-sur de Chile (Fascicularia) y Argentina (Tillandsia) (Zizka et. al. 2009). Se distribuye en distintas regiones climáticas, como el hábitat epífito de las selvas tropicales de América central y Brasil o regiones altamente xéricas en el sur del Perú, el altiplano boliviano y el norte de Chile (Benzing 2000).
La familia Bromeliaceae comprende alrededor de 3200 especies, agrupadas en 58 géneros
(Luther 2008) y se divide en 8 subfamilias: Tillandsioideae, Bromelioideae, Puyoideae,
Navioideae, Hechtioideae, Pitcairnioideae, Lindmanioideae y Brocchinioideae (Givnish et al. 2004). Dentro de cada subfamilia, el ordenamiento filogenético es relativamente conocido, y ha sido publicado por diversos autores, con diferencias menores (Terry et al.
1997, Crayn et al. 2000, 2004, Barfuss et al. 2005, Givnish et al. 2004, 2007, 2011, Horres et al. 2007, Rex et al. 2007, 2009, Schulte et al. 2009, Chew et al. 2010, Jabaily y Sytsma
2010, 2013).
La mayor parte de las especies de la familia Bromeliaceae presenta adaptaciones morfológicas y fisiológicas que les permiten desarrollarse en ambientes donde el agua es un recurso escaso, tales como el hábitat epifito en las zonas tropicales, sustratos con muy baja retención de agua o zonas desérticas/semidesérticas (Smith y Downs 1974, Benzing 2000).
La principal adaptación fisiológica es la ya mencionada fotosíntesis CAM, la que se considera presente en, aproximadamente, entre un tercio y la mitad de las especies de la familia (Lüttge 2004). Las adaptaciones morfológicas corresponden principalmente a la formación de tanques en el centro de la roseta, destinados a capturar y acumular el agua de
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lluvia, y al desarrollo de tricomas foliares absorbentes, cuya función es capturar y absorber el agua atmosférica (Smith y Downs 1974, Zotz y Winter 1994, Benzing 2000). Para otros atributos morfológicos que han sido descritos como respuestas a la aridez en plantas, tales como el desarrollo de hojas más gruesas, un mayor valor de LMA (razón masa foliar/área foliar) o la suculencia foliar, no se ha evaluado aún el posible valor adaptativo que puedan tener en Bromeliaceas, aunque éste no debe ser descartado a priori.
En la familia Bromeliaceae la aridez parece haber jugado un papel clave dentro de los procesos de diversificación (Benzing 2000), algo esperable debido a la alta exigencia que implica desarrollarse en ambientes áridos, considerados como un factor de gran influencia dentro de la evolución vegetal (Axelrod 1972, Arroyo et al. 1988). Si bien una parte considerable de las especies de la familia se desarrolla en hábitats áridos, hay taxa que se encuentran en hábitats menos áridos, como es el caso de las especies nativas de Chile centro- sur (32-43°S), que habitan en una zona geográfica donde el agua no es un recurso escaso
(DiCastri y Hajek 1976, Luebert y Pliscoff 2006) y, por ende, las adaptaciones mayores a la aridez no serían necesarias (Varadarajan 1990, Jabaily y Sytsma 2010). Un clado dentro de la familia Bromeliaceae que se encuentra en dicha zona geográfica, y presenta fotosíntesis
CAM en algunas de sus especies, es el género Puya.
Bromeliaceae y Puya en Chile.
En Chile la familia Bromeliaceae se distribuye entre el extremo norte del país y Chiloé. Está representada por 23 especies, 20 de las cuales son endémicas, agrupadas en 6 géneros pertenecientes a 4 de las 8 subfamilias actualmente reconocidas para la familia: 15
Deuterocohnia (Pitcairnioideae), Fascicularia, Greigia, Ochagavia (Bromelioideae), Puya
(Puyoideae) y Tillandsia (Tillandsioideae) (Zizka 1992, Zizka et al. 2009). El alto endemismo de la familia en Chile le otorga un particular interés a un grupo que, numéricamente, está poco representado en el país en comparación con otros territorios dentro de su rango de distribución (Smith y Downs 1974, Zizka et al. 2009). La mayor parte de las especies chilenas de la familia tiene hábito terrestre y no presenta las típicas adaptaciones morfológicas a la aridez de las Bromeliaceae, salvo las especies del género Tillandsia (Smith y Downs 1974, Dillon 1991, Zizka et al. 1999, Will y Zizka 1999, Zizka et al. 2002). La vía fotosintética CAM está presente en las especies que se desarrollan en la zona norte del país
(Deuterocohnia chrysantha, Puya boliviensis), en algunas especies de Puya y Tillandsia de la zona centro-sur (Medina 1974, 1990, Martin 1994), y está completamente ausente en
Greigia, Fascicularia y Ochagavia, las que alcanzan latitudes mayores, hasta los 43 °S
(Martin 1994).
Filogenéticamente, las especies que habitan las zonas central y centro-sur de Chile, salvo en el caso de Tillandsia, ocupan posiciones basales dentro de sus respectivas subfamilias
(Horres et al. 2007, Schulte et al. 2009, 2010, Chew et al. 2010, Jabaily y Sytsma 2010,
2013). Esto solía ser considerado contradictorio, tomando en cuenta la hipótesis aceptada durante mucho tiempo para la familia en general, y en particular para algunos de sus géneros, como Puya, de un origen en la región de las Guayanas y un posterior avance hacia el sur siguiendo la línea de los Andes (Varadarajan 1990, Givnish et al. 2007). Recientemente, sin embargo, a partir de datos moleculares se ha propuesto un centro de origen ubicado en la zona sur de Chile para las subfamilias Bromelioideae y Puyoideae, que incluyen a géneros como Greigia, Ochagavia, Fascicularia y Puya, (Jabaily y Sytsma 2013). En este contexto, 16
sería interesante determinar cómo la adopción de algunas adaptaciones a la aridez podría haber facilitado el avance de algunos de estos géneros hacia zonas más áridas, y estar limitando la mantención del rango o la expansión de éste dentro de zonas más húmedas, particularmente en el caso de Puya, género que habría adoptado la vía CAM con posterioridad a su separación de las Bromelioideae, antes de comenzar su avance hacia la zona de clima mediterráneo y, posteriormente, la región altoandina (Jabaily y Sytsma 2013).
Puya, único género dentro de la subfamilia Puyoideae, comprende 273 especies, siendo uno de los géneros más numerosos dentro de la familia Bromeliaceae (Luther 2008). Se distribuye entre Costa Rica y la zona centro-sur de Chile (aprox. 39º S). Es un género considerado altamente específico de hábitats áridos y semiáridos, siendo frecuente en las regiones xéricas de los Andes, a grandes altitudes (Varadarajan 1990). Todas sus especies son terrestres, y no presenta las adaptaciones morfológicas a la aridez antes mencionadas, típicas de la familia
Bromeliaceae. Por otro lado, la vía fotosintética CAM es bastante frecuente entre ellas
(Martin 1994, Crayn et al. 2004).
En Chile Puya está representado por 7 especies endémicas (P. alpestris, P. berteroniana, P. boliviensis, P. coerulea, P. chilensis, P. gilmartiniae y P. venusta). De éstas, una (P. boliviensis) es exclusiva de la zona norte del país, encontrándose restringida a la costa entre los 25 y 26 ºS, mientras las 6 restantes se distribuyen entre los 29 y 39 ºS aproximadamente, tanto en la costa como en el interior (Zizka et al. 2009). El grupo de especies chilenas de
Puya se considera notablemente disjunto, monofilético y filogenéticamente basal, habiéndose diversificado probablemente a partir de un linaje que se mantuvo a nivel del mar, aislado de otro linaje que siguió la cordillera de los Andes hacia el norte, a mayor altitud
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(Jabaily y Sytsma 2010, 2013). De los 11 centros de diversidad descritos por Varadarajan
(1990) para Puya, el único que se encuentra a nivel del mar y no alcanza grandes altitudes es el que se encuentra en Chile, en tanto los otros 10 se encuentran confinados a los Andes entre los 800 y 5000 msnm.
En la zona central y centro-sur del país, entre los 32 y 39 ºS, Puya convive con tres géneros basales en la filogenia de la subfamilia Bromelioideae: Greigia, Fascicularia y Ochagavia, así como con dos representantes del género Tillandsia (Will y Zizka 1999, Zizka 1992, Zizka et al. 1999, 2002, 2009). En esta región se enfrenta tanto a un clima mediterráneo como a la zona de transición entre los climas mediterráneo y templado-lluvioso, ubicada entre los 36 y
39 ºS, la que se caracteriza por mayores precipitaciones y menores temperaturas (Luebert y
Pliscoff 2006). Dicha zona de transición suele ser ocupada tanto por especies adaptadas a periodos considerables de sequía, típicos del clima mediterráneo, como por especies adaptadas a bajas temperaturas y altas precipitaciones, típicas del sur lluvioso. De las cinco especies de Puya que habitan entre los 32 y 39º S, tres presentan la vía fotosintética CAM
(Martin 1994, Crayn et al. 2004), la que se podría volver innecesaria en una zona geográfica donde la aridez se vuelve menos relevante al avanzar hacia el sur. Cabe destacar que la presencia de las especies CAM de Puya hacia el sur se vuelve progresivamente limitada, desapareciendo en la zona de transición. Se podría inferir entonces que, si bien la adopción de la vía CAM probablemente fue crucial para permitir el avance de Puya hacia el norte dentro de la zona de clima mediterráneo, su mantención en las poblaciones ubicadas en la zona de transición, más lluviosa, podría actuar como “lastre” y limitar la presencia del género al sur de dicha zona, ya sea por el costo en recursos que implica su mantención, como por su potencial correlación negativa con otros atributos que podrían ser beneficiosos para la planta. 18
Planteamiento del problema.
Restricciones a la presencia de Puya en el sur lluvioso: valor adaptativo de la vía CAM.
La mayor parte de las especies chilenas de la familia Bromeliaceae se encuentran en el centro- sur del país, en zonas donde la disponibilidad de agua no debería ser un factor limitante. En el caso de las especies chilenas de la subfamilia Bromelioideae, filogenéticamente muy cercana a Puya (Horres et al. 2007, Schulte et al. 2009), tres de ellas (Greigia sphacelata, G. landbeckii y Fascicularia bicolor) alcanzan las mayores latitudes para la familia, llegando hasta los 43º S aproximadamente, sin haber dejado de avanzar hacia el norte en la zona mediterránea (Zizka et al. 2009). En el caso de Puya, cuyo probable avance principal fue de sur a norte, cabe preguntarse por qué no tiene representantes al sur de los 39 °S, a pesar de que al menos dos de sus especies se encuentran en la lluviosa zona de transición (P. alpestris y P. chilensis).
Es importante considerar que las especies de Greigia y Fascicularia que se encuentran en latitudes mayores pertenecen a un clado basal en la filogenia de la subfamilia Bromelioideae, el cual se cree que es anterior a la aparición de la vía fotosintética CAM y otras adaptaciones a la aridez en la subfamilia, las cuales habrían aparecido al colonizar el hábitat epífito tropical
(Schulte et al. 2009, Chew et al. 2010). Las especies chilenas de Puya, en tanto, si bien también forman un clado basal en la filogenia del género, habrían adoptado la vía CAM casi inmediatamente después de su aparición, antes de su diversificación al resto del continente a través de los Andes (Crayn et al. 2004, Jabaily y Sytsma 2010, 2013). Esta adaptación les habría permitido, como ya se ha mencionado, avanzar hacia zonas más áridas, pero probablemente, en hábitats donde desaparece la aridez, desaparecen también las ventajas
19
asociadas a la adaptación a ésta. Además, no se debe dejar de lado el potencial efecto que las bajas temperaturas pueden tener sobre la fotosíntesis CAM. Se cree que temperaturas bajo los 5 °C alteran la conformación de la PEPC, inhibiendo o limitando su funcionamiento
(Buchanan-Bollig et al. 1984, Carter et al. 1995, Chinthapalli 2003). Por otro lado, se ha reportado que variaciones de temperatura día/noche superiores a 10 °C estimulan el funcionamiento de la vía CAM (Medina et al. 1977, Haag-Kerwer et al. 1992), variaciones que son bastante frecuentes en la zona desértica (Weischet 1975). En la zona centro-sur de
Chile, y particularmente al sur de los 36º S, tanto el aumento en las precipitaciones como la disminución de las temperaturas se pueden volver particularmente relevantes para cualquier especie que siga expresando la vía CAM.
Considerando lo expuesto anteriormente, la presencia de un núcleo de Puya en la zona centro- sur de Chile que presenta, entre sus especies, distintas vías fotosintéticas, puede permitir cuantificar la importancia de este atributo en hábitats donde la aridez no es un factor relevante, y donde la fotosíntesis CAM podría volverse innecesaria. Es importante preguntarse, entonces, cuál es el valor adaptativo de la vía fotosintética CAM para las especies chilenas de Puya, y cuál sería la consecuencia de la mantención del atributo respecto a los rangos de distribución de éstas. Considerando que la mantención de un atributo innecesario puede causar detrimentos sobre la adecuación biológica, y que la mayor parte de las Puya chilenas son CAM, se podría inferir que la mantención de dicha vía fotosintética, al avanzar hacia hábitats donde la aridez no es un factor relevante, podría hacer que este atributo actúe como un lastre y tenga efectos negativos sobre la presencia de las especies chilenas de
Puya en el sur del país. Tomando en cuenta que las temperaturas medias, máximas y mínimas disminuyen, y que las precipitaciones aumentan significativamente, al avanzar hacia el sur 20
en la zona central y centro-sur de Chile (Luebert y Pliscoff 2006), se puede inferir que la vía fotosintética CAM carecería de valor adaptativo para las poblaciones australes de las especies de Puya. Dichas poblaciones son las que enfrentan las condiciones de mayor humedad y menores temperaturas dentro del rango de distribución de sus respectivas especies, por lo que cualquier ventaja derivada de utilizar la vía CAM desaparece.
Esta tesis pone a prueba el valor funcional y adaptativo de la vía fotosintética CAM para la familia Bromeliaceae en general y para las especies nativas de Puya en particular, a distintas escalas, las que reflejan sus patrones de distribución a nivel continental y local.
Escala familiar (Capítulos 1,2)
A nivel de familia, las Bromeliaceae presentan atributos morfológicos (tanques, tricomas absorbentes) que han sido considerados clave para su diversificación, permitiéndole ocupar exitosamente el ambiente epífito, particularmente en los trópicos, donde la diversidad de la familia alcanza su mayor nivel (Smith & Downs 1974, Benzing 2000). Es esperable que la vía fotosintética CAM, atributo que ha demostrado su valor adaptativo en otras familias altamente diversificadas en el hábitat epifito (ej. Orchidaceae; Silvera et al. 2009), haya cumplido un rol similar para la familia Bromeliaceae, actuando como una innovación clave que facilitó su diversificación. Esto puede probarse a nivel de toda la familia, a lo largo de su rango completo de distribución (Hipótesis 1).
En Chile en tanto, la familia Bromeliaceae presenta una distribución que puede estar relacionada con el gradiente de humedad y temperatura existente en la zona central, donde
21
las precipitaciones aumentan, y las temperaturas disminuyen significativamente de norte a sur (Hajek and DiCastri 1975; DiCastri and Hajek 1976; Luebert y Pliscoff 2006). Cabría suponer que las especies CAM, más adaptadas a la aridez, se distribuyen principalmente al norte de los 37°S, entre la zona de clima árido y la zona de clima Mediterráneo, en tanto las especies C3, menos preparadas para enfrentar la aridez, deberían ser más frecuentes en la zona de clima templado-lluvioso. Las especies CAM facultativas, capaces de evitar el uso de la vía CAM en casos en que no es necesaria, probablemente tengan una distribución intermedia. Es probable que exista, entonces, una relación entre la latitud máxima alcanzada por cada especie, o las precipitaciones y/o la temperatura en dicho punto, y la vía fotosintética utilizada (Hipótesis 2).
Escala interespecífica (Capítulo 3)
Considerando que, como se mencionó anteriormente, se ha registrado variabilidad dentro de algunas especies en la expresión de la vía CAM a lo largo de gradientes de humedad, cabría esperar que en el gradiente de humedad presente en Chile centro-sur aquellas especies con rangos de distribución que alcanzan mayores latitudes, y por ende avanzan más hacia la zona húmeda y fría, presenten una mayor variabilidad en el uso de dicha vía fotosintética. Estas especies probablemente caigan dentro de la categoría de CAM-facultativas, con uso de la vía
C3 en las poblaciones más australes. Esto se puede probar utilizando a especies CAM que se distribuyen dentro del gradiente, y que tengan rangos latitudinales de distinta extensión y distinto alcance hacia el sur, probando la variación interpoblacional en la expresión de CAM en cada una de ellas (Hipótesis 3).
