La hibridación en el origen y extinción de especies en el género

Transcription

VNIVERSITM
© ^V a l e n c ia
VNIVERSITM ID V m e n c ix
Jardí Botánic
La hibridación en el origen y extinción de especies
en el género A n t i r r h i n u m (Scrophulariaceae):
el caso de las rupícolas amenazadas A . c h a r i d e m i ,
A. s u b b a e t i c u m y A. v a l e n t i n u m
Tesis Doctoral de
Elena Carrió González
Director
Dr. Jaime Güemes Heras
Valencia 2010
UMI Number: U 603067
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Jardl Botánic
D. Jaime Güemes Heras, Doctor en Biología y Conservador del Jardín
Botánico de la Universidad de Valencia-ICBiBE,
AUTORIZA
La presentación de la Tesis Doctoral titulada “La hibridación en el origen y
extinción de especies en el género Antirrhmum (Scrophulariaceae): el caso
de las rupícolas amenazadas A. charidemi, A. subbaeticum y A. valentinum”,
elaborada por Dña. Elena Carrió González bajo mi inmediata dirección y
supervisión en el Departamento de Botánica, y que presenta para la
obtención del grado de Doctor por la Universidad de Valencia.
'— 1
En Valencia a diecisiete de marzo de 2010
Esta memoria doctoral está dedicada a
P. P. T.
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Contenido
Introducciín
Hibridacicn: origen y extinción de especies
1
3
Origen Je especies
3
Extinciói de especies
7
Frecuercia de la hibridación interespecífica
El géneroAntirrhinum y los procesos de hibridación
11
12
El casode las rupícolas amenazadas A. charídemi, A. subbaeticum y
A. valertinum
15
Objetivo
23
Capítulos (3n inglés)
27
Chaptert A geographical pattern of Antirrhinum speciation since the
Pliocene kased on plastid and nuclear DNA polymorphism
27
1.1. Introduction
29
1.2. Materials and Methods
31
1.2.1.Sampling strategy and DNA sequencing
31
1.2.2.Plastid haplotype analysis
32
1.2.3.Phylogeneticanalysis
33
1.2.4. Dating lineage divergente
34
1.3. Resjlts
1.3.1.3lastid haplotypes analysis
34
34
1.3.2. ITS sequence variation
35
1.3.3. Phylogenetic analyses
37
38
1.4.
Discussion
41
1.4.1. Evidence for extensive hybridization in Antirrhinum
41
1.4.2. Pleistocene divergente oí Antirrhinum lineages
43
1.4.3. A geographical pattern of differentiation inEastern Iberia
44
Appendix (1.1)
47
C hapterll: Reproductive biology and conservaron implications in
Antirrhinum charídemi, A. subbaeticum and A. valentinum
53
2.1. Introduction
55
57
2.2.1. Study species and site
57
2.2.2. Flowering phenology
59
2.2.3. Floral biology: flowering duration, pollen viability and stigma
receptivity
60
2.2.4. Pollination treatments
61
2.2.5. Pollen-ovule ratio (P/O)
62
2.2.6. Inbreeding depresión
62
2.2.7. Statistical analysis
62
2.3. Results
63
63
2.3.2. Floral biology: flowering duration, pollen viability and stigma
receptivity
63
2.3.3. Pollinations treatments
64
72
2.3.5. Inbreeding depression
72
2.4. Discussion
73
2.4.1. Mating pattems
73
2.4.2. Limits on seed quantity and quality
74
2.4.3. Conservation implications
75
Chapterlll: Assessing the rísk of hybridization and introgression in a
rare endemic Antirrhinum species: the case of A. valentinum
79
3.1. Introduction
81
84
3.2.1. Plant species and study site
84
3.2.2. Experimental crosses
86
3.2.3. Pollen adherence, germination and tube growth
88
3.2.4. Hybrid reproductive capacity
88
3.3. Results
90
90
91
93
3.4. Discussion
95
3.4.1. Prezygotic and postzygotic reproductive barriere
95
97
3.4.3. Potential hybridization and introgression in theenvironment
98
100
Chapter IV: Evaluating species non-monophyly as a trait affecting
genetic diversity: the case of Antirrhinum charidemi, A. subbaeticum
and A. valentinum
101
4.1. Introduction
103
105
4.2.1. Sampling
105
4.2.2. DNA extraction, gene amplificationand sequencing
108
4.2.3. Data analysis
108
4.3. Results
111
4.3.1. Characteristics of nuclear and plastid sequences
111
4.3.2. Phylogenetic analysis
111
4.3.3. Analysis of plastid haplotypes
116
4.4. Discussion
4.4.1.
Testing monophyly at the species level
116
116
4.4.2.
Life history traits, monophyly and genetic diversity
Appendix (4.1)
119
122
Conclusiones
127
Referencias
131
Agradecimientos
157
j
¡
¡J l
Introducción
Introducción
HIBRIDACIÓN: ORIGEN Y EXTINCIÓN DE ESPECIES
El papel de la hibridación en el origen, mantenimiento y pérdida de la biodiversidad
ha sido sujeto de especulación y debate durante más de dos siglos. Linné ya
escribió en 1760 “es imposible dudar de que hay muchas especies producidas por
generación híbrida ...y consecuentemente, que muchas de las especies de plantas
de un género, que en principio eran una sola planta, han surgido por generación
híbrida”. Autores posteriores han mantenido el debate hasta nuestros días (Mendel,
1866; Grant, 1989; Arnold, 1997; Rieseberg, 1997). Mientras que algunos autores
han enfatizado, como Linné, el papel creativo de la hibridación en la formación de
especies (Stebbins, 1942; Anderson, 1949; Arnold, 1997), otros, en cambio, han
destacado su papel como fuerza evolutiva destructora, capaz de la desintegración
genética de especies (Ellstrand, 1992; Levin et al., 1996; Rhymer y Simberloff,
1996). Y sólo una minoría considera que la hibridación tiene un papel evolutivo
insignificante, bajo la visión de que es un fenómeno poco natural que principalmente
ocurre en lugares alterados por la actividad humana (Schemske, 2000).
Origen de especies
La idea de que la hibridación puede contribuir a incrementar la biodiversidad a través
de la creación de especies o formas nuevas está ampliamente aceptada, sobre todo
en el ámbito de las especiación vegetal (Arnold, 1993; Rieseberg, 1997; Rieseberg
et al., 1999; Ungerer et al., 1998). Una especie hibridógena puede formarse por el
aislamiento del híbrido natural de sus progenitores tras alcanzar la estabilidad
genética (Grant, 1989). La especiación por hibridación puede ser de dos tipos en
función del número cromosómico de la nueva especie: poliploide (alopoliploide)
caracterizada porque en los híbridos se produce una duplicación del genoma,
4
INTRODUCCIÓN
homoploide, en la que los híbridos mantienen el mismo número cromosómico que
tienen las especies progenitoras (Rieseberg y Willis, 2007).
El significado evolutivo de la especiación híbrida poliploide ha sido
más
estudiado que el de la homoploide (Chapman y Burke, 2007), pero todavía quedan
aspectos por revelar (Mallet, 2007). Este tipo de especiación se detecta con más
facilidad que la homoploide, porque implica un cambio en el número cromosómico
de las especies híbridas con respecto al de las especies progenitoras. Uno de los
mecanismos más conocidos por el que se pueden originar especies híbridas
poliploides es a través de la duplicación de los cromosomas somáticos de un híbrido
diploide (Rieseberg y Willis, 2007). Por ejemplo, en el híbrido diploide estéril entre
Prímula verticillata y P. floribunda, se produjo una duplicación del genoma de las
células vegetativas que dio lugar a tallos fértiles tetraploides. Posteriormente, este
híbrido, se reprodujo por autogamia, y generó un nuevo híbrido tetraploide, Prímula
kewensis, uno de los primeros híbridos poliploides descritos (Ramsey y Schemske,
1998; Mallet, 2007). El aislamiento reproductivo de los híbridos poliploides ocurre,
prácticamente, de manera directa debido a que los cruces entre estos híbridos y los
progenitores diploides fallan o producen progenie generalmente estéril, en parte,
porque presentan números cromosómicos diferentes (Grant, 1989).
Con frecuencia, los nuevos híbridos poliploides fracasan durante la etapa de
establecimiento debido a que sufren anormalidades meióticas y/o a la escasez de
cruzamientos apropiados en la población (Ramsey y Schemske, 1998; Soltis y Soltis,
2009). Sin embargo, otros factores ayudan a minimizar este fracaso. Por ejemplo, el
hecho de que los híbridos poliploides sean capaces de colonizar nuevos hábitats
disponibles y que tengan capacidad para la reproducción asexual, contribuye al éxito
durante las primeras etapas del estableciendo (Baack, 2005; Rausch y Morgan,
2005). En otras ocasiones, los híbridos poliploides presentan una alta fecundidad y
un modo de vida perenne, que les permite reproducirse en numerosas ocasiones a
lo largo de su ciclo de vida y les confiere una ventaja durante el establecimiento
(Rodríguez, 1996).
La especiación por hibridación poliploide se considera un evento relativamente
común, mucho más frecuente que la especiación por hibridación homoploide
(Rieseberg, 1997). Actualmente se conocen muchas especies poliploides de origen
INTRODUCCIÓN
5
híbrido (ej. Cardamine silana, en Perny et al., 2005; Achillea virescens, en Guo et al.,
2005; Saxífraga osloensis, en Nilsson y Jorde, 1998), incluso algunas de ellas se
han formado en los últimos 150 años, como Tragopogón mirus, T. miscellus
(Ownbey, 1950) o Spartina anglica (Ainouche et al., 2004); o se han originado, en
más de una ocasión, como Senecio cambrensis (Abbott y Lowe, 2004). Esta última
especie, se ha generado en dos ocasiones distintas, dando lugar a dos poblaciones
independientes (una en Escocia y otra en Gales), cada una sometida a diferentes
presiones de selección; aunque, actualmente, sólo persiste la población de Gales,
pues la población escocesa desapareció 20 años después de su descubrimiento
(Abbott y Lowe, 2004).
La especiación por hibridación homoploide es menos frecuente que la poliploide
porque los híbridos han de superar incompatibilidades genéticas o cromosómicas sin
el aislamiento reproductivo que confiere la poliploidía (Rieseberg, 1997; Barton,
2001). A diferencia de la especiación por hibridación poliploide, el proceso por el
cual los híbridos homoploides se convierten en reproductivamente aislados de sus
progenitores es menos obvio (Rieseberg, 1997; Wolfe et al., 1998).
Se han
propuesto dos vías por las cuales una forma híbrida homoploide puede alcanzar la
estabilidad genética y el aislamiento reproductivo (revisado en Buerkle et al., 2000):
en primer lugar, se ha sugerido que, aunque los híbridos de la primeras
generaciones suelen mostrar una fertilidad reducida, debido a desequilibrios en los
cromosomas, los cruzamientos entre estos híbridos, pueden dar a lugar a genotipos
nuevos,
cromosómicamente
equilibrados,
con
alta
fertilidad
y
aislados
reproductivamente de las especies progenitoras; en segundo lugar, se ha propuesto
que la estabilidad y aislamiento de los nuevos linajes híbridos dependerá de barreras
externas, es decir, si éstos son capaces de sobrevivir en ambientes distintos de los
de las especies progenitoras. Al parecer, ambos mecanismos han contribuido al
origen de la mayoría de las especies híbridas homoploides conocidas (Rieseberg,
1997).
Actualmente, se han documentado unos 20 casos claros de especies vegetales
híbridas homoploides (Rieseberg, 1997; Mallet, 2007), y de éstos, la situación más
investigada ha sido la de las tres especies híbridas de Helianthus, H. anomalus, H.
deserticola y H. paradoxus. Estas tres especies han derivado del cruce entre H.
6
INTRODUCCIÓN
annus y H. petiolarís, localizadas en Norteamérica. H. annuus vive en suelos
saturados en primavera y secos en verano, y H. petiolarís crece en suelos arenosos,
más o menos, secos. En cambio, los tres híbridos que producen estas dos especies,
han ocupado hábitats extremos si se comparan con los propios de las especies
pregenitoras: H. anomalus y H. deserticola muestran adaptaciones a la xericidad, y
habitan sobre dunas de arena, en zonas desérticas, en Utah y noreste de Arizona, y
en Utah y Texas, respectivamente; H. paradoxus vive sobre suelos salinos y
moderadamente pantanosos, en Texas y Nuevo México. El establecimiento de los
tres híbridos se ha visto facilitado por una rápida adaptación a los ambientes
extremos, que les ha proporcionado una ventaja selectiva y, además, ha funcionado
como un mecanismo efectivo de aislamiento (Buerkle et al., 2000; Lexer et al., 2003;
Gross y Rieseberg, 2005). La situación ilustrada en Helianthus, es la habitual en los
híbridos homoploides, es decir, todas las especies híbridas homoploides que se
conocen divergen ecológicamente de las especies progenitoras, y muestran, algún
grado de aislamiento ecogeográfico. En algunos casos, las especies híbridas
homoploides ocupan hábitats intermedios entre las dos especies progenitoras, y en
otros, han colonizado hábitats extremos (Gross y Rieseberg, 2005; Rieseberg y
Willis, 2007).
Por otra parte, en ocasiones, puede ocurrir que los nuevos híbridos
homoploides se crucen con distinta intensidad con las especies progenitoras, de
manera que las generaciones siguientes de linajes híbridos que se formen serán,
más o menos, parecidas genéticamente a las especies progenitoras. Si los
descendientes híbridos se cruzan de forma continua o muy frecuente con una de las
especies progenitoras se puede facilitar que unos pocos caracteres de una de las
dos especies que hibridan queden incluidos en el acervo genético de la otra especie.
Este proceso se conoce como hibridación introgresiva o introgresión (Soltis y Soltis,
2009) y se describió por vez primera a partir de los patrones de hibridación
observados en Iris (Riley, 1938). Uno de los ejemplos de hibridación introgresiva
mejor caracterizados es de /. fulva e /. hexagona. Por medio de análisis moleculares
(aloenzimas; RAPD -Random Amplication of Polymorphic DNA-, secuencias de ADN
nuclear y plastidial) se ha confirmado la presencia de genes de /. fulva en una
población de /. hexagona, situada a 10 km de la población más próxima de /. fulva\ y
INTRODUCCIÓN
7
la presencia de genes de I. hexagona en una población de I. fulva, situada a 25 km
de la población más cercana de I. hexagona (Arnold y Bennett, 1993). La hibridación
y la introgresión también han participado en la formación de I. nelsonii, derivado de
tres especies, I. fulva, I. hexagona e /. brevicaulls (Arnold, 1993).
Extinción de especies
El efecto negativo de la hibridación sobre la diversidad de especies se ha
investigado con mayor énfasis en las últimas dos décadas, de manera que en la
actualidad, la hibridación se considera un mecanismo evolutivo importante capaz de
extinguir o de mermar poblaciones o especies (Ellstrand y Elam, 1993; Rhymer y
Simberloff, 1996; Allendorf et al., 2001). Hay una serie de factores de tipo ecológico
y genético que influyen sobre el riesgo de extinción por hibridación en las plantas y
que son, entre otros, la magnitud de las barreras reproductivas que aíslan a los
táxones que hibridan, el vigor y la fertilidad de los híbridos, el tamaño relativo y
absoluto de las poblaciones, la estocasticidad demográfica o la tasa de crecimiento
de las poblaciones, la divergencia ecológica, la diversidad de alelos S, o los cambios
en la presión de la herbivoría o de los patógenos (Levin et al., 1996; Wolf et al.,
2001; Buerkle et al., 2003). Huxel (1999), Wolf et al. (2001) y Buerkle et al. (2003),
tras analizar la importancia relativa de estos factores, concluyeron que, en general,
los de mayor influencia sobre la tasa y probabilidad de extinción son las barreras
reproductivas, la divergencia ecológica y la abundancia de individuos. Dependiendo
de cómo operen todos los factores mencionados, el declive de una especie por
hibridación puede ocurrir a través de procesos de tipo genético o demográfico, y
ambos tipos pueden actuar de manera sinérgica (Levin et al., 1996; Wolf et. al.,
2001; Buerkle et al., 2003).
En particular, se ha observado que las plantas raras o amenazadas, al contar
con poblaciones más pequeñas, son más vulnerables a los efectos negativos de la
hibridación. Hay determinadas circunstancias que suponen un riesgo especial para
estas especies. Por ejemplo, si la especie rara entra en contacto con un congénere
cultivado o domesticado que se ha naturalizado (ej. Juglans en McGranahan et al.,
1988; Helianthus en Carney et al., 2000; Oryza en Song et al., 2004); o si la especie
rara contacta con otra especie nativa, normalmente abundante o incluso rara, como
consecuencia
de
la
ruptura
de
barreras
ecológicas
o geográficas
como
8
INTRODUCCIÓN
consecuencia de la alteración antropogénica del entorno (ej. Braya, en Parsons y
Hermanutz, 2006; Physaría, en Kothera et al., 2007). De todos los casos de especies
raras o amenazadas que entran en contacto con un congénere común,
probablemente la hibridación sólo suponga un problema para una parte de estas
plantas. Sin embargo, hay que tener en cuenta que la hibridación puede causar de
manera muy rápida la extinción de una especie amenazada, por lo que conviene
detectarla lo antes posible. En muchos casos, el flujo génico interespecífico resulta
obvio por la presencia de híbridos morfológicamente intermedios entre los
progenitores. Si los caracteres morfológicos no son tan evidentes,
es posible
investigar la hibridación por medio de métodos genéticos.
Extinción por procesos genéticos
El principal proceso genético capaz de ocasionar la pérdida de una especie por
hibridación, se conoce con el término de asimilación genética, e implica la
destrucción de la integridad genética de una especie y la incorporación de su
material genético al acervo génico de otra (Soltis y Gitzendanner, 1999), que
podríamos considerar como una compiloespecie (Harlam y deWet, 1963). Para que
esto se produzca es necesario que se formen híbridos parcial o totalmente fértiles y
con una fitness elevada (Wolf et al., 2001), puesto que son el puente para el flujo
génico entre los progenitores.
La diferencia en el tamaño poblacional de las
especies que hibridan es un punto clave del proceso (Ellstrand y Elam, 1993; Levin
et al., 1996): el taxon menos abundante del par tiene más probabilidades de ser
reemplazado por el otro, numéricamente superior. Cuanto mayor sea la diferencia
entre el tamaño poblacional de las especies que hibridan, mayor riesgo existirá de
que la especie menos numerosa sea asimilada genéticamente por su congénere
(Ellstrand y Elam, 1993). Carney et al. (2000) analizaron la composición genética y
morfológica de una población híbrida de girasoles (Helianthus) y la compararon con
datos tomados 50 años atrás; sus resultados mostraron que la población estudiada,
que ¡nidalmente estaba formada por individuos de H. bolanderi, una especie poco
abundante, se encontraba prácticamente compuesta por individuos híbridos muy
similares a H. annuus, especie localmente numerosa.
En otros circunstancias, la asimilación genética también puede suponer un
problema para una especie abundante que entra en contacto con un congénere
INTRODUCCIÓN
9
numéricamente inferior pero de mayor éxito reproductivo. Por ejemplo, Spartina
foliosa es una especie común y nativa de California, amenazada por la hibridación
con S. altemifolia, una especie invasora de marjales salinos de la bahía de San
Francisco, poco abundante, pero con mayor capacidad reproductiva masculina
(produce 21 veces más polen viable) y con una tasa mayor de crecimiento clonal
(Anttila et al., 1998). Los modelos de simulación que analizan la evolución de las
poblaciones simpátricas de ambas especies de Spartina revelan que S. foliosa,
podría extinguirse rápidamente, en sólo 3-20 generaciones (Wolf et al., 2001).
La hibridación puede provocar además que una de las especies que híbrida sea
genéticamente asimilada por los nuevos híbridos que se forman. Los híbridos
fértiles, con una fitness igual o superior a la de las especies progenitoras, se pueden
cruzar de manera intensiva con uno de los progenitores hasta provocar su extinción.
Argyranthemum coronopifolium es un endemismo amenazado de Tenerife (Moreno,
2008), que contaba con unas siete poblaciones (Levin et al., 1996). Debido a
actividades humanas en su territorio, en 1965, una de sus poblaciones entró en
contacto con A. frutescens, muy abundante a nivel local. En 1981, esta población,
originalmente constituida por A. coronopifolium, estaba compuesta por plantas
híbridas con distinto fenotipo y por unos pocos individuos puros de A. coronopifolium
(Levin et al., 1996). Situaciones similares se han documentado en: Lotus scoparius
ssp. traskiae (Listón et al., 1990), Cercocarpus traskiae (Rieseberg y Gerber, 1995) o
Morus rubra (Burgess et al., 2005).
En un extremo, menos probable, los híbridos pueden incluso causar, a través
de la asimilación genética, la extinción de las dos especies progenitoras. Hegde at
al. (2006) documentaron con evidencias genéticas, por primera vez en plantas, la
extinción local simultánea de dos especies. El híbrido entre Raphanus raphanistrum
y R. sativus, considerado una entidad evolutiva independiente, provocó la
desaparición local de los dos progenitores. Los resultados de un experimento que
estos autores desarrollaron en el invernadero demostraron que el híbrido presenta
una combinación específica de caracteres de sus progenitores que parece ser la
responsable de un comportamiento colonizador agresivo.
10
INTRODUCCIÓN
Extinción por procesos demográficos
Los procesos de tipo demográfico que se generan por la hibridación entre especies y
que pueden provocar un declive de las poblaciones se basan en una reducción de la
tasa de crecimiento de la población (Levin et al., 1996; Wolf et al., 2001). Cuando
una especie recibe polen de otra, este polen ajeno puede fecundar óvulos que
dejarían entonces de estar disponibles para la propia especie, y que de otra manera,
hubieran podido generar descendencia pura. Esto puede provocar una reducción en
la formación de la progenie pura, y consecuentemente la tasa de crecimiento de la
población puede disminuir hasta el punto de ser inferior al límite necesario para el
reemplazo de individuos en la población (Buerkle et al., 2003; Haygood et al., 2003).
El estudio de Burgess et al. (2008) en un par de especies de morera en Canadá,
Morus rubra, nativa amenazada, y Morus alba, invasora abundante, ilustra este
caso. Para M. rubra, la producción de semillas híbridas supone un gran coste
reproductivo, que va en detrimento de la producción de progenie pura. En parcelas
ubicadas en áreas donde cohabitan ambas especies y los híbridos entre ambas, se
observó que la mayoría de las semillas de M. rubra eran híbridas. Al eliminar las
plantas de M. alba y los híbridos de las parcelas, M. rubra no produjo más cantidad
de semillas, sino que aumentó el número de semillas puras.
Por otra parte, la hibridación también puede afectar al reclutamiento y
establecimiento de los táxones progenitores al incrementarse la competencia con los
híbridos por el hábitat disponible (Arnold et al., 1999, 2001). A pesar de la visión
tradicional de que los híbridos presentan una fitness generalmente baja comparada
con la de los progenitores, hay evidencias que muestran la situación contraria, lo que
sugiere que la
presencia de híbridos con mayor fitness durante la fase de
establecimiento de los progenitores, puede colocar a estos últimos en una situación
de desventaja (Burke y Arnold, 2001). Emms y Arnold (1997) realizaron una
experiencia de plantación de ejemplares de Iris fulva, I. hexagona y de híbridos entre
las dos en parcelas experimentales, que fueron elegidas por contener únicamente
individuos de I. fulva, o de I. hexagona, o bien, individuos híbridos, y que diferían en
características ecológicas. En cada parcela, transplantaron rizomas de los tres tipos
de plantas y evaluaron su desarrollo. Los resultados mostraron que los individuos
INTRODUCCIÓN
11
híbridos transplantados, una vez establecidos, superaban en fitness a las especies
progenitoras en alguno de los hábitats.
Frecuencia de la hibridación interespecífica
En la actualidad, aún no se dispone de información precisa sobre cuál ha sido la
incidencia de la hibridación ancestral en la naturaleza. Sin embargo, se conocen
bastante bien los procesos recientes de hibridación interespecífica. El indicador más
fiable para estimar la frecuencia de la hibridación contemporánea proviene del
análisis de las floras biosistemáticas de cada territorio (Rieseberg, 1997). Ellstrand et
al. (1996), a partir del análisis de cinco floras de diversas regiones del mundo,
estimaron que la frecuencia de los híbridos naturales comparada con el número total
de especies era de un 11%. Este valor variaba entre el 5,8% (Flora Intermontana de
Norteamérica) y el 22% (Flora de las Islas Británicas). Los híbridos naturales se
distribuían de manera irregular, con una clara tendencia taxonómica: sólo entre el 616% de los géneros y entre el 16%-34% de las familias presentaban uno o más
táxones híbridos. El análisis preliminar de los datos sobre la flora de la Península
Ibérica disponibles hasta el momento en Flora Ibérica (2009), mostró unos valores
que se situarían dentro del rango de los datos obtenidos por Ellstrand et al. (1996),
aunque próximos al límite superior en todos los casos. En concreto, en la Península
Ibérica, la frecuencia de híbridos naturales en relación con el número total de
especies fue del 21%. El 14% de los géneros y el 34% de las familias contenían al
menos un taxon híbrido. El caso de la flora de la Península Ibérica no es, pues,
distinto a la generalización establecida por Ellstrand et al. (1996). Los datos
disponibles sugieren que la hibridación contemporánea puede ser poco frecuente y
que hay ciertos grupos filogenéticos con una predisposición biológica a la formación
y mantenimiento de híbridos. Entre estos, en la Península Ibérica, destacarían los
géneros Limonium (Plumbagínaceae), con 107 especies y 141 híbridos, Sideritis
(Labiatae), con 31 especies y 35 híbridos, y Viola (Violaceae), con 29 especies y 30
híbridos (sin atender a los casos de Hieracium, Rubus y Taraxacum, por la compleja
interpretación que supone; Ellstrand et al. 1996).
12
INTRODUCCIÓN
EL GÉNERO ANTIRRHINUM Y LOS PROCESOS DE HIBRIDACIÓN
El género Antirrhinum L. está constituido por 25 especies distrituidas principalmente
por el mediterráneo occidental (sólo A. siculum y A. toruosum alcanzan el
mediterráneo oriental). La Península Ibérica es el centro de dixersificación. En este
territorio se presentan 23 de las 25 especies, de ellas, el 6£% son endemismos
ibéricos exclusivos (Güemes, 2009). El género forma parte de la familia
Scrophulariaceae, aunque recientemente se ha sugerido el orgen polifilético de la
familia y se ha propuesto su división en otras, de manera que Antirrhinum, bajo esta
visión, se incluiría dentro de Plantaginaceae (Olmstead et al., 2001).
El género Antirrhinum lo forman hierbas perennes o subarbustos, diploides (2n
= 16), que según su preferencia ecológica, pueden separarse, básicamente, en dos
grandes grupos. Por un lado, aproximadamente la mitad del géiero está constituido
por especies propias de terrenos removidos o pedregosos, bordes de caminos,
cascajos y muros, fisuras o rellanos de rocas, sobre substratos en su mayoría, de
naturaleza caliza o silícea (Güemes, 2009). La mayoría de estas especies tiene
numerosas poblaciones de gran tamaño y abarca un rango de distribución amplio
(Anthos, 2009). Sólo dos de estas especies {A. latifolium y A. linkianum) están
representadas en España por escasas poblaciones en el línite de su área de
distribución y se encuentran amenazadas en nuestro territorio, por lo que se
incluyeron en la Lista Roja de la Flora Vascular Española (Moreno, 2008). El resto
del género son principalmente rupícolas, que habitan en fisuras y rellanos de
roquedos verticales, sobre substrato, principalmente, de naturaleza caliza, y en
exposiciones ligeramente sombreadas (Güemes, 2009). Estas especies, en general,
cuentan con pocas poblaciones de escaso tamaño, y su área de distribución suele
ser pequeña (Bañares et al., 2004, 2006, 2009). En total, sor seis las especies
rupícolas de Antirrhinum {A. charidemi, A. lopesianum, A microphyllum, A.
pertegasii, A. subbaeticum, A. valentinum) amenazadas en España (Moreno, 2008).