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Escala interpoblacional: Selección fenotípica (Capítulo 4)
El valor adaptativo de un atributo puede estimarse determinando la ocurrencia de selección actuando sobre él (Phillips & Arnold 1989, Dudley 1996, Ackerly et al. 2000, Geber y Griffen
2003). Esto puede hacerse correlacionando el nivel de expresión del atributo con un estimador de fitness, en un grupo de individuos de una población determinada (análisis de selección fenotípica) (Lande & Arnold 1983, Endler 1986). Cabría esperar que, para aquellas especies CAM facultativas con poblaciones ubicadas en regiones de humedad contrastante, el uso de dicha vía fotosintética tenga efectos igualmente contrastantes sobre la adecuación biológica de los individuos. Así, para aquellas poblaciones ubicadas en zonas áridas y cálidas, la vía CAM tendría un efecto positivo sobre el fitness de los individuos que la expresen, en tanto en zonas donde la aridez no es relevante y las temperaturas son menores, su efecto sobre el fitness sería negativo, pudiendo incluso estar actuando como un “lastre” para la presencia de Puya al sur de los 37°S (Hipótesis 4).
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HIPÓTESIS
1.- La vía fotosintética CAM ha sido clave para la diversificación dentro de la familia
Bromeliaceae.
2.- Las especies chilenas de la familia Bromeliaceae que presentan fotosíntesis CAM alcanzan menores latitudes, a una misma longitud, que aquellas que utilizan la vía C3.
3.- En las especies CAM de Puya que habitan la zona centro sur de Chile, las poblaciones que ocupan zonas menos áridas expresarán menos la vía CAM que las poblaciones ubicadas a latitudes menores.
4.- La expresión de la vía CAM en Puya en las poblaciones ubicadas en zonas menos áridas afecta negativamente a la adecuación biológica de los individuos.
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OBJETIVOS
Objetivo general:
Determinar la importancia de la vía fotosintética CAM para la diversificación y distribución de las especies endémicas de Chile de la familia Bromeliaceae, particularmente las pertenecientes al género Puya.
Objetivos específicos:
1.- Determinar si la vía CAM actuó como una innovación clave que facilitó la diversificación de la familia Bromeliaceae (Capítulo 1).
2.- Determinar si existe relación entre la latitud máxima alcanzada por los miembros nativos de Chile de la familia Bromeliaceae y la vía fotosintética que presentan (Capítulo 2).
3.- Determinar, para 3 especies endémicas de Puya, si hay diferencias en la expresión de la vía fotosintética CAM entre poblaciones que se encuentran en latitudes distintas (Capítulo
3).
4.- Determinar si la expresión de la vía CAM tiene efectos negativos sobre la adecuación biológica, en individuos de una especie de Puya que se encuentran en una zona geográfica donde la aridez no es relevante (Capítulo 4).
5.- Inferir si la vía CAM es o no un “lastre” que limita la presencia de las especies chilenas de Puya en latitudes mayores a los 39º S, en Chile.
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CAPÍTULO I
En este capítulo se entregan resultados del análisis realizado para determinar si la vía fotosintética CAM fue una innovación clave para la familia Bromeliaceae. Los resultados demuestran que, efectivamente, la aparición de la vía CAM en diversos clados de la familia tuvo un efecto significativo sobre la diversificación de éstos. Los clados CAM son significativamente más diversos que sus contrapartes C3.
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CRASSULACEAN ACID METABOLISM PHOTOSYNTHESIS IN
BROMELIACEAE: AN EVOLUTIONARY KEY INNOVATION
IVÁN M. QUEZADA1 and ERNESTO GIANOLI1,2,3
1 Departamento de Botánica, Universidad de Concepción, Casilla 160-C Concepción, Chile
2 Departamento de Biología, Universidad de La Serena, Casilla 599 La Serena, Chile
3 Center for Advanced Studies in Ecology and Biodiversity (CASEB), P. Universidad
Católica de Chile, Santiago, Chile
Author for correspondence:
Ernesto Gianoli
Departamento de Biología, Universidad de la Serena
Casilla 599 La Serena, Chile
E-mail: [email protected]
Phone: +56-51-334637
Fax: +56-51-204383
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ABSTRACT
Crassulacean acid metabolism (CAM) is a photosynthetic pathway that significantly increases water use efficiency in plants. It has been proposed that CAM photosynthesis, which evolved from the ancestral C3 pathway, has played a role in the diversification of some prominent plant groups because it may have allowed them to colonize and successfully spread into arid or semi-arid environments. However, the hypothesis that CAM photosynthesis constitutes an evolutionary key innovation, thereby enhancing diversification rates of the clades possessing it, has not been evaluated quantitatively. We tested whether
CAM photosynthesis is a key innovation in the Bromeliaceae, a large and highly diversified plant family that has successfully colonized arid environments. We identified five pairs of sister groups with and without the CAM feature, including 31 genera and over 2000 species.
In all five cases, the clades with CAM photosynthesis were more diverse than their C3 counterparts. We provide quantitative evidence that the evolution of CAM photosynthesis is significantly associated with increased diversification in Bromeliaceae and thus constitutes an evolutionary key innovation. We also found preliminary evidence of an association between the CAM pathway and growth habit in bromeliads, with terrestrial species being more likely to show CAM photosynthesis than epiphytic species. To our knowledge, this is the first case of a physiological attribute shown to be a key innovation in plants.
KEYWORDS: adaptation – arid environments – bromeliads – diversification – epiphytism – niche – physiological traits – terrestrial habitat – water economy
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INTRODUCTION
Crassulacean acid metabolism (CAM) is a photosynthetic pathway widely recognized as a physiological adaptation of plants to aridity, and it is present in approximately 20000 species from at least 35 plant families (Cushman, 2001; Silvera et al., 2010). This mechanism involves nocturnal uptake of CO2 and its fixation into malic acid, which is stored in the vacuole and then decarboxylated during daytime for CO2 to be integrated into the Calvin cycle (Ting, 1985; Taiz & Zeiger, 2002). CAM photosynthesis allows plants to avoid stomatal opening during daytime, when the air temperature is higher and the probability of losing water by transpiration increases, thereby enhancing water use efficiency (Ehleringer
& Monson, 1993; Taiz & Zeiger, 2002). The CAM photosynthetic pathway is a distinctive attribute of large plant families such as Orchidaceae (Silvera et al., 2009) and Bromeliaceae
(Givnish et al., 2007), which are very successful in the epiphytic arid microenvironments of tropical forests, and Cactaceae, which are characteristic of arid ecosystems (Ogburn &
Edwards, 2009). The involvement of CAM photosynthesis in lineage diversification in arid ecosystems has often been suggested but rarely tested (Cushman, 2001; Silvera et al., 2010).
An exception to this is a recent study that showed evidence of correlated divergence in the photosynthetic pathway (with CAM evolving from C3) and epiphytism in Orchidaceae, which relates to orchid species diversification (Silvera et al., 2009).
The Bromeliaceae are a mostly Neotropical family (Smith & Till, 1998) composed of eight sub-families, 58 genera and 3200 species (Luther, 2008), with almost half of them being epiphytes (Gentry & Dodson, 1987) and approximately two-thirds showing CAM photosynthesis (Martin, 1994; Crayn et al., 2004). Adaptations to aridity in Bromeliaceae include the formation of tanks, a rosette-type growth that impounds water from rainfall
29
among compactly overlapping leaf bases, water-absorbing leaf trichomes that are able to capture atmospheric water vapour, and the CAM photosynthetic pathway (Smith & Downs,
1974; Benzing, 2000; Givnish et al., 2007). The CAM pathway evolved from the C3 ancestral state in Bromeliaceae several times, although reversals have also occurred (Givnish et al.,
2007). CAM bromeliads are mainly distributed in arid or semi-arid environments such as the high Andean ranges or deserts of Mexico and western South America, where they are primarily terrestrial (Smith & Downs, 1974; Crayn et al., 2004), or show the epiphytic habit in tropical forests, thus being exposed to low water availability (Benzing, 2000). It has been suggested that the evolution of CAM photosynthesis in Bromeliaceae, and the associated niche broadening to include drier habitats, should have stimulated the diversification of clades (Crayn et al., 2004; Givnish et al., 2007). Although several lines of evidence point to this hypothesis (Givnish et al., 2007), a quantitative verification is yet to be made.
Evolutionary key innovations are attributes whose appearance allows some taxonomic groups to successfully exploit formerly underused ecological niches, resulting in enhanced diversification rates of lineages (Simpson, 1953; Heard & Hauser, 1995). An outstanding example of key innovation in plants is the evolution of the flower, which is largely responsible for the enormous differences in species richness between angiosperms and gymnosperms (Stebbins, 1981). The association between putative key innovations and diversification rates can be evaluated by comparing the species richness in a clade possessing such a trait and a sister group (i.e. the most phylogenetically-related clade) that lacks it
(Slowinski & Guyer, 1993; Barraclough et al., 1998). Attributes shown to be key innovations within flowering plants include: annual growth habit, herbaceous life-form, climbing habit, floral nectar spurs, fleshy fruits, modifications of seeds that facilitate dispersal, and defensive
30
resin canals (Farrell et al., 1991; Eriksson & Bremer, 1992; Ricklefs & Renner, 1994;
Hodges, 1997; Dodd et al., 1999; Andreasen & Baldwin, 2001; Smith, 2001; Gianoli, 2004).
Interestingly, the possible role of physiological traits as key innovations in plants has not been addressed; studies have primarily focused on morphological attributes.
The Bromeliaceae include several particularly species-rich clades. Approximately one third of the species of the family are concentrated in eight genera (Aechmea, Dyckia,
Guzmania, Neoregelia, Pitcairnia, Puya, Tillandsia and Vriesea; Luther, 2008). It is possible to infer that one or more characters present in them have been responsible for their higher diversification rates compared to closely-related groups. CAM photosynthesis, along with other adaptations to aridity, has been mentioned as a possible key innovation for the diversification of Bromeliaceae by allowing the colonization and establishment in formerly nonsuitable arid or semi-arid habitats (Givnish et al., 1997; Benzing, 2000; Reinert et al.,
2003; Crayn et al., 2004; Givnish et al., 2007). The present study aimed to test the hypothesis that CAM photosynthesis is a key innovation for Bromeliaceae (i.e. to determine whether clades of bromeliads possessing the CAM pathway are more diverse than their sister groups with C3 photosynthesis). In addition, to estimate the independence of this hypothetical evolutionary pattern, we assessed whether the presence of CAM photosynthesis and epiphytism are correlated in Bromeliaceae, as has been shown for Orchidaceae (Silvera et al., 2009).
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MATERIALS AND METHODS
Valid genera of Bromeliaceae and their number of species were considered sensu Luther
(2008), excluding subspecies, varieties, hybrid genera and hybrid species. We carried out an extensive search of the available literature to determine the presence or absence of CAM photosynthesis in bromeliad species. Facultative CAM, constitutive CAM and species reported as “probably CAM” were classified as CAM species, whereas C3 and species reported as “probably C3” were classified as C3 species. A genus was considered CAM when the majority of the reported species was so classified. Importantly, we only considered cases where (1) the photosynthetic pathway for a whole genus was indicated in the text or in tables
(e.g. ‘all Greigia species are C3’) or (2) a given number of species within a genus were explicitly identified and their corresponding photosynthetic pathways informed in the text or in tables (e.g. Puya chilensis: CAM; Puya alpestris: C3) (i.e. we did not take into account studies merely asserting that a given number of species within a genus were C3 or CAM).
To determine sister groups within Bromeliaceae we used available phylogenies for the family (Crayn et al., 2004; Givnish et al., 2004, 2007) and for the main subfamilies
(Barfuss et al., 2005; Rex et al., 2009; Schulte et al. 2009; Jabaily & Sytsma, 2010). Genera with uncertain phylogenetic position or without enough information about the species’ photosynthetic pathway were not considered. The analysis was conducted on five contrasts, including 31 genera and 2015 species (Table 1).
To test the hypothesis that CAM clades are more diverse than their C3 sister groups, we used the method of Slowinski & Guyer (1993), which is based on a model of random speciation and extinction. For each of the five sister-group pairs, the probability that the clade with CAM photosynthesis has a species richness of r or greater was calculated by the
32
formula: Pc = p(r/n) + p(r + 1/n) + … + p(n – 1/n) = (n - r) / (n - 1), where n is the actual number of total species in both clades and r is the observed number of species in the clade with CAM photosynthesis. The natural logarithm (ln) of these independently calculated probabilities (Pc) was summed for the five pairs, and the result multiplied by -2 and tested against a chi-square distribution with 2k degrees of freedom, where k is the number of pairs evaluated (Fisher’s combined probability test; Sokal & Rohlf, 1995).
The independence of the evolution of CAM photosynthesis and the growth habit
(epiphytic versus terrestrial) was assessed via tables of contingency for each of four
Bromeliaceae clades where phylogenies were available and both the growth habit and the photosynthetic pathway were clearly identified at the species level (Table 2). Specifically, to test the explicit hypothesis that transitions from C3 to CAM photosynthesis are more probable on epiphytic clades than on terrestrial ones, we used the method of Sillén-Tullberg
(1993), which allows testing for the contingency of states in two discrete characters. The resulting probabilities (P-values) of the four independent chi-square tests were processed as described above (Fisher’s combined probability test).
RESULTS
We found that, in all five cases, the CAM clade had greater species richness than the corresponding sister clade with C3 photosynthesis (Table 1). The pattern of greater taxonomic diversification in CAM groups was statistically significant (χ2 = 27.18, d.f. = 10,
P = 0.002; Fisher’s combined probability test). Therefore, results validated the hypothesis that the CAM pathway is an evolutionary key innovation for Bromeliaceae. The contrasts
33
between sister groups showing different photosynthetic pathways included five of the eight
Bromeliaceae subfamilies (Table 1).
Assessment of the independence of the evolution of the CAM pathway and the growth habit included four clades (Table 2), with a total of 1627 species. In three cases, the transitions to CAM photosynthesis were much more common for the terrestrial taxa than for the epiphytic taxa (Table 2). Overall, there was a significant association between photosynthetic pathway and growth habit (χ2 = 25.36, d.f. = 8, P = 0.0013; Fisher’s combined probability test). Thus, evolution of the CAM pathway in Bromeliaceae is apparently not linked to the development of an epiphytic habit, instead being rather favoured by a terrestrial habitat.
DISCUSSION
We quantitatively verified the hypothesis that the CAM photosynthetic pathway constitutes an evolutionary key innovation for the Bromeliaceae. This finding supports earlier claims that the evolution of CAM photosynthesis, and the ensuing colonization of arid environments, has promoted taxonomic diversification in Bromeliaceae (Cushman, 2001;
Crayn et al., 2004; Givnish et al., 2007). The colonization of novel habitats, comprising potential “adaptive zones” where lineages are released from competitors, is one of the mechanisms by which a given attribute may promote diversification of clades (Heard &
Hauser, 1995). Several traits have proven to be evolutionary key innovations in plants, most of them being morphological or life-history traits (Eriksson & Bremer, 1992; Ricklefs &
Renner, 1994; Hodges, 1997; Dodd et al., 1999; Andreasen & Baldwin, 2001; Smith, 2001;
Gianoli, 2004). However, to our knowledge, no physiological character had been shown to
34
be a key innovation, with the closest case being the appearance of latex and resin canals that confer resistance against herbivores (Farrell et al., 1991). The paucity of such plant physiological attributes in the key innovation literature is more likely the result of a lack of sufficient information rather than to a minor role of physiological traits in plant adaptation to novel environments.
The number of contrasts included in the present study is close to the number of times
CAM photosynthesis supposedly arose within Bromeliaceae. According to Crayn et al.
(2004), this photosynthetic pathway appeared independently at least four times within the family. This statement is supported by other studies where four approximately defined monophyletic CAM clades were shown after mapping the presence or absence of CAM photosynthesis onto the Bromeliaceae phylogeny (Crayn et al., 2000; Reinert et al., 2003;
Givnish et al., 2007). We considered five CAM clades in the analysis, instead of four, because we chose to ‘split’ the Bromelioideae subfamily into two clades, in accordance with evidence that places Greigia, Ochagavia, and Fascicularia as a sister group to Puya (Rex et al., 2009; Jabaily & Sytsma, 2010) and Fernseea as a sister group of the CAM Bromelioideae
(Schulte et al., 2009). Although our number of contrasts may seem small compared to other similar studies (Gianoli, 2004), it reflects the actual number of appearances of CAM photosynthesis within the family.
The CAM pathway evolved from an ancestral C3 state during the evolution of
Bromeliaceae, probably being selected during the advance of different groups in the family into arid regions (Varadarajan & Gilmartin, 1988; Givnish et al., 2007). This pattern of origin and diversification of the CAM pathway in Bromeliaceae would be similar to that reported for Cactaceae (Ocampo & Columbus, 2010), as well as for Orchidaceae (Silvera et al., 2009),
35
a family that shares the dominance of the rather dry epiphytic habitat in tropical forests with
Bromeliaceae (Gentry & Dodson, 1987). The CAM pathway is considered to be a vital adaptation for plants living in arid or semi-arid environments (Cushman, 2001). For those
Bromeliaceae species that rely on foliar structures to obtain fog water and inhabit extremely dry environments, such as the rootless Tillandsia landbeckii that grows unattached on the
Atacama Desert sands, CAM photosynthesis is essential for survival (Rundel et al., 1997;
Rundel & Dillon, 1998). For terrestrial genera such as Puya, CAM photosynthesis appears to be a key feature for those species that live in the drier regions of its distribution range
(Benzing 2000).
The basal phylogenetic position of the C3 genera Brocchinia and Lindmania, both endemic to the Guayana Shield, implies that the probable origin of Bromeliaceae was in this warm and rainy region (Givnish et al., 2004, 2007). In the subsequent geographical expansion of the family, morphological and physiological adaptations to aridity were probably the main mechanisms behind its ecological and taxonomic diversification (Givnish et al., 1997, 2007).
Thus, the successful colonization of drier environments, such as the epiphytic habitat in tropical forests and semi-arid highlands in the Andes, was mediated by the appearance of tanks, absorbing trichomes and CAM photosynthesis (Givnish et al., 1997; Benzing, 2000).
This photosynthetic pathway probably allowed bromeliads to advance into extremely arid zones, such as deserts, by minimizing water loss during carbon assimilation (Ehleringer &
Monson, 1993). On the other hand, the CAM photosynthetic pathway also entails disadvantages. Thus, cold climate limits the distribution of CAM plants (Ehleringer &
Monson, 1993) mainly because of the sensitivity of PEPC, the carbon fixation enzyme, to low temperatures (Lambers et al. 1998). This could explain the scarcity of CAM plants at
36
higher latitudes in southern South America. In the case of Orchidaceae species, it has been established that the prevalence of CAM photosynthesis decreases with altitude (Silvera et al.,
2009).
A comprehensive study in the Orchidaceae revealed the involvement of CAM photosynthesis in the adaptive radiations within the family, and demonstrated correlated evolutionary divergence between photosynthetic pathways and plant growth habit, with
CAM epiphytic species being more prevalent at lower altitudes (Silvera et al., 2009). Our study found preliminary evidence suggesting that CAM photosynthesis is more likely to occur in terrestrial than in epiphytic clades within Bromeliaceae. This result is somewhat unexpected given that some of the most important and abundant genera of Bromeliaceae are mainly epiphytic and CAM (Smith & Downs, 1974; Martin, 1994). Although we cannot rule out the possibility that our noncomprehensive dataset is not representative of the whole family, there is also an explanation for such a pattern based on biogeographical and ecological issues. In the case of orchids and tropical bromeliads, such as the clade dominated by Tillandsia, the epiphytic habitat represents a low water environment where the water- conserving CAM strategy is advantageous, and the terrestrial habitat is associated with high soil moisture. By contrast, in the case of temperate bromeliads, which are mainly distributed in Andean highlands and deserts, the CAM pathway is associated with the terrestrial habitat, which is semi-arid or arid and much more challenging in terms of water economy than the epiphytic habitat. This is the case for Puya, Dyckia, Encholirium and Deuterocohnia, the
CAM representatives of their clades, which live in arid and semi-arid environments of South
America (Smith & Downs, 1974), and it is also the case for Hechtia, which is distributed in arid regions of North America (Smith & Downs, 1974; Crayn et al., 2004). Further research,
37
including a more comprehensive data set, containing a greater number of tropical groups, will shed light into the actual relationship between the evolution of CAM photosynthesis and growth habit in Bromeliaceae.
In the present study, we provide quantitative evidence indicating that the evolution of
CAM photosynthesis is significantly associated with increased diversification in
Bromeliaceae and thus constitutes an evolutionary key innovation for this family, as has been often stated. To our knowledge, this is the first case of a physiological attribute being shown to represent a key innovation in plants. We also found preliminary evidence of an evolutionary association between CAM photosynthesis and growth habit in the
Bromeliaceae, which deserves further investigation. Future studies that aim to explain the diversification of bromeliads should include, in addition to CAM photosynthesis, other characteristic adaptations to aridity in the family, such as tanks, absorbing trichomes and succulence.
ACKNOWLEDGEMENTS
Iván M. Quezada thanks CONICYT for a doctoral fellowship. We are thankful for the thoughtful comments made by C. M. Baeza and L. J. Corcuera that improved earlier versions of the manuscript.
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Table 1. Species richness contrasts between crassulacean acid metabolism (CAM) and C3 sister-groups in Bromeliaceae. Number Number of Sources
CAM genera of species C3 genera species Pc Billbergia - Lymania - Wittrockia - Edmundoa - 773 Fernseea 2 0.0026 1/2-6 Neoregelia - Nidularium - Canistrum - Araeococcus - [Bromelioideae] Quesnelia - Aechmea - Cryptanthus - Ortophytum - Ananas - Neoglazovia - Hohenbergia - Bromelia – Ursulaea [Bromelioideae] Encholirium - Dyckia – Deuterocohnia 170 Fosterella 30 0.1508 6-8/4-6, 9-10 [Pitcairnioideae] [Pitcairnioideae] Puya 273 Fascicularia - 38 0.1226 1, 8, 11/4, 6, [Puyoideae] Ochagavia – Greigia 10 [Bromelioideae] Hechtia 52 Glomeropitcairnia – 20 0.2817 6, 12/2-6, 10 [Hechtioideae] Catopsis [Tillandsioideae] Tillandsia 596 Racinaea 61 0.0929 6, 8, 12/2-6 [Tillandsioideae] [Tillandsioideae] Subfamilies are indicated in parenthesis; when both groups within a contrast belong to the same subfamily, the latter is indicated after the C3 genus or genera. Pc, probability that the CAM clade actually has the observed species richness or greater (Slowinski & Guyer, 1993; see Methods). Sources: phylogeny and species richness/photosynthetic pathway. 1, Schulte et al. 2009; 2, Medina 1974; 3, Griffiths & Smith 1983; 4, Martin 1994; 5, Pierce et al. 2002; 6, Crayn et al. 2004; 7, Givnish et al. 2004; 8, Rex et al. 2009; 9, Crayn et al. 2000; 10, Reinert et al. 2003; 11, Jabaily & Sytsma 2010; 12, Barfuss et al. 2005.
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Table 2. Contingency tables testing the association between the evolution of crassulacean acid metabolism (CAM) photosynthesis and growth habit in four Bromeliaceae clades.
Encholirium – Dyckia – Deuterocohnia – Fosterella – Pitcairnia – Pepinia [587 species]
C3→C3 C3→CAM P-value Phylogeny sources
Epiphytic 4 0 0.0463 [2], [4], [5]
Terrestrial 40 67
Puya – Fascicularia – Ochagavia - Greigia [311 species]
C3→C3 C3→CAM P-value Phylogeny sources
Epiphytic 1 0 0.7189 [1], [5], [6]
Terrestrial 102 51
Hechtia – Glomeropitcairnia - Catopsis [72 species]
C3→C3 C3→CAM P-value Phylogeny sources
Epiphytic 20 0 0.0001 [4], [7]
Terrestrial 2 33
Tillandsia - Racinaea [657 species]
C3→C3 C3→CAM P-value Phylogeny sources
Epiphytic 49 98 0.9362 [4], [5], [7]
Terrestrial 3 8
Total species richness in the clade is indicated in parenthesis. The number of branches in the phylogenies with C3→C3 and C3→CAM transitions for epiphytic and terrestrial taxa are shown (Sillén-Tullberg, 1993). P-values are calculated from Yates’ corrected chi-square distribution. Phylogeny sources are as indicated in Table 1.
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APÉNDICE AL CAPÍTULO I
46
47
48
49
Árboles filogenéticos construidos para el análisis de contrastes pareados. Se presentan por separado para cada par de clados hermanos generados para el análisis. Se indica la vía fotosintética para los géneros presentes en cada árbol.
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CAPÍTULO II
En este capítulo se entregan resultados de la comparación entre la vía fotosintética utilizada por las especies endémicas de Chile de la familia Bromeliaceae, la latitud máxima que alcanzan y algunas variables climáticas. Los resultados muestran que existe una correlación entre la vía fotosintética, la latitud máxima y la precipitación anual, pero no con la temperatura mínima. Al corregir por la filogenia mediante contrastes filogenéticamente independientes, ninguno de dichos factores mantiene una correlación significativa con la vía fotosintética. A pesar de esto, inferimos que los factores climáticos han modelado significativamente la distribución de la vía CAM en las bromeliáceas de Chile central, así como el probable efecto de otros factores no considerados en este estudio.
51
CRASSULACEAN ACID METABOLISM VARIES WITH LATITUDE IN CHILEAN BROMELIADS
Iván M. Quezada1, Ernesto Gianoli1,2
1Departamento de Botánica, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
E-mail address: [email protected]
2Departamento de Biología, Universidad de La Serena, Casilla 554, La Serena, Chile. E-mail address: [email protected]
Corresponding author: Iván M. Quezada.
Postal address: Departamento de Botánica, Universidad de Concepción, Casilla 160-C
Concepción, Chile.
Phone: +56 41 2203418
Fax: +56 41 2246005
E-mail address: [email protected]
52
ABSTRACT
Chilean bromeliads distribute along a highly variable latitudinal gradient. Within this gradient precipitation increases towards south and temperatures decrease on the same direction. Among Chilean bromeliads there are constitutive CAM, facultative CAM and C3 species. In this work we looked for correlations between the preferred photosynthetic pathway of each endemic Chilean bromeliad species with latitude, minimal temperature and annual precipitation. We found that CAM expression is correlated with all factors. However, since Chilean bromeliads are a closely related group, phylogenetic relationships needed to be accounted. For this we performed phylogenetically independent contrasts. After this phylogenetic correction, none of the alalyzed factors was correlated with CAM expression.
We infer that, although apparently phylogeny is the main driver behind CAM geographic distribution in Chilean bromeliads, the climate may have higher importance in a climatic region where vegetation has been modelled by it. We then suggest there is a possible correlation between latitude/environment and the expression of CAM in Chilean bromeliads that should be studied in the future.
53
INTRODUCTION
Plant distribution is directly affected by climate through physiological constraints on growth and reproduction, and indirectly by effects over competition (Shao and Halpin 1995; Sykes et al. 1996). Thus, the geographic range reached by a plant species can be influenced by the interplay between environmental conditions and individual abilities to tolerate these conditions, by way of local adaptation and/or phenotypic plasticity (Van Tienderen 1990,
Joshi et al. 2001). Latitude is a good predictor of climate in Chile. Climatic zones range from hyper-desert to boreal hyper-oceanic, with colder and wetter habitats with increasing latitude
(Hajek and DiCastri 1975; DiCastri and Hajek 1976; Schwedtfeger 1976; Luebert & Pliscoff
2006). This climatic gradient may be useful to understand variation in plant tolerance and adaptation to environmental conditions.
Plants from the Bromeliaceae family are present in a significant part of the Chilean latitudinal gradient, from 18°S to 43°S, and across arid, semiarid, Mediterranean and temperate-oceanic climatic regions (Zizka et al. 2009). Chilean bromeliads are closely related, and some of them form the basal clades of two of the main subfamilies of Bromeliaceae: Puyoideae and
Bromelioideae (Jabaily and Sytsma 2010, 2013). This group comprises 23 species, 20 of which are endemic (Zizka et al. 2009), and their geographic distribution is somewhat related to phylogeny at the genus or subfamily levels. For instance, Puya species are mainly distributed in the arid regions of the north and do not cross the boundary between
Mediterranean and temperate-oceanic climatic regions. On the other hand, species of the
Bromelioideae subfamily, from the genera Greigia, Ochagavia and Fascicularia, are the dominant bromeliads in the temperate region and reach the Mediterranean region (Zizka et
54
al. 2009). These differential distributions may be related to particular traits of the species/clades that granted them adaptation to the prevailing environments.
The CAM (Crassulacean Acid Metabolism) photosynthetic pathway is one of the most important adaptations of the Bromeliaceae, which are mainly distributed in arid and semiarid habitats (Benzing 2000). CAM photosynthesis is found in a substantial number of vascular plants apart from Bromeliaceae, such as Orchidaceae, Crassulaceae, and Cactaceae, all of which have successfully spread across semiarid, arid and hyperarid habitats (Nobel and
Bobich 2002; Pinto et al. 2006). CAM photosynthesis implies a temporal separation of photosynthetic phases: plants open their stomata at night, when CO2 is captured by PEPC, an enzyme present in CAM and C4 plants, and then stored as malic acid. This organic acid acts as a reservoir of CO2 for the rest of the photosynthetic process, which is carried out during daytime. This allows plants to keep their stomata closed during the day, when temperature, irradiation and chances of losing water via transpiration are higher, thus increasing its water use efficiency (Ting 1985; Dodd et al. 2002; Taiz and Zeiger 2002; Larcher 2003; Heldt
2005). CAM usage varies substantially among CAM-capable plants, including constitutive
CAM (always relying on CAM pathway), facultative CAM (“switching” between C3 and
CAM, or combining both, depending on the environment) and the more extreme CAM-idling
(recycling of respiratory CO2 via the CAM pathway: the plant keeps stomata closed day and night in extremely arid environments) (Ting 1985; Taiz and Zeiger 2002; Winter et al. 2008).
It has been noted that C4 and CAM pathways involve higher costs for plants than C3 photosynthesis. For C4 plants, processes such as the regeneration of PEP from pyruvate imply an extra cost in ATP (Ehleringer et al. 1997, Ehleringer and Cerling 2002). CAM plants have to face even higher costs, derived from the biochemical machinery and from the need of a 55
vacuolar storage system to keep malic acid until daytime (Forseth 2010). This increase has been estimated to elevate the overall ATP cost of the CAM pathway as high as twice the cost of C3 photosynthesis (Walker 1992, Furbank 1999). The expression of CAM is heavily influenced by water availability and temperature (Dodd et al. 2002). While water availability is the main driver for this physiological response, low temperatures are believed to affect it negatively by significantly reducing the performance of PEPC (Selinioti et al., 1986, Wu &
Wedding 1987).
Among Chilean bromeliads there are C3 and CAM species (Martin 1994, Crayn et al. 2004,
Quezada et al., unpublished). Within CAM species, there is variation in the expression of the pathway along the Chilean climatic gradient (Quezada et al., unpublished). Since temperature and water availability vary along this gradient, it may be expected that changes in CAM expression at the species level are correlated to climatic differences. However, despite the compelling functional arguments linking them, a pattern of covariation between climate and
CAM does not necessarily imply that Chilean bromeliads have undergone adaptive differentiation to their habitats. The shared phylogenetic history of these closely related species may also explain the presence or absence of CAM photosynthesis at the species level.
Thus, its expression can be a consequence of its adoption by an ancestor and not a direct response to recent climatic changes. Not accounting for this possibility when making correlations between traits in comparative data sets might lead to significant errors and therefore statistical correction is needed (Felsenstein 1985; Harvey and Pagel 1991).
The goal of this study was to determine whether there is a relationship between the latitudinal distribution of Chilean bromeliads and the photosynthetic pathway they use. We
56
hypothesized that species reaching higher latitudes (colder and wetter habitats) would tend to show the C3 pathway, while species distributed across the Mediterranean, semiarid and arid regions would rely on CAM. Those species reaching intermediate latitudes (higher latitudes within the Mediterranean zone) were expected to be facultative CAM. We also anticipated associations between CAM usage and the environmental variables that covary with latitude in Chile. Thus, we expected to find a significant influence of minimum temperature and annual precipitation on the use of CAM. Finally, to estimate the influence of phylogeny on the expression of CAM by Chilean bromeliads along the climatic gradient, we used phylogenetically independent contrasts (Felsenstein 1985).
MATERIALS AND METHODS
Study species and location data
We worked with 18 of the 23 Bromeliaceae species native to Chile (Table 1). We excluded
Ochagavia elegans and Greigia berteroi, both endemic to the Robinson Crusoe island
(33°38’ºS, 78°50’ºW), because of their location outside the latitudinal gradient of continental
Chile and their impossibility to advance towards north or south. We also excluded non- endemic bromeliads, some of which have a cosmopolitan distribution (e.g. Tillandsia usneoides) and therefore could add “noise” to the analysis.