Las especies
de Antirrhinum
tienen
flores zigomorias
agrupadas
en
inflorescencias, desde muy laxas a densas. La corola es personada de color blanco,
amarillo o purpúreo, y generalmente con venas purpúreas, con ui tubo más o menos
cilindrico que se prolonga en una giba basal donde se acumula el néctar (Sutton,
1988; Güemes, 2009). La mayoría de las especies son autoincompatibles (Sutton,
INTRODUCCIÓN
13
1988), con sistema de autoincompatibilidad del tipo gametofítico (Gruber, 1932,
1934), aunque se han documentado algunas excepciones (A siculum, en Harrison y
Darby, 1955; variedades cultivadas de A. majus, en Sutton, 1988; determinadas
poblaciones de A. cirrhigerum y A. linkianum, en Vieira y Charlesworth, 2002). Las
flores son polinizadas por insectos, principalmente, por especies de Hymenoptera,
Lepidoptera y Díptera (Rothmaler, 1956). El fruto es una cápsula bilocular, de ovoide
a globosa, de dehiscencia foraminal, que contiene numerosas semillas pequeñas,
sin estructuras de dispersión evidentes (Sutton, 1988).
Antirrhinum ha sido sujeto de numerosos estudios de genética de poblaciones
en los que la diversidad genética se ha analizado con las técnicas de aloenzimas y
de RAPD (Mateu-Andrés, 1999, 2004; Mateu-Andrés y Segarra-Moragues, 2000,
2003b; Jiménez et al., 2002; Torres et al., 2003; Mateu-Andrés y de Paco, 2006).
Estos análisis, hasta el momento realizados en 16 especies (7 rupícolas), se han
dirigido, en la mayoría de las ocasiones, al planteamiento de estrategias de
conservación.
Además, la relación entre la diversidad genética y el sistema
reproductivo ha sido investigada, pero con resultados ambiguos (Mateu-Andrés y
Segarra-Moragues, 2000; Jiménez et al., 2002; Mateu-Andrés y de Paco, 2006). En
otras ocasiones, la técnica de los aloenzimas se ha utilizado para establecer la
delimitación taxonómica y las relaciones entre especies próximas (en especies
relacionadas con A. graniticum y A. meonanthum, en Mateu-Andrés y SegarraMoragues, 2003a; y con A. majus y A. siculum, en Mateu-Andrés y de Paco, 2005).
En Antirrhinum, el aislamiento geográfico y ecológico ha contribuido a la
diferenciación y a la formación de endemismos locales en la Penísula Ibérica (Webb,
1971). Se ha sugerido que la diferenciación de muchos táxones habría ocurrido al
haber quedado éstos refugiados en áreas pequeñas y aislados de otros táxones por
medio de barreras de tipo geográfico o ecológico. Más recientemente, a estos
procesos evolutivos de aislamiento, se ha añadido la participación de la hibridación
ancestral, pues se ha sugerido su papel relevante en la formación de la mayoría de
las especies reconocidas (Vargas et al., 2004; Jiménez et al., 2005ab).
Por otra parte, también hay evidencias de que la hibridación opera actualmente
en la naturaleza, en parte, debido a que las barreras reproductivas entre las
especies son débiles (Rothmaler, 1956). Entre 1956 y 2003 se han descrito cinco
14
INTRODUCCIÓN
híbridos naturales (A australe x A. graniticum: A. x albanchezii Mateu; A.
controversum x A. mollissimum: A. x kretschmeri Rothm.; A. graniticum x A.
pulverulentum: A x segurae Fern. Casas; A. meonanthum x A. pulverulentum: A x
mazimpakae Fern. Casas; A. molle x A. majus: A x montserratii Molero & Romo;
revisado en Güemes, 2009), al menos hay otros cuatro híbridos descubiertos entre
1996 y 2009 y todavía sin describir (A. microphyllum x A. graniticum, J. Güemes y J.
Riera, com. pers.; A. microphyllum x A. majus, J. Güemes, com. pers.; A. pertegasiix
A. litigiosum, L. Sáez, com. pers.; A. pulverulentum x A. litigiosum, J. Güemes y J.
Fabado, com. pers.), y se conoce una amplia zona de hibridación activa, en los
Pirineos orientales, entre A. latifolium y A. majus (Whibley et al., 2006; Tastard et
al., 2008). Excepto A. australe x A. graniticum y A. latifolium x A. majus, el resto de
híbridos procede del cruce entre una especie rupícola, más o menos rara, y un
congénere no rupícola, más común y se han localizado en áreas de reciente
alteración antropogénica (próximos a conducciones y obras públicas). La actividad
humana en el territorio habitado por especies de Antirrhinum ha provocado la ruptura
del aislamiento geográfico y ecológico que mantenía la separación entre especies y
ha facilitado la coexistencia e hibridación entre táxones que antes eran alopátricos.
Los procesos de hibridación ancestral y reciente en el género, probablemente,
son los responsables de la dificultad en la delimitación de algunos ejemplares, lo
que ha fomentado la visión tradicional de Antirrhinum como género complejo y
conflictivo a nivel taxonómico. Baur (1932) ya califica con la expresión botanicorum
crux et scandalum a este género, para referirse a las dificultades taxonómicas que
presenta. Por este motivo, el reconocimiento o no de un taxon concreto ha variado
según el autor revisor del género. La sencilla comparación de los tratamientos de los
sucesivos monógrafos del género nos permiten adivinar su complejidad (Rothmaler,
1956; Stubbe, 1966; Webb, 1971; Sutton, 1988; Güemes, 2009). Los datos
moleculares basados en la secuenciación de ADN nuclear (ITS: Vargas et al., 2004)
y plastidial (tmT-trnL: Jiménez et al., 2005b) de las especies del género, no han
resultado útiles para aclarar conflictos taxonómicos ni resolver las relaciones
filogenéticas entre las especies.
INTRODUCCIÓN
15
El caso de las rupícolas amenazadas A. charidemi, A. subbaeticum y A.
valentinum
A. charidemi Lange, A. subbaeticum Güemes, Mateu & Sánchez Gómez y A.
valentinum Font Quer son tres endemismos del E y SE de la Península Ibérica,
filogenéticamente relacionados (Jiménez et al., 2005ab) y con características
ecológicas similares (hábitat, tamaño poblacional y rango de distribución). Las tres
especies son rupícolas, presentan un área de ocupación inferior a 15 km2 y cuentan
con pocas poblaciones (A. charidemi, cinco; A. subbaeticum, cuatro; A. valentinum,
cinco) de pequeño tamaño (Sánchez-Gómez et al., 2004; Carrió et al., 2006; Cueto
et al., 2009). Prácticamente toda la población de A. charidemi se encuentra en el
territorio del Parque Natural marítimo-terrestre Cabo de Gata - Níjar, en Almería.
Los individuos de esta especie se presentan aislados o en grupos a lo largo de un
hábitat continuo que abarca una extensión de unos 10-15 km lineales (Cueto et al.,
2009). Por otra parte, dos de las poblaciones de A. subbaeticum se encuentran en
los cañones de los ríos Mundo y Bogarra, en Albacete, a una distancia de unos 44
km de las otras dos poblaciones de esta especie, que se localizan en Benizar y en el
arroyo de Hondares, en Murcia (Sánchez-Gómez et al., 2004). Las poblaciones de
A. valentinum se distribuyen por las Sierras de Corbera, Buixcarró y Mondúber, en
Valencia, y mantienen una distancia de separación de entre 3 y 7 km (Carrió et al.,
2006). La Lista Roja de la Flora Vascular Española (Moreno, 2008) incluye A.
charidemi, A. subbaeticum y A. valentinum bajo las categorías en peligro crítico, en
peligro, y vulnerable, respectivamente.
La participación de la hibridación y sus consecuencias en la historia evolutiva
de las tres especies puede haber sido o puede ser muy diferente. Por un lado, las
tres especies ocupan áreas geográficas muy pequeñas, en zonas más o menos
aisladas y protegidas del contacto con otras especies. Sin embargo, recientemente,
A. valentinum ha entrado en contacto con A. controversum, un congénere común, no
rupícola, debido a la ruptura de barreras ecológicas por la actividad antropogénica
en el territorio de estas especies. En la actualidad, no hay información sobre el
potencial de hibridación entre A. valentinum y A. controversum. Por otro lado, hay
evidencias moleculares basadas en el análisis de RAPO en un grupo de especies de
Antirrhinum (A. charidemi, A. microphyllum, A. pertegasii, A. pulverulentum, A.
16
INTRODUCCIÓN
serpervirens, A. subbaeticum y A. valentinum) que apuntan hacia la hipótesis de que
en la formación de, al menos A. charidemi y A. subbaeticum, han intervenido
procesos de hibridación (Jiménez et al., 2005a).
Objetivo
Objetivo
El objetivo principal de esta memoria doctoral es investigar sobre los procesos de
hibridación en tres especies rupícolas amenazadas de Antirrhinum {A. charidemi, A.
subbaeticum y A. valentinum). La memoria doctoral se estructura en cuatro capítulos
con formato de manuscrito científico que pueden leerse de manera independiente.
La aproximación al objetivo general se aborda a partir del planteamiento de
cuestiones particulares en cada capítulo, donde se exponen y
discuten los
resultados.
El objetivo del primer capítulo es resolver las relaciones filogéneticas de A.
charidemi, A. subbaeticum y A. valentinum. Para ello, se plantea un análisis
filogenético de las especies del género (excepto A. martenii), basado en secuencias
de ADN nuclear (ITS) y plastidial (tmS-trnG, frnK-mafK) de varias poblaciones de
cada especie (excepto A. cirrhigerum).
Se utilizan marcadores moleculares
plastidiales no empleados con anterioridad en Antirrhinum y se amplia el número de
poblaciones muestreadas con respecto a las filogenias previas realizadas con
marcadores moleculares (nucleares: ITS, Vargas et al., 2004; plastidiales: tmT-trnL,
Jiménez et al., 2005b). En paralelo, se infieren las relaciones filogeográficas y se
estima la fecha de divergencia de los linajes del género.
El objetivo del segundo capítulo es analizar si existe una estrategia reproductiva
común y/o limitaciones reproductivas en A. charidemi, A. subbaeticum y A.
valentinum. En particular, se investiga sobre la biología reproductiva, la limitación
polínica (excepto en A. subbaeticum) y la depresión por endogamia en dos
poblaciones naturales de cada especie y se proponen estrategias de conservación.
Parte de esta información se utiliza para el desarrollo del capítulo tercero y cuarto.
26
OBJETIVO
El objetivo del tercer capítulo es analizar si existe potencial para la hibridación y
la introgresión entre A. valentinum y A. controversum. Se analizan las barreras
postpolinización entre las dos especies (precigóticas y postcigóticas: producción de
frutos y semillas, adherencia y germinación de polen en el estigma, desarrollo de los
tubos polínicos en el estilo, tasa de germinación y velocidad de las semillas) y se
examina la capacidad reproductiva de los híbridos artificiales que se generan por
hibridación experimental (producción de óvulos y polen, viabilidad del polen,
producción de frutos y semillas en cruces con otros híbridos y con las especies
progenitoras, tasa de germinación y velocidad de germinación de las semillas). Se
evalúan las posibles consecuencias de la hibridación entre las dos especies sobre la
conservación de A. valentinum.
El objetivo del cuarto capítulo es analizar la relación entre la diversidad
genética, el nivel de monofilia y las características biológicas y ecológicas de A.
charidemi, A. subbaeticum y A. valentinum. Para ello, se desarrolla un análisis
filogénetico de las especies del género (excepto A. martenii) basado en secuencias
de ADN plastidial (psbA-tmH, tm l-trnL) de varias poblaciones de cada especie
(excepto A. cirrhigerum). Se utiliza un marcador molecular plastidial (psbA-tmH) no
utilizado con anterioridad en Antirrhinum, y se amplia el número de poblaciones
muestreadas con respecto al estudio filogenético de ADN plastidial (tmT-tmL)
realizado por Jiménez et al. (2005b). Para cada especie, se examina la relación
entre el nivel de monofilia en la filogenia de ADN plastidial {psbA-trnH, tm l-trn l) de
este capítulo y de ADN nuclear (ITS) del capítulo primero, la diversidad genética (a
partir de los datos publicados por Mateu-Andrés y Segarra-Moragues, 2000; MateuAndrés, 2004), y las características biológicas y ecológicas (parte de esta
información procede del capítulo segundo).
Chapter I:
A geographical pattern of A n t i r r h i n u m speciation
since the Pliocene based on plastid
and nuclear DNA polymorphisms
GEOGRAPHICAL PATTERN OF SPECIATION
29
1.1. INTRODUCTION
The genus Antirrhinum L. contains 25 species primarily distributed throughout the
western Mediterranean (Fig. 1.1). The circumscription of Antirrhinum ¡nto these
species represents the result of more than 250 years of taxonomic effort. Linné
(1753) considered 28 species in Antirrhinum, of which only A. majus and A. molle are
currently retained in this genus. Increasing numbers of species were proposed in
further publications by Willdenow (1800; four species) and Bentham (1846; eight
species). The complex phenotype delimitation is reflected in the unstable
classification of certain populations ¡nto different taxa and in the different numbers of
species recognized by more recent authors: 23 in Rothmaler (1956); 24 in Stubbe
(1966); 17 in Webb (1972); and 20 in Sutton (1988). This illustrates an ongoing
discussion of taxonomic entities, which are characterized by a combination of few
morphological characters (11) shared by two or more species (Vargas et al., 2004).
Based on the distribution of key morphological characters, such as plant
indumentum, leaf and bract shape, and branching patterns, Rothmaler (1956) and
Webb (1972) envisioned isolation-contact-isolation processes during the dry and
wet episodes of the Ice Ages, resulting in hybridization and subsequent character
sharing.
Despite the lack of taxonomic consensus and the fact that species boundaries
are difficult to define, researchers have not been impeded in investigating evolution in
Antirrhinum. The inheritance of floral development in A. majus was studied by Darwin
(1876) and Mendel (1866), and the pioneering work of Bateson, Wheldale and
particularly Baur established Antirrhinum as a model species group from the
beginning of twentieth century onwards (Schwarz-Sommer et al., 2003). Baur was
also one of the first researchers to appreciate the potential of evolutionary genetics,
and used Antirrhinum species and mutant lines to ¡dentify and map genes
responsible for differences in flower colour and morphology (Stubbe, 1966). The
combination of classical genetics using inbred lines and new genetic technologies
(Harrison and Carpenter, 1979) has resulted in a conceded collaborative effort
between
several
research
groups to describe
a
model
system
of floral
morphogenesis (Coen et al., 1986; Endress, 1992; Schwarz-Sommer et al., 2003;
Whibley et al., 2006)
30
Medíterranean Sea
Atlantic Ocean
10°W
5°W
0o
5°E
Figure 1.1. Geographical distributlon of the 25 Antirrhinum species. (a) Species with broader
distributions (A. controversum, A. cirrhigerum, A. latifolium, A. litigiosum, A. majus, A.
meonanthum, A. siculum, A. tortuosum) and A. mollissimum. (b) Species primarily distributed in
the Iberian Península (A. australe, A. braun-blanquetii, A. charidemi, A. graniticum, A. grosii, A.
hispanicum, A. linkianum, A. lopesianum, A. microphyllum, A. molle, A. pertegasii, A.
pulverulentum, A. sempervirens, A. subbaeticum, A. valentinum) and A. latifolium. All species
were sampled, except for the narrowly distributed endemic A. martenii (northern Africa), which
was search but not found. Dashed lines divide the Iberian Península ¡nto the four quadrants
used in the phylogeographical analysis.
31
Research effort in Antirrhinum also includes ¡ntraspecific, population genetic
studies, with 16 out of 25 species examined by means of a range of molecular
markers (Mateu-Andrés, 1999, 2004; Mateu-Andrés y Segarra-Moragues, 2000,
2003; Jiménez et al., 2002; Torres et al., 2003; Mateu-Andrés y de Paco, 2006).
Considerable population diversity has been uncovered, as evidenced by a wide
range of gene heterozygosity valúes {Ht 0.03-0.52) from co-dominant allozymes
(Mateu-Andrés, 1999; Mateu-Andrés and de Paco, 2006), and similar high levels of
diversity revealed by dominant, fingerprinting techniques (Jiménez et al., 2005a).
This diversity has been shown to be both congruent (Jiménez et al., 2005a) and
incongruent (Vargas et al., 2004; Jiménez et al., 2005b) with currently recognized
taxonomic species.
The body of knowledge on the taxonomy, population genetics and genetic
control of organ morphogenesis in Antirrhinum is in contrast to the limited information
available regarding phylogenetic relationships and phylogeographical patterns.
Limited resolution obtained in previous phylogenetic studies of Antirrhinum species
based on nuclear ribosomal ITS (Vargas et al., 2004) and plastid (Jiménez et al.,
2005b) DNA sequences leaves open the question of whether complex and recent
evolutionary processes or unsuitable sampling and molecular markers are
responsible for the poor patterns observed. Here, we performed phylogenetic,
phylogeographical and lineage divergence dating analyses bn an extended sample of
Antirrhinum species using nuclear (ITS) and plastid (trnS-tmG, fmK-mafK)
sequences to address speciation and divergence times in a morphologically complex
Mediterranean system.
1.2. MATERIALS AND METHODS
1.2.1. Sampling strategy and DNA sequencing
A total of 96 individuáis representing 24 of the 25 Antirrhinum species were sampled
(Appendix 1.1). The number of populations sampled per species depended on their
distribution and ¡ntraspecific variation. Particular effort was made to associate
species distribution, morphological forms and taxonomic nomenclature by analysing
material from 10 localities (locus classicus) where plants were first collected for the
32
original species descriptions. Based on previous phylogenetic results, the tribe
Antirrhineae (i.e. 29 genera including Antirrhinum) ¡s monophyletic (Vargas et al.,
2004) and closely related to the Plantaginaceae (Olmstead et al., 2001). In
agreement with these results, outgroup sequences of genera closely related to
Antirrhinum
(Acanthorrhinum,
Pseudomisopates,
Misopates,
Gambelia,
Chaenorhinum) and sequences of Plantago and Digitalis were generated and ^
analysed (Appendix 1.1).
Total DNA was extracted from silica-dried material using the CTAB (cetyl j
trimethyl ammonium bromide) method as in Vargas et al. (2004) or DNeasy Plant
Mini Kits (Qiagen, Valencia, CA, USA). Polymerase chain reactions (PCRs) were !
í
performed on a Perkin-Elmer PCR System 9700 (Fremont, CA, USA) or a MJ
Research (Waltham, MA, USA) thermal cycler. We obtained and analysed 83 trnKmalK, 83 trnS-trnG and 87 ITS sequences (Appendix 1.1). Standard primers were
used for amplification of the trnK-matK spacer (fmK-3914F, maíK-1470R) (Johnson
and Soltis, 1994) and the trnS (GCU)-frnG (UCC) spacer (Hamilton, 1999). After 1-3
min denaturation at 94°C, PCR conditions were: 28-30 cycles of 1 min at 94°C, and
1-2 min at 50-58°C, followed by an extensión of 10 min at 72°C. One microlitre of
dimethyl sulfoxide (DMSO) at 99.9% was included in each 25-pL reaction. Amplified
producís were cleaned using spin filter columns (PCR Clean-up Kit, MoBio
Laboratories, Carlsbad, CA, USA) following the manufacturera protocols. Cleaned
Products were then directly sequenced using dye terminators (Big Dye Terminator
ver. 2.0, Applied Biosystems, Little Chalfont, UK) following the manufacturéis
protocols and run into polyacrylamide electrophoresis gels (7%) using an Applied
Biosystems Prism Model 3700 automated sequencer. PCR primers were used for
cycle sequencing. Sequence data were assembled and edited using the program
SEQED (Applied Biosystems, Foster City, CA, USA). Procedures used for DNA
sequencing of the ITS región are given in Vargas et al. (2004). IUPAC (International
Union of Puré and Applied Chemistry) symbols were used to represent nucleotide
ambiguities.
1.2.2. Plastid haplotype analysis
The 83 concatenated sequences of the trnK-ma1K and trnS-tmG spacers were
aligned by hand, given the low number of indels across sequences. The number of
33
Antirrhinum plastid haplotypes and relationships among them were inferred using the
software TCS ver. 1.21 (Clement et al., 2000). The program implements a statistical
parsimony approach using the algorithm described in Templeton et al. (1992) to
construct haplotype networks. The máximum number of differences among
haplotypes, as a result of single substitutions, was calculated with 95% confidence
limits, and treating gaps as missing data. Given the distribution of Antirrhinum
species (Fig. 1.1), we used six geographical areas (recognized biogeographical
areas) to link haplotypes and geography: four Iberian quadrants (north-eastern, NE;
north-western, NW; south-eastem, SE; south-western, SW) as divided by the
geographical coordinates 40° N/5° W; northern Africa; and Europe (excluding Iberia)
plus western Asia (Appendix 1.1).
1.2.3. Phylogenetic analyses
The plastid and nuclear ribosomal DNA (nrDNA) sequence data sets were analysed
using máximum parsimony (MP) and Bayesian inference (Bl) approaches. Parsimony
analyses were performed in PAUP* (Swofford, 2002) using a heuristic search
replicated 1000 times with random taxon-addition sequences, tree bisectionreconnection (TBR) branch swapping, the options MulTrees and Steepest Descent in
effect, and holding 100 trees at each step. To evalúate the internal support of each
clade, 100 bootstrap replicates (Felsenstein, 1985) were performed using equal
weights, the TBR swapping algorithm with 10 random additions of taxa per bootstrap
replícate, and 100 trees held at each step. The appropríate model of nucleotide
substitution for Bl was determined by the hierarchical likelihood ratio test (hLRT) and
the Akaike information criterion (AIC) implemented using MrModeltest ver. 1.1b
(Posada and Crandall, 1998; Nylander, 2002). Bayesian inference analyses were
conducted in MrBayes ver. 3.2.1 (Ronquist and Huelsenbeck, 2003). Two identical
searches with three million generations each (chain temperature = 0.2; sample
frequency = 100) were performed. In both runs, probabilities converged on the same
stable valué, approximately after generation 500,000 in the plastid and 300,000 in the
nuclear analyses. A 50% majority-rule consensus tree was calculated using the sumt
command to yield the final Bayesian estímate of each phylogeny. We used posterior
probability (PP) as an estímate of robustness.
34
1.2.4. Dating lineage divergence
No reliable fossil data of Antirrhinum and relatives (the tribe Antirrhineae) are known.
We therefore used the estimated age (38-48 Ma) of the split between the
Plantaginaceae and Antirrhinum as obtained by Wikstrom et al. (2001). As outgroup j
sequences could not be reliably aligned for the ITS and trnS-trnG regions, we used
the plastid trnK-matK data set to estímate lineage divergence. Tree topology and
branch lengths were estimated for this data set under the Bl approach. We used the
same parameters as above. Probabilities converged on the same stable valué after
approximately generation 500,000 in the two Bayesian runs.
To check the constancy of substitution rates, we used the Langley and Fitch
(LF) test (Magallón and Sanderson, 2005). As the nuil hypothesis of constant rate
was rejected, we used the penalized likelihood method (PL, Sanderson, 2002)
implemented in r8s ver. 1.71. PL was implemented with the truncated Newton (TN)
algorithm. Initial results were obtained under the following parameters: cvstart = 0.5;
cvinc = 0.5; cvnum = 10 with cross-validation enforced. The rate smoothing with the
lowest cross-validation scores was selected, and the dating procedure was repeated
with the following parameters: collapse; num_time_guesses = 5 and nurrwestarts =
5. Cross-validation suggested 100,000 as the best smoothing parameter. Branching
order and branch lengths from 1000 Bayesian trees (500 trees from each run),
sampled every 5000 generations after stationarity, were analysed to obtain means
and standard deviations of clade ages (Hughes and Eastwood, 2006). As
recommended in the r8s manual, we pruned the extra outgroup (Callitriche brutia) in
order to infer the root node of the real tree.
1.3. RESULTS
1.3.1. Plastid haplotypes analysis
The aligned trnS-trnG and trnK-matK Antirrhinum sequences were 672 and 1350
base pairs long, respectively. We detected 51 haplotypes (with no gap recoded), of
which 37 are exclusive to single accessions (Appendix 1.1). The 51 haplotypes were
connected in a star-like network with no more than seven inferred mutational
35
changes to connect any pair of haplotypes (Fig. 1.2). Outgroup (Misopates,
Gambelia, Acanthorrhinum) sequences were disconnected. A low number (27) of
missing haplotypes (extinct or not found) were inferred to connect all sampled
haplotypes (51). The interior haplotype 1 is shared by samples of five species (A.
braun-blanquetii, A. graniticum, A. linkianum, A. litigiosum, A. lopesianum) from three
geographical areas of Iberia (SE, NE, NW). Haplotype sharing was also observed in
samples of the following species groups: A. charídemi-A. mollissimum (haplotype 37
from SE Iberia), A. grosii-A. meonanthum (haplotype 30 from NW Iberia), A.
latifolium-A. majus-A. molle (haplotype 11 from NE Iberia), A. majus-A. molle
(haplotype 12 from NE Iberia) and A. pulverulentum-A. subbaeticum (haplotype 25
from NE and SE Iberia).
The eight major haplotype clades (formed by two or more haplotypes) depicted
in the network analysis are not necessarily related to species assignation (Fig. 1.2;
Appendix 1.1). Interestingly, the limited exclusiveness of haplotypes associated with
species ñames contrasts with the geographical distribution of haplotypes. SE Iberia
harbours the highest number of haplotypes (21), followed by NE Iberia (18), NW
Iberia (5), N Africa (4), Europe excluding Iberia (4), and SW Iberia (3). Admittedly, the
two areas (SE and NE Iberia) with a high number of haplotypes were sampled more
intensively, albeit they also contain a higher number of species. Phylogeographical
results suggest a complex pattern in NE Iberia, given the number of unrelated
haplotype clades (I, II, V, VI), of which one (V) contains four samples from two more
geographical areas and haplotypes (25, 29) shared by two different geographical
areas (Fig. 1.2; Appendix 1.1). Clade III also reveáis a certain phylogeographical
complexity, with an association of haplotypes from SW Iberia, Europe and N Africa.
Cohesiveness is, in contrast, observed in 18 of 21 haplotypes from SE Iberia (Fig.
1.2 ).