For each species we determined the highest latitude where it has been reported, from two viewpoints: the “maximum coastal latitude” and the “maximum absolute latitude”. The maximum coastal latitude is the southernmost population of each species that has been
57
reported along Chilean coastline, which is important because the Pacific Ocean has a significant influence in Chilean plants’ distribution since it acts as a climatic buffer
(Zinsmeister 1978). In addition, since some Bromeliaceae species reach higher latitudes inland than in the coastline (e.g. Puya berteroniana), we also considered the “maximum absolute latitude”, which is the southernmost location where each species has been found.
Information on the location of each species was collected from herbaria (CONC, SEL), personal observations by the first author and published reports (Dillon 1991, Will & Zizka
1999, Zizka et al. 1999, 2002, 2009).
Photosynthetic pathway
We classified species into three categories: C3, CAM-f (facultative CAM) and CAM-c
(constitutive or “obligate” CAM). In ambiguous cases (species reported as CAM and C3 by different authors) species were considered facultative (CAM-f). This disagreement is probably due to sampling sites with different climatic conditions, among other factors, a matter that has been shown to lead to confusion with facultative CAM species (Pierce et al.
2002, Quezada et al. unpublished). Information on the photosynthetic pathway of all the studied species was obtained from bibliographical sources (mainly Martin 1994 and Crayn et al. 2004) and data previously collected by the authors and not yet published (Table 1). In cases where no information was available for a particular species, if the genus had been classified as a whole as C3, CAM-f or CAM-c, then the species was classified accordingly
(e.g. Greigia, mentioned as completely C3; see Crayn et al. 2004).
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Climatic data
We obtained data of annual precipitation and minimum temperature of the coldest month for all the maximum “absolute” latitude points of occurrence of each species. Data for both climatic factors was obtained from the BIOCLIM database of WORLDCLIM (Hijmans et al.
2005), using a GIS software package. We preferred maximum absolute latitude instead of maximum coastal latitude because inland populations in central-southern Chile face more variable conditions than coastal populations, even at the same latitude, due the abovementioned effect of the Pacific Ocean. Thus, when maximum absolute latitude was higher than maximum coastal latitude, we believe climatic conditions at the first are more representative of the most extreme conditions each species is adapted to tolerate.
Data analysis
CAM categories were assigned a value (C3 = 1; CAM-f = 2; CAM-c = 3) to facilitate statistical analyses. First, for each species we evaluated the relationship between CAM category (dependent variable) and the following predictors: maximum coastal latitude, maximum latitude, annual precipitation in maximum latitude, and minimum temperature of the coldest month in maximum latitude. This was assessed using one-way ANOVAs (factors:
CAM category vs. latitude or climatic variable) followed by post-hoc procedures (Fisher’s
LSD test). Second, to account for the potential effect of phylogenetic relations in the development and usage of the CAM pathway in a closely related group as the Chilean bromeliads, we performed phylogenetically independent contrasts (Felsenstein 1985) on all the previously compared variables. To construct the phylogenetic tree necessary for the analysis (Fig. 1), we considered the most recent phylogenetic trees for Bromeliaceae that
59
explicitly include the Chilean species or genera (Barfuss et al. 2005, Horres et al. 2007,
Givnish et al. 2007, 2011, Schulte et al. 2009, Jabaily & Sytsma 2010, 2013), and combined them to form a single tree. Contrasts were caucluated by hand, since no available software package is able to calculate contrasts when one variable is categorical with more than 2 categories. Four comparisons were made contrasting the photosynthetic pathway and each climate/latitude dataset. For each comparison, a t-test was performed to determine the significance of the difference on the climate or latitude values between groups of contrasts that had a photosynthetic pathway change on their phylogenetic root and those that had not.
RESULTS
Photosynthetic pathway vs. latitude and climatic variables
ANOVAs indicate that photosynthetic pathway is significantly influenced by maximum absolute latitude, maximum coastal latitude and annual precipitation at maximum absolute latitude (p < 0.005 in all cases). On the other hand, minimum temperature of the coldest month at maximum absolute latitude did not have a significant influence over CAM use (p =
0.07) (Table 2). LSD test shows a significant difference between maximum latitudes and precipitation levels reached by CAM and C3 species (Fig. 2).
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Phylogenetically independent contrasts
After applying the phylogenetic correction, all t-test showed no significant difference between groups of contrasts with and without pathway change (Table 3). For each comparison, 17 contrasts were detected.
DISCUSSION
Phylogenetically independent contrasts show no significant influence of latitude, precipitation or temperature over the expression of CAM or C3 photosynthesis. This suggests that the early adoption of CAM by an ancestor, prior to the advance to the north of a significant part of Chilean bromeliads, is the main reason for CAM modern geographic distribution on the group. These results are not surprising, since the close relationship that exists between Chilean bromeliads located in the Mediterranean and Temperate climatic zones, as happens with the abovementioned P. alpestris/P. berteroniana clade (Horres et al.
2007, Schulte et al. 2009, Chew et al. 2010, Jabaily and Sytsma 2010, 2013). However, its implications should be taken with caution. In a geographic region where climate has such an impact on modelling the distribution and diversity of vegetation (Gasith and Resh 1999), it is reasonable to expect that climatic conditions have great influence on the selection of phenotypic attributes with adaptive value. The seeming lack of relationship between climatic variables and photosynthetic pathway on Chilean bromeliads after phylogenetic correction may be, then, a consequence of the geographically isolated events of speciation undergone by early Chilean bromeliads, and of the multiple adoption of CAM by a small and very
61
closely related group of species (Jabaily and Sytsma 2010, 2013), all of which could mask the effects of local adaptation.
Although phylogenetically independent contrasts apparently discard the importance of the climatic variables acting over the geographic distribution of photosynthetic pathways in
Chilean bromeliads, the influence of temperature, latitude and especially precipitation should not be discarded. In addition, the effect of other factors (ecological, historical, climatic) may be taken into account. In this particular case, other factors that may influence CAM expression such as light, salinity or nutrient availability (Lüttge 2004), could be having a significant effect over its distribution. This because Chilean bromeliads, in particular those living along the coastline, tend to occupy poor, rocky soils on cliffs facing the ocean, thus exposed to high salinity and irradiance (Zizka et al. 2009).
Considering direct comparisons (ANOVA) between climatic factors, latitude and CAM expression, results suggest that the photosynthetic pathway used by Chilean endemic
Bromeliaceae species is related to moisture conditions underlying latitude variation along the
Chilean climatic gradient (Hajek and DiCastri 1975; DiCastri and Hajek 1976; Schwedtfeger
1976, Arroyo et al. 1988). Apparently, there is a decrease in the use of CAM in species that reach higher latitudes, where precipitation is higher, while species distributed in lower latitudes, where conditions are drier, tend to rely on the CAM pathway (Table 1; Fig. 2). This relationship is supported by our results, which show a significant influence of latitude and precipitation on the photosynthetic pathway used by each species.
Annual precipitation at the maximum absolute latitude had a significant influence on the expression of CAM. The six species with southern limits located at the drier sites (i.e. below
62
200 mm/year) were CAM, five of them CAM-c. C3 species became dominant when annual precipitation at the maximum absolute latitude was above 500 mm/year (nine C3 species, two
CAM-f species, and one CAM-c species within this range). The decrease in the use of CAM following the increase in rainfall seems clear. It has been previously reported that low water availability is the main driver of the occurrence of CAM in plant species and populations
(Winter and Ziegler 1992, Sayed and Hegazy 1994, Borland et al. 1996, 1998; Cushman and
Borland 2001), and our results are concordant with these reports. CAM increases the overall water use efficiency of plants, allowing them to fix up to 10 times more CO2 per transpired mol of H2O than C3 plants (Lüttge 2004), and this feature is extremely relevant for plants inhabiting zones where precipitation is as low as 200 mm/year or less. The fact that no C3 species of our list is found in the semiarid region might indicate that the CAM pathway may be crucial for terrestrial bromeliad species in these conditions. On the other side, the three
CAM species reaching locations with annual precipitation above 500 mm/year belong to
Puya. Two of them (P. venusta and P. chilensis) are facultative-CAM, and live on rocky cliffs facing the ocean (Zizka et al. 2009). Puya chilensis is the Chilean Puya species with the longest latitudinal range, along which its populations face significantly different moisture conditions, with the southernmost populations being, in fact, more C3 than CAM (Quezada et al., unpublished). The third species, Puya berteroniana, seems to be constitutive-CAM according to isotopic measurements (Crayn et al. 2004), and no inter-population variation has been found in the expression of CAM along its geographic range (Quezada, unpublished).
This species lives on the coast in the northern half of its geographic range, and moves towards pre-Andean slopes on the southern half (Zizka et al. 2009). At the southern limit of its distribution, P. berteroniana populations reach altitudes of ca. 1200 m.a.s.l., where
63
precipitation during summer is scarce, and during winter a significant part of it stays as snow or ice (Hijmans et al. 2005, Luebert and Pliscoff 2006). Considering water availability is relatively low along most of the species range, relying in the CAM pathway is probably a good strategy.
The other factor we evaluated, low temperature, did not show a significant effect ofer CAM expression, by a slight margin (p = 0.07). Temperature variation along the Chilean coastline is not as big as could be expected, due to the buffer effect of the Humboldt current
(Zinsmeister 1978). This probably makes temperature less effective over CAM expression than precipitation, at least in coastal Chile, where Chilean bromeliads have part, if not all, of their geographic range (Zizka et al. 2009). Temperature has been suggested as a possible negative factor for the CAM pathway, mainly because PEPC decreases its performance under cold conditions (Wu & Wedding 1987, Chinthapalli 2003). This has been extensively studied for C4 plants, which apparently lose efficiency under 15 °C (Selinioti et al. 1986, Potvin and
Simon 1990, Chinthapalli et al. 2003), although there are some opposite results (see Krall and Edwards 1993 and Du et al. 1999). For CAM plants, on the other hand, the effect of low temperature on PEPC is not clear (e.g. see Buchanan-Bollig et al. 1984), although chilling temperatures (0-3 °C) are believed to alter its performance by changing CAM-PEPC ultrastructure and/or promoting its phosphorylation (Carter et al. 1995, Chinthapalli 2003).
While C4 plants grow on warm tropical and subtropical regions, CAM plants can be found on arid environments and even deserts (Taiz and Zeiger 2002), where there is a significant difference in day/night temperatures. In the Atacama desert day temperature can rise up to
30 °C and night temperature can fall down to 10 °C or less on the same day (Weischet 1975).
An appreciable day/night temperature difference (10–20 °C) enhances CAM operation 64
(Medina et al. 1977, Haag-Kerwer et al. 1992). Although the relationship between CAM expression and cold temperature was not significant in our analysis, a certain trend can be observed on our database (Table 1) where sites with minimum temperatures above 5 °C were mostly occupied by CAM species, and C3 species prevailed in sites with minimum temperatures below 5 °C. Probably chilling events (below 5 °C) can influence the distribution of CAM in Chilean bromeliads, which is concordant with the theory that puts the tolerance limit of CAM-PEPC on the chilling range (Buchanan-Bollig et al. 1984). Specifically, minimum temperature of the coldest month in our 18 sites ranged between -3.4 °C
(Ochagavia andina, C3) and 10 °C (Puya boliviensis, CAM). Five species of our set face minimum temperatures below 0° C at the coldest month. Four of those are C3 species and only Puya berteroniana is CAM-c (Table 1). Also, four of our studied species reach latitudes with minimum temperatures within the 0-5 °C range at the coldest month, three of which are
C3, being the only exception Tillandsia landbeckii (CAM-c). Why do these two CAM species behave contrary to their group? T. landbeckii lives on the Atacama desert sands, with no functional roots and thus being totally fog-dependant for water uptake (Pinto et al. 2006,
Latorre et al. 2011). In coastal Atacama desert temperature difference between night and day is around 10-20 °C (Weischet 1975), which probably enhances the CAM process as we mentioned above. Given its morphological characteristics and its physiological requirements, the fact that this is a CAM species seems to be an advantage, even facing chilling temperature events at the coldest month. A similar scenario is faced by southernmost populations of Puya berteroniana on pre-Andean slopes where day/night variation on temperature is probably significant, especially in summer, and as we mentioned above, moisture conditions probably favored the adoption of CAM despite the possible negative effects of below-zero minimum
65
temperatures. However, P. berteroniana is not the only Chilean bromeliad to live on pre-
Andean slopes under such climatic conditions, but is the only CAM bromeliad to do so. The other species that can be found on similar sites (Ochagavia andina, Greigia pearcei, Puya alpestris, Ochagavia carnea) are all C3. The fact that these species are C3 and P. berteroniana is CAM might be a reflection of phylogenetic history, more than a physiological response to aridity/temperature. Species from the Bromelioideae subfamily (O. andina, O. carnea, G. pearcei) are believed to have originated prior the adoption of CAM by the central-Chilean clade of the family (Crayn et al. 2004). The other C3 species, Puya alpestris, advances less to the north, not reaching the drier part of the Mediterranean region (Zizka et al. 2009). Both
P. berteroniana and P. alpestris are closely related, being referred as a “species complex” by some authors, with P. berteroniana apparently diverging from P. alpestris populations
(Jabaily & Sytsma 2010, Schulte et al. 2010), and even being considered as one single species divided in two subspecies (Zizka et al. 2013). The fact that P. berteroniana is probably a lineage formed from P. alpestris might suggest a late adoption of CAM by the first, while advancing towards north after separating from the latter. This adoption probably helped the new species to reach the semiarid region around 29°S.
Climatic variables have been previously reported as good predictors of plant species distribution (Shao and Halpin 1995; Sykes et al. 1996; Stephenson 1998; McKenzie et al.
2003; Gavin and Hu 2006). On the other hand, latitude itself is not an environmental factor that can directly influence the expression of CAM. This has been observed in other studies, where latitude turns out to be a better predictor of phenotypic variation than the measured climatic variables (Naya et al. 2011, 2012; Molina-Montenegro and Naya 2012). According to these studies, this probably happens because latitude is a better predictor of long-term 66
climatic regimes than values provided by weather stations or, in this case, Worldclim data interpolations.
The adoption of the CAM pathway has been proven to be an evolutionary key innovation for
Orchidaceae (Silvera et al. 2009) and Bromeliaceae (Quezada & Gianoli 2011). Other adaptations to drought in Bromeliaceae, such as tanks and absorptive trichomes, are considered crucial for the expansion of the family within the epiphytic tropical habitat (Smith and Downs 1974, 1977, 1979; Benzing 2000). CAM, being the main adaptation to drought in terrestrial bromeliads, was probably a very important feature for the expansion of the geographic range of species living on arid and semiarid environments. For Chilean bromeliads we can infer that the adoption of CAM while advancing towards north from their possible southern origin (Jabaily & Sytsma 2010, 2013) was crucial for the occupation of semiarid regions in the Mediterranean climate zone and for their advance to arid regions, a movement that was decisive for the diversification of genus like Puya, which radiated along the Andes high plains and slopes (Varadarajan 1990). The seemingly higher importance of phylogenetic relationships, more than the effect of climate, for the distribution of photosynthetic pathways in Chilean bromeliads is a subject that should be accounted for in future studies. This might be done broadening the scope of geographic distribution of CAM and C3 pathways to other families within the Mediterranean region of central Chile, or expanding the studied range to a bigger part of the continental distribution of Bromeliaceae.
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TABLES
Table 1: The 18 study species (SP) and the analysed variables: Photosynthetic pathway (PP), Maximum coastal latitude (MCL), Maximum “absolute” latitude (MAL), Annual precipitation at maximum “absolute” latitude (APP) and Minimal temperature of coldest month at maximum “absolute” latitude (MTCM). Latitudes are expressed in decimal degrees, precipitation in mm, temperature in celsius degrees. Sources for photosynthetic pathway data are included.
SP PP MCL MAL APP MTCM Sources of PP data Fascicularia bicolor C3 42.277 42.641 2326 3.7 Crayn et al. 2004 Greigia landbeckii C3 41.911 42.5 2254 4.7 Crayn et al. 2004 Greigia pearcei C3 39.918 40.152 1426 -2.3 Crayn et al. 2004 Greigia sphacelata C3 42.604 42.604 1974 5.7 Crayn et al. 2004 Ochagavia andina C3 36.528 36.528 952 -3.4 Crayn et al. 2004 Ochagavia carnea C3 37.237 38.353 1889 -1.1 Crayn et al. 2004 Ochagavia litoralis C3 33.484 34.207 561 5.9 Crayn et al. 2004 Martin 1994, Puya alpestris C3 36.746 38.59 1333 -1.9 McWilliams 1970, Crayn et al. 2004 Puya coerulea C3 35.131 36.447 919 4.2 Crayn et al. 2004 Martin 1994, Puya chilensis CAM-f 37.246 37.246 1233 5.8 Quezada et al. (unpubl.) Quezada et al. Puya gilmartiniae CAM-f 31.563 31.563 171 8 (unpubl.) Crayn et al. 2004, Puya venusta CAM-f 33.458 33.458 569 7.6 Quezada et al. (unpubl.) Deuterocohnia CAM-c 25.469 25.469 14 8.4 Crayn et al. 2004 chrysantha Crayn et al. 2004, Puya berteroniana CAM-c 33.398 35.116 999 -2.6 Quezada et al. (unpubl.) Puya boliviensis CAM-c 25.749 25.749 21 10 Martin 1994 Tillandsia geissei CAM-c 30.185 30.185 101 8.2 Martin 1994 Crayn et al. 2004, Tillandsia landbeckii CAM-c 30.669 30.855 162 2.5 Latorre et al. 2011 Tillandsia tragophoba CAM-c 24.933 24.933 14 9.6 Martin 1994
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Table 2: ANOVA F-values and significance of interactions between photosynthetic pathway variation and all the compared factors.
Factor F p-value
Maximum coastal latitude 16.217 0.0001
Maximum absolute latitude 16.851 0.0001
Annual precipitation at maximum absolute latitude 10.556 0.001
Minimum temperature at the coldest month at maximum absolute 3.086 0.075 latitude
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Table 3: Student’s t-test t and p values for the phylogenetically independent contrast performed on the four comparisons.
Factor t p-value
Maximum coastal latitude 0.067 0.947
Maximum absolute latitude -0.358 0.725
Annual precipitation at maximum absolute latitude -0.135 0.893
Minimum temperature at the coldest month at maximum absolute -0.129 0.899 latitude
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FIGURES
Figure 1: Phylogenetic tree of the Chilean species of Bromeliaceae, constructed for use in the phylogenetycally independent contrasts (see text for references). Photosynthetic pathway is indicated for each species. 82
CAM-c b CAM-c b
CAM-f a CAM-f b
C3 a C3 a Photosynthetic pathway Photosynthetic Photosynthetic pathway Photosynthetic
0 1020304050 0 500 1000 1500 2000 2500 Maximum coastal latitude Annual precipitation at maximum absolute latitude
CAM-c c CAM-c a
CAM-f b CAM-f a
C3 a C3 a Photosynthetic pathway Photosynthetic pathway Photosynthetic
0 1020304050 0246810 Maximum absolute latitude Minimum temperature of the coldest month at maximum absolute latitude
Figure 2: Distribution of photosynthetic pathway categories along latitude, temperature and precipitation gradients. Bars represent mean values. Bars represent standard error. Letters beside error bars represent significant differences between means as given by Fisher’s LSD test.
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CAPÍTULO III
En este capítulo se comparó la variación interpoblacional en la expresión de CAM para 3 especies nativas de Puya: P. berteroniana, P. chilensis y P. venusta. De ellas, solo P. chilensis mostró una disminución significativa en la expresión de CAM al avanzar hacia el sur en el gradiente latitudinal, como resultado de los dos métodos utilizados para medir la actividad fotosintética. Por lo mismo, en el artículo que conforma este capítulo se entregan solo los datos para esta especie (ver Apéndice de este capítulo para figuras de P. venusta y
P. berteroniana), enfocándose además en el valor metodológico que implica el uso de dos tipos de análisis distintos, en forma y foco. La población más austral de P. chilensis mostró valores indicativos de fotosíntesis C3, con leve uso de CAM, en tanto en el resto de las poblaciones muestreadas, el aumento en el uso de CAM es gradual, de sur a norte.
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LATITUDINAL VARIATION IN THE DEGREE OF CRASSULACEAN ACID METABOLISM IN PUYA CHILENSIS: ADVANTAGES OF THE USE OF TWO ANALYTICAL METHODS
Iván M. QuezadaA, Gerhard ZotzB, Ernesto GianoliA,C
ADepartamento de Botánica, Universidad de Concepción, Casilla 160-C, Concepción, Chile. E-mail address: [email protected]
BFunctional Ecology Group, Institute of Biology and Environmental Sciences, University of Oldenburg, Box 2503, D-26111, Oldenburg, Germany. E-mail address: gerhard.zotz@uni- oldenburg.de
CDepartamento de Biología, Universidad de La Serena, Casilla 554, La Serena, Chile. E-mail address: [email protected]
Corresponding author: Iván M. Quezada. Postal address: Departamento de Botánica, Universidad de Concepción, Casilla 160-C Concepción, Chile. Phone: +56 41 2203418 Fax: +56 41 2246005 E-mail address: [email protected]
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Summary Text for the Table of Contents
Understanding how plants survive on arid and semi-arid environments is an important topic for plant ecology. Mainstream methods of detection of Crassulacean Acid Metabolism
(CAM) may lead to wrong conclusions when used on their own. Our results show that CAM expression can vary for populations living under different environmental conditions, and that a wide sampling scheme, together with a combination of analytical methods, can be crucial for a correct detection of CAM species.
Abstract
Crassulacean acid metabolism (CAM) is a photosynthetic pathway found in many plant species from arid and semiarid environments. Originally, CAM plants were identified via the determination of nocturnal acidification. Later, isotopic measurements have allowed researchers to detect this photosynthetic pathway in extensive samples of plants. However, isotopic methods alone may fail to distinguish between C3 species and species with a limited degree of CAM activity (facultative CAM species). A combination of isotopic measurements and other methods has been proposed to deal with this lack of resolution. On the other hand, studies usually do not account for inter-population differences in photosynthetic pathway.
This may be especially problematic for species growing under highly variable climatic conditions. We used Puya chilensis, a putative facultative CAM species, to study among- population variation in the expression of the CAM pathway within its distributional range using both nocturnal acidification and stable isotope measurements. We studied 5 populations of P. chilensis along its latitudinal range, which spans about 800 km with 86
substantial variation in climatic conditions, ranging from semi-arid to temperate oceanic climates. Results of both analytical methods indicated that CAM decreased with latitude.
However, even in the southern populations, where δ13C values were indicative of C3 metabolism, we found some measurable nocturnal acidification. Results suggest that P. chilensis populations use C3 and CAM to varying degrees depending on environmental conditions. We stress the value of using two methods, and samples from different populations, to get more reliable information on the photosynthetic pathway for “probable
CAM” plant species that face varying climatic conditions within their distributional ranges.
Introduction
Crassulacean acid metabolism (CAM) is a photosynthetic pathway found in a substantial number of vascular plants. This physiological adaptation to drought stress is very frequent in the Cactaceae, Orchidaceae, Crassulaceae and Bromeliaceae, which have successfully spread across a variety of arid and semiarid habitats such as tropical forest canopies, rocky cliffs in semi-arid regions or even desert sands (Benzing 2000; Nobel and Bobich 2002; Pinto et al.
2006). While C3 plants capture CO2 exclusively during the day, the CAM pathway implies a temporal separation of photosynthetic phases: plants open their stomata at night, when they capture and store CO2 in malic acid. This organic acid acts as a reservoir of CO2 for the rest of the photosynthetic process, which is carried out during daytime. This allows plants to keep their stomata closed during the day, when temperature, irradiation and chances of losing water via transpiration are higher (Ting 1985; Taiz and Zeiger 2002; Larcher 2003; Heldt
2005). However, the difference between CAM and C3 plants may be seen as gradual rather
87
than categorical, since even obligate CAM plants can take up part of their CO2 during the day, and plants classified as C3 can show some degree of nighttime CO2 capture under certain circumstances (Pierce et al. 2002; Zotz 2002).
CAM plants are usually classified as obligate or facultative (Winter et al. 2008). Obligate
CAM plants are considered almost totally dependent on the CAM pathway, while facultative
CAM plants show different degrees of CAM usage and are able to induce, or at least to up- regulate, CAM under stress (Ting 1985; Taiz and Zeiger 2002; Winter et al. 2008). A modern method of analysis based on different discrimination rates for 13C of RUBISCO and PEPC enzymes has proven to be an efficient tool to determine the photosynthetic pathway of plants
(Farquhar et al. 1989; Dawson et al. 2002). However, facultative or weak CAM plants are rarely detected with this method and are typically classified as C3 (Pierce et al. 2002; Winter and Holtum 2002; Silvera et al. 2005; Winter et al. 2008). A combination of analyses (i.e. isotopic measurements combined with nocturnal acidification and/or diel gas exchange measurements) has been proposed as a solution to reduce the chance of error in sorting species according to their photosynthetic pathway, in particular for tropical species (Pierce et al. 2002; Silvera et al. 2005). Even this combined approach might miss CAM species and classify them as C3, especially when species grow over a wide geographical range with high climatic variation. Thus, different populations of a facultative CAM species are possibly classified as C3 species at the wet end of the gradient and as CAM at the dry end. Such variation will easily go unnoticed in larger surveys.
Bromeliaceae are a good example for this general problem. Since the 1970’s several studies of the photosynthetic pathways in this family have been published. Methods used to
88
distinguish between C3 and CAM species varied from classic diel gas exchange or nocturnal acidification measurements (e.g. Medina 1974; Winter et al. 1992; Loeschen et al. 1993) to isotopic methods (e.g. Medina et al. 1977; Griffiths 1984; Crayn et al. 2004) and combination of different methods (Pierce et al. 2002). Distinguishing C3 and CAM species in this large family has allowed researchers to understand the evolution of CAM photosynthesis (Crayn et al. 2004) and even identify CAM as a key innovation that facilitated lineage diversification
(Quezada and Gianoli 2011). However, in large families such as Bromeliaceae and
Orchidaceae information on the photosynthetic pathway of a given species usually comes from samples taken from a single population or cultivar (e.g. Silvera et al. 2005), and/or from individual herbarium material (e.g. Crayn et al. 2004; Silvera et al. 2009, 2010). This may increase the chance of underestimating the number of CAM species if significant among- population variation exists.
Puya is a bromeliad genus that spreads along the Andes (Smith and Downs 1974). It is believed to have originated in central-southern Chile (Jabaily and Sytsma 2010, 2013), and its diversification probably took place while advancing into arid and semiarid landscapes, such as the Mediterranean climate zone in central Chile and arid/semiarid slopes and cliffs of the Andes mountains (Varadarajan 1990). This genus is well adapted to drought, with thick leaves that show a degree of succulence (Smith and Downs 1974) and the adoption of CAM photosynthesis by some of its species (Crayn et al. 2004). A Puya species with a significantly wide geographical/climatic range is Puya chilensis, which is endemic to Chile. This species is distributed in coastal plains between 29°30’S and 37°S, with a gap between 33°30’S and
35°30’S, which may be due to an actual absence of populations or lack of data (Zizka et al.
2009). This latitudinal range is the longest of all Chilean Puya species (Zizka et al. 2009) and 89
seems to be limited in the south by the presence of humid temperate rainforest. Consequently,
P. chilensis populations experience highly diverse environmental conditions, which become increasingly humid from north to south: semiarid climate, Mediterranean-type climate, and the rainy transition zone between Mediterranean and Temperate Oceanic climates (Hajek and
DiCastri 1975; DiCastri and Hajek 1976; Schwedtfeger 1976). There is a ten-fold increase in mean annual precipitation between the northern and southern limits of the species range, and a pattern of decreasing mean temperatures towards the south (Hajek and DiCastri 1975). This species has been classified either as C3 (Griffiths 1984) or CAM (Medina et al. 1977), a discrepancy which has been noted earlier (Martin 1994), but remains unresolved. The environmental gradient along the species geographic range could be reflected in corresponding differences in the degree of CAM activity in P. chilensis populations, which led us to expect a significant decrease in the use of the CAM pathway, or a switch to C3, at higher latitudes in central Chile. We propose that P. chilensis is a facultative CAM species, and suggest that limited sampling has led to the current uncertainty, which could be reduced with a wider sampling scheme and a combination of analytical methods. The goals of this work were: i) to verify that Puya chilensis is indeed a facultative CAM species, ii) to demonstrate the utility of a combined analytical approach for CAM determination in a species with a wide geographical range, and iii) to determine whether the expression of the CAM pathway in Puya chilensis decreases with latitude.
90
Material and Methods
We chose two methods to determine the photosynthetic pathway: determination of nocturnal acidification and stable isotopes measurement. These methods complement each other when the purpose is to avoid the bias of the isotopic measurements; it has been recommended to combine the latter with other methods (Pierce et al. 2002). The two methods differ in the time scale they measure. Determining the accumulation of malic acid during one individual night reflects the plant performance under the climatic conditions of the day/night cycle at which measurements were taken. Isotopic analysis, on the other hand, gives information on the photosynthetic pathway over the life cycle of the sampled tissue, thus integrating plant responses over long time scales (Farquhar et al. 1989; Adams and Grierson 2001; Dawson et al. 2002).
Five coastal populations of Puya chilensis in central Chile were chosen for our analyses (Fig.
1): Canela (31°15’S), Los Vilos (31°53’S), Pichidangui (32°09’S), Punta Curaumilla
(33°05’S) and Caleta Lenga (36°45’S).
Nocturnal acidification measurements
Sampling was carried out in August 2011, during the southern hemisphere winter season, when differences in precipitation along the distributional range of P. chilensis are more marked (Hajek and DiCastri 1975; DiCastri and Hajek 1976) (see “Expected” column in
Table 1). Fifteen plants per population were randomly chosen and samples were collected from mature leaves of these plants at two different times: just before dusk and just before dawn. Extract from the collected leaf material was tritrated using 0,1N NaOH to estimate the
H+ concentration of the tissues. Results from leaves collected at dusk and dawn from the 91
same plant were compared to obtain a measure of the variation of acid content between night and day (∆H+) for each individual (modified from Silvera et al. 2005).
Isotopic discrimination
Leaves from 5 plants per population were collected during February 2012. To avoid the influence that leaf development has on δ13C values, only mature leaves were collected, as for the measurements of nocturnal acidification. Leaf material was dried at 80°C for 48 h and ground to a fine powder. Samples were analyzed for carbon stable isotopic composition
(δ13C) with an isotope ratio mass spectrometer (Department of Terrestrial Ecosystem
Research, Faculty of Life Sciences, University of Vienna) as described by Wanek et al.
(2002). 13C/12C ratios were calculated relative to the Pee Dee belemnite standard using the relationship:
δ13C (‰) = [(13C/12C in sample)/(13C/12C in standard) – 1] x 1,000
Although CAM and C3 are not absolute categories, no alternative to this categorization has been proposed yet (see Zotz 2002). Therefore, for analytical reasons we had to place a boundary between both pathways, and chose a classification for the obtained isotopic values.
Samples with values from -33 to -22.1‰ were classified as C3, and those with values ranging from -22 to -12‰ were classified as CAM (Ehleringer and Osmond 1989). The difference in sampling dates for the two analytical methods should not cause a bias because isotopic measurements reflect the photosynthetic pathway across the entire leaf lifespan (see above) and leaves of Puya species are long-lived (Augspurger 1985; Sgorbati et al. 2004).
92
Data analysis
Data obtained from both nocturnal acidification and isotopic discrimination methods were analyzed using statistical software packages STATISTICA™ 10 (Statsoft) and
SIGMAPLOT™ 12.3 (Systat). To determine the level of inter-population variation detected by each analytical method we performed one-way ANOVAs followed by LSD tests comparing all the sampled populations. To evaluate the variation in CAM expression with latitude we performed regression analyses, including all sampled populations for each method.
Results
All sampled populations had statistically similar acid levels at dusk (population means between 0.069 and 0.071 mmol H+ / g FW; p > 0.05 LSD test, for all comparisons). Mean
ΔH+ values per population ranged between 0.446 and 0.493 mmol H+ /g FW. Nocturnal acidification decreased significantly with latitude (R2 = 0.16, p <0.01), a pattern that was mostly due to the southernmost population (Caleta Lenga; 36°45’S), which showed significantly lower ΔH+ values compared to 3 of the other 4 populations (Fisher LSD test, p
<0.05, Fig. 2).
The mean δ13C values in the 5 populations varied by 5‰. Consistent with the acidification data, tissue δ13C became increasingly negative with latitude (R2 = 0.59, p <0.001; Fig. 2).
The northern populations, Canela (31°15’S) and Los Vilos (31°53’S), had δ13C values within the range usually accepted as indicative of CAM. Central populations had δ13C values beyond
93
the CAM range, but near -22‰, its lower limit. At the southern extreme, the δ13C values of
Caleta Lenga (36°45’S) were clearly within the C3 range and significantly different from the other 4 populations (Fisher LSD, p <0.001).