1.3.2. ITS sequence variation
The ITS sequences (ITS-1+5.8S+ITS-2) ranged between 586 and 603 base pairs in
Antirrhinum, resulting in an aligned matrix of 145 variable and 80 parsimonyinformative characters. All accessions are considered ITS functional copies, rather
36
20
27
25
44
NWIberia
O SWIberia
^
NEIberia
® SEIberia
NAfrica
47
Europe(cxcluding Iberia)
45
(j
morethantwo arcas
Figure 1.2. Statistical parsimony network of 51 plastid haplotypes found in Antirrhinum, as
defined on the basis of plastid trnS-trnG/trnK-matK sequences. Lines represent single
mutational steps; black, small circles are inferred haplotypes not found in any sample. Colours
in the inset refer to geographical areas (see text). Román numbers indícate major plastid
lineages with two or more haplotypes. Haplotype distribution across species is as follows: A.
australe (17, 42, 43); A. braun-blanquetii (1, 7); A. charidemi (37, 49); A. cirrhigerum (15); A.
controversum (34, 35, 36, 40); A. graniticum (1, 29); A. grosii (30, 31); A. hispanicum (38, 39,
41); A. latifolium (11); A. linkianum (1, 8); A. litigiosum (1, 5); A. lopesianum (1, 6); A. majus (9,
10, 11, 12); A. meonanthum (29, 30); A. microphyllum (24, 26); A. molle (11, 12); A.
mollissimum (37); A. pertegasii (4); A. pulverulentum (3, 25, 27, 28, 50, 51); A. sempervirens (2,
13, 14); A. siculum (21, 22, 23); A. subbaeticum (25, 32, 33); A. tortuosum (16, 18, 19, 20); and
A. valentinum (44, 45, 46, 47, 48) (see Appendix 1.1).
37
than pseudogenes, because they are similar not only to other Antirrhineae species
(Vargas et al., 2004), but also to other plant taxa. The number of variable/parsimonyinformative sites was distributed as follows: 80/42 in ITS-1, 2/2 in 5.8S and 63/36 in
ITS-2. In our data set, 105 of the 145 variable sites contained at least one IUPAC
symbol, reflecting more than a single nucleotide. Fifty-nine electropherograms of the
87 Antirrhinum accessions displayed double peaks of similar height (nucleotide
additivities). The number of additivities per accession varied from one to 16
(Appendix 1.1). Although it could not be determined, in some cases, whether doublepeak patterns were the result of sequencing artefacts, equimolar proportions of
alternative nucleotide peaks are ¡nterpreted in many accessions as the presence of
different ITS copies. We observed a limited number of sequencing artefacts versus a
general pattern of múltiple ITS copies contained in single Antirrhinum samples
because: (1) two alternative nucleotides at the same site were widely present across
59 of the 87 accessions; (2) most ambiguous nucleotides of these 59 accessions
were at the 80 parsimony-informative positions; (3) only two of the 164 sites of the
5.8S showed additivities, as expected from a highly conserved región; (4) previous
clones of ITS producís of A. iitigiosum (population 2) revealed alternative nucleotides
at seven additive sites (Vargas et al., 2004); and (5) controlled hybrids between A.
pulverulentum x A. controversum displayed complex additivity patterns, including the
maintenance of some of the parental additivities and the generation of new ones
(data not shown). Múltiple ITS copies in a single individual can be derived by DNA
mutation in the ITS región (ITS divergence) or by the merging of different nrDNA
copies into single genomes (hybridization) followed by failure in conceded evolution
(Arnheim, 1983; Whittall et al., 2000). The fact that accessions from isolated
populations (A. grosii, A. lopesianum, A. siculum) displayed no or lower additivities
than those from populations in areas with a high number of Antirrhinum species (A
controversum 6, A. Iitigiosum 2, A. majus 8, A. mollissimum 2, A. pertegasii 2, A.
tortuosum 2) led us to conclude that it is hybridization that is primarily responsible for
this nucleotide additivity pattern (Appendix 1.1).
1.3.3. Phyiogenetic analyses
The number of variable/parsimony-informative characters was 55/26 in the trnS-trnG
and trnK-matK sequence matrix of Antirrhinum accessions. The models of
38
substitution selected by MrModeltest (Posada and Crandall, 1998; Nylander, 2002)
were GTR+G (trnS-trnG), GTR (trnK-malK) and GTR+I+G (ITS). Both Bl and MP
analyses of the plastid and ITS sequence data produced a low number of wellsupported groups (Fig. 1.3; results not shown for ITS). The plastid analysis retrieved
Antirrhinum accessions as monophyletic (Fig. 1.3, 1.4). The Bl tree based on plastid
sequences revealed only 10 species groups supported by posterior probability valúes
equal to or higher than 95 (Fig. 1.3). The MP analysis of plastid sequences rendered
237 most-parsimonious trees of 142 steps [consistency índex (Cl) = 0.94; retention
índex (Rl) = 0.97] and even lower levels of support, with only four species groups in
clades with bootstrap valúes > 75% (Fig. 1.3). Irrespective of support valúes, no
monophyletic groups including all conspecific samples of Antirrhinum were retrieved
in the two analyses, except for the two A. pertegasii accessions. In contrast,
numerous clades include exclusively or primarily accessions from the six
geographical areas (Fig. 1.2, 1.3). All major plastid (species) lineages include
samples from SE or NE Iberia. Neither MP ñor Bl analyses of ITS sequences,
however, found major accession groupings; instead a large, unresolved polytomy
was retrieved (results not shown), similar to previous findings using a more limited
sample (Vargas et al., 2004). The high number of ITS most-parsimonious trees
(91,100) and low consistency index (0.57) and retention index (0.77) also revealed a
serious failure to obtain a cladogenetic pattern in Antirrhinum.
The assumption of the hypothesis of equivalent rates of sequence evolution across
lineages was rejected (x = 218.37, d.f. = 96). Estimates of divergence times using the
PL method are shown in Fig. 1.4. Our results indícate that, after the divergence
between the Plantaginaceae-Scrophulariaceae and the tribe Antirrhineae (Wikstróm
et al., 2001), the divergences of lineages of Antirrhineae (21.49 ± 4.27 Ma) and
Antirrhinum (4.10 Ma to present; error term truncated) probably took place in the
Miocene and Pliocene-Pleistocene, respectively. Given the large number of trees
with remarkably different topologies, it is not possible to propose a single
phylogenetic hypothesis, and thus to estímate divergence times within Antirrhinum
(Fig. 1.4). All analyses are, however, congruent with the differentiation of current
Antirrhinum post-dating the Miocene (Fig. 1.4).
39
----------Misopates orontium
Gambeha speciosa
i------- A meonanihum 2 NE
I
A. ganiticum 3 S W
I |— A. grosi: 1 SW
í5'—|— A. grosii 2 SW
*— A. meonanihum 1 SW
---------- A microphyllum 1 NE
A microoh'vllum 2 NE
A microphyllum 3 NE
100 r- A. pulverulentum I NE
L A pulverulentum 5 NE
,'nn i— A pulverulentum 4 NF
|— A subhaeticum I SE
'— A subbacticum 2 SE
p A. subbaeticum 3 SE
L- A. subbaeticum 4 SE
A controversum 1 SE
A controversum 2 SE
A controversum 5 SE
A controversum 6 SE
100 r- A controversum 1 SE
63 1- A controversum 8 SE
A charidemi 1 SE
A chande mi 2 SE
A. charidemi 3 SE
A. charidemi 4 SE
A. charidemi 5 SE
A. charidemi 7 SE
A valentmum 4 SE
A mollissimum I SE
A mollissimum 2 SE
2 SE
C AA valentmum
valentinum 3 SE
r- A valentinum 1 SE
L A valentmum 5 SE
hispanicum 1 SE
C- AA hispanicum
h is n a m r u m 2
1 SE
r A pulverulentum 3 NE
J00_
A. pulverulentum 7 NE
A.
australe
1
SE
C A a u s tr a le 3 SE
63
— A controversum 3 SE
— A controversum 4 SE
100
— A hispanicum 3 SE
— A. latifolium 4 NE
i— A. siculum 3 F.
{ r A siculum 1 E
>- A siculum 2 E
— A braunblanquetn ] NW
— A braun-blanquetii 2 NW
— A. sempenirens 2 NE
— A. pulverulentum 2 NE
— A. pulverulentum 6 NE
— A majus 2 NE
— A Iitigiosum I NE
— A. Iitigiosum 2 SE
— A. linkianum 1 NW
— A lopexianum 3 NW
— A granituum 1 SE
— A granmcum 2 NE
i- A. pertegasii 1 NE
A pertegasii 2 NE
J00_
• A linkianum 2 SW
• A linkianum 3 SW
C A lopesianum 1 NW
A lopesianum 2 NW
— A majus 3 NE
— A majus 5 NE
— A sempen-irens 1 NE
— A sempen irens 3 NE
— A molle 2 NE
86
_ 100_
100
¡z= iA SSféí
Mi
maius 4 NE
A.
A
A.
A.
A.
A
A.
A
A
A
A.
A.
A.
A.
A
A
molle I NE
latifolium 1 NE
latifolium 2 NE
latifolium 3 NE
australe 2 SW
siculum 5 NA
cirrhigerum 1 NA
tortuosom 2 NA
tortuosum 4 NA
lortuosum 7 NA
tortuosum 8 NA
tortuosum 1 E
tortuosum 3 E
lortuosum 5 E
tortuosum 6 E
tortuosum 9 E
Figure 1.3. Phylogenetic analysis of plastid trnS-trnG/trnK-matK sequences of 24 Antirrhinum
species based on the 50% majority consensus tree of the Bayesian inference analysis.
Numbers above branches are posterior probability valúes. Numbers below branches show both
branch agreement with the strict consensus tree and bootstrap support > 50%. One dash (-)
below branches indicates bootstrap support < 50%, whereas two dashes (— ) indícate
disagreement between the máximum parsimony (MP) strict consensus tree and the Bl tree.
Sample coding as in Appendix 1.1. Geographical abbreviations: SE, south-eastern Iberia; SW,
south-western Iberia; NE, north-eastern Iberia; NW, north-western Iberia; NA, northern Africa;
E, Europe (excluding Iberia) and western Asia.
40
Eocene
Olifioccne
Miocene
100
*¡antago lanceolata
Ijgitans thapsi
haenorhmum crassifolium
tambelia speciosa
fisopates bronttum
seuaom isppates rivas-m artinezii
microt
Constraints:
Maxage = 48 Ma
Minage = 38 Ma
21.49 ± 4 .2 7 M a
7 .2 5 ± 3 .0 4 M a
-C
f
Eocene
Olifiocene
raruthum 1
ramticum 2
raun-blanquetii 2
Miocene
Figure 1.4. Chronogram based on the Bayesian consensus tree and the penalized likelihood
(PL) analysis of the trnK-matK sequences. Branch lengths represent millions of years (Ma). A
previous estimated date of the Plantaginaceae-Scrophulariaceae and the tribe Antirrhineae split
at 38-48 Ma (Wikstrom et al., 2001) was used to implement the analysis. Sample coding as in
Appendix 1.1. A vertical, grey strip indicates the establishment of the current Mediterranean
climate in the Mediterranean basin.
41
1.4. DISCUSSION
1.4.1. Evidence forextensive hybridization in Antirrhinum
The phylogenetic results show a limited number of monophyletic groups of
Antirrhinum accessions related to sections and species. High levels of homoplasy
(correspondence of characters acquired as the result of parallel, reversal or
convergent evolution), lineage sorting (persistence of ancestral polymorphism
through speciation events) and reticulation (non-hierarchical gene transfer) can
produce similar phylogenetic results (Linder and Rieseberg, 2004). The parsimony
estimates of Cl (0.94) and Rl (0.97) obtained from the parsimony analysis of plastid
sequences ¡Ilústrate that the levels of homoplasy found in our data set reach similarly
low valúes to those of other angiosperm groups (Álvarez and Wendel, 2003).
Differentiating between hypotheses of lineage sorting and hybridization is, however,
extremely difficult because both of these processes can generate incongruent
phylogenetic patterns, and thus additional sources of evidence are needed (Linder
and Rieseberg, 2004). Our results of plastid and ITS sequence polymorphism are
better explained by the historical hypothesis of extensive hybridization in Antirrhinum.
The ITS región is a multicopy DNA región, and the presence of two or more ancient
copies may be obscured by processes of conceded evolution, which opérate by
recombinational mechanisms occurring during meiosis (Whittall et al., 2000). In
contrast, failure in ITS copy fixation allows the possibility of detecting additive
patterns (both parental ribotypes present), which have been historically related to
polyploidy and homoploid hybridization (Arnheim, 1983; Sang et al., 1995; Vargas et
al., 1999; Whittall et al., 2000). Nucleotide additivity sites may constitute evidence for
reticulation under the assumption that polymorphism has resulted from the merging
of divergent ITS repeats in a single genome. Our ITS sequence analysis strongly
supports múltiple events of acquisition of different ITS copies. In fact, the analysis
herein performed using an extended sample (87 accessions from 24 of the 25
species) fits previous predictions of extensive failure in nucleotide fixation across
Antirrhinum populations (Vargas et al., 2004).
Múltiple ITS copies in single samples displaying additive patterns have been
widely related in literatura to recent hybridization in angiosperms (Arnheim, 1983;
Fuertes-Aguilar et al., 1999; Bailey et al., 2003; Nieto-Feliner and Rosselló, 2007).
42
Indeed, the lack of monophyly of plastid haplotypes hto species (Jiménez et al.,
2005b; see below) and disagreement between plastid (Fig. 1.3) and nuclear (Vargas
et al., 2004; Langlade et al., 2005) tree-based topologies for some clades further
support a severe failure to retrieve cladogenetic processes. Although broad
reticulation is difficult to demónstrate conclusively in all instances, genetic and
morphological results from Antirrhinum accumulated in recent years clearly fit into the
hybridization model previously described in angiosperms because of: (1) the
difficulties in recovering any synapomorphy in cladistic analyses based on a small
number of intermedíate morphological characters (Sutton, 1988; Vargas et al., 2004;
see also McDade, 1990); (2) the low resolution in parsimony analyses of nrDNA
sequences (this chapter; Vargas et al., 2004; see also Soltis et al., 2008); (3) the
significant conflict between ITS, plastid and morphological results (Vargas et al.,
2004; Jiménez et al., 2005b; see also McDade et al., 2005); (4) the additivity patterns
from dominant (RAPD, Jiménez et al., 2005a) or co-dominant (ITS sequences,
Appendix 1.1; see also Soltis et al., 2008) molecular markers; (5) the cióse
relationship between geography and haplotype dístribution (Fig. 1.2; see also
Fuertes-Aguilar and Nieto-Feliner, 2003); and (6) the high number of ITS additivities
in overlapping geographical areas of certain species (Fig. 1.1, Appendix 1.1; see also
Fuertes-Aguilar and Nieto-Feliner, 2003).
The viability of interspecific hybrids further supports a scenario of extensive
hybridization (Baur, 1932; Mather, 1947). With the exception of A. siculum, all the
species are interfertile (Langlade et al., 2005). The ease with which consecutive
generations can be obtained in artificial crossings indicates a lack of genetic barriers
between species (Rothmaler, 1943; Thompson, 1988; Xue et al., 1996). Indeed, F1
generations between species displaying the most extreme phenotypes have been
used to quantify leaf (Langlade et al., 2005) and flower (Whibley et al., 2006) gene
expression. The importance of hybridization in Antirrhinum has also been identified
by the description of 'unilateral hybridization', which can be made only in one
direction, namely, when the self-fertile species of the pair is used as female parent
and the self-incompatible one as male (Harrison and Darby, 1955). Natural hybrids
have been easily detected in the field where two or more species occasionally meet
(Rothmaler, 1956). Our intensive fieldwork studies over the last 15 years have
43
contributed to the finding of four new ¡nterspecific hybrids in addition to the five
previously described (J. Güemes,
L. Sáez,
P. Vargas, unpublished data).
Hybridization processes may also have contributed to historical taxonomic
disagreement (Sution, 1988). Individuáis of a hybrid zone ¡nvolving A. majus and A.
latifolium display intermedíate and novel flower colour phenotypes found only in the
Pyrenees (Whibley et al., 2006). Our sequence analyses are congruent with
hybridization processes in a large area in the eastern Pyrenees, where two (I, II)
haplotype lineages meet (Fig.
1.1, 1.2), and individual samples contain a
considerable number (2-10) of ITS additivities (A. latifolium 1 and 2, and A. majus 1,
3, 5 and 6; Appendix 1.1). We suggest that this pattern of interfertility observed now
in Pyrenean populations has been historically predominant in Antirrhinum, given that
self-incompatibility in the majority of Antirrhinum species favours hybridization
(Sutton, 1988; Mateu-Andrés and de Paco, 2006).
Molecular markers can therefore provide unambiguous ’footprints' of ancient
hybridization events (haplotype diversity significantly related to geography, but
unrelated to species boundaries) and recent hybridization contacts (limited nucleotide
additivity homogenization). However, identifying which ancestral lineages (taxa) have
hybridized is not possible, given the complicated pattern of our results. The question
remains of whether hybridization is the only process historically involved in
Antirrhinum speciation.
1.4.2. Pleistocene divergence of Antirrhinum lineages
Phylogeography explores the lineage relationships among populations of the same
(or closely related) species in a geographical context (Avise, 2000). Antirrhinum, with
24 species sampled in this study, displays low levels of genetic diversity. In fact,
haplotype number (51) and one single network retrieved in the phylogeographical
analysis (Fig. 1.2) parallel phylogeographical results at the populational level of
particular species (Albadalejo et al., 2005; Koch et al., 2006; Besnard et al., 2007).
The fact that 51 haplotypes have been found in Antirrhinum and only 27 have been
inferred to be missing (extinct or not found) per se indicates: (1) that our sample is
suitable for defining haplotype clades; (2) cióse species relatedness; and (3) short
divergence times.
44
Hypothetical pre-Pliocene differentiation oí Antirrhinum, based on ITS sequence
variation (Vargas et al., 2004) and Rothmaler's (1956) arguments, ¡s not supported
by our calibrated plastid phylogeny. The PL dock method using trnK-matK
sequences is largely congruent with a differentiation process of extant Antirrhinum
lineages since the Pliocene (Fig. 1.4). These estimates of divergence times are ;
consistent with previous estimates based on CYCLOIDEA-like genes (Gübitz et al., í
2003). The increasing number of studies of Mediterranean plants reveáis evidence of
active differentiation in the Pliocene and Pleistocene, as coincident with the onset of \
the Mediterranean climate (Conti, 2007; Guzmán and Vargas, 2008, for Cistus). j
However, we add a cautionary note that our estimated divergence times were I
calibrated on an estimated time of divergence between Plantaginaceae and
Antirrhinum from Wikstrom et al. (2001). Topological irresolution and short branch
length at the base of plastidial and nuclear phylogenies are again congruent with
recurrent hybridization but also with a slow rate of sequence change and with
Antirrhinum radiation. This could be interpreted given a large number of species (25)
originating in a short period of time (Fig. 1.4).
1.4.3. A geographical pattern of differentiation in eastern Iberia
The haplotype distribution and plastid DNA (cpDNA) network (Fig. 1.2) give clear
evidence that geography has been the major factor in Antirrhinum differentiation. In
contrast to disassociation between haplotypes and species assignation, geographical
cohesión is observed. South-eastern Iberia appears to be a main centre of
differentiation, as revealed by the highest figures of haplotypes/haplotype clades
(21/4), closely followed by NE Iberia (18/4), and then by Europe excluding Iberia
(4/2), N Africa (4/1), NW Iberia (5/1) and SW Iberia (3/1) (Fig. 1.2; Appendix 1.1). In
addition, eastern Iberia harbours the highest number of species (15 of the 25
Antirrhinum species), indicating a relatively long history of Antirrhinum differentiation
and speciation since the establishment of the Mediterranean climate. These results
were not unexpected. Habitat requirements (rocky soils) for most Antirrhinum species
are found to be particularly abundant in the mountains of eastern Iberia. Within the
Mediterranean región (Myers et al., 2000), SE Iberia is a hotspot for diversity as
manifested not only by the high number of species and high level of endemism
(Sáinz Ollero and Moreno Saiz, 2002), but also by the accumulation of divergent
45
lineages within different angiosperm groups (Quercus, Lumaret et al., 2005; Hederá,
Valcárcel et al., 2003; Phlomis, Albadalejo et al., 2005; Arenaria, Valcárcel et al.,
2006).
Limited distributions of seven species (Fig. 1.1b), including recognition of six
endangered species (Moreno, 2008), additionally suggests that geography has had
an important role in Antirrhinum evolution. Geographical speciation may have taken
place in Iberian mountains, followed by secondary contacts, with range expansions
and contractions reflecting climate fluctuations in the Quaternary (Hewitt, 2000). The
mountainous eastern Iberia offers a large area within which to infer the evolutionary
causes of either historical or recent isolation of two endangered species (A.
subbaeticum,
A.
valentinum)
(Fig.
1.1b).
Chorological,
phylogenetic,
phylogeographical, population genetic, morphological and ecological data are
consistent with the following scenario. As naturalness of Antirrhinum has been
demonstrated in the analysis of the tribe Antirrhineae (Vargas et al., 2004; P. Vargas
and A. Forrest, unpublished data), an Antirrhinum ancestor may have diverged into
múltiple lineages at least by the Pliocene (Fig. 1.4). Geographical speciation may
have taken place in Iberian mountains, followed by secondary contacts promoted by
the climatic episodes of the Pliocene-Pleistocene (Fauquette et al., 1999), as
Rothmaler (1956) and Webb (1971) have already proposed. Some evidence for this
is found in the limited species distributions in numerous cases (Fig. 1.1) and the
ancestral retention of plastid haplotypes in particular clades related to endemic
areas. We have substantial evidence for ancient contacts in one species, A.
subbaeticum, because it shows the convergence of two plastid lineages (V, VIII) (Fig.
1.2), but a low number of additivities (only 0-1 ITS) (Appendix 1.1), which is a signal
of lack of recent hybridization (Nieto-Feliner and Rosselló, 2007; Soltis et al., 2008).
In contrast, a most recent isolation process is inferred in A. valentinum because this
species has five haplotypes in the same clade (VI) and a more considerable number
(0-5) of ITS additivities. Secondary contacts in A. valentinun may have resulted in
the increase of allozyme gene alíeles (Mateu-Andrés and Segarra-Moragues, 2000)
and haplotype diversity (Appendix 1.1). Similar morphological phenotypes, as a result
of further geographical isolation of populations in restricted areas (Fig. 1a), may be
the cause of the undisputable taxonomic recognition of A. valentinum and A.
46
subbaeticum. In fact, genetic phenotypes (RAPDs) unequivocally discriminated
populations into these two species (Jiménez et al., 2005a).
Optimal ecological conditions may also have been important in Antirrhinum
evolution (Whibley et al., 2006). In addition to isolated rocky habitats in the western
Mediterranean región, territorial bees such as Anthidium spp. pollinating the
characteristic persónate flower of Antirrhinum species (Glover and Martin, 1998;
Torres et al., 2003; Vargas, 2007) should be further explored to infer the most recent
processes of disruptive selection and speciation.
47
Appendix 1.1.
Antirrhinum material used for ITS, tmS-tmG and fmK-mafK sequencing of 96 samples. Population numbers are given in
brackets after species ñames. Geographical abbreviations: SE, south-eastem Iberia; SW, south-western Iberia; NE, north-eastern Iberia; NW,
north-westem Iberia; NA, northem Africa; E, Europe (excluding Iberia) and westem Asia. Localities in bold indícate sites where the material was
collected for the first plant description (locus classicus). Voucher abbreviations: BdB, Valencia database; ECA, Elena Carrió's collection
numbers; JG, Jaime Güemes' collection numbers; PV, Pablo Vargas’ collection numbers; JRV, Jesús Riera's collection numbers; MA,
herbarium of the Royal Botanic Garden of Madrid; MS, María Santos' collection numbers; GB, Gianluigi Bacchetta's collection numbers; LM,
Leopoldo Medina's collection numbers; VAL, herbarium of the Botanical Garden of Valencia. Collection vouchers in bold indícate plant material
used in Vargas et al. (2004), from which nomenclature is followed except for A. controversum (= A. barrelieri). Asterisks (*) after GenBank
accession numbers of ITS sequences refer to those from Vargas et al. (2004).
Taxon
A.
A.
A.
A.
australe Rothm. (1)
australe Rothm. (2)
australe Rothm. (3)
braun-blanquetii Rothm. (1)
Geographical area/locality
SE/Spain: Albacete, Yeste
SW/Spain: Cádiz, Benaocaz
SE/Spain: Granada, Castríl
NW/Spain: Asturias, Cuetu
L’Abeyera
A. braun-blanquetii Rothm. (2) NW/Spain: León, Oblanca
A. braun-blanquetii Rothm (3) NW/Spain: Palencia, Cervera
de Pisuerga
A. charidemi Lange (1)
SE/Spain: Almería, C. Gata,
La Lobera
Sabinal
Santa Cruz
Vela Blanca
Sabinal
Collection
voucher
GenBank accession no.
(ITS/frnS trnGItrnK-matK)
Number of
ITS
additivities
0
0
4
6
Plastid
haplotype
no.
42
17
43
1
JG4077
ECA49
VAL 140895
JRV5333
EU677194
EU677195
AY731273*
EU677196
EU673477
EU673478
EU673479
EU673480
EU717968
EU717969
EU717970
EU717971
JRV5177
VAL35121
EU677197
AY731269*
EU673481
—
EU717972
0
0
7
—
132PV05(1)
EU677198
EU673482
EU717973
0
37
24PV05(16)
-
EU673483
EU717974
—
37
136PV05(2)
EU677199
EU673484
EU717975
0
37
137PV05(7)
—
EU673485
EU717976
—
37
24PV05
—
EU673486
EU717977
—
37
48
Appendix 1.1. (cont.).
EU717978
Number of
ITS
additivities
—
Plastid
haplotype
no.
37
EU717979
0
49
0
0
-
EU717980
14
-
3
4
6
34
40
—
EU717983
EU717984
2
8
36
34
EU673496
EU673497
EU717985
EU717986
3
2
35
35
AY731283*
EU673498
EU717987
7
1
JG4101
EU677208
EU677209
ECA54
ECA77/VAL37 AY731281 *
EU673499
EU673500
EU673501
EU717988
EU717989
EU717990
3
0
2
1
29
31
276PV063
BdB 14
120PV99
EU677210
FJ487614
AY731286*
EU673502
EU673503
EU673504
EU717991
EU717992
EU717993
0
5
0
30
39
38
ECA40
EU677211
EU673505
EU717994
0
41
Taxon
Collection
voucher
(ITS/frnSfrnG/frnK-mafK)
Santa Cruz
Vela Blanca
SE/Spain: Almería, C. Gata
NA/Morocco: Doukkala-Abda,
El Jadida
SE/Spain: Albacete, Villa de
Ves
SE/Spain: Alicante, Jalón
SE/Spain: Almería, Berja
SE/Spain: Granada,
Bérchules
SE/Spain: Valencia, Bolomor
SE/Spain: Valencia,
Carcagente
SE/Spain: Valencia, Chella
SE/Spain: Valencia, Xeresa,
Colom
SE/Spain: Madrid,
Fuentidueña del Tajo
NE/Spain: Soria, Caltojar
SW/Spain: Huelva, Aracena
NW/Spain: Ávila, El Trampal
136PV05 (7)
—
EU673487
137PV05(5)
FJ487611
EU673488
VAL37158
VAL111299
AY731282*
EU677200
-
VAL 145152
AY731272*
-
BdB 47
ECA37
BdB 15b
EU677201
EU677202
EU677203
EU673491
EU673492
—
EU717981
EU717982
JG4001
BdB 29
EU677204
EU677205
EU673494
EU673495
JG4067
BdB 2
EU677206
EU677207
JG4009
A. cirrhigerum Welw. ex
Ficalho (1)
A. controversum Pau (1)
A. graniticum Rothm. (1)
A. grosii Font Quer (1)
A. hispanicum Chav. (1)
NW/Spain: Ávila, Guisando
SE/Spain: Granada, Juviles
SE/Spain: Granada, Veleta
road
SE/Spain: Granada, Vélez de
Benaudalla
r\Af\
EU673489
15
49
Taxon
Collection
voucher
{YTSItrnS trnGItrnK-ma fK)
A. latifolium Mili. (1)
NE/Spain: Girona, Collada de
Toses
NE/Spain: Lérida, Bapá
NE/Spain: Lérida, Martinet
E/ltaly: Piamonte, Cuneo
NW/Spain: La Coruña,
Cedeira
SW/Portugal: Peniche, Cabo
Carvoeiro
SW/Portugal: Trafaria,
Almada
SW/Portugal: Cintra
NE/Spain: Teruel, Griegos
SE/Spain: Valencia, Serra
SE/Spain: Zaragoza,
Nuévalos
NW/Portugal: Braganga,
Alfaiéo
JG4142
EU677212
A.
A.
A.
A.
latifolium Mili. (2)
linkianum Boiss. (1)
A. linkianum Boiss. (2)
A.
A.
A.
A.
linkianum Boiss. (4)
Iitigiosum Pau (1)
Iitigiosum Pau (2)
Iitigiosum Pau (3)
A. lopesianum Rothm. (1)
A.
A.
A.
A.
majus L.
majus L.
majus L.
majus L.
(1)
(2)
(3)
(4)
VAL144658 AY731274*
JG4139
MS781
EU677213
S. Ortiz (s.n.) EU677214
EU673506
EU717995
Number of
ITS
additivities
2
EU673507
EU673508
EU717996
EU717997
-
EU717998
1
1
-
EU673510
-
Plastid
haplotype
no.