Discussion
Plants with CAM are mostly distributed in regions with low water availability (e.g. Cactaceae in arid and semiarid areas), while C3 plants are usually found in temperate and cold regions
(Ting 1985; Lüttge 2004). In tropical and subtropical regions, CAM plants –usually epiphytic– are mainly found at drier sites, but they are present throughout the whole moisture range (Zotz and Hietz 2001). This ecological breadth can be considered a consequence of the flexibility of the CAM pathway (Lüttge 2004). The genus Kalanchöe (Crassulaceae) in
Madagascar represents a remarkable example for the influence of the photosynthetic pathway on the geographic (and climatic) range of groups within a clade. Madagascar has a marked climatic gradient, from perhumid-hot to subarid-hot climates (Kluge et al. 1991). There,
Kalanchöe subsections Kitchingia, Bryophyllum and Eukalanchoe are present and show differences in photosynthetic pathway and the degree of CAM activity. Species belonging to
Kitchingia usually rely on C3 photosynthesis, but a few species are capable of performing
CAM photosynthesis to some extent. Kitchingia species are restricted to the per-humid sections of the island, mainly rainforests and evergreen montane forests. Species from
Eukalanchoe, all obligate CAM, are concentrated in the arid southwestern extreme of the island. Species from Bryophyllum, which are facultative CAM that tend to rely on CAM but are more flexible in this “switching”, can be found on a wider geographical and climatic scale
94
(Kluge et al. 1991; Lüttge 2004). At an intraspecific level, Scarano et al. (2002) found significant variation in acid accumulation levels between populations of Aechmea bromeliifolia (Bromeliaceae) living under different light and flooding regimes in southern
Brazil. There, plants from shaded sites had significantly lower ΔH+ values. In addition, while all populations had δ13C values within the CAM range, these values were lower on plants from drier sites. These intraspecific differences have also been noted on species cultivars, a pattern probably related to artificial selection of varieties (Ehleringer 1990). Our results are concordant with studies addressing variations in CAM expression within and between species along different gradients and between seasons, with its expression being induced under drought conditions (Winter et al. 1978; Earnshaw et al. 1987; Zotz and Tyree 1996;
Herrera et al. 2010; Winter et al. 2008, 2011).
In Chile, rainfall increases considerably and progressively from north to south (Hajek and
DiCastri 1975; DiCastri and Hajek 1976). P. chilensis has the widest latitudinal range (almost
8 degrees of latitude in total, albeit not continuous) among the seven Chilean species of Puya, and it is the only species that spreads from the semiarid region to the temperate Oceanic climatic region (Zizka et al. 2009). Previous work (Griffiths 1984; Medina et al. 1977) suggested variable use of nocturnal CO2 uptake in this species and we hypothesized that this was directly related to the diverse environmental conditions within its geographical range.
Results show a clear pattern of variation in the use of the CAM pathway for P. chilensis along its latitudinal range of distribution. Acidity level at dusk in P. chilensis was statistically similar for all sampled populations. It was higher than dusk values reported for the amphibious plant Littorella uniflora (Robe and Griffiths 2000), and Opuntia elatior (Winter
95
et al. 2011), and it was similar to the ones reported for Tillandsia usneoides under different experimental conditions (Martin et al. 1985), and for strong-CAM Clusia species, in which a low background level of acidity apparently makes possible to perform a stronger CAM activity (Holtum et al. 2004). For Puya chilensis, inter-population differences in acidity are due to different rates of nocturnal acid accumulation. These rates are similar to those reported for other bromeliads under drought or high irradiance conditions, such as Guzmania monostachia (Freschi et al. 2010) and Tillandis usneoides (Martin et al. 1985). Although even the southernmost population of P. chilensis, which experiences moister growth conditions, showed some nocturnal acidification, δ13C values indicate that the importance of
CAM activity to overall carbon gain is probably negligible (Winter and Holtum 2002). Taken together, our results indicate that P. chilensis is able to use C3 and CAM metabolism to varying degrees depending on environmental conditions. Plants from the southernmost population still accumulated a limited amount of organic acids at night during the 2011 winter season, even when water availability should not be a limiting factor in this region. This may reflect a process of recycling respiratory CO2 that frequently occurs within leaves (e.g.
Griffiths et al. 1989), and/or the time scale spanned by nocturnal acidification measurements, which measure the activity of the plant during a specific night of a given season. The southernmost population (Caleta Lenga) received unusually low rainfall during the 2011 winter season (Table 1), which may have led to a higher use of CAM than usual. Even short periods without rain lead to a significant loss of moisture in the rocky soils where these individuals of P. chilensis grow (Quezada, pers. obs.). Thus, the documented nocturnal acidification is more indicative of the flexible use of CAM in P. chilensis in general; the δ13C values suggest that CAM hardly plays a role under the moist growing conditions at the
96
southern end of the species´ distribution. Other environmental factors that change within the latitudinal gradient, such as temperature, may have some influence on CAM expression in P. chilensis. It has been shown that temperature, in particular a significant difference between day/night temperatures, improves CAM performance, and that chilling temperatures reduce the performance of the main CAM enzyme (PEPC) (Medina et al. 1977; Haag-Kerwer et al.
1992; Carter et al. 1995; Chinthapalli 2003). At the location of the southernmost P. chilensis population, temperatures may fall below 0 °C during winter nights, which can effectively reduce the efficiency of CAM photosynthesis. However, this effect is probably less important compared to the effect of low water availability. Day/night temperature variation probably had little effect on the different levels of CAM expression between the studied coastal populations of P. chilensis because thermal variation is similar along the latitudinal gradient due to the ‘buffer’ effect of the Humboldt oceanic current (Zinsmeister 1978).
Our study contributes unambiguous evidence that Puya chilensis is a facultative CAM plant species. It highlights the importance of the use of multiple methods with material from several locations, as previously suggested by other authors (Pierce et al. 2002; Silvera et al.
2005), in this case for plants living on large and marked environmental gradients. Both methods used here showed differences between populations, but the fact that samples came from populations that live under different climatic conditions enabled us to clearly state that the study species is facultative CAM. Had we only relied on nocturnal acidification measurement from samples of any of the chosen populations, we would have considered P. chilensis to be obligate CAM. The same would have happened had we used both methods but only with samples from the north. In contrast, if we had only relied on isotopic
97
measurements from samples of the southernmost population, we would have labeled P. chilensis a C3 species.
All of the above illustrates the importance of a wide sampling scheme, and the use of more than one analytical method, to ascertain whether a plant is C3, obligate or facultative CAM.
Pierce et al. (2002) sampled a large number of tropical bromeliad species combining isotopic measurements with nocturnal gas exchange measurements. Their results show that isotopic measurement alone can underestimate the number of CAM species by overlooking those performing intermediate levels of CAM, or those switching between both. Despite some reported variations of δ13C values within species (e.g. Kluge et al. 1991), the advantages of a sampling scheme including different populations of a widely distributed species had not been explicitly shown.
Conclusions
This work presents clear evidence that Puya chilensis is a facultative CAM species, showing a decrease in the expression of the CAM pathway with increasing latitude. Also, we have shown that collecting data from different populations growing under contrasting environmental conditions increases the reliability of methodological approaches, and thus the resulting conclusions in terms of the photosynthetic pathway of a given species. Future studies in large environmental gradients should consider the potential drawbacks of the most popular methods used in screening studies, and the importance of sampling plants growing under different environmental conditions. Our study species, Puya chilensis, may not be unique with respect to its substantial variation in the use of CAM. 98
Acknowledgements
We thank Paula Aguayo, Leonor Quezada and David Aguayo for their valuable help during fieldwork. We are grateful to Patricio Novoa (Jardín Botánico Nacional, Chile) for his help in locating Puya populations and to Wolfgang Wanek (University of Vienna) for providing the isotope data. This research is funded by Doctoral (21090071) and Doctoral Thesis
Support (24110160) grants by CONICYT, Chile (I.M.Q.).
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TABLES
Table 1: Values of mean annual precipitation (mm) for 2011 and average precipitation
(mean of the last 30 years) at the closest meteorological stations to the sampling sites, from north to south (Based on Instituto Nacional de Estadísticas, 2012a, 2012b). Distance in km between stations and sampling sites is indicated.
Station 2011 Average Distance from Sampling site
154 Km N of Canela La Serena 158,6 78,5 225 Km N of Los Vilos
97 Km S of Pichidangui Valparaíso 291,9 372,5 15 Km NE from Punta Curaumilla
Concepción 760,4 1110,1 12 Km SE from Caleta Lenga
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FIGURES
Figure 1: Geographical location of the five studied populations of Puya chilensis.
108
0,52 (I)
a a 0,50 a
0,48 / g FW) / g +
b 0,46 ab (mmol H + H
0,44
0,42 30 31 32 33 34 35 36 37 38 -18 (II) a
-20 a
-22 C (‰) 13
b b c -24
-26
30 31 32 33 34 35 36 37 38 Latitude (°S)
Figure 2: (I) Variation of nocturnal acid accumulation for the 5 populations. Means ± S.E.
(n = 15 plants per population) are given, along with the fitted regression curve. (II) δ13C
109
(‰) measured for the 5 populations. Means ± S.E. (n = 5 plants per population) are given, along with the fitted regression curve. Statistically different populations have different lowercase letters (see Section 3 for detailed results).
110
APÉNDICE AL CAPÍTULO III.
0,60 a
0,58
0,56 / g FW) +
0,54 H+ (mmol H (mmol H+
0,52
0,50 31,35 32,15 33,09 Latitud (°S)
0,65 b 0,60
0,55
/ g FW) 0,50 +
0,45 H+ (mmol H
0,40
0,35
0,30 31,56 32,15 32,55 Latitud (°S)
Variación interpoblacional en la acumulación nocturna de ácido málico (ΔH+) en a) Puya berteroniana y b) Puya venusta. Se grafican las medias con su error estándar y la curva de regresión lineal ajustada.
111
-18,5 a -19,0
-19,5
-20,0
-20,5 C (‰) 13 -21,0
-21,5
-22,0
-22,5 31,35 32,15 33,09 33,6 Latitud (°S)
-22,0 b -22,5
-23,0
-23,5 C (‰) 13
-24,0
-24,5
-25,0
-25,5 31,56 32,15 32,55 Latitud (°S)
Variación interpoblacional en los valores de δ13C (‰) en a) Puya berteroniana y b) Puya venusta. Se grafican las medias con su error estándar y la curva de regresión lineal ajustada.
112
CAPÍTULO IV
En este capítulo se evaluó el valor adaptativo de la expresión de la vía CAM en dos poblaciones de Puya chilensis: una ubicada en la zona norte y otra cerca del extremo sur de su rango de distribución. Para esto se construyeron gradientes de selección, los que mostraron que, en el norte, la expresión de CAM tiene efectos positivos en el fitness de los individuos, en tanto en la zona sur, más húmeda, la expresión de CAM tiene efectos negativos sobre el fitness.
113
PHENOTYPIC SELECTION ON CAM PHOTOSYNTHESIS IN PUYA CHILENSIS: WATER AVAILABILITY AS A MAIN DRIVER
Iván M. QuezadaA, Alfredo SaldañaA, Ernesto GianoliA,B
ADepartamento de Botánica, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
E-mail address: [email protected]
BDepartamento de Biología, Universidad de La Serena, Casilla 554, La Serena, Chile. E-mail address: [email protected]
Corresponding author: Iván M. Quezada.
Postal address: Departamento de Botánica, Universidad de Concepción, Casilla 160-C
Concepción, Chile.
Phone: +56 41 2203418
Fax: +56 41 2246005
E-mail address: [email protected]
114
ABSTRACT
Puya chilensis, a facultative CAM species, distributes along a latitudinal gradient of precipitation. The CAM phptosynthetic pathway is a well known adaptation to aridity, but no negative effect of its expression under moist conditions has been proven yet. We tested the adaptive value of CAM expression, along with some morphological attributes related to drought tolerance, in two P. chilensis populations living under different moisture regimes.
Only CAM expression had a significant correlation with our fitness estimator. This correlation was positive in the dry (northern) population, and negative in the moist (southern) population. We infer the possible effect of CAM expression over P. chilensis ability to spread into moister regions. Probably, for plants living in moister areas CAM is only a resource sink, that will impair their chances to spread to southern regions.
115
INTRODUCTION
Water availability is a key factor for plant development and diversification. The geographical range of plant species is sometimes related to moisture, or to the ability to tolerate arid or semiarid conditions (Royce and Barbour 2001; Hampe 2005; Engelbrecht et al. 2007; Eckhart et al. 2010). Aridity has been considered a key factor for plant diversification (Axelrod 1972,
Arroyo et al. 1988), and some of the adaptations plants have developed to tolerate low water availability have been crucial for the diversification of plant families (e.g. Silvera et al. 2009,
Quezada & Gianoli 2011).
One of the most prominent adaptations to aridity is the CAM (Crassulacean Acid
Metabolism) photosynthetic pathway. This pathway exists in several vascular plant families that have successfully diversified on a wide range of habitats ranging from semiarid to hyperarid (e.g. Orchidaceae, Crassulaceae and Bromeliaceae) (Nobel and Bobich 2002; Pinto et al. 2006). The CAM pathway implies a temporal separation of photosynthetic phases, in which plants open their stomata and capture CO2 at night, storing it as malic acid in cellular vacuoles. The rest of the photosynthetic process is carried out during daytime. This separation allows plants to keep their stomata closed during daytime, when chances of losing water via transpiration are higher (Ting 1985; Taiz and Zeiger 2002; Larcher 2003; Heldt 2005). There is a variety of levels of CAM expression among CAM-capable plants. Its usage ranges from facultative CAM to constitutive CAM plants, up to the extreme CAM-idling plants, which recycle respiratory CO2 and keep stomata closed day and night in extremely arid environments (Ting 1985; Taiz and Zeiger 2002; Winter et al. 2008).
116
The use of CAM has been proven to be crucial for the diversification of different groups of plants in arid and semiarid habitats (e.g. see Silvera et al. 2009 for Orchidaceae, and Quezada
& Gianoli 2011 for Bromeliaceae). There are reports of a positive correlation between CAM expression and fitness for facultative-CAM plants (e.g. Winter and Ziegler 1992, Sayed and
Hegazy 1994, Taisma and Herrera 1998, 2003), and the plasticity on its expression its believed to be of adaptive value, in particular for species that face variable climatic conditions
(Lüttge 2004, Herrera 2009). However, the CAM pathway probably requires more biochemical “machinery” to work than C3 considering its extra steps in CO2 capturing-storing and the involvement of a second enzyme (PEPC), responsible for the CO2 capture (Taiz and
Zeiger 2002, Lüttge 2002). Thus, it could become a significant resource sink under high moisture conditions, where it may be unnecessary. Spending resources into a trait no longer necessary could impose a negative effect on the performance of a plant, reducing its fitness
(see Guimaraes et al. 2008, Johnson 2009). To address this issue, the change in the adaptive value of the CAM pathway under different moisture conditions can be measured in a species able to “switch” between C3 and CAM pathways, in populations located in contrasting habitats in terms of soil moisture. A high adaptive value for CAM could be expected for plants living in arid or semiarid conditions, and a lower, or negative adaptive value for plants living in mesic or wet sites. To determine the adaptive value of a trait under a given environment, it is necessary to estimate its influence on plant fitness (Phillips and Arnold
1989, Dudley 1996, Ackerly et al. 2000, Geber and Griffen 2003). Phenotypic selection analyses can be used to test for these adaptive hypotheses (Lande and Arnold 1983, Endler
1986).
117
The genus Puya (Bromeliaceae) includes facultative CAM species and shows intraspecific variation in the use of CAM (Martin 1994, Herrera et al. 2010, Quezada et al. unpublished).
This genus is distributed from Costa Rica to central Chile, and comprises strictly terrestrial species with fully functional roots that lack the typical morphological adaptations to aridity found in other bromeliad groups (Smith & Downs 1974, Benzing 2000, Varadarajan 1990).
However, this genus is very well adapted to aridity, growing in arid and semiarid regions of the Andes high plains and the Mediterranean climate region of Chile (Smith & Downs 1974,
Benzing 2000, Zizka et al. 2009). Seven Puya species are endemic to central Chile and four of them have been classified as CAM (Martin 1994, Crayn et al. 2004). Within these CAM species, only P. chilensis ranges from the semiarid region (29°30’S) to the rainy transition zone between Mediterranean and Temperate-Oceanic climates (37°S), being the Puya species with the longest latitudinal range in Chile (Zizka et al. 2009). Consequently, P. chilensis populations experience contrasting environmental conditions, which become increasingly humid from north to south (Luebert and Pliscoff 2006). There is a ten-fold increase in mean annual precipitation between the northern and southern limits of the species range (Hajek and DiCastri 1975). It has been shown that P. chilensis populations in markedly different habitats express different levels of CAM photosynthesis, and that CAM usage at the species level decreases with increasing latitude (Quezada et al., unpublished). In this context, to test if the long latitudinal and climatic range faced by P. chilensis populations imposes differential selective pressures on the CAM pathway that might lead to a significant change on its adaptive value from north (dry) to south (wet), can help to understand the functional bases underlying to its latitudinal distribution pattern.
118
This study addresses phenotypic selection on CAM photosynthesis, and on leaf traits related to plant functional responses to low moisture, of P. chilensis plants from two populations located in sites with divergent moisture regimes. Regarding the CAM pathway, as it could become less useful and a resource sink under high moisture conditions, we hypothesized that, for plants of the southern population, use of CAM would have negative effects on fitness. On the contrary, for plants living on a northern, drier population, we expected that CAM expression would be positively associated with fitness. We also anticipated that plants from the northern population would express higher levels of CAM than their southern counterparts, due to the greater aridity they experience. Finally, we expected positive selection on leaf thickness and leaf mass per area (LMA) in the dry site, as increased values of these traits are considered adaptations to arid conditions (Wright et al. 2004, 2005, 2006). We included these leaf traits in the analysis because the correlation between traits target of selection may explain observed adaptive syndromes or lack thereof (Lande and Arnold 1983, Etterson and Shaw
2001).
MATERIALS AND METHODS
Study sites and data collection.
Two populations of Puya chilensis were sampled: Los Vilos (31°53’S, 71°30’W) and Caleta
Chome (36°46’S, 73°12’W). The northern population, Los Vilos, is located on a hillside approximately 2 km from the coastline, facing the ocean. The southern population, Caleta
Chome, is located on the coastline, on steep slopes and rocky cliffs. In both sites, P. chilensis forms large populations of aggregated rosettes of approximately 1 m height. Mean annual 119
precipitation in Los Vilos and Caleta Chome is ca. 200 mm and ca. 1300 mm, respectively
(Hajek & DiCastri 1975, Hijmans et al. 2005).
A total 47 plants in Los Vilos and 60 plants in Caleta Chome were selected and marked. All of them were adult plants, which were due to flower during the upcoming months, and lived under similar light and humidity conditions within their respective populations. Sampling for functional traits took place during winter season (August 2012), when moisture differences between both sites are higher, and it is easier to recognize plants that are close to flowering.
We estimated CAM activity measuring nocturnal acidification by collecting leaf samples from mature leaves before dusk and before dawn of the same night. Extract from the collected leaf material was tritrated using 0,1N NaOH to estimate the H+ concentration of the tissues.
Results from leaves collected at dusk and dawn from the same plant were compared to obtain a measure of the variation of acid content between night and day (∆H+) for each individual
(modified from Silvera et al. 2005). This method allows to estimate CAM activity in a particular moment, and in this case it allowed us to relate CAM activity during winter to the immediately upcoming flowering event. Leaf thickness and leaf dry weight were measured using standard equipment (scale, digital caliper) and LMA was calculated by dividing the whole leaf mass by the area of its adaxial side. In order to estimate fitness, in late September and late November 2012 we counted the number of flowers produced by the same individual plants in both populations. Both dates were chosen according to each population’s previously observed flowering time (Quezada, pers. obs.). On each plant, flowers were carefully counted and photographed for backup purposes.
120
Data analysis
To characterize both populations, and detect any difference in the expression of measured traits, we compared their means between populations using t-tests. We also calculated the correlation among traits, from Pearson correlation matrix, to account for any possible collinearity among traits. To test the adaptive hypothesis we calculated selection gradients for each population, which assessed the covariance between the fitness estimator and the standardized functional traits (Mean = 0; SD = 1). Selection gradients estimate the effect of selection on the focal trait, independent of selection on correlated traits included in the analysis (Lande & Arnold 1983). Linear selection gradients (β) estimate the magnitude of directional selection; nonlinear selection gradients (γ) estimate the curvature of the selection function (stabilizing or disruptive) (Lande & Arnold 1983). Directional and nonlinear selection gradients were obtained from linear and quadratic regression of relative number of flowers on functional traits, respectively (Multiple Regression, Polynomial Regression,
Statistica 10, Statsoft Inc.). We estimated both types of selection gradients for leaf thickness, leaf dry weight, LMA and ∆H+. Quadratic coefficient results were dismissed (data not shown) because they seemed to reflect a change in the slope of the fitness function rather than the occurrence of minimum/maximum fitness values, thus they did not reflect true stabilizing/disruptive selection (Mitchell-Olds & Shaw 1987).
RESULTS
The four measured functional traits showed significant differences in their expression between populations of Puya chilensis (Table 1). Mean values were higher for all traits in the 121
northern population (Los Vilos). Thus, plants in the northern population showed thicker and heavier leaves, with a higher LMA, and a higher value of ∆H+. Number of flowers per plant, on the other hand, was not significantly different between populations (p > 0.46). Correlation between traits varied between populations. In Los Vilos LMA had a significant positive correlation with both leaf thickness and leaf dry weight (p < 0.01). In Caleta Chome leaf thickness had a significant negative correlation with leaf dry weight and a significant positive correlation with LMA (p < 0.001) (Table 2). Because none of these correlations was high enough to reach collinearity (correlation coefficients > 0.80) all traits entered into the phenotypic selection analysis.
Of all the traits included in the phenotypic selection analysis, only ∆H+ showed a significant association with relative reproductive effort (number of flowers) in both populations. Thus, for ∆H+ there are clear differences on its effect over fitness between both populations: directional selection was positive in Los Vilos and negative in Caleta Chome (Fig. 2; Table
3). Morphological traits did not have any significant influence on the plant fitness estimator nor showed correlations with ∆H+.
DISCUSSION
The differences in the expression of all measured traits between both populations are in accordance with the expected functional responses for sites of contrasting aridity. These results might be related to the different environmental factors both populations face. Los
Vilos plants grow under lower moisture, higher mean annual temperatures and higher irradiation than those in Caleta Chome (Hajek and DiCastri 1975; Luebert and Pliscoff 2006). 122
Regarding the morphological traits we included, to our knowledge their correlation with drought in Bromeliaceae has not been studied yet. Thicker leaves have been associated to low moisture conditions (Westoby et al. 2002; Wright et al. 2004; Poorter et al. 2009), low nutrient availability (e.g. Chabot and Hicks 1982) and high irradiance (Niinemets 2001).
LMA is usually correlated to leaf thickness, as plants with thicker leaves have higher
LMA/lower SLA values (Fonseca et al. 2000; Wright et al. 2004). Also, the construction of leaves with a high LMA require more structural investment per unit of leaf area (Wright et al. 2004), which probably means higher leaf weight. Sclerophyllous plants that inhabit arid and semi-arid regions have thicker, high LMA leaves (Schulze et al. 1998; Fonseca et al.
2000; Niinemets 2001; Wright et al. 2001). These traits are considered adaptations in these species that allow leaf function under very dry conditions (Wright et al. 2004), where LMA rises following the increase on density per unit of area. Thicker leaves have more densely packed cells, which added to a smaller transpiring leaf surface (low SLA, the same as high
LMA) and sunken stomata (Raven 2005) could reduce the chance of losing water via evapotranspiration, improving their water use efficiency (Poorter et al. 2009). Despite these well documented correlations between our measured morphological traits and low water availability, our results show no significant correlation between these traits and our fitness estimator. It is possible that this lack of adaptive value is due the scale of the analysis.
Phenotypic selection analyses measure selection at an individual level. While our results show a significant increase of these traits between populations, which could suggest adaptation to local conditions, it is possible that variation within each population was not significant enough to detect an effect over fitness.
123
We found that ∆H+ was the only trait that had a significant correlation with plant fitness estimates, both in Los Vilos and Caleta Chome populations. Directional selection on ∆H+ showed a clear pattern for both populations. For Los Vilos population, the expression of
CAM had a positive effect on fitness, an expected correlation since this population lives in a semiarid region where precipitation is scarce (ca. 200 mm/year) and the CAM pathway is a good choice to endure these conditions. On the other hand, for Caleta Chome population the expression of CAM had a negative effect on fitness. This negative correlation may be due the reduced need of CAM in a wetter environment (between 700 - 1300 mm/year), which then may become a resource sink, spending resources that could be directed to reproductive effort. These results confirm our adaptive hypothesis for the CAM pathway under low moisture, and are concordant with those reported by Winter and Ziegler (1992) in
Mesembryanthemum crystallinum, which showed a correlation between reproductive output and ΔH+ under drought and salinity conditions. Sayed and Hegazy (1994) found an increase in both ΔH+ and fecundity in Mesembryanthemum nodiiflorum during the dry season. Both
Mesembryanthemum species are facultative CAM, and the induction of CAM under stressful conditions is probably an advantage. A similar correlation was reported for Talinum triangulare under both laboratory and field conditions (Taisma and Herrera 1998, 2003), a correlation that could be explained by the increase in water use efficiency and other morphological changes (Herrera 2009). On the other hand, to our knowledge, no cases of negative correlation between CAM and fitness have been reported. This might be due to the fact that in mesic or wet environments the focus on physiological adaptation rarely considers
CAM photosynthesis. We would expect similar outcomes as the one herein reported if CAM were measured in these environments.
124
The adoption of CAM happened at least three times within Bromeliaceae (Crayn et al. 2004).
Chilean bromeliads, in particular those with a probable origin in central-southern Chile
(Jabaily and Sytsma 2010, 2013) may have adopted this pathway while advancing towards north. CAM probably became an advantage for species of Puya that originated after the radiation to the high Andean arid and semiarid slopes along South America (Varadarajan
1990). For species or populations living in places where water availability is high, the CAM pathway seems to be unnecessary. In the case of Puya chilensis, being facultative CAM is clearly an advantage since it allows this species to grow under the wide spectrum of climatic conditions of central Chile, but for individual plants living in the wetter southern extreme of the species’ geographic range, expressing this attribute is not adaptive. Interestingly, the fact that plants in both studied populations differed phenotypically but produced similar numbers of flowers might suggest that plants on each population are equally adapted to their habitat.
Thus, functional adjustments have assimilated the overall effect of the environment on individual plants. Attributes other than CAM might explain the apparent local adaptation of southern P. chilensis plants, attributes that may acquire higher importance than the photosynthetic pathway in the overall performance of the individuals.
While crassulacean acid metabolism has been proved crucial for the diversification of families like Bromeliaceae and Orchidaceae (Silvera et al. 2009; Quezada and Gianoli 2011), its expression under not appropriate conditions might have negative effects over fitness. Our results could lead to expect that the use of CAM in the southern extreme of the geographic range of Puya chilensis is being slowly removed by natural selection. Further work should estimate genetic variation for CAM in southern populations in order to assess the magnitude of evolutionary response to selection for reduced CAM (see Geber and Griffen 2003). 125
Nevertheless, if the climate change scenario of increased aridity for central Chile is verified
(Fuenzalida et al. 2007) the selective forces herein reported should intensify in the north but relax in the south of P. chilensis distribution.
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FIGURES
1,3 1,8 a b
1,2 1,6
1,1 1,4
1,0 1,2
0,9 1,0 Reproductive effort Reproductive Reproductive effort
0,8 0,8
0,7 0,6 -3 -2 -1 0 1 2 3 -2-101234
Figure 1: Linear relationship between reproductive effort (flower production) and nocturnal acidification (∆H+) in (a) Los Vilos and (b) Caleta Chome. Both relationships were statistically significant (see text).
134
TABLES
Table 1: Mean values (± SE) of functional traits and number of flowers of Puya chilensis in two populations differing in mean annual precipitation (Los Vilos is the drier population).
Trait Los Vilos Caleta Chome
Leaf thickness (mm) 1.956 ± 0.072 1.464 ± 0.036
Leaf dry weight (g) 12.529 ± 0.224 7.735 ± 0.137
LMA 0.059 ± 0.002 0.035 ± 0.0003
∆H+ 0.653 ± 0.011 0.159 ±0.009
Number of flowers 94.191 ± 1.543 96.483 ± 2.497
Los Vilos: N = 47; Caleta Chome: N = 60. All functional traits showed significant differences between populations (t-test, p < 0.001). Number of flowers did not show significant difference between populations (t-test, p = 0.466).
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Table 2: Correlation between measured functional traits of Puya chilensis in both populations.
Leaf thickness Leaf dry weight LMA ∆H+
Leaf thickness (mm) --- 0.26 0.39** -0.14
Leaf dry weight (g) -0.47*** --- 0.39** -0.24
LMA 0.43*** -0.21 --- -0.10
∆H+ 0.04 -0.0009 -0.15 ---
Pearson coefficients of correlation are shown for both Los Vilos (above the diagonal) and Caleta Chome (below the diagonal) populations. ** P < 0.01 *** P < 0.001
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Table 3: Linear standardized selection gradients of Puya chilensis flower production against measured traits in both populations.
Trait β
Los Vilos Caleta Chome
Leaf thickness -0.026 ± 0.014 -0.009 ± 0.027
Leaf dry weight 0.005 ± 0.014 0.016 ± 0.025
LMA 0.018 ± 0.015 0.006 ± 0.025
∆H+ 0.066 ± 0.013*** -0.112 ± 0.022***
Los Vilos: N = 47; Caleta Chome: N = 60. The overall model for each population was significant (Los Vilos: R2 = 0.41, P < 0.001; Caleta Chome: R2 = 0.33, P < 0.001). *** P < 0.001
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DISCUSIÓN GENERAL
138
DISCUSIÓN
La importancia de la vía fotosintética CAM para las plantas que habitan en zonas áridas y semiáridas ha sido reconocida por diversos autores y en diversos grupos vegetales
(Ehleringer y Monson 1993, Cushman 2001, Taiz y Zeiger 2002, Silvera et al. 2010). La vía
CAM ha demostrado ser clave para la diversificación de algunos grupos de plantas (ej.
Orchidaceae, Silvera et al. 2009), además de ser un atributo plástico que puede, en algunas especies, expresarse bajo determinadas condiciones con un efecto positivo sobre la adecuación biológica (Winter y Ziegler 1992, Sayed y Hegazy 1994, Taisma y Herrera 1998,
2003).
En esta investigación se evidenció el valor adaptativo de la vía CAM para una familia de gran tamaño y distribución geográfica, y su relación con las variables ambientales desde distintas escalas: i) como una innovación clave para la diversificación de la familia y su expansión geográfica; ii) como un atributo cuya expresión al nivel de especie se relaciona con la latitud, en una zona geográfica con un marcado gradiente latitudinal de precipitaciones y temperatura; iii) como un atributo plástico, cuya expresión entre poblaciones varía de acuerdo a las necesidades de los individuos, y iv) como un potencial “lastre” (selección negativa) para aquellos individuos que lo expresan en áreas geográficas donde, teóricamente, no es necesario hacerlo. Cada capítulo de la presente tesis pone a prueba el valor de la vía
CAM en alguna de dichas escalas. Nuestros resultados, en términos generales, demuestran que dicha vía fotosintética ha sido de gran importancia para la diversificación de la familia
Bromeliaceae en ambientes áridos y semiáridos, que su expresión puede guardar directa
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relación con las condiciones ambientales y que, bajo determinadas circunstancias, el expresarla puede ser desventajoso para la planta, en términos de adecuación biológica.
¿Puede la vía fotosintética CAM favorecer significativamente la diversificación de un grupo vegetal?
En Capítulo I del presente trabajo de tesis se determinó que la adopción de la vía fotosintética
CAM fue una innovación clave dentro de la familia Bromeliaceae. Dicha familia es frecuente en hábitats xéricos (epífitos y terrestres) de la región Neotropical. Nuestros resultados demuestran que la adopción de la vía CAM por la familia facilitó su diversificación taxonómica, algo previamente asumido por otros autores (Cushman 2001, Crayn et al. 2004,
Givnish et al. 2007), aunque nunca probado de forma cuantitativa. Es muy probable que la vía CAM haya sido seleccionada durante el avance del grupo hacia regiones áridas
(Varadarajan y Gilmartin, 1988; Givnish et al. 2007), algo similar a lo propuesto para las
Cactaceae (Ocampo y Columbus, 2010) y Orchidaceae (Silvera et al. 2009). Al distribuirse algunos grupos de la familia en hábitats áridos, la vía CAM se habría vuelto esencial, ya sea en hábitats terrestres o epifitos, más aun para aquellas especies que dependen de la niebla para sobrevivir (Rundel et al. 1997, Rundel y Dillon 1998, Benzing 2000).
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¿Existe relación entre la latitud y la expresión de CAM en un gradiente latitudinal marcado?
En Chile existe un marcado gradiente ambiental, que va desde un desierto híper-árido en el norte a un clima antártico-boreal en el extremo sur (Luebert y Pliscoff 2006). La marcada variación ambiental, generalmente relacionada con el avance latitudinal de la distribución de las especies, puede ser útil para comparar los niveles de expresión de una adaptación como la vía CAM bajo distintas condiciones. Las Bromeliaceae chilenas se encuentran distribuidas en parte importante de dicho gradiente, particularmente en las zonas de clima mediterráneo y templado-oceánico, e incluyen especies C3, CAM facultativas y CAM constitutivas.