11
5
11
11
1
ALQ3435
EU677215
EU673511
EU717999
1
8
ALQ4877
—
EU673512
EU718000
-
8
VAL144655
ECA74
VAL144656
VAL31598
AY731278*
EU677216
AY731271*
AY731277*
-
EU718001
EU718002
0
6
7
0
-
EU673513
EU673514
—
EU673515
EU718003
EU677217
EU673516
EU718004
3
6
EU677218
EU673517
EU718005
2
1
FJ487615
FJ648325
AY731280*
FJ487616
EU673518
EU673519
EU673520
EU673521
EU718006
EU718007
EU718008
EU718009
10
8
8
0
11
9
12
10
F. Amich & S.
Bernardo
(s.n.)
F. Amich & S.
NW/Portugal: Vimioso,
Cargao
Bernardo
(s.n.)
F. Amich & S.
NW/Spain: Salamanca,
Bernardo
Corporario
(s.n.)
NE/Spain: Barcelona, Gréixer JG4150
NE/Spain: Huesca, Panticosa JG4108
NE/Spain: Lérida, Valle d’AránVALI44657
NE/France: Hérault, St.
230PV06
Chinian
1
5
6
50
Taxon
Collection
voucher
(YTSItrnS trnGItrnK-matK)
A. majus L. (5)
NE/France: Pyréneés
Orientales, Salses
NE/Spain: Gerona, La Molina
NW/Spain: Ávila, S. Gredos,
El Tremedal
NE/Spain: Soria, Río Lobos
VAL 39727
FJ487613
EU673522
273PV06
149PV99
EU677219
AY731284*
-
JG4098
A. majus L. (6)
A. meonanihum Hoffmans. &
Link (1)
A. meonanthum Hoffmans. &
Link (2)
A. microphyllum Rothm. (1)
NE/Spain: Cuenca, Buendía JG4024
NE/Spain: Guadalajara,
JG4021
Bolarque
JG4023
Sacedón
VAL40051
Entrepeñas
A. molle L. (1)
NE/Spain: Barcelona,
JG4143
Riguréixer
A. molle L. (2)
NE/Spain: Huesca, Sopeira VAL35176
A. mollissimum Rothm. (1)
SE/Spain: Almería, Benizalón ECA29
SE/Spain: Almería, Caballar ECA32
gorge
SE/Spain: Almería, Sierra de VAL37143
Gádor
NE/Spain: Castellón, Cova
JG4092
A. pertegasii Rotmh. (1)
Fosca
NE/Spain: Castellón, Solá
JG4091
d'en Brull
NE/Spain: Castellón
JG
A. pulverulentum Lázaro Ibiza NE/Spain: Cuenca, Hoz de
ECA28
Beteta
(1)
A. pulverulentum Lázaro Ibiza NE/Spain: Guadalajara, DurónJG4027
(2)
EU718010
Number of
ITS
additivities
10
EU673530
EU718011
7
8
EU677220
EU673531
EU718012
2
29
EU677221
EU677222
EU673532
EU673533
EU718013
EU718014
0
3
24
24
EU677223
EU673534
EU718015
7
26
2
“
AY731267*
Plastid
haplotype
no.
12
-
30
FJ487612
EU673524
EU718016
6
11
AY731268*
EU677224
EU677225
EU673525
EU673526
EU673527
EU718017
EU718018
EU718019
1
4
8
12
37
37
AY731275*
—
EU677226
EU673528
EU718020
0
4
EU677227
EU673529
EU718021
8
4
EU673535
EU718022
0
0
27
EU673536
EU718023
0
3
EU677228
EU677229
EU677230
0
-
51
Taxon
A. pulverulentum Lázaro
(3)
(4)
(5)
(6)
(7)
A. sempen/irens Lapeyr.
A. sempervirens Lapeyr.
A. sempervirens Lapeyr.
A. siculum Mili. (1)
Ibiza NE/Spain: Guadalajara, La
Peregrina
Ibiza NE/Spain: Guadalajara,
Peralejos Truchas
Ibiza NE/Spain: Teruel,
Tramacastilla
Ibiza NE/Spain: Guadalajara,
Alcorlo
Ibiza NE/Spain: Zaragoza,
Nuévalos
(1)
NE/Spain: Huesca, Bielsa
(2)
NE/Spain: Huesca, Plan
(3)
NE/Spain: Huesca, Panticosa
E/ltaly: Sicily, Catania
E/ltaly: Sicily, Messine
Collection
voucher
(ITS/írnSfrnG/frnK-mafK)
JG4035
EU677231
EU673537
ECA26
EU677232
ECA71
EU718024
Number of
ITS
additivities
3
Plastid
haplotype
no.
51
EU673538
EU718025
4
25
EU677233
EU673539
EU718026
1
28
JG4028
EU677234
EU673540
EU718027
5
50
VAL31592
AY731279*
EU673541
EU718028
1
28
EU677235
EU677236
AY731270*
EU677237
AY731276*
EU673542
EU673543
EU673544
EU673545
EU673546
EU718029
EU718030
EU718031
EU718032
EU718033
4
0
0
3
1
13
2
14
23
22
FJ648327
EU673547
EU718034
1
21
EU673549
EU718035
3
0
EU677239
EU673550
EU718036
0
25
EU677240
EU673551
EU718037
1
33
EU677241
EU673552
EU718038
1
32
FJ487617
EU677242
EU673553
EU673554
EU718039
EU718040
10
16
16
18
FJ487618
EU673555
EU718041
5
16
JG4114
JG4116
VAL145148
GB66/06
VAL 119899/
JG3019
E/ltaly: Sicily, Siracusa
VAL 178308/
JG3437
NA/Morocco: Oriental, Zegzel 192PV00
SE/Spain: Albacete,
JG4081
Bogarra, El Batán
JG4084
SE/Spain: Albacete, Los
Vizcaínos
SE/Spain: Murcia, Benízar
JG4068
A. subbaeticum Güemes,
Mateu & Sánchez Gómez (1)
SE/Spain: Murcia, Hondares BdB 227
A. tortuosum Bosc ex Vent. (1) E/ltaly: Sardinia, Cagliari
GB136/06
ALQ3441
A. tortuosum Bosc ex Vent. (2) NA/Morocco: West Rif,
Talembot
A. tortuosum Bosc ex Vent. (3) E/Turkey: Sulcuk, Efeso
GB316/06
EU677238
AY731287*
-
-
25
52
Taxon
Collection
voucher
A. tortuosum Bosc ex Vent. (4) NA/Morocco: Taza-AI
188PV06
Hoceima, Taza
A. tortuosum Bosc ex Vent. (6) E/ltaly: Ancona, Sirolo
VAL 39871
A. tortuosum Bosc ex Vent. (7) NA/Morocco: Taza-AI
MA 643294/
Hoceima, Tazzeka
201PV06
A. tortuosum Bosc ex Vent. (8) NA/Morocco: Tadla-Azilal,
MA 746269/
Ighir
LM3678
A. tortuosum Bosc ex Vent. (9) E/Turkey: Bursa lli, Gemlik
164PV06
A. valentinum Font Quer (1)
SE/Spain: Valencia, Bolomor BdB 229
SE/Spain: Valencia, Buixcarró JG4002
SE/Spain: Valencia, Font del BdB 8
Cirer
SE/Spain: Valencia, La Drova JG4004
SE/Spain: Valencia, Peña
BdB 1
Colom
Acanthorrhinum ramosissimum Morocco: Mid Atlas
JG 3284-2
Rothm.
Chaenorhinum crassifolium
Spain: Huesca, Sopeira
JG
(Cav.) Lange
Digitalis thapsi L.
Spain: Madrid, Colmenar
61PV07
Viejo
VAL 145156
Gambelia speciosa Nutt.
Botanischer Garten BerlinDahlen
VAL 145155
Misopates orontium (L.) Raf.
Spain: Valencia, Serra
62PV07
Plantago lanceolata L.
Spain: Madrid, Colmenar
Viejo
Spain: Ávila, Sierra de Gredos377PV99
Pseudomisopates rivasmartinezii (Sánchez Mata)
Güemes
EU718042
Number of
ITS
additivities
2
Plastid
haplotype
no.
18
(nSItrnS trnG/trnK-matK)
FJ487619
EU673556
AY731285*
-
1
-
-
EU673558
EU718043
-
20
-
EU673559
EU718044
-
19
FJ648326
EU677243
EU677244
EU677245
EU673560
EU673561
EU673562
EU673563
EU718045
EU718046
EU718047
EU718048
5
0
0
8
16
47
44
45
EU677246
AY39799*
EU673564
EU673565
EU718049
EU718050
2
3
48
46
-
-
EU718051
-
-
-
-
EU718052
-
-
—
—
EU718053
—
~
—
EU718054
-
-
—
-
EU718055
EU718056
-
-
EU718057
-
-
—
—
-
-
Chapter II:
Reproductive biology and conservation implications
¡n A n t i r r h i n u m c h a r i d e m i , A. s u b b a e t i c u m
and A. v a l e n t i n u m
REPRODUCTIVE BIOLOGY
55
2.1. INTRODUCTION
The reproductive biology of a plant species affects, at least to some extent, its
reproductive success (Rymer et al., 2005) with important consequences for the
viability of the populations (Evans et al., 2003). Therefore, knowledge of a species’
reproductive biology provides important insights when designing conservaron
programs for endangered plants (Weller, 1994; Bosch et al., 1998; Weekley and
Race, 2001; Young et al., 2002). The degree to which a plant is dependent on
pollinators to produce seeds may be decisive for its reproductive success. In
situations of pollinator scarcity or low potential mates, oblígate outcrossers may yield
few seeds (Steffan-Dewenter and Tscharntke, 1999; Hendrix and Kyhl, 2000), fewer
than required to maintain the population (Lamont et al., 1993). In similar
circumstances, selfers can reach higher seed production (Totland and SchulteHerbrüggen, 2003); however, self-seeds might be affected by inbreeding depression
(Fischerand Matthies, 1997; Kephart et al., 1999).
The existence of divergent mating systems within cióse species has frequently
been observed (Schemske and Lande, 1985) and many genera that contain selfincompatible species also include species that are self-compatible (Vogler et al.,
1998; Charlesworth, 2006). This observation would suggest that the breeding system
can respond rapidly to natural selection (Jain, 1976), being strongly ¡nfluenced by
several ecological factors like fragmentation, pollinator or resource availability, plant
density, number of flowers per plant, or pollination movements (Chen, 2000;
Franceschinelli and Bawa, 2000). It is thought that repeated limitations in compatible
mates and/or pollinators may favour the evolution of self-compatible and selfing
forms, providing reproductive assurance (Baker, 1955; Carpenterand Recher, 1979;
Affre et al., 1995; Herlihy and Eckert, 2002).
Antirrhinum L. is a genus of 25 perennial species primarily distributed in the
western Mediterranean región (Sutton, 1988) with 68% of the species endemic to the
Iberian Península and believed to be of Pleistocene origin (Chapter I). Many taxa
grow exclusively in crevices on rocks, usually showing a restricted distribution, with
low population size. A total of eight species are listed as endangered in the Red List
of Spanish Vascular Flora (Moreno, 2008). Antirrhinum flowers produce néctar and
56
pollen as rewards for ¡nsect visitors, being mainly pollinated by Hymenoptera,
Lepidoptera and Díptera (Rothmaler, 1956; personal observation). Most of these
species are considerad self-incompatible (Sutton, 1988) and self-compatibility has
only been reported in Antirrhinum siculum (Harrison and Darby, 1955) and the
cultivated varieties of Antirrhinum majus (Sutton, 1988), whereas Antirrhinum
cirrhigerum and Antirrhinum linkianum are partially self-compatible, including both
self-compatible and self-incompatible populations (Vieira and Charlesworth, 2002).
However, knowledge of the reproductive biology of Antirrhinum species is quite
limited and only two of the endangered species have been studied, both in the
greenhouse (Torres et al., 2002; Mateu-Andrés and Segarra-Moragues, 2004).
However previous assays within the genus suggest that the breeding system may be
affected by changes in plant growth conditions (Stubbe, 1966), therefore field data
are needed.
Although studies into the reproductive biology of Antirrhinum species are scare,
genetic diversity has been thoroughly evaluated by allozyme and RAPD analysis in
some taxa of the genus (16), including all the endangered species (Mateu-Andrés,
1999, 2004; Mateu-Andrés and Segarra-Moragues, 2000, 2003b; Jiménez et al.,
2002; Torres et al., 2003; Mateu-Andrés and de Paco, 2006). Correlations between
genetic diversity and the reproductive system in Antirrhinum are unclear. A strong
relationship has been found between both parameters in some taxa (Jiménez et al.,
2002; Mateu-Andrés and de Paco, 2006), but not in others (Mateu-Andrés and
Segarra-Moragues, 2000). Accordingly, in-depth studies of the reproductive biology
oí Antirrhinum species are necessary to establish the relationship between levels and
patterns of genetic variation and the reproductive system within the genus.
We investigated the reproductive biology (flowering phenology, floral biology,
breeding system) and potential limits on seed quantity and quality (pollen limitation,
inbreeding depression) in natural populations of the endangered Antirrhinum
charidemi, Antirrhinum subbaeticum and Antirrhinum valentinum. These three
species are closely related (Jiménez et al., 2005ab) and share many ecological
characteristics (similar habitat, low population, narrow distribution) (Pérez-García et
al., 2003; Sánchez-Gómez et al., 2004; Carrió et al., 2006), but it is unknown whether
they share a common reproductive strategy. Although the three species are legally
57
protected, so far no conservation plan has been proposed for them. This study
attempts to address the following specific questions: (a) what is the reproductive
biology of A. charidemi, A. subbaeticum and A. valentinum? and, do the three
species show different mating systems?; (b) is seed quantity and quality of the three
species limited by pollen limitation and/or inbreeding depression?; (c) what are the
main recommendations to the future conservation plans for these three species?
2.2.1. Study species and sites
A. charidemi, A. subbaeticum and A. valentinum are small shrubs, reaching a height
of 50 cm. These species have flowers grouped in inflorescences which can be
numerous per plant and the number of flowers per ¡nflorescence varíes between the
three taxa (Table 2.1). The flowers have a characteristic persónate corolla, being
pinkish in A. charidemi and A. subbaeticum, and whitish in A. valentinum. The length
of the corolla tube ¡ncreases from A. charidemi to A. subbaeticum to A. valentinum
(Table 2.1). All three species have four didynamous stamens and an erect style
positioned between the two pairs of stamens of different length (Güemes, 2009). The
fruit is a bilocular capsule containing many small seeds without specific dispersal
organs that are spread short distance by boleochory (sensu Vittoz and Engler, 2007).
Seeds of A. subbaeticum are smaller than those of A. charidemi and A. valentinum
(Table 2.1). The chromosome number in the three species is 2n = 16 (Diosdado et
al., 1994; Boscaiu et al., 1997; Coy et al., 1997). Differences in allozyme diversity
among these taxa have been reported: A. charidemi had high intraspecific genetic
diversity and little differentiation among populations; A. subbaeticum exhibited low
intraspecific diversity and high differentiation among populations; and A. valentinum
had high level of intraspecific genetic diversity and high population divergence (Table
2 .2 ).
A. charidemi, A. subbaeticum and A. valentinum are endemics of the E and SE
of the Iberian Península (Fig. 2.1), growing in rocky places. Each covers an area of
less than 15 km2 and, to date, a few small populations are known for each taxon:
58
populations of A charidemi occupy a large and almost continuous habitat along a 1015 km long mountain range, being divided by a gap of less than 1 km long, whereas
those of A. subbaeticum are distributed in two regions 44 km apart and A. valentinum
populations are from 3-7 km apart. The common threats to A. charidemi, A.
subbaeticum and A. valentinum include habitat loss or degradation (Pérez-García et
al., 2003; Sánchez-Gómez et al., 2004; Carrió et al., 2006). A. charidemi has
maintained a stable population size, or has done over the last thirty years (Sáinz
Ollero and Hernández-Bermejo, 1979; Hernández-Bermejo and Pujadas, 1999).
However, in the last twelve years, human activities such as abusive herbarium
collection, buildings, grazing pressure or fires have reduced the population size in A.
subbaeticum and A. valentinum by approximately half (Jiménez et al., 2002; Güemes
et al., unpublished). A. charidemi and A. subbaeticum are listed as critically
endangered and endangered, respectively, and A. valentinum as vulnerable
(Moreno, 2008).
Table 2.1. Number of flowers per inflorescence (range), length of the corolla tube (range), seed
size (range), start and end date of blooming and flowering duration (mean) for A. charidemi, A.
subbaeticum and A. valentinum.
Flowers /
Flowering
Corolla tube
Seed size
Date of
Inflorescence
length (mm)
(mm)
blooming
A. charidemi
1 -1 5a
16-258
0.6-0.83
March-Julyb
6b
A. subbaeticum
3 -1 2a
14-16a
0.5-0.6“
April-Juneb
7b
A. valentinum
5 -1 0a
11-15“
0.6-0.8a
March-Mayb
7b
Species
duration
(days)
a Güemes, 2009;6 This work.
For each species we studied populations (Fig. 2.1; Table 2.3) selected as
containing many accessible individuáis. Fieldwork was carried out during the optimal
flowering period of each species, specifically from: February to July of 2005 for A.
charidemi; April to July of 2000 for A. subbaeticum; and February to May of 2004 for
A. valentinum.
59
Table 2.2. Genetic diversity reported for A. charidemi, A. subbaeticum and A. valentinum.
Species
He
Ht
Hs
Gst
A. charidemi
0.080-0 117
0.103
0.094
0.054
Reference
Mateu-Andrés and
Segarra-Moragues (2000)
A. subbaeticum
0.000-0.024
0.070
0.010
0.850
A. valentinum
0.029-0.092
0.178
0.068
0.481
Mateu-Andrés (2004)
Mateu-Andrés and
Segarra-Moragues (2000)
2.2.2. F lo w e rin g p h e n o lo g y
To s tu d y th e flo w ering pheno logy, several in dividuá is of A. ch a rid e m i (50 plants), A.
su b b a e tic u m (20 plants) and A. valentinu m (20 plants) w ere random ly tagged before
flo w e rin g . T here afte r, the p opula tions w e re visited w eekly until the last flo w e r o f the
last m a rke d plant w as no lo n ge r open. For each visiting day, a p e rcentag e o f
flo w e rin g plants w as obtaine d by dividing the nu m b e r o f m arked plants that had open
flow e rs by the total nu m ber o f tagged in dividuá is.
•O O ,
Figure 2.1. Map of the Iberian Península showing the geographic distribution of the known
populations of A. charidemi, A. subbaeticum and A. valentinum. Black symbols indícate the
studied populations. Populations codes are: SA, Sabinar; VE, Vela Blanca; BE, Benizar; PO,
Potiche; BO, Bolomor; CO, Colom .
60
Table 2.3. Population size, geographic coordínate and altitude of the populations investigated of
A. charídemi, A. subbaeticum and A. valentinum.
Species
Population
Population
size
Geographic
coordínate
Altitude
(m.a.s.l.)
Sabinar
158a
36°45'N/02°10'W
300
Vela Blanca
150a
36°43'N/02°09'W
215
A. subbaeticum
Benizar
37b
38°15'N/01°59'W
980
Potiche
500b
38°33,N/02°10'W
780
A. valentinum
Bolomor
120c
39°03'HI0Q°WW
100
Colom
284°
39°00'N/00°13'W
230
A. charídemi
8 Mateu-Andrés and Segarra-Moragues (2000); b Sánchez-Gómez et al. (2004); c Carrió et al.
(2006).
2.2.3. Floral biology: flowering duration, polten viability and stigma receptivity
Floweríng duration, pollen viability and stigma receptivity were studied ¡n one
population of each species (Vela Blanca, Potiche, Bolomor). Ten plants were
randomly selected and marked. To quantify flowering duration, ten flowers on each
marked plant were tagged before opening and monitored daily until withering. To
investígate variation in pollen viability in relation to pollen age, one floral bud from
each marked plant was bagged prior to opening in order to prevent subsequent
cross-pollen contamination in the anthers. Flowers were bagged using nylon net
bags with a 0.5 x 0.5 mm mesh that prevented insect pollinators from accessing
flowers but allowed normal flower development. Pollen from each flower was
collected at 2, 24, 48, 72, 96, 120 and 144 h after anther dehiscence, and incubated
(25 °C, 4 h) in GK médium (3.2 mM H3B03, 0.8 mM MgS04, 1 mM KN03, 1.3 mM
Ca(N03)2 and 0.29 M sucrose) (Wilson et al., 1997). Pollen grains were observed
under a microscope (400*) and germinated grains were distinguished by a pollen
tube measuring at least double the length of the grain diameter (Stiehl-Alves and
Martins-Corder, 2007). The proportion of viable pollen was determined on 500 grains
per sample. To analyze stigma receptivity, eight flowers on each marked plant were
bagged and emasculated before anther dehiscence. For each plant, 2-, 24-, 48-, 72-,
96-, 120- and 144-h oíd flowers were hand cross-pollinated. The flowers were
61
removed 24 h after pollination and fixed in ethanol-acetic acid (1:1). Before
observation under UV light on a BX40 Olympus microscope with epifluorescence (URFL-T), the pistils were washed, softened (1 M KOH, 15 min, 60 °C) and stained in
0.01% decolorized aniline blue overnight. Stigma receptivity was checked through
the presence of pollen tubes on the stigmatic surface, by the callóse fluorochrome
reaction (Martin, 1959).
Five pollination treatments were applied on plants from the two populations of each
species (Table 2.4): (1) autonomous self-pollination (bagged flowers not handled
further); (2) hand self-pollination (bagged flowers were hand pollinated with pollen
from the same flower): (3) hand cross-pollination (bagged flowers were emasculated
before anther dehiscence and hand pollinated with pollen from flowers of different
plants growing 7-10 m away); (4) supplementary pollination (unbagged flowers were
hand pollinated with pollen from flowers of different plants growing 7-10 m away); (5)
control (marked flowers were left for open pollination). Flowers were bagged as
above (see pollen viability) and the bags were removed following the completion of
anthesis of each treated flower in order to minimize the effects of bagging on fruit
formation. Hand pollinations were done 2-3 days after flower opening, and pollen
was applied to stigmas until their surface was saturated. The effectiveness of each
treatment was evaluated in terms of fruit set (ratio of fruits to treated flowers), seed
set (ratio of seeds to the average number of ovules estimated as below) and seed
weight.
Moreover, to check the degree of self-incompatibility, the pollen tube
germination and growth was analyzed in one population of any species that yielded a
modérate amount of fruits after the hand self-pollination treatment (A. charídemi in
Sabinar: A. valentinum in Colom). To do this, bagged flowers were hand self- or
cross-pollinated as above. Ten flowers for each pollination type were collected 24,
48, 72 and 96, 120 and 144 h after pollination, and fixed in ethanol-acetic acid (1:1).
Samples were prepared and stained with aniline blue as above (see stigmatic
receptivity) and the growth of the pollen tubes in the style was observed under UV
light by the callóse fluorochrome reaction (Martin, 1959).
62
Estimates of the breeding systems were obtained by the pollen-ovule ratio (P/O)
according to Cruden (1977). For the two populations of each species, an average
P/O valué was calculated based on the number of pollen grains and the number of
ovules from ten flowers chosen at random (one flower per plant). Pollen quantity was
obtained based on pollen removed from indehiscent anthers (two per flower) placed
¡n one mi of detergent water and opened with a lancet. Ten subsamples of 10 pl were
scored on slides and pollen grains were counted under a BX40 Olympus optical
microscope (400*). The average number of pollen grains per anther was estimated
and multiplied by four to determine the pollen production per flower. The ovary was
dissected with a needle, and the ovules counted under a Wild stereomicroscope
(120x) to obtain the average ovule number.
Inbreeding depression was estimated in the two A. subbaeticum populations, the only
species studied that was markedly self-compatible. A mean valué of inbreeding
depression was calculated from the valúes obtained in the two populations studied
for three reproductive traits (fruit set, seed set, seed weight) as 5 = 1 - (ws/w0),
where ws is the average fitness of selfed progeny from hand self-pollination, and w0
is the average fitness of outcrossed progeny from hand cross-pollination (Schemske
and Lande, 1985; Charlesworth and Charlesworth, 1987). A cumulative valué of
inbreeding depression was also obtained for the set of the fitness ratio of the three
reproductive traits (fruit set [a], seed set [b], seed weight [c]) according to 6 = 1 [(Wsa/Woa) x (wsb/w0b) x (wSc/w0c)] (Husband and Schemske, 1996). Inbreeding
depression, ranges from zero to one, where zero indicates no inbreeding depression,
and positive valúes indícate that outcrossed progeny are fitter than the selfed
progeny, whereas negative valúes mean the opposite.
2.2.7. Statistical analysis
All the analyses were performed with the programme package SPSS v. 15.00 (SPSS
Inc., Chicago, II, USA). For each species, the variation in the percentage of pollen
viability in connection with pollen age was analyzed by the Kruskal-Wallis test, as the
63
data could not be normalized. The effects of the pollination treatments on seed set
and seed weight within each plant were analyzed separately for each population of
each species. Seed set data were arcsin squareroot transformed and seed weight
data were log 10 transformed to meet the normality assumption. Pairwise differences
among treatments were detected using Generalized Linear Model and the pollination
treatment nested within individual plant was included in the model. The tests
performed were: hand cross-pollination vs. control and supplementary pollination vs.
control in A. charídemi and A. valentinum; hand self- vs. hand cross-pollination, hand
self-pollination vs. control and hand cross-pollination vs. control in A. subbaeticum.
2.3. RESULTS
Start and end dates of blooming differed between the species studied, but were
similar among populations of the same species, being the flowering period thus twice
as long in A. charídemi as in A. subbaeticum and A. valentinum (Fig. 2.2, 2.3, 2.4;
Table 2.1). Moreover, there was a high percentage of flowering plants throughout
most of the flowering period of A. charídemi, with over 50% of sampled plants in
flower from early March-mid April until mid-June. A. subbaeticum and A. valentinum
had a short flowering peak with more than half the marked plants in bloom during
May and April, respectively (Fig. 2.2, 2.3, 2.4).
2.3.2. Floral biology: flowering duration, pollen viability and stigma receptivity
Flowers of A. charídemi, A. subbaeticum and A. valentinum open sequentially from
the base to the apex of the inflorescences, frequently having more than one flower
open each day. The floral buds of all three species started to open in the early
morning, and remained open for several days until the corolla fell. The flowering
duration lasted between 6 and 7 days and varied among the three species (Table
2.1). The anther dehiscence occurred on the first day the flower opened in A.
charídemi and A. valentinum, and on the day after the flower opened in A.
subbaeticum and for the three species, the percentage of pollen viability remained
high at 2, 24, 48, 72, 96, 120 and 144 h after anther dehiscence and was not
64
significantly affected by pollen age (Table 2.5). For all three species, the stigmas of
2-, 24-, 48-, 72-, 96-, 120- and 144-h oíd flowers were receptive.
For both populations of A. charídemi and A. valentinum, fruit set under the
autonomous and hand self-pollination treatment was low, compared with that under
hand cross-pollination (Fig. 2.5, 2.7). However, fruit set results differed markedly
between the populations of either species, due to the existence of three self-fertile
individuáis in one population of each species (Sabinar in A. charídemi; Colom in A.
valentinum). These produced a large quantity of fruits in both self-pollination
treatments (Table 2.6). For the
two A. charídemi populations, the comparison
between the control and supplementary pollination indicated similar fruit set and no
significant differences in seed set and seed weight (Fig. 2.5; Table 2.7). For A.
valentinum, the control treatment compared to hand supplementary pollination
resulted in higher fruit set and in a significantly higher seed set in only one population
(Bolomor), and control seeds were not heavier than those obtained by supplementary
pollination in either of the populations (Fig. 2.7; Table 2.7).
«Sabinar -
-
«Vela Blanca
120 i
100
-
80 en
s
0)
$
e
60 40 20 -
&
.<&
J&
Date
Figure 2.2. Flowering phenology of A. charídemi in Sabinar {N = 10) and Vela Blanca (N = 40).
N, number of plants treated.
65
Table 2.4. Range of flower numbers treated per plant, number of plants treated and number of flowers treated per population of A. charídemi, A.
subbaeticum and A. valentinum in the pollination experiment.