Nuestra aproximación, en el Capítulo II de esta tesis, involucró comparar la vía fotosintética utilizada por cada una de las especies endémicas de Chile de la familia Bromeliaceae con la latitud máxima que alcanzan y con los valores, a dicha latitud, de los dos factores climáticos que pueden tener una mayor influencia sobre la expresión de la vía CAM: precipitación y temperatura mínima. La latitud máxima alcanzada fue considerada a nivel de costa y en términos absolutos (la máxima latitud alcanzada por la especie, independientemente de su cercanía a la costa). Esto porque la influencia del Océano, en particular de la Corriente de
Humboldt, en la distribución de las plantas en la costa de Chile es significativa (Zinsmeister
1978), y es probable que el efecto “amortiguador” que dicha corriente tiene sobre el clima costero influenciara la distribución de las bromelias endémicas, y con ellas de la vía CAM.
En principio, nuestros resultados muestran que existe una relación significativa entre la latitud máxima alcanzada, absoluta y en la costa, y las precipitaciones sobre la vía fotosintética utilizada por las bromeliáceas endémicas de Chile. Sin embargo, al ser las
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Bromeliaceae chilenas un grupo filogenéticamente muy emparentado y con importantes niveles de hibridación (Givnish et al. 2007, 2011, Jabaily y Sytsma 2010, 2013, Zizka et al.
2013), era pertinente descartar la influencia de la filogenia en dichas correlaciones. Para esto aplicamos contrastes filogenéticamente independientes (Felsenstein 1985), cuyos resultados muestran que ninguno de los cuatro factores estudiados está significativamente correlacionado con la vía fotosintética. Este resultado sugiere que es la filogenia, y no el clima, lo que ha modelado la distribución de CAM en las bromeliáceas endémicas de Chile.
Sin embargo, no se debe descartar el efecto que los distintos factores climáticos que varian a lo largo del gradiente latitudinal chileno pueden haber tenido sobre la selección de un atributo con tanto valor adaptativo como dicha vía fotosintética. En una región geográfica donde el clima ha sido el principal modelador de la vegetación (Gasith y Resh 1999), es muy probable que éste haya ejercido una presión selectiva que llevó al uso de la vía fotosintética más adecuada para cada condición local, en etapas tempranas del proceso de diversificación de la familia en Chile central.
La relación entre la expresión de CAM y la latitud era esperable, considerando la marcada variación climática que existe dentro del gradiente latitudinal. Sin embargo, la latitud en sí misma no es un factor que influya directamente en el fenotipo de los organismos, sino que son los factores climáticos que covarían con ella los que lo hacen. En este sentido, otros autores que han llegado a conclusiones similares han planteado que es muy probable que la latitud refleje mejor las variaciones ambientales que los datos entregados por las estaciones meteorológicas, algo que aplica también a los datos interpolados de WORLDCLIM que utilizamos en este capítulo, los cuales posiblemente no reflejen el espectro completo de variación climática a lo largo del gradiente (Naya et al. 2011, 2012; Molina-Montenegro and 142
Naya 2012). Por otro lado, tampoco se debe descartar la influencia de otros factores que afectan la expresión de la vía CAM, como la salinidad, la radiación solar o la disponibilidad de nutrientes (Lüttge 2004). Las bromelias chilenas que se distribuyen en la costa generalmente lo hacen en quebradas rocosas, de difícil acceso y que enfrentan directamente al océano (Zizka et al. 2009), quedando expuestas a altos niveles de radiación y al spray salino.
Además, no debemos descartar un probable efecto de las precipitaciones y la temperatura dentro del gradiente estudiado. En éste, tanto la temperatura como las precipitaciones varían con la latitud (Hajek y DiCastri 1975, Luebert y Pliscoff 2006). Las bajas temperaturas son un factor relevante para el funcionamiento de la vía CAM, pues temperaturas en el rango de los 0 a 5 °C pueden afectar significativamente el desempeño de la PEPC, enzima encargada de la captura de CO2 (Buchanan-Bollig et al. 1984). Además, variaciones día/noche de 10 °C o más se considera que mejoran el funcionamiento de la vía CAM, actuando como un inductor (Medina et al. 1977, Haag-Kerwer et al. 1992). Si bien la temperatura media desciende al aumentar la latitud dentro del gradiente climático chileno, en la costa las diferencias de temperatura y las oscilaciones día/noche no son tan pronunciadas como al interior del territorio, debido al ya mencionado efecto de la Corriente de Humboldt
(Zinsmeister 1978). Por lo mismo, si bien se registra un descenso en las temperaturas medias y mínimas en la costa, al aumentar la latitud, este descenso, y las oscilaciones térmicas asociadas, probablemente no sean tan significativos para la expresión de CAM como pueden ser las precipitaciones. En cuanto a las precipitaciones, al aumentar la latitud el incremento de éstas a lo largo de la costa chilena es sostenido (Hajek y DiCastri 1975, Hijmans et al.
2005). Entre los dos extremos del rango comprendido por las latitudes máximas alcanzadas 143
por la especie que avanza menos hacia el sur (Tillandsia tragophoba) y la que alcanza mayores latitudes (Fascicularia bicolor) hay una diferencia en las precipitaciones medias anuales que supera los 2000 milímetros, registrándose un aumento constante al avanzar hacia mayores latitudes. Se sabe que la vía CAM es principalmente una adaptación fisiológica a condiciones de aridez, que mejora significativamente la eficiencia en el uso del agua de la planta (Ehleringer y Monson 1993, Lüttge 2004). Por lo mismo, el expresarla puede ser ventajoso para aquellas especies que se encuentran restringidas a la zona norte, más árida.
De hecho, del listado de especies trabajado en este capítulo, ninguna especie C3 tiene como latitud máxima alguna localidad donde las precipitaciones anuales sean menores a 200 mm/año. De cualquier forma, sea cual sea el factor ambiental que se encuentra detrás de la distribución de CAM en las bromelias chilenas, al parecer hay un efecto ambiental sobre ésta, aparentemente enmascarado por efecto de la filogenia, lo cual indica una posibilidad cierta de adaptación al ambiente. Es probable que la adopción de la vía CAM haya significado una ventaja en el avance hacia el norte de las especies chilenas de Bromeliaceae, a partir de su probable punto de origen en Chile centro-sur (Jabaily y Sytsma 2013). Esto se puede ejemplificar con el caso de Puya berteroniana, especie CAM que se cree se habría originado a partir de poblaciones de Puya alpestris, una especie C3 (Schulte et al. 2010, Jabaily y
Sytsma 2010, 2013). Se puede inferir que P. berteroniana adoptó la vía CAM luego de separarse de P. alpestris, lo que le habría permitido avanzar más hacia el norte y diversificarse en las planicies áridas y semiáridas alrededor de los 29°S. Una mayor eficiencia en el uso del agua, asociada a una mayor tolerancia a la sequía, habría sido clave para el avance hacia el norte de dicha especie.
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¿Hay variación intraespecífica en el uso de CAM?
La existencia de una relación entre latitud y el uso de CAM en las Bromeliaceae chilenas es relevante, aun cuando no se tenga total claridad de los factores ambientales que se encuentran detrás de ésta. Al indagar a nivel intraespecífico, comparando entre poblaciones de tres especies endémicas de Chile del genero Puya (P. berteroniana, P. chilensis y P. venusta), se pudo determinar que en una de ellas, Puya chilensis, existe una disminución clara y significativa en el uso de CAM a medida que se avanza hacia el sur en el gradiente latitudinal
(Capítulo 3). El que solo dicha especie haya alcanzado niveles de variación significativos puede estar relacionado con que es la que presenta el rango latitudinal más amplio dentro de las especies chilenas de Puya, y es la única que tiene poblaciones en los extremos norte y sur de la zona de clima mediterráneo (Zizka et al. 2009). Nuestros resultados muestran como, en un gradiente de aridez, Puya chilensis cambia progresivamente su vía fotosintética de C3 a
CAM al avanzar hacia zonas más áridas. Otros autores han reportado variación intraespecífica en el uso de CAM bajo distintas condiciones ambientales (Ehleringer 1990,
Scarano et al. 2002, Herrera et al. 2010). Nuestros resultados demostraron claramente que
Puya chilensis es una especie CAM facultativa, y que, dentro del marcado gradiente latitudinal chileno, puede existir variación intraespecífica en el uso de la vía CAM.
La plasticidad en la expresión de la vía CAM, que va desde aquellas plantas que la utilizan solo ante condiciones de aridez, a las que son permanentemente CAM y hasta aquellas que mantienen los estomas cerrados las 24 horas del día, recirculando CO2 respiratorio, se cree que tiene un valor adaptativo (Herrera 2009). Esto implicaría necesariamente que las plantas
CAM facultativas se encontrarían siempre en ventaja ante las constitutivas, y que, por
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ejemplo, Puya chilensis tiene una ventaja por sobre otras especies de Puya que sean CAM constitutivas, lo que le puede haber permitido alcanzar un rango geográfico mayor. Sin embargo, según otros autores, es la inducción de la expresión de la vía CAM lo que cuenta con valor adaptativo en plantas CAM facultativas (Winter y Ziegler 1992, Sayed y Hegazy
1994, Taisma y Herrera 1998, 2003), pero no el hecho de que el atributo sea plástico. Para
Puya chilensis podemos inferir que la capacidad de expresar la vía CAM es ventajosa en la parte norte de su rango de distribución, pero consideramos pertinente evaluar si lo era en el extremo sur, lo que se hizo en el Capítulo 4 de la presente tesis.
¿Tiene la vía CAM valor adaptativo para los individuos de Puya que habitan en zonas menos áridas?, ¿o la vía CAM es un “lastre” que afecta la presencia de Puya al sur de los 39°S?
Considerando que la expresión de la vía CAM en Puya chilensis varía con la latitud, y por ende con la disponibilidad de agua, a lo largo de la costa de Chile central, buscamos determinar si el utilizar dicha vía fotosintética a mayores latitudes tendría algún efecto sobre el fitness de los individuos, esperando que en un ambiente húmedo dicho efecto fuera negativo (Capítulo 4).
Nuestros resultados verificaron dicha hipótesis, pues mostraron que la expresión de CAM en zonas donde la aridez no es un factor relevante tiene efectos negativos sobre el fitness de los individuos, mientras que en las zonas más áridas el efecto sobre el fitness es positivo. En la zona norte el utilizar la vía CAM implica una mayor eficiencia en el uso del agua, optimizándose el uso de un recurso escaso, y siendo favorecidos aquellos individuos que 146
incurrieron en el gasto de recursos que significa utilizar dicha vía fotosintética (Walker 1992;
Furbank 1999; Forseth 2010). En la zona sur, en tanto, el usar la “maquinaria” bioquímica que hace funcionar a la vía CAM es un gasto que se volvería innecesario debido a que el agua no es un recurso escaso. Si bien, como mencionamos anteriormente, se han documentado efectos positivos de la expresión de CAM sobre el fitness, no encontramos registros de reportes previos que mencionen efectos negativos. Este atributo suele considerarse una adaptación ventajosa, sobre todo en condiciones de escasez de agua (Ehleringer y Monson
1993, Taiz y Zeiger 2002) y, en el caso de las especies CAM facultativas, se asume que bajo condiciones de humedad la planta siempre volverá a la fotosíntesis C3. En este sentido, nuestros resultados son novedosos y apuntan a que la vía CAM puede seguirse expresando en algunos individuos aunque ya no sea necesaria, convirtiéndose en un gasto innecesario de recursos. No tenemos registro de reportes que señalen a la vía CAM como un atributo con efectos negativos sobre el fitness bajo determinadas condiciones ambientales.
Siguiendo ese criterio, es muy probable que la vía CAM en las poblaciones más australes de
Puya chilensis, particularmente aquellas ubicadas en la zona de transición entre los climas mediterráneo y templado-lluvioso (37-38°S), esté teniendo un efecto “lastre”, es decir, se haya vuelto un simple gasto de recursos que no mejora, por sí mismo, el desempeño de los individuos, tal como ocurre en otros casos (ver Zangerl et al. 1991, Fuentes y Schupp 1998 o Verdú y García-Fayos 2001, 2002 para la producción de frutos partenocárpicos en Pistacia lenticus; o ver Guimaraes et al. 2008 o Johnson 2009, respecto a la producción de megafrutos destinados a la dispersión de semillas por megafauna actualmente extinta). En verano la población más austral de Puya chilensis enfrenta condiciones de mayor aridez y temperatura
(DiCastri y Hajek 1975), por lo que el que sus individuos sean capaces de utilizar la vía CAM 147
puede ser ventajoso, lo que se opondría, a priori, a nuestra calificación de “lastre” para el atributo. Sin embargo, el que algunos individuos se mantengan expresando dicha vía en invierno, a pesar del efecto negativo sobre su propia adecuación biológica, implica como resultado global que la vía CAM tiende a mantenerse en parte de la población, a pesar de las condiciones ambientales, y que, de avanzar la especie hacia zonas más húmedas, el efecto
“lastre” de la vía CAM sería mayor. Es probable que en esas zonas más húmedas la pérdida de agua, incluso en temporada estival, sea mucho menor que en la zona de transición, por lo que la expresión de CAM se volvería completamente innecesaria. Sin embargo, este punto no puede ser probado in situ, ya que no se registra presencia de Puya chilensis, ni de ninguna
Bromeliaceae terrestre CAM, al sur de los 38°S.
En general, y respecto al valor adaptativo de la vía fotosintética CAM para Puya, pregunta central de este trabajo de tesis, podemos afirmar que la adopción de dicha vía fotosintética ha sido clave para la diversificación y el establecimiento del género a lo largo de todo su rango de distribución y, localmente, en la zona de clima mediterráneo de Chile central. Sin embargo, dicho valor adaptativo se pierde bajo condiciones húmedas, convirtiéndose la vía
CAM en un gasto innecesario de recursos que afecta negativamente a la adecuación biológica de los individuos que la expresan, al menos en Puya chilensis, y que podría, teóricamente, limitar su presencia en regiones donde el agua es un recurso abundante. Estos resultados podrían considerarse como referencia para otras especies CAM-facultativas que ocupan rangos geográficos altamente variables en términos de humedad. El evaluar el efecto que el mantener en funcionamiento la vía CAM, bajo condiciones no áridas, puede tener sobre la adecuación biológica, puede ser una aproximación de gran valor para comprender la
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variabilidad intrapoblacional de dicha vía fotosintética, los procesos de selección natural que la afectan y sus efectos sobre los patrones de distribución de las especies CAM facultativas.
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CONCLUSIONES
Los resultados de esta tesis sostienen la idea de que la vía CAM es un atributo con valor adaptativo, plástico, y cuya expresión puede ser perjudicial en condiciones distintas a las
áridas o semiáridas. En particular, nuestros resultados indican que:
I.- La aparición de la vía fotosintética CAM dentro de la familia Bromeliaceae fue clave para su diversificación, permitiéndole expandir su rango geográfico significativamente. Esto apoya nuestra primera hipótesis, y da por cumplido nuestro primer objetivo específico.
II.- Existe cierta relación entre la vía fotosintética utilizada por las especies de
Bromeliaceae endémicas de Chile, ubicadas dentro del gradiente latitudinal, la latitud máxima que éstas alcanzan, en la costa y el interior del territorio, y las precipitaciones. Si bien la latitud en sí misma no es un factor ambiental con efectos directos sobre la expresión de atributos en plantas, inferimos que factores ambientales, entre ellos las precipitaciones, pueden estar modelando la distribución geográfica de las vías fotosintéticas en las bromeliáceas chilenas, aún cuando los resultados sugieren que es la filogenia del grupo la principal influencia. Los resultados dan, en parte, soporte a nuestra segunda hipótesis, y dan por cumplido nuestro segundo objetivo específico.
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III.- De las especies CAM de Puya que habitan la zona centro-sur de Chile, solo Puya chilensis mostró una variación significativa en el uso de la vía CAM con la latitud. Dicha especie pasa de una clara dependencia de la vía CAM en el norte de su rango de distribución al uso de la vía C3 en su límite sur. Estos resultados apoyan nuestra tercera hipótesis, y dan por cumplido nuestro tercer objetivo específico.
IV.- La expresión de la vía CAM en la zona árida tiene efectos positivos sobre el fitness de individuos de Puya chilensis, en tanto su expresión en el límite sur de su rango de distribución tiene efectos negativos. Si bien la expresión de la vía CAM en la zona de transición mediterráneo-templada puede ser útil en la temporada cálida y seca, el que algunos individuos mantengan su expresión durante la temporada húmeda, a pesar del efecto negativo que ésta tiene sobre el fitness, permite inferir que la vía CAM podría actuar como un “lastre” para Puya chilensis en caso de encontrarse más al sur de lo que efectivamente se encuentra.
Esto da soporte a nuestra cuarta hipótesis y da por cumplidos los objetivos específicos 4 y 5.
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