Species
Population
A. charídemi
Autonomous
self-pollination
Hand
self-pollination
Hand
cross-pollination
Supplementary
pollination
Control
6-11, 12, 100
Sabinar
6-8, 11,80
5-8, 11,70
3-1 0,12 , 60
5-7, 10, 58
Vela Blanca
4-5, 23,100
4-8, 18, 108
4-8, 21, 90
5-7, 17, 96
9 -1 1 ,2 3 , 222
A. subbaeticum
Benizar
2-3,10, 25
1-2, 10, 19
1-2, 10, 19
-
4-6 , 10, 50
Potiche
3-4, 10,32
3 -4 ,1 0 ,3 1
3 -4 ,1 0 ,3 1
-
4 -6 , 10, 50
A. valentinum
Bolomor
12-14, 9, 120
8-12, 9, 90
9-10, 10, 93
7-11, 10, 85
10-12, 11, 122
Colom
8-10, 10, 90
7-11, 10, 90
7-10, 9, 73
5-8, 9, 60
12-14, 10, 139
Table 2.5. Pollen viability of A. charídemi, A. subbaeticum and A. valentinum at 2, 24,48, 72, 96,120 and 144 h after anther dehiscence (a.a.d.)
and results of the Kruskal-Wallis test comparing valúes within a line. P-values are not significant at the 5% level.
,
Pollen viability (%)
2 h a.a.d.
24 h a.a.d
48 h a.a.d.
72 h a.a.d.
96 h a.a.d.
120 h a.a.d.
144 h a.a.d.
A. charídemi
96.32 ±00.47
95.95 ±00.39
96.18 ±00.61
95.14 ±01.23
94.00 ±01.38
93.94 ±01.11
93.89 ±00.57
9.834
0.132
A. subbaeticum
92.62 ± 00.97
92.98 ± 0146
92.01 ± 01.28
90.55 ± 00.66
90.95 ±01.11
90.18 ± 00.76
89.64 ± 00.95
7.079
0.314
A. valentinum
94.00 ±00.65
94.33 ±00.80
92.40 ±01.40
93.40 ±00.98
91.38 ±01.22
90.60 ±01.41
91.00 ±01.10
8.234
0.221
66
Benizar 120
-|
100
-
-
'P otic h e
80 60 40 20
-
V
_CV
-CV
-CV
-CV
_CV
_CV
-CV
Date
Figure 2.3. Flowering phenology of A. subbaeticum ¡n Benizar (A/ = 10) and Colom (N = 10). N,
number of plants treated.
B o lo m o r-
-
'C olom
120 -i
100 -
80 60 40 20 -
^
-C$>V
.< &
-C & -< & .< &
_ (&
.< &
.< &
Date
Figure 2.4. Flowering phenology of A. valentinum in Bolomor (Af = 1 0 ) and Colom (A/= 10). N,
number of plants treated.
67
Table 2.6. Fruit set and seed set for self-fertile individuáis of A. charídemi (Sabinar) and A.
valentinum (Colom) in the pollination treatment experiment (N, number of flowers treated).
No.
individual
Autonomous
self-pollination
Seed
set
Hand
self-pollination
N
Fruit
set
3024-90
8
0.375
0.635
3142-90
8
0.500
0.772
763-92
8
0.375
1.155
8
0.417
0.854
Hand
cross-pollination
Seed
set
N
Fruit
set
0.889
1.052
10
0.800
0.849
0.778
0.883
11
0.909
0.719
1.000
1.052
12
0.889
0.995
Fruit
set
Seed
set
N
Fruit
set
8
0.250
0.852
9
8
0.500
0.784
9
0.500
1.005
10
0.417
0.880
N
Control
Seed
set
A. charídemi
Mean
1.000
1.053
0.903
0.873
0.564
A. valentinum
1
12
0.417
0.217
15
0.467
0.373
8
0.875
0.605
10
0.900
3
10
0.300
0.319
10
0.200
0.382
9
0.889
0.813
11
0.909
0.675
7
10
0.500
0.252
14
0.500
0.510
10
0.900
1.084
13
0.923
0.690
0.405
0.262
0.389
0.421
0.888
0.834
0.911
0.643
Mean
For the two A. subbaeticum populations, fruit set was lower under autonomous
self-pollination than under hand self- and cross-pollination (Fig. 2.6). Similar fruit set
and no significant differences were found in seed set or seed weight between the
hand self- and hand cross-pollination treatments (Table 2.7). Control flowers resulted
in higher seed set and similar seed weight compared to hand self- or hand crosspollinated flowers (Table 2.7).
Differences in growth rate of self- and cross-pollen tubes were observed in both
A. charídemi and A. valentinum. In both species, cross-pollen tubes started to
penétrate into the ovules 48 h after the hand cross-pollination and they had already
entered the majority of the ovules (70-90% of the total ovules) 144 h after hand
cross-pollination. In contrast, the self-pollen tubes were seen in both species in the
first third of the style 48 h after the hand self-pollination and remained arrested there
144 h after self-pollination. Self-pollen tubes were occasionally observed in the last
part of the style, but never in the ovules.
68
Table 2.7. Results of the generalized linear models comparing the effects of the pollination
treatment on seed set and seed weight in A. charídemi, A. subbaeticum and A. valentinum.
Significance effects at the 5% level are in bold type.
Population
Pollination treatment
Seed weight
Seed set
df
F
P
df
F
P
Hand cross-pollination
vs. control
20
0.716
0.402
16
0.713
0.385
Supplementary
pollination vs. control
18
1.044
0.209
16
0.211
0.499
vs. control
43
0.713
0.453
40
1.417
0.040
Supplementary
39
0.673
0.465
40
0.485
0.497
Hand self- vs. hand
cross-pollination
14
0.533
0.290
11
0.988
0.168
Hand self-pollination vs.
control
17
1.851
0.031
15
1.361
0.079
vs. control
17
3.248
0.002
15
0.912
0.187
Hand self- vs. hand
cross-pollination
18
1.617
0.051
19
0.561
0.299
Hand self-pollination vs.
control
19
6.111
0.000
19
0.561
0.302
vs. control
18
4.800
0.000
19
0.990
0.166
vs. control
20
0.617
0.447
20
1.190
0.138
Supplementary
20
0.104
0.010
20
0.708
0.405
vs. control
18
0.744
0.379
18
0.753
0.375
Supplementary
18
0.926
0.274
18
0.410
0.492
A. charídemi
Sabinar
Vela Blanca
A. subbaeticum
Benizar
Potiche
A. valentinum
Bolomor
Colom
69
■
■mil i -iiuiVeai'!
Sabinar
Vaia Blanca
Population
Population
■
Autonom ous self-pollination
U H a n d self-pollination
13890155^94^
H H a n d cross-pollination
■
S u p plem entary pollination
33 Control
Sabalar
Vala Blanca
Population
Figure 2.5. Fruit set, seed set and seed weight (mg) of A. charídemi resulting from pollination
treatments testing for autonomous self-pollination, hand self- and hand cross-pollination,
supplementary pollination and control. Bars are means and vertical lines above bars are
standard errors.
70
Population
Population
■
A utonom ous self-pollination
m H a n d self-pollination
■
S u p plem entary pollination
■
control
Population
Figure 2.6. Fruit set, seed set and seed weight (mg) of A. subbaeticum resulting from
pollination treatments testing for autonomous self-pollination, hand self- and hand crosspollination and control. Bars are means and vertical lines above bars are standard errors.
Bolomor
Population
Colom
Population
■
4145845399
71
Autonom ous self-pollination
Q ]H a n d self-pollination
H
Su pplem entary pollination
8 8 Control
Population
Figure 2.7. Fruit set, seed set and seed weight (mg) of A. valentinum resulting from pollination
treatments testing for autonomous self-pollination, hand self- and hand cross-pollination,
supplementary pollination and control. Bars are means and vertical lines above bars are
standard errors.
y
®
;
72
The pollen and ovule numbers per flower and P/O ratios for the species are shown in
Table 2.8. The mean number of pollen grains decreased from A. charídemi to A.
valentinum to A. subbaeticum, and the mean number of ovules increased from A.
charídemi to A. valentinum to A. subbaeticum. The P/O ratios suggest that A.
charídemi and A. valentinum are oblígate outcrossers and A. subbaeticum is a
facultative outcrosser, according to Cruden’s categories (1977).
Table 2.8. Number of pollen grains per flower and ovules and the P/O ratio for A. charídemi, A.
subbaeticum and A. valentinum. Valúes are means ± s.e.
Species
A. charídemi
A. subbaeticum
A. valentinum
Population
Pollen grains/flower
Ovules/flower
P/O
Sabinar
285733.30 ± 70535.27
242.33 ± 20.12
1181.56 ±271.83
Vela
Blanca
486768.60 ± 32399.87
232.64 ± 42.34
2126.00 ± 1438.25
Mean
386250.95 ± 51467.57
237.48 ±21.23
1653.78 ±855.04
Benizar
179803.00 ±29649.00
321.30 ±26.40
560.05 ± 82.90
Potiche
189669.00 ±21536.00
347.50 ± 43.50
555.10 ± 100.40
Mean
184736.95 ±51467.57
334.40 ± 34.95
557.57 ±91.65
Bolomor
203320.00 ± 66652.86
192.40 ±29.77
1065.28 ±333.52
Colom
376731.70 ±91729.42
313.14 ±74.79
1251.89 ±413.20
Mean
290025.50 ±79191.14
252.77 ± 52.28
1158.58 ±373.36
The mean level of inbreeding depression of A. subbaeticum was 0.028 for fruit set
(Benizar: 5 = 0.140; Potiche: 5 = -0.084); -0.179 for seed set (Benizar: 5 = -0.379;
Potiche: 5 = 0.021); and 0.100 for seed weight (Benizar: 5 = 0.142; Potiche: 5 =
0.058). The mean cumulative valué of inbreeding depression was -0.007 (Benizar: 5
= -0.017; Potiche: 5 = -0.002). In all cases these valúes were near zero, indicating
the absence of inbreeding depression, at least in these initial phases of the
reproductive cycle.
73
2.4. DISCUSSION
2.4.1. Mating pattems
In order to produce fruits A. charídemi, A. subbaeticum and A. valentinum need
pollinators to bring pollen to their stigmas. The pollination assay revealed that A.
charídemi and A. valentinum are all highly self-incompatible with only 25-30% of the
plants producing fruits by autonomous self-pollination. On the other hand, although
individuáis of A. subbaeticum are highly self-compatible, they yielded low quantity
fruits in autonomous self-pollinated flowers. This may be due to the anther-stigma
separation (herkogamy) observed during flower handling, since the reproductive
structures are functional at the same time.
Self-incompatibility has been considered unstable within Antirrhinum (Stubbe,
1966). This is supported by the pollination results obtained in A. charídemi and A.
valentinum as self-incompatibility broke down in particular individuáis from Sabinar
and Colom that produced fruits and seeds when self-pollinated manually, and also
yielded fruits and seeds by autonomous self-pollination. Our results for A. valentinum
contrast with those of Mateu-Andrés and Segarra-Moragues (2004) obtained under
greenhouse conditions. These authors did not find self-fertile individuáis in Colom
where we found a 30% of self-fertile plants. However, they found a 6% of self-fertile
plants in Bolomor, whereas the present study found none, although during the
second year of their study only the 1.8% was self-fertile. Observations in other
genera revealed that self-incompatibility may vary with environmental and physical
conditions (Vogler et al., 1998; Stephenson et al., 2000) and can break down with
floral age or low prior fruit set as occurs in Solanum carolinense (Stephenson et al.,
2003) or Witheríngia solanaceae (Stone et al., 2006).
Several aspects of the reproductive biology of A. charídemi, A. subbaeticum and
A. valentinum suggest different mating systems. The three species have many
functional flowers open on the same day within a plant and, therefore, a flower is
highly likely to receive self-pollen from flowers of the same plant (geitonogamous
pollination). This is unlikely to result in fruits in A. charídemi and A. valentinum, given
their high self-incompatibility. A. subbaeticum is self-compatible and preliminary
observations of pollinators of this species indícate that its main pollinators (55.9% of
74
total visits by Megachilidae and Apidae) visit múltiple flowers on an individual plant
before moving to another plant. Self-compatibility combined with the predominantly
within-plant movement of pollinators, suggest that inbreeding may be common in this
species. Thus A. charídemi and A. valentinum are mainly outcrossers, while A.
subbaeticum has a mixed mating system. These results are consistent with the P/O
valúes of the three species.
Our results obtained for the mating systems of the three species are partially
congruent with their genetic diversity. Early reports of allozyme diversity showed that
A. subbaeticum had less total genetic diversity and was more genetically structured
than the outcrossers A. charídemi and A. valentinum (Table 2.2). Previous RAPD
data
also
indicated
differentiation
in A.
lower overall
genetic diversity and
subbaeticum compared
to the
higher population
outcrosser Antirrhinum
microphyllum (Jiménez et al., 2002; Torres et al., 2003). The effect of geitonogamy in
A. subbaeticum could indícate the level and pattern of genetic diversity was more
similar to that of selfing species than outcrossing ones. On the other hand, the two
outcrossers A. charídemi and A. valentinum differed in distribution of genetic diversity
since the genetic structure was markedly higher in A. valentinum than in A.
charídemi. The distribution of genetic diversity in A. valentinum contrasts with its
outcrosser mating system and could be better explained by other ecological factors,
such as flowering duration and habitat continuity (Mateu-Andrés and SegarraMoragues, 2000). Thus, although a strong correlation has been suggested between
genetic diversity and reproductive biology within Antirrhinum (Mateu-Andrés and de
Paco, 2006), our results do not support this.
2.4.2. Limits on seed quantity and quality
There is no evidence to suggest that A. charídemi is pollen limited, since
supplementary pollination in open flowers failed to increase fruit set, seed set and
seed weight. However, in A. valentinum, supplementary pollination considerably
increased both fruit (33%) and seed (14%) production in the small population
(Bolomor). This is less than half that of the Colom population size, suggesting that
pollen limitation could be due to small population size. Small populations of selfincompatible plants may be especially vulnerable to pollen limitation (Byers, 1995;
75
Campbell and Husband, 2007) and this is supported by observations of reduced
seed production in small populations of Lythrum salicaria (Agren, 1996) and
Brunsvigia radulosa (Ward and Johnson, 2005). Moreover, differences in pollen
limitation between the A. valentinum populations may be magnified by the
occurrence of self-fertile individuáis in the Colom population.
Pollen limitation in A. subbaeticum was not examined by supplementary
pollination, but seed quantity and quality were not affected by pollen limitation in
either population, since control flowers had high fruit and seed set, which were not
significantly lower than in hand cross-pollinated flowers. Seed quantity and quality
may be lowered by inbreeding depression in self-compatible species (Brown and
Kephart, 1999; Evans et al., 2003), but this did not occur in the populations of A.
subbaeticum studied. Successive generations of selfing over long periods of time
may have purged deleterious recessive mutations, leading to decreased inbreeding
depression (Lande and Schemske, 1985; Charlesworth and Charlesworth, 1987).
Species entirely dependent on pollinators for fruit set may reduce seed production
due to a lack of pollinator visits (Agren, 1996; Alexandersson and Agren, 1996).
Given that pollinators are essential to seed production in A. charídemi, A.
subbaeticum and A. valentinum, further studies into their pollinators are required in
order to plan efficient conservation strategies. Studies of this type should be a priority
for A. valentinum, since scarcity of pollinators is more likely in this species for two
reasons: firstly, all populations are located near citrus-growing areas, where
pesticides are commonly used (Hernández et al., 2008), which may affect pollinator
communities and lead to similar situations to those described by Allen-Wardell et al.
(1998) or Kearns et al. (1998); and secondly, the blooming period of A. valentinum
occurs in the early spring, when poor weather conditions can influence pollinator
activity (Baker et al., 2000; Alonso, 2004).
A. charídemi and A. valentinum are self-incompatible species and consequently
depend on the availability of compatible mates for reproduction. Small populations of
self-incompatible species are vulnerable to extinction if the availability of mating
types (S-alleles) falls below a certain threshold (Luijten et al., 2002). Natural
76
populations of A. charídemi and A. valentinum exceeding 25 individuáis are thought
to be necessary to maintain sufficient S-allele diversity (Byers and Meagher, 1992).
Drastic reductions of the population size in A. charídemi could be counteracted with
high gene flow among populations due to extended flowering and almost continuous
habitat. In the event of a reduction in A. valentinum numbers, the populations may
have
difficulties
in
successfully
cross-pollinating
due
to
high
population
fragmentation, which can lead to population decline. If necessary, reinforcement or
reestablishment should be performed with an appropriate number of plants (up to 25
individuáis). The low number of individuáis used to form new populations of A.
charídemi thirty years ago (Sáinz Ollero and Hernández-Bermejo, 1979) could be the
reason why this conservation strategy failed. Reinforcements or reestablishments
should be performed with seeds collected from plants, maintaining a sufficient
distance between them to guarantee the availability of compatible mates. The data
shows effective Crossing of plants 7-10 m apart. A. valentinum population
reinforcements should avoid the use of material from other locations, as high genetic
differentiation among individuáis of different populations (Mateu-Andrés and SegarraMoragues, 2000) could cause outbreeding depression in mixed populations. Indeed,
the incidence of outbreeding depression can be predicted by comparing fruit and
seed set from hand cross-pollination in the present study (using pollen from the same
population) and that obtained by Mateu-Andrés and Segarra-Moragues (2004) (using
pollen from another population).
Self-fertile plants may produce seeds when compatible mates and/or pollinators
become limited (Baker, 1955; Herlihy and Eckert, 2002). In this respect, MateuAndrés and Segarra-Moragues (2004) proposed evaluating the role of self-fertility in
maintaining A. valentinum populations where self-fertile plants are present, in order
to design an appropriate conservation strategy. However, as self-compatibility seems
to be influenced by environmental and/or physical factors in A. charídemi and A.
valentinum, conservation strategies should not rely on the chance that self-fertile
individuáis will produce seeds. Furthermore, A. valentinum yields low quantity of
seeds by autonomous and hand self-pollination compared to hand cross-pollination,
suggesting that the overall self-fertile individuáis would probably not produce enough
self-seeds to maintain a viable population.
77
Pollen limitation in the reduced population of Bolomor of A. valentinum could be
due to severa! causes such as a low rate of pollinator visits, a lack of compatible
pollen donors (mate limitation) (Byers, 1995; Campbell and Husband, 2007) and/or
excessive deposition of self-incompatible pollen on stigmas by pollinator movements
between flowers of the same plant, which may clog the stigma preventing crosspollination (Tepedino et al., 2007). Nonetheless, as pollen limitation may vary from
one year to the next due to the environmental variability (Dudash and Fenster, 1997;
Baker et al., 2000), this should be monitored for several years in order to evalúate
the long-term impact on reproduction; moreover, the factors involved in this process
should be quantified before specific proposals can be made for species conservation
(Hirayama et al., 2007).
It is believed that geitonogamy increases as population size decreases since the
quantity of cross-pollen is expected to diminish accordingly (Hirayama et al., 2007).
Thus, recent reductions in population size of A. subbaeticum (Jiménez et al., 2002)
may have increased the inbreeding rate, thereby exacerbating the risks associated
with inbreeding. The short and long-term negative impacts of inbreeding include
inbreeding depression and loss of genetic diversity (Barret and Kohn, 1991;
Huenneke, 1991; Evans et al., 2000). Population size reduction does not appear to
lead to high inbreeding depression, at least in terms of seed quantity and quality.
Preliminary studies also revealed the absence of inbreeding depression on seed
germination in Benizar (5 = -0.279). On the other hand, current levels of A.
subbaeticum genetic diversity are low (Jiménez et al., 2002; Mateu-Andrés, 2004),
which is congruent with frequent inbreeding. It seems unlikely that reduced genetic
diversity could be negatively affecting the current reproduction in the populations
studied, since reproductive output does not appear to be diminished. Thus, although
it is important to conserve large population sizes, small population sizes do not have
a dramatic effect on the actual reproduction of this species. Severe reductions in
population size should not be tackled by applying translocation strategies as
outbreeding depression may occur in individuáis of different populations given the
high genetic differentiation among them (Jiménez et al., 2002; Mateu-Andrés, 2004).
Seeds used in reinforcements or reestablishments can be collected from plants
without the need to keep a minimal separation distance between them.
Chapter III:
Assessing the risk of hybridization and ¡ntrogression
¡n a rare endemic A n t i r r h i n u m species:
the case of A. v a l e n t i n u m
RISK OF HYBRIDIZATION AND INTROGRESSION
81
3.1. INTRODUCCION
Hybridization has long been recognized to play an ¡mportant role in plant evolution
(Arnold, 1997; Rieseberg, 1997), having both positive and negative outcomes on
species biodiversity (Rieseberg, 1991; Buerkle et al., 2003). This may increase
biodiversity by the creation of novel adaptations (Rieseberg, 1991; Johnston et al.,
2004) or the origin of new hybrid lineages (Arnold, 1997; Rieseberg, 1997).
Conversely, hybridization may reduce biodiversity by contributing to species decline
through genetic assimilation by congeners or hybrid lineages (Carney et al., 2000;
Hegde et al., 2006) or by demographic processes that may opérate via a reduction in
reproductive fitness (Levin et al., 1996; Burgess et al., 2008) or via ¡ncreased
competition for suitable sites (Levin et al., 1996; Burgess and Husband, 2006). Thus,
the negative impact that hybridization and ¡ntrogression may cause on the survival of
rare taxa has became a matter of concern for conservation botanists (Rhymer and
Simberloff, 1996; Allendorf et al., 2001). Particular concern arises when this occurs
as result of human-mediated habitat alterations (Ferdy and Austerlitz, 2002; Lamont
et al., 2003) as anthropogenic hybridization has led to the extinction of rare plant
populations (Levin et al., 1996). Another ¡mportant factor to consider when
conserving rare taxa in risk of hybridization is the speed with which this mechanism
operates. Simulation models demónstrate that hybridization is perhaps the most
rapidly acting genetic threat to endangered species as it may cause extinction within
five generations.
Therefore, cases of hybridization between a rare and a more
common congener should be assessed without delay (Wolf et al., 2001; Burkle et al.,
2003).
Even ¡f evidence of hybridization and ¡ntrogression is not visible, the
evaluation of gene-exchange potential between rare and common taxa may
contribute to designing accurate conservation strategies, thus preventing these
processes (Parsons and Hermanutz, 2006; Kothera et al., 2007).
The impact of hybridization between a rare taxa and a common congener will
depend on the degree of reproductive isolation between the two species, which is
related to a variety of ecological and genetic factors (Levin et al., 1996; Rhymer and
Simberloff, 1996; Carney et al., 2000). Reproductive isolation barriers are often
divided into pre- and postpollination mechanisms (Tiffin et al., 2001). Prepollination
isolation between species (exclusively prezygotic) may arise due to ecological,
82
behavioural, and temporal differences or divergent floral traits (Mayr, 1963; Hodges
and Arnold, 1994). Postpollination isolation between species (pre- or postzygotic)
includes, among others, interactions between male and female tracks, low viability of
hybrids, or hybrid sterility (Dobzhansky, 1937; Muller, 1942; Schluter, 1994).
Prepollination barriers may be fragile, as they can be effective under stable
environmental conditions, but break down easily if there are environmental changes;
postpollination barriers, by contrast, are considered independent of the environment
and are relatively robust (Grant, 1992).
Antirrhinum is a western Mediterranean genus comprising 25 perennial and
diploid (2n = 16) species (Sutton, 1988; Güemes, 2009). Ancestral hybridization has
been considered as the main evolutionary mechanism within the genus, according to
several studies based on morphological (Webb, 1971; Sutton, 1988) and molecular
data (Vargas et al., 2004; Jiménez et al., 2005; Chapter I). Geographical and
ecological isolation has presumably led to the differentiation and formation of local
endemic species in the Iberian Península (Webb, 1971), with 68% of the species
being exclusive to this territory (Webb, 1971; Sutton, 1988; Güemes, 2009). All the
species have a characteristic persónate corolla with minor distinctions among
species in shape and size (Sutton, 1988; Güemes, 2009). The flowers are insect
pollinated and the majority of species are outcrossers, mainly self-incompatible
(Sutton, 1988; Mateu-Andrés and de Paco, 2006). Differences in flower colour
¡ntensity and pattern might led to significant pollinator selection, which may have
contributed to the reproductive isolation and the gene-flow limitation among species
(Hodges and Arnold, 1994; Schwinn et al., 2006). Nevertheless, the reproductive
barriers between species are not strong, and it is possible to obtain hybrids through
artificial interspecific pollinations (e.g. Hackbarth et al., 1942; Rieger, 1957; Stubbe,
1966).
Approximately half of the species of the genus have a restricted distribution, live
on calcareous or siliceous surfaces in rupicolous habitats, with few populations
comprising a small number of plants. At least six of these Antirrhinum species are
considered endangered under the IUCN criteria (Moreno, 2008). The rest of the
species have a widespread distribution, with numerous populations of high
population size, and are generally linked to disturbed environments, frequently being
83
found ¡n land drifts, roadsides, slopes or removed substrates (Güemes, 2009).
Habitat disturbance through anthropogenic activities, particularly by opening up the
access to previously isolated territories, has led to recent contact between formerly
allopatric species. Especially evident is the closeness between species pairs formed
by one rare rupicolous species and one common species, typical of disturbed sites.
This situation has led to the formation of seven new hybrids in such sites the last 2030 years, none of which could have arisen in nature. Three of these hybrid plants
have been reported previously and described in the literature: A. molle x A. majus {A.
x montserratii Molero & Romo, described in 1988); A. pulverulentum x A. graniticum
(A. x segurae Fern. Casas, described in 1981); and A. pulverulentum x A.
meonanthum {A. x mazimpakae, Fern. Casas, described in 1981). The other four
hybrids have been found during our revisión of herbarium collections and are
considered hybrids because of their intermedíate morphological characters between
the two putative parental taxa found in the collection area: A. microphyllum x A.
graniticum (VAL37124, J. Güemes and J. Riera, 1997); A. microphyllum x A. majus
(VAL40042, J. Güemes, 1998); A. pertegasii x A. litigiosum (BCB/sn, L. Sáez and A.
Buira, 2008); A. pulverulentum x A. litigiosum (VAL35096, J. Güemes, 1995;
VAL181164, G. Mateo and V.J. Arán,
1996; VAL182364, J. Fabado and
colaborators, 2006).
Antirrhinum valentinum is a rare species from the E of the Iberian Península with
five known populations, occupying an area of less than 10 km2, with less than 1,500
effectives (Carrió et al., 2006). This species was considered common in the past
(Borja, 1948), but has undergone severe reductions in population size during the last
50 years, with its current distribution range practically restricted to the Sierra del
Mondúber. Consequently, the species is considered endangered (Moreno, 2008) and
is ¡ncluded in the Valencian Catalogue of Protected Flora (DOCV Decreto 70/2009).
Antirrhinum controversum is a common species from the E and SE of the Iberian
Península with hundreds of known populations, occupying an area of more than
100,000 km2, probably with hundreds of thousands of specimens (Anthos, 2009).
The two species have an overlapped distribution range; however, their populations
rarely co-occur since they tend to be separated by ecological preferences: A.
valentinum lives in limestone crevices on shady, exposed rock surfaces whereas A.
64
controversum thrives ¡n areas of natural and anthropogenic disturbance such as
rocky altered lands or roadside verges, upon calcareous soils exposed to the sun
(Güemes, 2009). In our field surveys conducted during the last seven years, we have
detected three mixed populations of these two species with the presence of A.
controversum in human-mediated disturbed sites (Fig. 3.1), all of which were related
to recent changes in land use.
Antecedents of hybridization within Antirrhinum, promoted by anthropogenic
habitat disturbance in other territories, led us to evalúate hybridization and
¡ntrogression potential between A. valentinum and A. controversum in order to predict
potential threats to A. valentinum. In particular, we address the following questions:
1) how ¡mportant is postpollination reproductive isolation (prezygotic and postzygotic)
between A. valentinum and A. controversum; 2) if hybridization between the two
species leads to the formation of hybrid plants, could these hybrids successfully
reproduce with other hybrids and with the parental species? Furthermore, according
to the data obtained from evaluating the potential risk of hybridization and
¡ntrogression between the two species, recommendations are made for the
conservation and managementof A. valentinum populations.
3.2.1. Plant species and study site
A. valentinum is a dwarf shrub with decumbent stems (12-35 cm) and lax
¡nflorescences (5-10 flowers). It has small whitish flowers (11-15 mm), with the
superior lip perpendicular to the corolla tube and blooms from March to May. A.
controversum is a perennial herb with erect stems (40-120 cm) and dense
¡nflorescences (10-40 flowers). Its flowers are pinkish and bigger (16-24 mm), with
the superior lip oblique with respect to the corolla tube and flowering lasts from
February to October (Güemes, 2009). Both species are outcrossers, fundamentally
self-incompatible (Mateu-Andrés and Segarra-Moragues, 2004; Chapter II; E. Carrió
and J. Güemes, unpubl. res.) and are mainly pollinated by Apis and Bombus species
(Hymenoptera), but also by other sorts of insects ¡ncluding species of Psithyrus and
Xylocopa (Himenoptera) or Anthophora (Díptera) (Sutton, 1988; Mateu-Andrés and
85
S e g a rra -M o ra g u e s , 2004; pers. obs.). The fru it o f both sp e cie s is a bilocular capsule
co n ta in in g m a n y sm all seeds w ith o u t specific dispersa! organs, w hich are spread a
sh o rt d is ta n c e by bo leoch ory (sen su V ittoz and E ngler, 2007).
T he e xp e rim e n ts w ere perform ed at the B otanical G arden o f the U niversity o f
V a le n cia
b e tw e e n 2004 and
2008 from
m aterial co llected
in a natural m ixed
p o p ula tion (B o lo m o r’s G ully, 3 9 °0 3 'N /0 0 °1 4 'W , 100 m .a.s.l.; Fig. 3.1). All individuáis
o f A . co n tro v e rs u m detected in this locality w ere e stablishe d in recent hum anm ed ia te d d istu rb e d sites. T ab le 3.1 gives data con ce rn in g th e reproductive features
o f A. va le n tin u m and A. co ntro versum in this popula tion taken from previous field
stu d ie s (C h a p te r II; E. C arrió and J. G üem es, unpubl. res.).
Figure 3.1.
Map of the Iberian Península with the geographic dístribution of the known
populatíons of A. valentinum (☆). Red symbols indícate A. valentinum populations where A.
controversum individuáis have been
(Bolomor's Gully).
detected.
Black cycle indicates the studied population
86
A. valentinum and A. controversum seeds were collected from 20 plañís of each
species in the Bolomor’s Gully population in 2003 and grown in pots in the
greenhouse. Before flowering, adult plants were transferred to the pots to an
Table 3.1. Reproductive traits of A. valentinum and A. controversum ¡n the Bolomor’s Gully
population in 2004 (mean ± standard deviation). N1t number of plants considered; N2i number of
flowers considered in the population.
n2
A. valentinum
A. controversum
Pollen size (pm3)
10
10
16528,12b
11614,45b
Pollen
grains/flower
10
10
203320.00±66652.86a
1096100.00±245176b
Style length
(mm)
10
73-123
5.50±0.04b
9.26±0.09b
Ovules/flower
10
10
192.40±29.77a
414.90±78.70b
Date of blooming
10-15
-
March-May8
March-Juneb
Flowering
duration (days)
10-15
90-100
7a
8b
Open pollination
11-20
122-635
42.43±11.90% fruit seta
83.76±2.87% fruit setb
70.61 ±4.13% seed set*
94.54±54.67% seed setb
Autonomous
9-20
120-671
0% fruit set8
0% fruit setb
9-20
90-181
0% fruit seta
0% fruit setb
10-20
93-178
75.00±11.54% fruit seta
85.34±23.22% fruit setb
77.73±7.73% seed seta
91.32±34.23% seed setb
self-pollination
Hand
self-pollination
Hand
cross-pollination
BChapter II; bE. Carrió and J. Güemes (unpubl. res.).
experimental garden plot. One fertile plant from each maternal family was chosen at
random to perform the following experimental crosses: 1) interspecific pollination. in
which emasculated flowers were hand pollinated with pollen from the other species;
87
2) intraspecific pollination. in which emasculated flowers were hand pollinated with
pollen from the same species. Pollinations were done by applying pollen to the
stigma until the surface was saturated on the 2-3 day after flower opening, since this
period is within the floral phase in which the stigma ¡s receptive (Chapter II; pers.
obs.). Prior pollination, flowers were emasculated before anther dehiscence and
bagged using nylon net bags with 0.5 x 0.5 mm opening to avoid access to flowers
by insect pollinators and normal development of the flowers. The bags were removed
when anthesis finished in order to minimizing the effects of the bags on fruit
formation. The number of treated flowers was: 142 (3-5 per plant) in A. valentinum
and 187 (5-7 per plant) in A. controversum, for interspecific pollination; and 140 (3-5
per plant) in A. valentinum and 170 (5-7 per plant) in A. controversum for intraspecific
pollination. For each plant, the effectiveness of each experimental cross was
evaluated in terms of fruit set (ratio of fruits to treated flowers), seed set (ratio of
seeds to the average number of ovules estimated as below, see the hybrid
reproductive capacity experiment) and seed mass. For each species, fruit set, seed
set and seed mass were compared between ínter- and intraspecific pollinations with
Mann-Whitney’s U-test using SPSS 17.0 (SPSS, Inc., Chicago, llinois, USA).
Seeds obtained in the inter- and intraspecific crosses in A. valentinum and A.
controversum were used to study the seed germination rate and speed. For each
pollination treatment, four replicates of 50 seeds were incubated in 0.6% agar in an
M-153 Sanyo chamber with a constant temperature of 20°C and 12 h photoperiod.
Preliminary germination tests in both species revealed an optimal germination for
these conditions (E. Estrelles, University of Valencia, Spain, unpubl. res.). The
number of germinated seeds (radíele protruded more than 2 mm from the seed coat)
was checked every day for 30 days and, for each experimental pollination type, we
calculated a ratio of seed germination and the Germination Speed Index (GSI) of
Maguire (1962). When this assay finished, ungerminated seeds were dissected to
check for the presence of the embryo and germinated seeds from interspecific
crosses were transferred into pots in the greenhouse and were cultivated for use in
the hybrid reproductive capacity experiment (see below). For each species, seed
germination rate and speed were compared between inter- and intraspecific
pollinations with the Student’s f-test using SPSS 17.0.
88
A. valentinum and A. controversum were analyzed for pollen adherence and
germination in the stigma and pollen tube growth into the style. To do this, bagged
flowers of both species were intra- and interspecifically pollinated as above and were
collected 12 h later (to examine pollen adherence and germination) and 24, 48, 72
and 96 h later (to analyze pollen tube growth). Pollinated pistils were fixed in glacial
acetic acid: 95% ethanol ( 3 : 1 ) for 24 h, rinsed in de-ionized water, and softened in
1M KOH at 60° C for 15 min. Once softened, they were stained in 0.01% decolorized
aniline blue overnight (modified from Martin, 1959), mounted in a drop of stain,
squashed with a cover slip and observed under UV light on a BX 40 Olympus
microscope with epifluorescence (U-RFL-T). The pollen tubes on the stigma and in
the style can be detected by the callóse fluorochrome reaction (Martin, 1959). For
pollen adherence, we quantified the number of pollen grains adhered to the stigma,
using normal transmission microscopy (50-70 flowers per cross type for each
species). For pollen germination, we counted the number of germinated and
ungerminated pollen grains in the stigma and, then, we obtained the percentage of
pollen germination (50-70 flowers per cross type for each species). For pollen tube
growth, we estimated the ratio of the length of the main wave of pollen tubes (c. 80%)
and the style length at each observational time period, using a calibrated ocular
micrometer (20-25 flowers per cross type and observational time period for each
species). Pollen adherence and germination in the stigma, and pollen tube growth
were compared between the interspecific and intraspecific pollinations for each
species using the Mann-Whitney’s L/-test with SPSS 17.0.
The hybrid reproductive capacity was estimated only on hybrids from interspecific A.
controversum crosses (A. controversum [$] x A. valentinum [c?]) because no hybrid
plants were obtained when A. valentinum acted as the female parent. To examine
the relative fertility of hybrids, the number of pollen grains and ovules per flower, and
the pollen viability was estimated in 45 hybrid plants and in 15 cultivated parental
plants of each puré taxa. One flower per plant was selected to obtain the number of
pollen grains and ovules per flower. Pollen was removed from indehiscent anthers
(two per flower) that were placed in one mi of detergent water and opened with a
89
lancst. Ten subsamples were scored on slides and pollen grains were counted under
a BX 40 Olympus optical microscope (400x). The ovules were counted after the
dissection of the ovary under a Wild stereomicroscope (120x). To investígate pollen
vlablity, one flower bud from each plant was bagged prior anther dehiscence in order
to pievent posterior pollen contaminaron in the anthers. Pollen was collected on the
thirdday after flower opening and incubated (25°C, 4 h) in GK médium (Wilson et al.,
1997). Pollen grains were observed under a stereomicroscope (250x) and
germinated pollen grains were distinguished by a pollen tube of at least double the
lengt» of the grain diameter. The rate of viable pollen was determined in 500 grains
per sample. The amount of pollen and ovules and pollen viability was compare
among the three plant types (A. valentinum, A. controversum, A. controversum [$] x
A. valentinum
[$]) using one-way ANOVA and the Tukey test for subsequent
comparisons with SPSS 17.0.
In addition, in order to estímate the level to which hybrids are reproductively
isolaed and are able to cross with other hybrids and with the parental species, we
made seven experimental crosses using flowers (3 to 10 per plant) from 30 hybrid
plants and 30 puré plants: (1) hybrid (S) x A. valentinum (9), in which pollen from
hybrid plants was used to pollinate emasculated A. valentinum flowers; (2) hybrid (c?)
x A. controversum ($), in which pollen from hybrid plants was used to pollinate
emasculated A. controversum flowers; (3) hybrid (?) x A. valentinum (S), in which
pollen from A. valentinum was used to pollinate emasculated flowers of hybrid plants;
(4) tybrid (?) x A. controversum ($), in which pollen from A. controversum was used
to pdlinate emasculated flowers of hybrid plants; (5) autonomous self-pollination, in
which flowers of hybrid plants were bagged and left to spontaneous pollination; (6)
hand self-pollination, in which flowers of hybrid plants were pollinated with their own
pollen; (7) hand cross-pollination, in which emasculated flowers of hybrid plants were
pollinated with pollen from other hybrid plants. Pollinations were done on previously
bagged flowers following the method used in the experimental crosses (see above).
For each plant, the fruit set, seed set, seed mass, seed germination rate and speed
was quantified as previously described and results were compared among
experimental crosses with the Kruskal-Wallis test using SPSS 17.0. Subsequent
90
Mann-Whitney pairwise comparisons between treatments were also made with
Bonferroni significance correction (Sokal and Rohlf, 1995).
3.3. RESULTS
Different results were found in the experimental crosses assay depending on the
species pollinated (Table 3.2). For A valentinum, the fruit set and the seed set did
not vary significantly with respect to the pollen source applied to the flowers (fruit set:
N = 3 0 , U = 88.50, P = 0.768; seed set: N = 43, U = 225.00, P = 0.884) but seed
mass was statistically higher in intraspecific crosses than in interspecific crosses (N 59, U = 117.50, P = 0.000). By contrast, for A. controversum, fruit set and seed set
was significantly lower in interspecific crosses than in intraspecific crosses (fruit set:
N = 4 4 , U= 127.00, P = 0.010; seed set: N = 24, U = 24.00, P = 0.003) but the type
of pollination did not affect seed mass {N = 19, U = 19.00, P = 0.781).
Table 3.2. Fruit set (%), seed set (%), seed mass (mg), seed germination rate (%) and seed
germination speed (GSI) of A. valentinum and A. controversum in interspecific and intraspecific
pollinations (mean ± standard deviation).
Fruit set
Seed set
Seed mass
Germination
rate
Interspecific
pollination
69.25±8.84
77.10±10.23
0.030±0.005
0
Intraspecific
pollination
74.78±9.14
77.85±7.66
0.059±0.003
29.0±5.1
2.66±0.21
Interspecific
pollination
38.57±8.46
21.02±7.54
0.074±0.014
34.0±4.1
10.52±1.64
Intraspecific
pollination
83.08±2.37
69.60±14.59
0.082±0.004
48.0±6.1
9.33±0.69
Experimental
cross
GSI
A. valentinum
A. controversum
91
Both species had low rate of seed germination in the two experimental cross
types (Table 3.2). The germination rate of the intraspecific A. valentinum seeds was
29%, while seeds from interspecific crosses in this species did not germinated and
the posterior dissection of these seeds showed none of them had either an embryo
or an endosperm inside. Seed germination rate in A. controversum varied between
34% in seeds from interspecific crosses to 48% in those from intraspecific
pollinations, and no statistically significant difference were found in the germination
rate between both seed types (t = 1.131, P = 0.301). All ungerminated seeds from
inter- and intraspecific A. controversum crosses and from intraspecific A. valentinum
crosses were found to contain a normal embryo. On the other side, speed of seed
germination varied depending on the maternal species (Table 3.2). Seeds from
intraspecific A. valentinum pollinations had a low germination speed compared to A.
controversum seeds obtained in the two types of crosses, for which there were no
significant differences (f = -0.523, P = 0.620).
Pollen adherence and germination results are shown in Fig. 3.2. For A. valentinum,
the number of pollen grains adhering in the stigma was similar between pollination
treatments (N = 95, U = 768, P = 0.470) while pollen germination was significantly
higher in intraspecific crosses than in interspecific crosses (N = 79, U = 388.5, P =
0.004). For A. controversum, no significant variation between cross types was found
in pollen grain adherence {N = 103, U = 733, P = 0.287) and germination in the
stigma {N = 107, U = 877.5, P = 0.266).
92
A Miltn firu m
A oortrow rsum
S pecies
A *»*ntinum
A centro w rstm
Species
Figure 3.2. Pollen adherence (a) and pollen germination in the stigma (b) for A. valentinum and
A. controversum in the interspecific (grey bars) and intraspecific pollinations (black bars). Bars
are means and vertical lines above bars are standard errors. Significant differences between
crosses within species for Mann-Whitney’s LMest: **P < 0.01.
A n alysis o f pollen tube grow th revealed d ifferent results depen ding on the
spe cie s (Fig. 3.3). For each observation tim e in A. valentinum , no d iffe re n ce s
betw een p o llina tio n tre a tm e n ts w ere found in the proportion o f the style reached by
the pollen tubes. F o r A. co ntroversum , pollen tu b e grow th w as higher in in te rsp e cific
than in in tra sp e cific cro sse s at 24 and 48 h a fte r pollination. H ow ever, th e tu b e s of
both pollen typ es reached a sim ilar proportion o f the style (ca. 85% ) 72 h after
pollination.
In th e
next 24 h, the m ain w ave o f in terspecific pollen tu b e w as
su spend ed in the sam e strength o f the style w hile in tra sp e cific pollen tu b e s w ere
observed at the b a se o f the style.
24
48
72
96
Hours after pollination
24
48
72
93
96
Hours after pollination
Figure 3.3. Pollen tube growth for A. valentinum (a) and A. controversum (b) in interspecific
(grey bars) and intraspecific pollinations (black bars) at 24, 48, 72 and 96 h after pollination.
Bars are means and vertical lines above bars are standard errors. Significant differences
between crosses within species at each time of observation for Mann-Whitney’s O-test: **P <
0.01; *P < 0.05.
3.3.3. H y b rid re p ro d u ctive c a p a c ity
The n u m b e r o f pollen g ra in s per flow e r w as d iffe re n t am ong A. valentinum , A.
co n tro v e rs u m and hybrid pla nts (F = 12.623, P = 0 .000) (Table 3.3). A. valentinum
had less pollen grains than A. co n tro versum and the hybrids, fo r w hich the quantity of
pollen g ra in s w a s sim ila r (P = 0.977). The nu m b e r o f ovules w as also d iffe re n t
am ong the th re e typ es o f plants (F = 26.891, P = 0.000), being higher in A.
co n tro ve rsu m than in A. va len tinu m and the hybrids, w hich produced a sim ilar
q u a ntity o f o vule s (P = 0.703). All ovules seem ed w ell form ed, indicating potential
fertility. In all the cases, pollen via bility w as high, ranging from 84.45% to 94.04% .
H ow ever, th is p a ra m e te r varied sign ifican tly am ong the three plant types (F = 12.905,
P = 0.0 00 ) sin ce the hybrid plants w ere found to produce less viable pollen grains
than A. va le n tin u m and A. controversum .
94
Table 3.4. Reproductive capacity of hybrid plants (A
controversum [$] x A. valentinum [á1]) based on fruit set (%), seed set (%), seed mass
(mg), seed germination rate (%) and seed germination speed (GSI) obtained in the experimental crosses (mean ± standard deviation). Valúes
with identical letter do not differ statistically at the 0.05 level (Mann-Whitney’s U-test with Bonferroni significance correction).
Experimental cross
Fruit set
Seed set
Seed mass
Germination
GSI
rate
Hybrid (<$) x A.
valentinum ($)
37.5±0.3a
63.4±9.1ab
0.034±0.003ab
63.3±13.7a
5.99±1.17a
Hybrid {¿) x A.
controversum (9)
86.0±0.4C
69.0±10.8ab
0.040±0.002b
45.0±7.7a
4.20±1.06a
Hybrid
(9) x A. valentinum (á1)
56.2±0.6b
83.0±12.0b
0.024±0.002a
48.3±11.3a
4.23±1.21a
Hybrid
(9) x A. controversum (<$)
81.9±2.1c
74.0±8.0ab
0.039±0.002b
58.0±12.0a
5.41 ±1,9a
0
-
-
-
-
2.3
-
-
-
-
33.3±21.7a
41.0±5.0a
0.031 ±0.001ab
52.25±13.5a
5.74±0.62a
Autonomous self-pollination
Hand self-pollination
Table 3.3. Pollen grains per flower, pollen viability (%) and ovules per flower for A.
95
valentinum,
A. coniroversum and hybrids {A. controversum [$] x A. valentinum [$]). Data are mean ±
standard deviation. Valúes with ¡dentical letter do not differ statistically at the 0.05 level (Tukey’s
test).
Plant type
Pollen grains/flower
Pollen viability
Ovules/flower
A. valentinum
291820.00±76650.00a
96.74±0.46a
219.80±16.00a
A. controversum
838 868.97±68 966.92b
94.04±0.83a
346.04±24.41b
808 480.00±174 486.10b
84.45±2.82b
211.60±6.69a
Hybrid
Different results were found depending on the type of pollination assayed (Table
3.4). Hybrids did not yield fruits by autonomous self-pollination and only formed a few
fruits by hand self-pollination. When hybrids were crossed with pollen from other
hybrids or from the parental species, the fruit set obtained was between 33% and
81%, whereas seed set was between 41% and 83%. Crosses in A. valentinum and
A. controversum using pollen from hybrid plants also resulted in fruits and seeds
(37% - 86% in fruit set; 63% - 69% in seed set). However, significant amongpollination variation was found in fruit and seed production (fruit set: N = 95, F =
82.168, P = 0.000; seed set: N = 82, F = 10.746, P = 0.030). The type of cross was
also observed to significantly affect seed mass (N = 76, F = 14.153, P = 0.007), but
no differences were detected in seed germination rate (N = 20, F = 1.009, P = 0.407)
and speed (N = 20, F = 3.594, P = 0.849).
3.4. DISCUSSION
3.4.1. Prezygotic and postzygotic reproductive barriers
Our results reveal an asymmetrical prezygotic reproductive barrier between A.
valentinum and A. controversum, when the last species acted as the female parent.
Although both species yield fruits and seeds in interspecific pollinations, the fruit and
seed set in A. valentinum was significantly higher than in A. controversum.
Differences in reciprocal crosses between Antirrhinum species were also reported by
Harrison and Darby (1955), Hudson et al. (2008), and Thompson (1988), who
96
documented a máximum difference in fruit set of 0.21 in the cross between A.
controversum and A. hispanicum. In the present case, the lower fruit and seed set in
the interspecific A.controversum pollinations is the result of a prezygotic barrier.
Pollen grains of A. valentinum had high adherence and germination in the A.
controversum stigma, although the adherence was lower in comparison to those with
conspecific A. controversum pollen. However, a great proportion of the A. valentinum
pollen grains were unable to reach the last portion of the style in A. controversum
(Fig. 3.3), and therefore, many ovules could not be fecundated. This blocking of
pollen tube growth may be due to energy deficiency. In many studies within
taxonomic groups, species with the shorter styles have been observed to have
smaller pollen size (or volume) (e.g. Williams and Rouse, 1990; Roulston et al., 2000;
Aguilar et al., 2002) and this has been associated with smaller pollen grains storing
less resources and developing shorter pollen tubes (Williams and Rouse 1990;
Torres, 2000). A. valentinum has both shorter style (40% lower) and smaller pollen
volume (30% lower) than A. controversum (Table 3.1). The majority (80%) of A.
valentinum pollen grains used up their energy resources 72 h after being deposited
on the A. controversum stigma, and this period might not be enough to develop
pollen tubes that reached the ovules. Nevertheless, this is not a total reproductive
barrier and, therefore, A. controversum yield modérate levels of fruit and seeds in the
interspecific crosses.
We also found a strong asymmetrical postzygotic barrier between A. valentinum
and A. controversum, which limited hybridization when the former species was the
female parent. All interspecific crosses in A. valentinum gave rise to unviable seeds.
In contrast, hybrid seeds from interspecific A. controversum crosses were highly
viable with similar mass, germination rate and speed compared to the intraspecific A.
controversum offspring. Asymmetric postzygotic barriers have been less frequently
reported in angiosperms, and they result from nuclear-cytoplasmic or triploid
endosperm interactions, among others reasons (Tiffin et al., 2001; Turelli and Moyle,
2007). In A. valentinum, the hybrid seed unviability detected here was related with
the fact that all these seeds lacked the embryo and the endosperm, as we could
observe by seed dissection after the germination test. Anatomical studies of the
seeds of several species of Scrophulariaceae have shown that mature seeds of
97
Antirrhinum species are usually formed by an embryo and an endosperm of
approximately the same volume (Juan et a!., 2000). The failure of hybrid seeds is
known to be a common early interspecific barrier among many plant species (Turelli
and Moyle, 2007) and has been reported in Arabidopsis (Brassicaceae) (Bushell et
al., 2003; Josefsson et al., 2006), Chamaecrísta (Leguminosae) (Costa et al., 2007),
Lens (Leguminosae) (Abbo and Ladizinsky, 1994), or Solanum (Solanaceae) (Lester
and Kang, 1998). This is often due to the abnormal development or the abortion of
hybrid endosperm or, less commonly, of the hybrid embryo (Bushell et al., 2003;
Gutierrez-Marcos et al., 2003). The growth of the embryo and the endosperm during
early seed development are interdependent processes (Ungru et al., 2008), that is,
the failure in one of these processes may have an effect on the other, and therefore it
may be impossible to distinguish between the primarily cause of hybrid failure by
simple observation of the seeds. Thus, further studies are needed to detect the main
cause of hybrid seed unviability in A. valentinum.
Hybrids may frequently exhibit a low reproductive capacity due to sterility or inability
to produce embryos (Rieseberg and Carney, 1998; Cruzan and Arnold, 1999). Within
Antirrhinum, fertility in artificial hybrids has been documented by several authors
(Baur, 1932; Hackbarth etal., 1942; Rothmaler, 1956; Rieger, 1957; Stubbe, 1966).
Moreover, Rothmaler (1956) reported fully viable seeds in experimental hybrids and
Mateu-Andrés and Boscaiu (2003) indicated a high percentage of germination (78%)
in seeds produced by the natural hybrid A. x albanchezii. These data are congruent
with the reproductive capacity results found in hybrids in this study. Hybrids survived
until flowering in the greenhouse (c. 85% of the transplanted seedlings) and
produced a quantity of pollen grains similar to the female parent and ovule production
approximately equal to the male parent. They also had high pollen viability, although
this was lower than in the progenitors, and developed well-formed ovules. These
hybrid plants, although self-sterile, could be successfully crossed with other hybrids
plants (33% in fruit set; 41% in seed set) and with both parental species, reciprocally
(37% to 86% in fruit set; 63% to 83% in seed set). Moreover, seeds obtained in these
crosses had, almost in all the cases, a higher germination rate than puré seeds from
parental species.
98
3.4.3. Potential hybridization and introgression in the environment
A. valentinum and A. controversum had substantial reproductive barriere against
hybridization, but a modérate production of viable hybrids was possible when A.
controversum acted as the female parent (c. 40% in fruit set and c. 20% in seed set
in interspecific crosses). Our data represent the potential máximum hybridization rate
between these two species under experimental conditions. In nature, it is frequent to
find ecológica!, temporal or behavioural barriere that also contribute to restrict
hybridization among species: large separation distance between plants (depending
on pollinator range), asynchronous flowering periods, different pollinatore or failure in
hybrid establishment (Kay, 2006; Marques et al., 2007; Martin and Willis, 2007; Yang
et al., 2007). In A. valentinum and A. controversum, these kind of barriere have not
been studied in depth, but evidence suggests they are not strong. In the studied
population, both species are in physical proximity (range 5-200 m), have a long
overlapping flowering period (three months; Table 3.1), although with different peak
bloom, and they also share common visitors, since our preliminary observations in
the natural population revealed both species were visited by bees all day long.
Moreover, given the short dispersal ability of Antirrhinum seeds (Sutton, 1988), the
hybrid seeds produced by A. controversum would be dispersed within the maternal
environment. The behavioural pattern of these seeds, in terms of germination speed,
was similar to A. controversum puré seeds, suggesting both types of seeds having
equal opportunities to establish themselves. On taking these data together with our
results, we expected to find that some hybridization between A. valentinum and A.
controversum currently occurs in nature. However, this prediction does not conform
to our observations of hybrids in fíeld conditions and we have not detected hybrid
plants in any of the three natural mixed populations known.
One possible explanation for the lack of hybrids in nature could be that contact
between A. valentinum and A. controversum is so recent that more time is needed for
hybridization to occur.
Almost 80 years ago, Baur (1932) reported A. valentinum
populations being completely isolated with plants of this species never co-occurring
with other congénere. Other early floristic studies of the territory of A. valentinum did
not detect mixed populations either (Borja, 1948; Rivas Goday, 1956). Our revisión of
the Antirrhinum material conserved in the main ibero-levantine herbaria (MA and
99
VAL) reflects that A. valentinum and A. controversum did not co-occur until the
1980s. More recently, Mateo and Figuerola (1985) cited the presence of A.
controversum 5 km from A. valentinum populations. Three herbarium specimens
(VAL 61926, VAL 111272 and VAL 111274) are testimony of the presence of A.
controversum in territories before A. valentinum. During our preliminary field studies
on A. valentinum, we have observed that recent human-related habitat disturbance
(road construction and clearing vegetation to prevent fires, extensión of citri-culture
and archaeological works) has lead to the formation of open areas in scrubland
surrounded the rocky outcrops where A. valentinum inhabits and, currently,
individuáis of A. controversum colonize these grounds.
Even if F1 hybrids are rare in nature, introgression through backcrossing can
occur (Arnold, 1997; Rieseberg, 1997; Arnold et al., 1999; Broyles, 2002). In this
study, the high reproductive capacity in the hybrids raises the possibility for
introgression between A. valentinum and A. controversum, but this might be severely
limited if the hybrids experience limited opportunities for reproducing. Estimating the
consequences of this mechanism would require further research to be developed
under field conditions and this falls outside the scope of this study. However, there
are several factors that might increase the chances of hybrid plants reproducing.
First, in the garden conditions of our study, hybrid flowering occurred simultaneously
in both parental species and we hypothesize this is also likely in nature. Although
variation in environmental factors can have a dramatic effect of flowering initiation in
Antirrhinum (Adams et al., 2003), our garden conditions seem similar to those of the
natural population. This is supported by the fact that A. valentinum and A.
controversum flowered at the same time in both the garden and field conditions
during this study. Second, hybrid plants are likely to attract pollinators, since they
produce numerous flowers per inflorescence (10.77, mean; 1-37, range; which is less
than A. controversum, but more than A. valentinum) with néctar (pers. obs.) and a
high quantity of pollen grains (less than A. controversum, but more than A.
valentinum). In fact, we have detected pollinators making movements among flowers
of hybrid plants and between flowers of hybrids and A. controversum in our
observations made in the Botanical Garden, where the fruit set found in open
pollinations of hybrid plants was 83±12%.
100
A. valentinum has high genetic allozyme differentiation among populations (MateuAndrés and Segarra-Moragues, 2000), therefore other A. valentinum populations with
different genetic composition could
have different degrees of reproductive
compatibility with A. controversum. This has been seen between populations of
Raphanus sativus and R. raphanistrum, which showed a variation in potential for
hybridization across different geographic regions (FitzJohn et al., 2007). Accordingly,
we recommend the evaluation of the potential for hybridization and introgression
between A. valentinum and A. controversum in other mixed populations. Otherwise,
A. valentinum is considered an endangered species (Moreno, 2008), threatened
mainly by the habitat loss or degradation and by recent reductions in population size
(Carrió et al., 2006), and hybridization and introgression with A. controversum should
be considered as an additional potential threat to A. valentinum persistence. Contact
between the two species seems recent but hybridization and introgression may pose
a future problem, especially in the event that the two species continué or increase
contact with each other.
Small plant populations are considered particularly
vulnerable by interspecific gene flow because it may create disruptions in local
adaptations that prove detrimental to the population (Soltis and Gitzendanner, 1999).
To prevent possible consequences of hybridization or introgression in A. valentinum
we suggest the habitat occupied by A. valentinum should be legally protected
(current protection
¡ncludes
only two
populations).
Furthermore,
vegetation
restoration should be carried out in human-related disturbed habitats of this species,
together with elimination procedures of A. controversum inhabiting these territories.
These measures should not be taken in the naturally disturbed areas surrounding A.
valentinum populations, even though potential for hybridization and introgression
exist. Speciation through hybridization has played an important role in plant
biodiversity (Ellstrand and Elam, 1993; Ellstrand et al., 1996) and our conservation
actions might prevent the occurrence of this evolutionary process.
Chapter IV:
Evaluating species non-monophyly as a trait
affecting genetic diversity: the case of A n t i r r h i n u m
c h o ' i d e mi , A. s u b b a e t i c u m and A . v a l e n t i n u m
NON-MONOPHYLY AND GENETIC DIVERSITY
103
4.1. INTRODUCTION
In what are now considered classic articles, Hamrick and co-workers (Hamrick et al.,
1979, 1992; Loveless and Hamrick, 1984; Hamrick and Godt, 1989, 1996)
summarized available information on genetic diversity within and among plant
populations to establish which life history traits had a profound and significant effect
on the level and distribution of diversity observed. Significant effects were described
in eight out of 12 traits examined (Hamrick et al., 1979) and subsequent papers have
lead to similar conclusions based upon a wide range of currently available molecular
techniques (Nybom, 2004). However, Hamrick et al. (1992) and Hamrick and Godt
(1996) also pointed out that genetic diversity maintained by a species is a function
not only of its life history traits (which explains, for example, about 34% of variation
detected in woody species [Hamrick et al., 1992]) but must also depend heavily on
the species ecological and evolutionary history.
In these reviews, the unit of study is almost invariably the taxonomic species
(sensu Mayden, 1997). In many countries, the taxonomic species is currently the
basÍG unit of assessment for conservation, and is the subject of the majority of
conservation legislation and conservation management planning protocols (Isaac et
al., 2004; Mace, 2004; Garnett and Christidis, 2007). However, studies of genetic
diversity may be of limited interpretative valué if the study species is poorly known
and an implicit assumption is made that the species populations form a natural
(monophyletic) group, which means they have a single origin being more closely
related to each other than to any population of a different species (Doyle, 1992; Funk
and Omland, 2003). If the study species is an artificial population assemblage, then
the level and distribution of genetic diversity may not reflect population genetic
processes within an assumed natural group (Arnold, 1997; Rieseberg, 1997). If no
knowledge of species monophyly is available, then interpretation of the pattems of
diversity observed will be incomplete: population genetic structure may primarily
reflect the causes of species non-monophyly rather than population diversity, life
traits and ecological variables (Hamrick et al., 1979). This could seriously impede
interpretation of the levels of genetic diversity within taxonomic species.
104
Species evolutionary history can be inferred with increasingly sophisticated
molecular phylogenetic analyses, using complementary loci from up to three different
plant genomes (Savolainen and Chase, 2003; Hughes et al., 2006). There are
several
reasons
why
non-monophyletic
groups
are
recovered
in
such
reconstructions. These can be of a technical and/or analytical nature (e.g. inadequate
phylogenetic signal in the markers used, analysis of unrecognised paralogous genes,
¡mperfect taxonomy, widespread hybridization processes in angiosperms or the
detection of previously undetected cryptic taxa) (Goodwillie and Stiller, 2001;
Treutlein et al., 2003; Álvarez et al., 2005). Having detected such sources of error,
suitable phylogenetic analyses are essential to observe the signal of evolutionary
events that have impacted on lineage diversification and therefore species
monophyly. For instance, it has been documented that hybridization with Bertya
rosmarínifolia increased levels of genetic diversity in one of the three populations
known of the endangered B. ingramii (Fatemi and Gross, 2009).
The genus Antirrhinum L. consists of 25 perennial species primarily distributed
throughout the western Mediterranean basin, with 23 species occurring in the Iberian
Península (Sutton, 1988; Güemes, 2009). Phylogenetic reconstructions have
generally ¡ncluded single accessions of each taxon, and have revealed little
¡nfrageneric structure (Vargas et al., 2004; Jiménez et al., 2005) with some authors
proposing that speciation in Antirrhinum is recent, geographically structured, and
reticulate (Webb, 1971; Jiménez et al., 2005; Chapter I). Antirrhinum species are
perennial herbs or small shrubs having identical chromosome number (2n = 16), with
most species considered self-incompatible and entomophylous (Rothmaler, 1956;
Sutton, 1988). Many species are either geographically restricted or narrow endemics,
with a total of eight species included in the Red List of Spanish Vascular Flora
(Moreno, 2008). Genetic diversity within Antirrhinum has been extensively evaluated
(Table 4.1), allowing comparative assessment of genetic diversity between species
with similar life history traits.
In this article, we used nuclear (ITS) and plastid (psbA-trnH, trnT-trnl) gene
sequences from múltiple individuáis of 24 species of Antirrhinum (single accession of
A. cirrhigerum) to investígate whether population genetic structure is related to the
level of monophyly detected in three endemic snapdragon species from south east
105
Iberia (A charídemi, A. subbaeticum, A. valentinum). Specifically, we tested whether
populations of the three species form monophyletic groups using nuclear ITS and
plastid sequences, and whether monophyly gives insights into evolutionary histories
not apparent from genetic diversity estimates alone. The three species selected to
test the hypothesis of non-monophyly affecting levels of genetic variation have similar
life history traits but show remarkably different levels of allozyme diversity (Table 4.1,
4.2). They share six out of the eight life history traits shown to have a significant
effect on genetic diversity (Hamrick et al., 1979; Table 4.2). These species differ in
their mating system: A. charídemi and A. valentinum are mainly outcrossers due to
self-incompatibility, while A. subbaeticum is a self-compatible species with a mixed
mating system (Chapter II). Further, A. subbaeticum is not considered a ‘narrow’
endemic as it is represented by two groups of populations separated by ca. 44 km
with an extent of occurrence of ca. 200 km2, although its area of occupancy is less
than 15 km2, whereas A. charídemi and A. valentinum are restricted to areas of 10-15
km and 14 km in length, respectively. Differences in allozyme genetic diversity
among these taxa have been reported (Table 4.1): A. charídemi had a high degree of
intraspecific genetic diversity and little differentiation among populations; A.
subbaeticum exhibited low levels of intraspecific genetic diversity and high
differentiation among populations; and A. valentinum had a high level of intraspecific
genetic diversity and high population divergence (Mateu-Andrés and SegarraMoragues, 2000; Mateu-Andrés, 2004). A. charídemi and A. subbaeticum are listed
as endangered and A. valentinum as vulnerable in the Red List of Spanish Vascular
Flora (Moreno, 2008). A clear taxonomic delimitation in the three species has been
recognised in all revisions of the Antirrhinum species (Rothmaler, 1956; Sutton,
1988; Güemes, 2009).
4.2.1.Sampling
A total of 88 accessions representing 24 Antirrhinum species were sampled for
phylogenetic reconstructions (Appendix 4.1). A rare species, A. martenii (Font Quer)
Rothm., was not ¡ncluded in the study, because only the type material is available
106
Table 4.1. Reproductive system and details of population genetic parameters estimates for 16 species of Antirrhinum. Rep = reproductive
system: SI = self-incompatible, SC = self-compatible, pSC = partially self-compatible; Marker = genetic markers used in original study; N pops =
number of populations studied in original study; HT = total diversity at the species level; GSr = alíele frequency differences among populations
averaged across populations; D-HW = number of loci that deviate from Hardy-Weinberg equilibrium in at least one population, compared to the
number of loci interpreted.
Taxon
Rep
Reference
Marker
Ht
G st
D/HW
Reference
Allozyme
N
pops
5
A. charídemi
SI
Chapter II
0.103
0.054
4/21
Allozyme
3
0.190
0.132
2/13
Mateu-Andrés and SegarraMoragues (2000)
Mateu-Andrés and de Paco (2006)
A. cirrhigerum
pSC
A. graniticum1
SI
Vieira and Charlesworth
(2001)
(2001)
Allozyme
3
0.130
0.110
0/14
A. graniticum2
Allozyme
3
0.090
0.080
0/14
A. graniticum3
Allozyme
10
0.190
0.210
3/14
Allozyme
8
0.140
0.093
2/13
Mateu-Andrés and
Moragues (2003b)
Mateu-Andrés and
Moragues (2003b)
Mateu-Andrés and
Moragues (2003b)
Mateu-Andrés and
Allozyme
6
0.160
0.176
2/13
Allozyme
5
0.280
0.148
2/13
A. latifolium
SI
A. linkianum
pSC
A. litigiosum
SI
Mateu-Andrés and de Paco
(2006)
(2001)
(2006)
SegarraSegarraSegarrade Paco (2006)
107
i a u a e H, i , \ L , u n t . } .
Taxon
Rep
Reference
Marker
A. lopesianum
A. majus
SI
SI
Allozyme
Allozyme
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
SI
(2006)
Torres et al. (2002)
N
pops
1
4
Allozyme
Allozyme
RAPD
Allozyme
Allozyme
Allozyme
Allozyme
Allozyme
RAPD
Allozyme
4
4
7
4
5
8
3
4
15
Allozyme
5
microphyllum
microphyllum
microphyllum
mollissimum
pertegasii
pulverulentum
siculum
subbaeticum
subbaeticum
tortuosum4
A. valentinum
SI
SI
se
se
SI
SI
Carrió et al. (unpublished)
Harrison and Darby (1955)
Chapter II
(2006)
Chapter II
Ht
Gst
D/HW
Reference
0.193
0.190
na
0.100
na/14
1/13
0.520
0.469
0.188
0.280
0.080
0.300
0.030
0.070
na/14
2/13
na/14
2/14
7/14
2/13
0/14
0.200
0.040
0.056
0.076
0.110
0.060
0.230
0.270
0.850
0.822
0.081
Jiménez et al. (2002)
0.178
0.480
0/21
-
-
7/13
Mateu-Andrés and SegarraMoragues (2000)
1' 2' 3Treated as A. graniticum Rothm. ssp. ambiguum (Lange) Mateu & Segarra, A. graniticum Rothm. ssp. brachycalyx D.A. Sutton and A.
graniticum Rothm. ssp. graniticum in Mateu-Andrés & Segarra-Moragues (2003b), respectively; 4Genetic diversity data of A. tortuosum induded
A. australe populations, according to Mateu-Andrés and de Paco (2006), therefore, estimates for the former species may be artificially elevated
and may account for the high number of loci deviating from Hardy-Weinberg equilibrium.
108
(Rothmaler, 1956). Sampling strategy was based on morphological studies (Güemes,
2009) and a criterion of collecting at the locus classicus of each species (see also
Chapter I). The number of individuáis sampled of each species range from two to
eight (single accession of A. cirrhigerum), being from geographically sepárate
populations (Appendix 4.1). Two individuáis of the related genera Misopates and
Pseudomisopates (M. orontium and P. rivas-martinezii) were used as outgroup
samples, according to a previous phylogenetic study (Vargas et al., 2004).
4.2.2. DNA extraction, gene amplifícation and sequencing
DNA was extracted from 20-25 mg of leaf material using the DNeasy Plant Mini Kit
(Qiagen) following the manufacturéis protocol. DNA was amplified vía polymerase
chain reaction (PCR) on a Perkin Elmer PCR System 9700 thermal cycler. The psbAtrnH intergenic spacer was amplified using the primers of Hamilton (1999) and the
tmT-tmL intergenic spacer using primers a and b of Taberlet et al. (1991). Reactions
included 1 p\ of dimethyl sulfoxide (DMSO) at 99.9% in each 25 ¡A reaction. The
thermocycling profile consisted of initial denaturation at 94°C for 5 mins, followed by
30 cycles of denaturation at 94°C for 1 min, annealing for 2 mins (50°C for tmT-tmL,
58°C for psbA-tmH) and extensión at 72°C for 2 mins, followed by a final extensión at
72°C for 10 mins and storage at 4°C. Amplified products were purified using spin filter
columns (MoBio Laboratories) following the manufacturéis protocol, and directly
sequenced with dye terminators (Big Dye Terminator v. 2.0, Applied Biosystems)
using an Applied Biosystems Prism Model 3700 automated sequencer. GenBank
accession numbers are given in Appendix 4.1.
4.2.3. Data analysis
Two matrices were constructed to perform phylogenetic analyses: one with 90
sequences (88 of Antirrhinum, two of the outgroup) of each plastid región (psbA-trnH,
tmT-tmL)] and the other one with 71 sequences (69 of Antirrhinum, two of the
outgroup) of the nuclear ribosomal ITS región (Appendix 4.1). All ITS sequences
were obtained from two previous phylogenetic analyses (Vargas et al., 2004; Chapter
I). Sequences were corrected and aligned manually using BioEdit Sequence
Alignment Editor 7.0.9 (Hall, 1999). Alignments were adjusted manually to minimize
the number of gaps following the logic of Kelchner (2000).
NON-MONOPHYLY AND GENETIC DIVERSITY"
109
Table 4.2. Details of twelve life history traits (Hamrick et al., 1979) for the three studied species
(Antirrhinum charídemi, A. subbaeticum, A. valentinum).
A. charídemi
A. subbaeticum
A. valentinum
1
Taxonomic status
Dicots8
Dicots8
Dicots8
2
Geographic range
Narrow8
Regional8
Narrow*
3
Generation length
Perennial8
Perennial8
Perennial8
4
Mode of reproduction
Sexual1
Sexual5
Sexual1
5
Mating system
Outcrosser8
Mixed mater8
Outcrosser8
6
Pollination mechanism
Entomophylous8
Entomophylous8
Entomophylous8
102-103
seeds/plant7
Boleoanemocory1
102-103
seeds/plant7
Boleoanemocory5
102-103
seeds/plant7
Boleoanemocory1
2n=162
2n=164
2n=163
Late1
Late5
Late1
Mesic1
Mesic5
Mesic1
Non cultivated1
Non cultivated5
Non cultivated1
7
Fecundity
8
Seed dispersal
mechanism
9
Chromosome number
10
Successional stage
11
Habitat type
12
Cultivated status
1 Sutton (1988);2 Diosdado et al. (1994);3 Boscaiu et al. (1997);4 Coy et al. (1997);5 SánchezGómez et al. (2004); 8Carrió et al. (Chapter II); 7 Carrió et al. (unpublished);8 Güemes (2009);
The eight life history traits identified by Hamrick et al. (1979) as significantly affecting the level
and distribution of genetic variation, and those that differ among the studied species, are shown
in bold.
Phylogenetic analyses were performed using Máximum Parsimony (MP) as
¡mplemented in PAUP* (Swofford, 2002) and
Bayesian
Inference (Bl) as
implemented in MrBayes ver. 3.1.2 (Ronquist and Huelsenbeck, 2003). MrModeltest
ver. 2.1 was used to determine appropriate models of sequence evolution for each
dataset (Posada and Crandall, 1998; Nylander, 2004) via bottom-up strategy of
hierarchical likelihood ratio test and Akaike Information Criterion (AIC; Akaike, 1979).
When different evolutionary models were obtained by different criteria, each dataset
110
was analyzed under both models. The analyses based on different models displayed
the same topologies differing only in support valúes, thus the tree topologies and
clade supports presented were those obtained from applying the evolutionary model
selected by AIC. Pairwise sequence divergence for both psbA-tmH and trnT-trnL
were calculated under the common model retrieved by Mr. Modeltest (GTR), uslng
the program PAUP* (Swofford, 2002).
For MP analysis, an heurlstlc search was conducted with 100 times randomaddition sequences, tree-blsection reconnection (TBR) branch swapping, and the
Multrees and Steepest Descent options ¡n effect. All trees collected were combined
and used as starting trees, with MulTrees on and no tree limit (these trees were then
swapped to completion) and Sub-tree-Pruning-Regrafting (SPR; Salamin et al.,
2003). Internal support was assessed using 1000 replicates with simple taxon
addition and SPR branch swapping, but permitting only ten trees per replícate to be
held (Chase et al., 2003).
For Bl both data matrices were run for three million generations (four MCMC,
chain temperatura = 0.2; sample frequency = 100). A 50% majority rule consensus
tree was calculated from the pooled sample using the sumt command to yield the
final Bayesian estímate of phylogeny. Internodes with posterior probabilities £ 95%
were considerad statistically significant (Ickert-Bond and Wen, 2006).
As previous studies have identified different copies of ITS within individual
accessions, and interpreted these as evidence of the failure of conceded evolution
after hybridization in the evolutionary history of recently evolved lineages (FuertesAguilar et al., 1999), the number and position of ITS additivities was assessed.
To determine relationships among combined plastid haplotypes, a statistical
parsimony analysis was conducted using the algorithm described in Templeton et al.
(1992) as implemented in TCS ver. 1.21 (Clement et al., 2000). Gaps were coded as
missing data.
111
4.3. RESULTS
4.3.1. Characterístics of nuclear and plastid sequences
Detailed ¡nformation on the nuclear and plastid sequences obtained is given in Table
4.3. Within Antirrhinum, ITS sequence divergence ranges from 0.00% to 3.73%. The
psbA-tmH and trnl-tmL sequence divergence varied from 0.00% to 3.71%. Mean
sequence divergence within A. charidemi, A. subbaeticum and A. valentinum was
0.00%, 0.96(±0.37)% and 0.82(±0.53)% for ITS, and 0.05(±0.06)%, 0.78(±0.60)%
and 0.13(±0.07)% for the combined plastid regions, respectively.
Table 4.3. Sequence characteristics obtained from the analysis of ITS, psbA-trnH and tmJtmün Antirrhinum. 8 Akaike Information Criterion (Akaike, 1979).
ITS
psbA-tmH
tmJ-trnL
Length range (bp)
578-595
228-299
613-624
Aligned length (bp)
599
311
636
No. variable/informative characters
109/49
37/31
30/20
Máximum sequence divergence (GTR)
3.90%
8.88%
2.17%
Informative indels (no. bp)
2(1-16)
12(1-12)
7(1-7)
Mean G+C content
60.6%
28.6%
27.2%
Simplest model8
GTR+I+G
GTR
GTR+I+G
4.3.2. Phylogenetic analysis
The Bl and MP analysis of ITS sequences yielded similar topologies, with Bl
displaying higher support valúes (Fig. 4.1). The Bl analysis using GTR as the
simplest model of DNA evolution reached equilibrium after 35,000 generations. Six
species displayed either monophyly or ¡dentity of ITS sequences: the four accessions
of A. charidemi (¡dentical sequences), the four accessions of A. subbaeticum, with
112
100% posterior probability (PP) and 56% bootstrap valué (BS), and the five
accessions of A. valentinum, with 100% PP and 73% BS; well-supported
monophyletic groups were also retrieved for A. braun-blanquetii (99% PP, 69% BS)
and A. sempervirens (100% PP, 83% BS); the two accessions of A. mollissimum
form a monophyletic group with 91% PP but low BS support (< 50% BS). Within A.
subbaeticum two sub-groups were identified: one was well supported (100% PP,
94% BS), while the other was weakly supported (88% PP, < 50% BS).
Nucleotide additivity in ITS (i.e. double peaks in the chromatograms) was
present in 53 of the 69 accessions oí Antirrhinum. These additivities occurred at 35 of
the 49 parsimony-informative sites. No additivities were detected in the four
accessions of A. charidemi, while two of the four accessions of A. subbaeticum
showed a single additivity, and three of the five accessions of A. valentinum showed
eight, two and three additivities sites, respectively (Table 4.4).
A majority rule consensus tree for plastid sequences obtained from Bl under the
GTR (psbA-fmH) and GTR+I+G (tmT-trnL) model is shown in Fig. 4.2. The Bl
analysis reached equilibrium after 180,000 generations. Plastid sequence data do not
clearly support monophyly of any of the species of Antirrhinum. However, all
accessions of A. charidemi were located in a well-supported clade (98% PP, 64%
BS) that also included a single accession of A. mollissimum. In A. subbaeticum, two
accessions (3 and 4) had identical sequences (Appendix 4.1), but were unresolved
regarding to other accessions. The other two accessions (1 and 2) of A. subbaeticum
were identical to a single accession (4) of A. pulverulentum (Appendix 4.1), and were
located in a well-supported clade (100% PP, 86% BS). All accessions of A.
valentinum were located in a large clade, with high 83% PP and low BS support (<
50%), containing accessions of six other species. In A. valentinum, accession 4
cluster together with accession 3 of A. hispanicum (90% PP, 85% BS), while the
other accessions (1 ,2 ,3 and 5) are not resolved into strongly supported groups. The
plastid sequence analysis therefore revealed lack of monophyly for populations of the
three species.
-fU
113
" — A. au nrale ( í)
A ausbato (3)
—— A auslrale (2)
A controversum (1)
— — A. controversum (2)
A controversum (5)
A. controversum (7)
A controversum (8)
A gramucum (2)
A latrfohurr, (2)
A Unkianum (1)
A meonanthum (1)
A moHe (1)
A. moka (2)
—
A üraun-bJanqueta (1)
A braun-bianquetii (2)
A lopesianum (3)
A chandemi (1)
A charidemi (2)
—
A charidemi (3)
A charidemi (4)
—
A cirrhtgerum (1)
A. tortuosum (6)
A tuspanicum (3)
A. mollissimum (1)
A mollissimum (2)
A controversum (3)
A controversum (4 J
—
A controversum (6)
A pertegasu <1)
A p e rte y a *' (21
A putveruhntum 11)
A pulverulentum (2)
A gra nlicum (3)
A grosu (1)
A gmsa (2)
A hispamcom «2)
A laMohum (1)
—
—
A
A
A
A
latilohum (5)
Unkianum (2 1
litigiosum 11)
lopasionum (2)
A
A
A
A
A
majos (3)
majos (6)
nmcroQhyfíum (2)
putvaruJantum (3)
pulvem lenlum (5)
A pulvorulantum (6)
A putvamkíntum <4)
—
—
A
A
A
A
A
meonanfíuim (2)
nvcmphyUum ( 1)
rrvcrap/W tam (3)
sampervtrans (1)
sam parvm ns (3)
A
A.
A
A.
A
sicukjm (1)
sicukim (5)
subbaeiioum (1)
subbaalKum {21
subbaeticum (3)
A subbaeticum (4)
A tortuosum (2)
A. valentinum 11)
A valentinum (2)
A. valentinum (3)
A valentinum (5)
A valentinum |4)
A graruticum
A siculum (2)
(1)
A Migiosum (2)
M oronbum
P. nvas-martmezn
Figure 4.1. 50% majority consensus tree from Bl analysis of ITS sequences of Antirrhinum
based on the GTR model. Above branches: posterior probabilities; below branches: bootstrap
support > 50% of the strict consensus tree of 223 MP trees (Cl = 0.55; Rl = 0.71; 277 steps).
Incongruence between the Bl tree and the MP strict consensus tree or bootstrap support < 50%
are indicated with a hyphen (-) below branches. Population numbers are given in brackets after
species ñame (see Appendix 4.1).
114
Table 4.4. Polymorphic sites in ITS sequences of A. charidemi, A. subbaeticum and A.
valentinum. Numbers refer to the aligned sequences. Population numbers are given in brackets
after species ñame (see Appendix 4.1)
Taxon
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
charidemi (1)
charidemi (2)
charidemi (3)
charidemi (4)
subbaeticum (1)
subbaeticum (2)
subbaeticum (3)
subbaeticum (4)
valentinum (1)
valentinum (2)
valentinum (3)
valentinum (4)
valentinum (5)
Taxon
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
charidemi (1)
charidemi (2)
charidemi (3)
charidemi (4)
subbaeticum (1)
subbaeticum (2)
subbaeticum (3)
subbaeticum (4)
valentinum (1)
valentinum (2)
valentinum (3)
valentinum (4)
valentinum (5)
Taxon
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
charidemi (1)
charidemi (2)
charidemi (3)
charidemi (4)
subbaeticum (1)
subbaeticum (2)
subbaeticum (3)
subbaeticum (4)
valentinum (1)
valentinum (2)
valentinum (3)
valentinum (4)
valentinum (5)
0
1
7
C
A
0
1
9
C
0
3
7
C
0
2
1
C
T
T
0
4
1
G
0
4
0
C
0
4
5
G
G
0
8
0
G
0
8
1
A
0
8
5
T
0
9
4
G
T
T
T
T
C
C
C
C
G
G
G
A/G
G
G
G
G
G
C
C
G
G
G
G
G
G
G
T
A
C/G
C/T
C/T
T
C/G
1
1
3
C
T
1
3
6
C
1
2
5
C
G
A
A
A
A
A
4
1
2
G
A
A
A
A
A
A
A
A
A
4
3
8
C
1
4
5
A
1
6
3
C
G
T
T
T
A/G
A/G
A
1
7
3
C
T
T
T
T
T
T
4
4
6
A
4
4
8
C
G
G
A/G
T
G
T
T
T
T
T
T
T
T
T
1
7
4
C
1
8
6
C
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
4
4
9
G
2
1
3
C
A
3
5
6
A
1
0
9
C
-
G
G
-
-
C/T
3
6
4
C
3
7
8
C
T
C/T
C/T
T
A/G
G
C/T
5
3
4
C
T
T
A
A/G
2
2
6
C
0
9
5
5
4
9
T
5
5
1
C
G
G
G
G
A
A
A/G
G
A
T
T
T
T
T
T
T
T
T
5
5
2
C
T
5
5
6
C
A
A
A
A
A
A
5
8
3
C
5
8
9
T
T
T
T
T
T
T
T
T
T
T
T
96
100
98
100
86
58
96
57
64
85
H .
100
92
115
- A. BUZtfálG (1)
- A australe (3)
- A chandem i (1)
- A chandem i (2)
- A. charidem i (3)
- A chandem i (4)
- A m ollissimum (1)
- A controversum (3)
- A htspantcum (1)
- A htspantcum (2)
- A htspantcum (3)
- A valentinum (4)
- A m ollissim um (2)
- A m ollissim um (3)
- A polverulentum (3)
- A polverulentum f6)
- A valentinum (1)
- A valentinum (2)
- A valenünum (3)
- A valentinum (5)
- A austraie (2)
- A braun-blanquebi (1)
- A braun-blanquotit (2)
- A Unkianum ( 1)
- A M ig A m m ( 1 1
- A Htgtosum (2)
- A lopesianum (3)
- A majus (2)
- A majus (3)
- A majus (5)
- A microphytlum (2)
1
- A penegasti 11)
1
- A pertegasií (2)
- A motte (2)
- A sempervtrens (1)
- A. ctrrhtgerum (1)
- A gramOcum (1)
i---------------------------- A granihcum (2)
- A tatdoüum (1)
- A majus
- A majus (4)
- A. majus (6)
- A molkt (1)
- A lablohum (2)
1---------------------------- A lattlolium (3)
- A Unkianum (2)
- A Unkianum (3)
----------------------------- A stcukim { i )
1---------------------------- A tuctrium (2)
- A letifoéum (4)
- A loposianum (1)
- A lopestanum (2)
- A sicuhjm (5)
- A toriuosum (1)
- A to riuosum (2)
- A to riuosum (3)
- A to riuosum (4)
- A to riuosum (5)
- A to riuosum (6)
- A toriuosum (7)
- A to riuosum (8)
- A controversum 17)
- A qrantticum (3)
- A meonanthum (2)
- A g ro tsi (1)
- A meonanthum (1)
- A. grossi (2>
- A microphytlum (1)
- A. m tcropliyllum (3)
- A pulverulenium (4)
- A subbaeticum (1)
- A subbaeitcum (2)
- A pulverulenium (5)
- A latifolium (5)
- A stcuium (3)
- A stcuhtm (4)
- A subbaeticum ( i )
- A subbaeticum (4)
- M orontrum
- P nvas-martinezu
Figure 4.2. 50% majority consensus tree from Bl analysis of combined psbfk-trnH and trnJ-trnL
sequences of Antirrhinum based on the GTR (psbA-trnH) and GTR+I+G (frnT-fmL) models.
Above branches: posterior probabilities; below branches: bootstrap support > 50% of the strict
consensus tree of 69,300 MP trees (Cl = 0.79; Rl = 0.89; 200 steps). Incongruence between the
Bl tree and the MP strict consensus tree or bootstrap support < 50% are indicated with a
hyphen (-) below branches. Population numbers are given in brackets after species ñame (see
Appendix 4.1).
116
4.3.3. Analysis of plastid haplotypes
Thirty-nine plastid haplotypes were identified in the 88 Antirrhinum accessions
analyzed, with a total of 11 haplotypes shared between congeneric accessions
(Appendix 4.1). The statistical parsimony analysis of the plastid región yielded a
single network with two haplotypes connected by a single step in A. chandemi, two
divergent haplotypes in A. subbaeticum separated by 12 steps, and five haplotypes
connected by one or two steps in A. valentinum (data not shown).
4.4. DISCUSSION
4.4.1. Testing monophyly at the species level
ITS accessions of the three study species of Antirrhinum formed three monophyletic
groups (Fig. 4.1), which supports the morphological and taxonomical boundaries of
these species. In contrast, analyses of the plastid sequences did not reveal
monophyly for any of the study species, potentially reflecting different evolutionary
histories. Several processes have been proposed as potential mechanism for
retrieving non-monophyletic groups at the species level estimated for nuclear and
plastid phylogenies. Particularly in plants, lack of congruence between nuclear and
plastid data sets and the fact that identical haplotypes are shared among unrelated
species suggest that lineage sorting, hybridization or a combination of both
processes may be responsible (Hardig et al., 2000; Under and Rieseberg, 2004).
In A. charidemi, a shared history for populations is suggested by plastid
sequences as they occur in the same clade, have high similarity, and were
connected by a single mutational step in the network analysis (results not shown).
The occurrence of a single accession of A. mollissimum in this clade may reflect
hybridization between these species as they are geographically proximal (the same
haplotype shared by A. charidemi and A. mollissimum, in Chapter I). A more complex
evolutionary history is interpreted in the case of A. subbaeticum. This species clearly
consists of two related lineages, as recognised not only in ITS sequence analysis but
also in allozyme (Mateu-Andrés, 2004) and RAPD (Jiménez et al., 2002) analyses.
These populations also show some evidence of the effects of inbreeding, with very
117
low allozyme and RAPD diversity. However, our plastid reconstruction reveáis
topological incongruence with the ITS tree, including a cióse relationship between
two populations of A. subbaeticum and one of A. pulverulenium. The low number of
ITS additivities in A. subbaeticum accessions (Table 4.4) does not give support for
recent hybridization events and the fact that a high number of characters (12)
sepárate the two plastid haplotypes is difficult to explain by mechanisms such as
lineage sorting. Nevertheless, these two mechanisms (hybridization vs. lineage
sorting) have been historically invoked as responsible for topological incongruence
between genome phylogenies (Doyle, 1992; Rosenberg, 2002; Degnan and
Rosenberg, 2009). As recommended (Maddison and Knowles, 2006), a sample
increase of populations and DNA regions and the use of coalescence methods do
not alleviate the problem of lineage sorting. Thus, ancient hybridization between
these populations of A. subbaeticum and A. pulverulenium (see also Fig. 4.2) or a
related ancestral taxon is a more parsimonious explanation for the distribution of
these haplotypes, resulting in a non-monophyletic group for plastid sequences of A.
subbaeticum and a weakly supported ITS group. In A. valentinum, despite strongly
supported monophyly for ITS sequences, plastid haplotypes were unresolved due to
a low number of informative characters rather than character incongruence. Although
the five haplotypes are differentiated by only one or two mutational steps, they are
shared with four other species that are widespread in south east Iberia. This makes it
difficult to distinguish unequivocally between the effects of lineage sorting and
hybridization as an explanation for non-monophyly. However, evidence from intraindividual ITS variation supports recent or contemporary hybridization in A.
valentinum: three of the five accessions of this species contain 10 ITS additivities
(Table 4.4).
Hybridization has been historically suggested as significant in Antirrhinum
evolution because: (1) plants display intermedíate morphological characters in nature
(Rothmaler, 1956; Webb, 1971); (2) reproductive barriere between any species pair
are weak in nature and under experimental conditions (Baur, 1932; Rothmaler, 1956;
Sutton, 1988; Xue et al., 1996); (3) the high number of ITS additivities are common in
overlapping geographical areas of many species (Chapter I); (4) additivity patterns
are well documented through analysis of ITS clones (Vargas et al., 2004; Vargas et
118
al. unpublished) and fingerprints (Jiménez et al., 2005). Moreover, nuclear and
plastid phylogenies are incongruent and haplotype variation reflects geography rather
than species taxonomy (Chapter I). This scenario is not unexpected in genera that
have undergone recent diversification since the onset of the Mediterranean climate 23 mya, as reported for Antirrhinum (Chapter I), which additionally has some species
still ¡nvolved in hybrid zones in the Pyrenees (Whibley et al., 2006).
The question remains as to whether lack of single ancestry characterises
Antirrhinum species or is a common pattem in angiosperms. Syring et al. (2007)
reviewed 16 studies from four representative journals covering diverse taxonomic
groups. Of the 460 taxa considered 170 (37%) could be evaluated for monophyly due
to múltiple accessions being sampled in phylogenetic reconstructions, and of these
53% were non-monophyletic. As the review of Syring et al. (2007) was small and
considered only articles that ¡ncluded múltiple accessions of at least some taxa, and
that these taxa were a priorí closely related, we have compiled similar but more
broad-ranging data from three further sources (articles cited in Gitzendanner and
Soitis [2000], 378 taxa cited in the Genetical Flora of the British Isles by Squirrell et
al. [2006], and 172 articles published in six leading journals between 2000-2007).
Studies examining plant species for population genetic diversity were examined on a
taxon by taxon basis to assess whether each taxon could be assessed for monophyly
based upon published phylogenetic reconstructions, and how many of these species
were monophyletic. Data from 270 articles identified 634 species (328 genera, 117
families) and of these 109 (16.6%) could be assessed for monophyly due to a
generic phylogeny containing múltiple accessions of the study taxon being available.
Of those taxa for which monophyletic status could be deduced from the published
phylogeny (N = 92), 57 (62%) were monophyletic for all markers assayed. This brief
review demonstrates that species non-monophyly is rarely considered for specific
taxa assessed for population and conservaron genetic purposes, due primarily to the
fact that múltiple accession generic phylogenies are not available for the taxa under
study. When non-monophyly is assessed, ¡t appears to be taxonomically widespread
(detected here in 19 families representing conifers [1], monocots [5] and dicots [13])
and perhaps more common than generally recognised in both plant (38%; this study)
and animal taxa (23% non-monophyly in mtDNA; Funk and Omland, 2003). In
119
support of the widespread occurrence of plant species non-monophyly, a recent DNA
barcoding study by Fazekas et al. (2008) determined that 31-39% of plant species
assessed were non-monophyletic using a combination of plastid loci. All these results
reflect that lack of monophyly is a more common pattern than generally recognised in
angiosperms and can dramatically influence assessments of genetic diversity.
4.4.2. Life history traits, monophyly and genetic diversity
Hamrick et al. (1979) indicated that significant differences in levels and patterns of
genetic diversity existed between species with different life history traits. Accordingly,
we should expect to find a comparable level and distribution of genetic variation
between species with similar life history traits where taxa are closely related.
However, previous allozyme and RAPD analysis revealed markedly different levels of
genetic diversity within and among populations of the three species herein evaluated
(Mateu-Andrés and Segarra-Moragues, 2000; Mateu-Andrés, 2004) despite the
similarity of their life history traits (see Table 4.2). Genetic diversity differences may
therefore reflect additional sources of variation in the evolutionary history of the
species concerned.
For out-crossing perennial species, the expectation is that species with lower
overall diversity (Ht) will have higher differentiation among populations (Gsr) and vice
versa. This trend is broadly apparent in Antirrhinum species (see Table 4.1), with
some notable exceptions. Narrow endemic species such as A. charidemi and A.
microphyllum both harbour relatively high levels of diversity with a corresponding low
estímate of population differentiation, whereas A. siculum shows a low level of
diversity and high population differentiation (Mateu-Andrés, 1999; Mateu-Andrés and
Segarra-Moragues, 2000; Torres et al., 2003; Mateu-Andrés and de Paco, 2006).
The latter species is self-compatible whereas the former two are self-incompatible,
therefore the differences can be explained by variation in breeding system.
Exceptions to this trend include A. pertegasii, which has both low diversity and little
differentiation among populations, and A. pulverulentum and A. valentinum which
both have médium to elevated overall diversity and the highest levels of
differentiation among population in Antirrhinum (see Table 4.1). Diversity estimates
for the majority of Antirrhinum species fall between these extreme examples.
120
In addition, out-crossing species tend to show higher levels of within-population
variation and lower levels of differentiation among populations (Hamrick and Godt,
1996) so the high level of differentiation (G s r= 0.480) in A. valentinum is intriguing.
This level of diversity is not apparent from ITS sequences as it is in A. subbaeticum.
Other species in the same plastid clade have comparable levels of H t {A.
mollissimum 0.280; A. pulverulenium 0.300), but lower levels of G sr (A. mollissimum
0.110; A. pulverulenium 0.230) compared to A. valentinum. It has been suggested
that A. valentinum maintains a high genetic diversity as consequence of a large
population size in the past (currently 1332 individuáis) due to the fact that the species
was common just 50 years ago (Mateu-Andrés and Segarra-Moragues, 2000).
However, this short time period would not allow development of high G sr estimates
due to fragmentation, suggesting that gene flow among populations is restricted by
an altérnate mechanism. This may also account for a reduction in inter-specific gene
flow explaining why A. valentinum is the only species represented in this plastid clade
to attain monophyly for ITS sequences. Levels of genetic diversity can be shaped by
natural hybridization, wherein hybrids may exhibit elevated levels of genetic diversity
resulting from the mixing of parental genomes (Rieseberg and Wendel, 1993; Arnold,
1997). High diversity in species of this clade may be the result of historical
introgression among divergent lineages (see Doyle, 1992).
A. charidemi and A. valentinum share the eight life history characteristics with
effect on genetic variation (Table 4.2). The differences estimated for population
genetic parameters between these species can be largely explained through
differences in gene flow among populations (five separated populations in A.
valentinum vs. continuous distribution of populations in A. charidemi) and the
potential effects of introgression in A. valentinum. While genetic diversity in A.
subbaeticum is undoubtedly ¡nfluenced by its different reproductive strategy and
geographic range, the extreme plastid sub-division suggests that historical processes
have also played a role in shaping current diversity.
In summary, the impact of reticulation involved in species formation should be
included in the list of factors responsible for influencing levels of genetic diversity.
Indeed, lack of monophyly and incongruence between plastid and nuclear
phylogenies provide an essential framework to detect reticulation processes. The 12
121
life history traits described by Hamrick and collaborators (1979) are not necessarily
the most significant for assessing levels of genetic diversity within species. Careful
attention should be paid to monophyly to elucídate the potential impact of
hybridization on estimates of genetic variation.
122 NON-MONOPHYLY AND GENETIC DIVERSITY
Appendix 4.1. List of studied material including sample number (¡n brackets after species ñame), locality, voucher (herbarium initial or collector
number), ITS, psbA-trnH and trnJ-trnL GenBank accession numbers, and plastid haplotype identifier. Hap = Plastid haplotype identifier (psbA-tmH,
tml-trni.)', A,8GenBank accession numbers published in Vargas et al. (2004) and cited in Chapter I, respectively.
Taxon
Locality
Voucher
ITS
GenBank
psbA-frnH
GenBank
trnT-ím L
GenBank
Hap
Antirrhinum L.
A. australe Rothm. (1)
Spain: Albacete, Yeste
JG4077
EU6771940
forthcoming
forthcoming
1
Spain: Cádiz, Benaocaz
ECA49
EU6771958
forthcoming
forthcoming
2
Spain: Granada, Castril
VAL140895
AY731273 a
forthcoming
forthcoming
1
A. braun-blanquetii Rothm. (1)
Spain: Asturias, Cuetu L'Abeyera
JRV5333
EU6771968
forthcoming
forthcoming
3
A. braun-blanquetii Rothm. (2)
Spain: León, Oblanca
JRV5177
EU6771978
forthcoming
forthcoming
3
Spain: Almería, cerro de La Lobera
132PV05(1)
EU6771988
forthcoming
forthcoming
4
A. chandemi Lange (2)
Spain: Almería, barranco del Sabinar
VAL37158
AY731282 a
forthcoming
forthcoming
5
Spain: Almería, cerro de Santa Cruz
136PV05(2)
EU6771998
forthcoming
forthcoming
5
Spain: Almería, Vela Blanca
137PV05(5)
FJ4876118
forthcoming
forthcoming
5
A. cirrhigerum Welw. ex Ficalho
(1)
Morocco: Doukkala-Abda, El Jadida
VAL111299
EU6772008
forthcoming
forthcoming
6
Spain: Albacete, Villa de Ves
VAL145152
AY731272 a
forthcoming
forthcoming
7
Spain: Alicante, Jalón
BdB47
EU6772018
forthcoming
forthcoming
8
Spain: Almería, Berja
ECA37
EU6772028
forthcoming
forthcoming
1
Spain: Granada, Bérchules
BdB15b
EU6772038
forthcoming
forthcoming
1
Spain: Valencia, Bolomor
JG4001
EU6772048
forthcoming
forthcoming
8
Spain: Valencia, Carcagente
BdB29
EU6772058
forthcoming
forthcoming
8
Taxon
Locality
Voucher
Spain: Valencia, Chella
Spain: Xeresa, Colom
JG4067
GenBank
ITS
EU677206^
GenBank
psbA-trnH
forthcoming
GenBank
trnT-frnL
forthcoming
8
BdB2
EU6772078
forthcoming
forthcoming
9
Spain: Madrid, Fuentidueña del Tajo
JG4009
AY731283a
forthcoming
forthcoming
10
Spain: Soria, Caltojar
JG4101
EU6772088
forthcoming
forthcoming
11
Spain: Huelva, Aracena
ECA54
EU6772098
forthcoming
forthcoming
22
Spain: Ávila, El Trampal
ECA77
AY731281a
forthcoming
forthcoming
12
Spain: Ávila, Guisando
276PV06
EU6772108
forthcoming
forthcoming
13
Spain: Granada, Juviles
BdB14
-
forthcoming
forthcoming
1
Spain: Granada, Veleta
120PV99
AY731286a
forthcoming
forthcoming
1
Spain: Granada, Vélez de Benaudalla
ECA40
EU6772118
forthcoming
forthcoming
14
Spain: Girona, Collada de Toses
JG4142
EU6772128
forthcoming
forthcoming
15
Spain: Lérida, Bapá
2E4PV99
AY731274a
forthcoming
forthcoming
16
Spain: Lérida, Martinet
JG4139
-
forthcoming
forthcoming
16
Turkey: Bursa lli, Gemlik
164PV06
-
forthcoming
forthcoming
10
Italy: Piamonte, Cuneo
MS781
EU6772138
forthcoming
forthcoming
17
Spain: La Coruña, Cedeira
SO (s.n.)
EU6735108
forthcoming
forthcoming
18
Portugal: Peniche, Cabo Carvoeiro
IS(ALQ3435)
EU6772158
forthcoming
forthcoming
19
Portugal: Trafaria, Almada
IS(ALQ4877)
-
forthcoming
forthcoming
20
A. litigiosum Pau (1)
Spain: Teruel, Griegos
ECA74
EU6772168
forthcoming
forthcoming
3
A. litigiosum Pau (2)
ECA44
AY731271a
forthcoming
forthcoming
3
Portugal: Braganga, Alfaiáo
FA & SB (s.n.)
-
forthcoming
forthcoming
10
Hap
i
Taxon
Locality
Voucher
Portugal: Vimioso, Cargao
FA & SB (s.n.)
GenBank
ITS
EU677217“
GenBank
psbA-frnH
forthcoming
GenBank
trnT-frnL
forthcoming
Hap
3
10
Spain: Salamanca, Corporario
FA & SB (s.n.)
EU6772188
forthcoming
forthcoming
A. majus L. (1)
Spain: Barcelona, Gréixer
JG4150
-
forthcoming
forthcoming
15
A. majus L. (2)
Spain: Huesca, Panticosa
JG4108
-
forthcoming
forthcoming
3
A. majus L. (3)
Spain: Lérida, Valle d'Arán
JG26/8/99
AY731280a
forthcoming
forthcoming
3
A. majus L. (4)
France: Hérault, St. Chinian
230PV06
-
forthcoming
forthcoming
21
A. majus L. (5)
France: Pyréneés Orientales, Salses
VAL39727
-
forthcoming
forthcoming
3
A. majus L. (6)
Spain: Gerona, La Molina
273PV06
EU6772198
forthcoming
forthcoming
15
A. meonanthum Hoffmans. & Link
(1)
A. meonanthum Hoffmans. & Link
(2)
Spain: Ávila, Gredos
149PV99
AY731284a
forthcoming
forthcoming
12
Spain: Soria, Río Lobos
JG4098
EU6772208
forthcoming
forthcoming
22
Spain: Cuenca, Buendía
JG4024
EU6772218
forthcoming
forthcoming
23
Spain: Guadalajara, Bolarque
JG4021
EU6772228
forthcoming
forthcoming
24
Spain: Guadalajara, Sacedón
JG4023
EU6772238
forthcoming
forthcoming
25
A. molle L. (1)
Spain: Barcelona, Riguréixer
JG4143
FJ4876128
forthcoming
forthcoming
15
A. molle L. (2)
Spain: Huesca, Sopeira
JG4117
AY731268a
forthcoming
forthcoming
3
Spain: Almería, Benizalón
ECA29
EU6772248
forthcoming
forthcoming
5
Spain: Almería, Caballar
ECA32
EU6772258
forthcoming
forthcoming
1
MA427229
-
forthcoming
forthcoming
1
Spain: Almería, Dalias
A. pertegasii Rothm. (1)
Spain: Castellón, Cova Fosca
JG4092
EU6772268
forthcoming
forthcoming
26
A. pertegasii Rothm. (2)
Spain: Castellón, Solé d’en Brull
JG4091
EU6772278
forthcoming
forthcoming
26
Taxon
Locality
A. pulverulenium Lázaro Ibiza (1)
Spain: Cuenca, Hoz de Beteta
A. pulverulentum Lázaro Ibiza (2)
Spain: Guadalajara, Durón
Voucher
ECA28
GenBank
ITS
EU677229“
GenBank
psbA-trnH
forthcoming
GenBank
trnT-frnL
forthcoming
Hap
27
JG4027
EU6772308
forthcoming
forthcoming
11
Spain: Guadalajara, La Pelegrina
JG4035
EU6772318
forthcoming
forthcoming
28
Spain: Guadalajara, Peralejos Truchas
ECA26
EU6772328
forthcoming
forthcoming
29
Spain: Teruel, Tramacastilla
ECA71
EU6772338
forthcoming
forthcoming
30
Spain: Guadalajara, Alcorlo
JG4028
EU6772348
forthcoming
forthcoming
28
A. sempervirens Lapeyr. (1)
Spain: Huesca, Bielsa
JG4114
EU6772358
forthcoming
forthcoming
31
Spain: Huesca, Plan
JG4116
-
forthcoming
forthcoming
3
Spain: Huesca, Panticosa
JG4107
AY731270a
forthcoming
forthcoming
3
Italy: Sicily, Catania
GB66/06
EU6772378
forthcoming
forthcoming
32
Italy: Sicily, Messine
VAL119899
AY731276a
forthcoming
forthcoming
32
33
Italy: Sicily, Siracusa
VAL178308
-
forthcoming
forthcoming
Malta: Siggiewi, Ghar Lapsi
VAL39295
-
forthcoming
forthcoming
34
Morocco: Oriental, Zegzel
192PV00
EU6772388
forthcoming
forthcoming
35
A. subbaeticum Güemes, Mateu
& Sánchez Gómez (1)
A. toriuosum Bosc ex Vent. (1)
Spain: Albacete, El Batán
JG4081
AY731287a
forthcoming
forthcoming
29
Spain: Albacete, Los Vizcaínos
JG4084
EU6772398
forthcoming
forthcoming
29
Spain: Murcia, Benízar
JG4068
EU6772408
forthcoming
forthcoming
36
Spain: Murcia, Hondares
BdB227
EU6772418
forthcoming
forthcoming
36
Italy: Sardinia, Cagliari
GB136/06
-
forthcoming
forthcoming
10
Appendix 4.1. {cont.).
Taxon
Locality
Voucher
GenBank
ITS
EU677242“
GenBank
psbA-frnH
forthcoming
GenBank
trnT-frnL
forthcoming
Hap
forthcoming
10
A. tortuosum Bosc ex Vent. (2)
Morocco: West Rif, Talembot
IS(ALQ3441)
Turkey: Sulcuk, Efeso
GB316/06
-
forthcoming
10
Morocco: Taza-AI Hoceima, Taza
188PV06
-
forthcoming
forthcoming
10
Italy: Latina, Norma
VAL142945
-
forthcoming
forthcoming
10
Italy: Ancona, Si rolo
VAL39871
AY731285a
forthcoming
forthcoming
10
Morocco: Taza-AI Hoceima, Tazzeka
MA643294
-
forthcoming
forthcoming
10
Morocco: Tadla-Azilal, Ighir
MA746269
-
forthcoming
forthcoming
10
Spain: Valencia, Bolomor
BdB229
EU6772438
forthcoming
forthcoming
1
Spain: Valencia, Buixcarró
JG4002
EU6772448
forthcoming
forthcoming
37
Spain: Valencia, Font del Cirer
BdB8
EU6772458
forthcoming
forthcoming
38
Spain: Valencia, La Drova
JG4004
EU6772468
forthcoming
forthcoming
14
Spain: Valencia, Peña Colom
BdB1
AY39799a
forthcoming
forthcoming
39
forthcoming
forthcoming
forthcoming
forthcoming
forthcoming
forthcoming
forthcoming
forthcoming
Misopates Raf.
M. orontium (L.) Raf.
VAL145155
AY731260a
Pseudomisopates Güemes
P. rivas-martinezii (Sánchez
Mata) Güemes
Spain: Ávila, Sierra de Gredos
377PV99
AY731262a
-
Conclusiones
4?a InZ*
t 7T
Conclusiones
Del estudio de los procesos de hibridación en A. charidemi, A. subbaeticum y A.
valentinum se pueden establecer las siguientes conclusiones:
1) Las relaciones filogéneticas de las especies de Antirrhinum no han podido ser
clarificadas en base a los análisis moleculares de las secuencias de ADN nuclear
(ITS) y plastidial (tmS-tmG, fmK-mafK, tmT-tmL, psbA-trnH). Los procesos
evolutivos de hibridación ancestral y reciente podrían constituir el principal
mecanismo distorsionador de la filogenia de las especies de Antirrhinum.
2) Los análisis de las secuencias de ADN plastidial (tmK-matK) indican que los
procesos de diferenciación de los linajes de Antirrhinum ocurrieron a partir del
Plioceno, lo que coindice con el comienzo del estableciemiento del clima
Mediterráneo.
3)
Además de la hibridación, la especiación geográfica ha tenido un papel
importante en la diferenciación de las especies de Antirrhinum, con el sudeste de la
Penísula Ibérica como principal foco de diferenciación. En la evolución del género, a
la especiación geográfica, originada principalmente en en el seno de las montañas
Ibéricas, se añadirían posteriores contactos secundarios entre especies promovidos
por los episodios de cambio climático durante el Plioceno-Pleistoceno.
130
CONCLUSIONES
4) Las eviencias moleculares apoyan procesos de hibridación en A. subbaeticum
(hibridación ancestral) y en A. valentinum (hibridación reciente), y posiblemente en
A. charidemi.
5) La diversidad genética de A. charidemi, A. subbaeticum y A. valentinum está
afectada por las características biológicas y ecológicas de cada especie, pero
también por su historia evolutiva, en la que han intervenido los procesos de
hibridación.
6) Actualmente,
las barreras reproductivas (postpolinización: precigóticas y
postcigóticas; viabilidad de híbridos de primera generación) entre A. valentinum y la
especie común A. controversum son substanciales pero no absolutas, por lo que
existe potencial para la hibridación e introgresión entre las dos especies. Si el
contacto entre A. valentinum y A. controversum se intensifica o prolonga en el futuro,
la
hibridación
podría
suponer
una
amenaza
seria
para
A.
valentinum,
principalemente por el riesgo de asimilación genética.
7) Las similitudes biológicas y ecológicas entre A. charidemi, A. subbaeticum y A.
valentinum no se corresponden con una estrategia reproductiva común, y sugiere
precaución a la hora de establecer planes de conservación comunes para especies
afines.
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Agradecimientos
Gracias al Dr. Jaime Güemes, por su dedicación en la dirección de esta tesis,
su tiempo, y por su apoyo en todo momento, dentro y fuera de esta tesis; al
Dr. Pablo Vargas, por sus comentarios, ideas y colaboración en el capítulo I y
IV; al Dr. Juan Jiménez y al Dr. Alan Forrest, por sus contribuciones en el
capítulo II y IV, respectivamente; al Dr. Patricio García-Fayos, por su
asesoramiento estadístico en los capítulos II y III; a Fabiola Barraclough por
revisar el texto en inglés; a la Dr. María Bosch y a la Dr. Rosario Gil, por
acogerme con tanta familiaridad durante mis estancias;
al personal del
Jardín Botánico de la Universidad de Valencia y del Real Jardín Botánico de
Madrid; a mis compañeros de Valencia, Madrid y Barcelona; a la Consejería
de Medio Ambiente (Junta de Andalucía) y a la Conselleria de Medi Ambient,
Aigua, Urbanisme i Habitatge (Generalitat Valenciana), por permitir la
investigación en las poblaciones de A. charídemi y A.
valentinum,
respectivamente; a la Dirección General Española de Investigación Científica
y Técnica, por financiar esta memoria doctoral a través del proyecto
REN2002-02434/GLO y de la beca de Formación de Personal Investigador
BES2003-0387.
Y gracias a mis amigos biólogos, botánicos y no botánicos; a P.G. y a
J.M.S. (dónde estés); y a E.B, por el Yelcho.
UNIVERSITAT DE VALENCIA
FACULTAT DE CIÉNCIES BIOLÓGIQUES
R e u n i d o el T r i b u n a l q ue s u s c r i b e , en el día de la fecha
a c o r d ó o t o r g a r a e s t a T e s i s D o c t o r a l de
D / D a ELENA CARRIÓ GONZÁLEZ
la c a l i f i c a c i ó n de
V a l e n c i a , a . . . 1. . .
de
juJio.
de ...2010
EL/LA SECRETARIO/A,
EL/LA PRESIDENTE/A
Dr. D. iu li Cau apé Castells
Dr. D. César Blanché i Verges
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La hibridación en el origen y extinción de especies en el género

Transcription

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