CHARACTERIZATION OF SEED DORMANCY
OF NOLANA (SOLANACEAE) IN THE COASTAL
ATACAMA DESERT OF CHILE
JOSEFINA HEPP CASTILLO
SANTIAGO - CHILE
2019
Pontificia Universidad Católica de Chile
Facultad de Agronomía e Ingeniería Forestal
CHARACTERIZATION OF SEED DORMANCY
OF NOLANA (SOLANACEAE) IN THE COASTAL
ATACAMA DESERT OF CHILE
Josefina Hepp Castillo
Tesis
para obtener el grado de
Doctor
en Ciencias de la Agricultura
Santiago, Chile, agosto 2019
Tesis presenteda como parte de los requisitos para obtener el grado
de Doctor en Ciencias de la Agricultura, aprobada por el
Comité de tesis
_____________________
Prof. Guía, Samuel Contreras Escobar
_____________________
Prof. Co-Guía, Pedro León Lobos
__________________
Prof. Informante 1, Gloria Montenegro Rizzardini
___________________
Prof. Informante 2, Miguel Gómez Ungidos
Contents
Chapter I. General introduction.......................................................................................... 8
1.1 Definition of desert .................................................................................................... 8
1.1.1 The coastal Atacama Desert in Chile ............................................................. 9
1.2 Germination in desert environments .................................................................... 11
1.3 Dormancy mechanisms and their ecological importance ................................. 12
1.3 The genus Nolana ................................................................................................... 15
Hypothesis ...................................................................................................................... 17
Objectives........................................................................................................................ 18
a.
General objective ................................................................................................ 18
b.
Specific objectives ............................................................................................... 18
References ...................................................................................................................... 27
Chapter II. Characterization of seed dormancy of twelve Chilean species of Nolana
(Solanaceae) from the coastal Atacama Desert ........................................................... 31
Introduction ..................................................................................................................... 31
Materials and methods .................................................................................................. 33
Results and discussion ................................................................................................. 38
Conclusions .................................................................................................................... 47
References ...................................................................................................................... 47
Chapter III. Current and future patterns of distribution of Nolana species
(Solanaceae) from the coast of Chile and their relation to seed dormancy ............. 51
Introduction ..................................................................................................................... 51
Materials and methods .................................................................................................. 53
Results and discussion ................................................................................................. 59
Conclusions .................................................................................................................... 66
References ...................................................................................................................... 67
Chapter IV. Comparative fruit anatomy and morphology of twelve species of
Nolana (Solanaceae) of the coastal Atacama Desert, Chile....................................... 77
Introduction ..................................................................................................................... 77
Materials and methods .................................................................................................. 79
Results and discussion ................................................................................................. 83
Conclusions .................................................................................................................... 95
References ...................................................................................................................... 95
Chapter V. A new endemic species of Nolana (Solanaceae-Nolaneae) from near
Iquique, Chile ...................................................................................................................... 98
Introduction ..................................................................................................................... 99
Materials and Methods .................................................................................................. 99
Description .................................................................................................................... 100
References .................................................................................................................... 104
Annexes ......................................................................................................................... 107
Chapter VI. A study of fruit development in Nolana paradoxa (Solanaceae) from
the coast of Chile ............................................................................................................. 115
Introduction ................................................................................................................... 115
Materials and methods ................................................................................................ 116
Results and discussion ............................................................................................... 118
Conclusions .................................................................................................................. 125
References .................................................................................................................... 125
VII. General conclusions ................................................................................................. 127
AGRADECIMIENTOS
Esta tesis fue financiada por CONICYT a través de la Beca de Doctorado en Chile N°
21130176.
Han sido muchas las personas que me han acompañado a lo largo de estos años. En primer
lugar, quiero agradecer a los profesores de mi comité de tesis; a mi tutor, Samuel Contreras,
por guiarme en todo el proceso, por su confianza y disponibilidad, por sus enseñanzas, su
ética y paciencia. A Pedro León, co-tutor de esta investigación, por haber sido el primero
en motivarme a realizar el doctorado y facilitar la vinculación con el Jardín Botánico de Kew;
a Gloria Montenegro, por ser un ejemplo de lo que se puede hacer como mujer en ciencias;
y a Miguel Gómez, por compartir conmigo sus conocimientos, sus libros y su amistad.
También a Eduardo Olate, por haberme impulsado en el inicio.
Al profesor Michael Dillon, por ayudarme con la taxonomía de Nolana y compartir
muchísima información y fotografías; a Patricio Pliscoff, por enseñarme sobre nichos,
modelación y proyecciones. A los curadores y al personal de los herbarios SGO y CONC,
por permitir la revisión y fotografía de colecciones, en particular a Gloria Rojas, Víctor
Ardiles y Alicia Marticorena. A Vanezza Morales por su ayuda con la descripción de
especies. A Wolfgang Stuppy, por posibilitar la pasantía en el Banco de Semillas del Milenio.
A todos los que estuvieron conmigo en UK: a mis amigos Paulina Hechenleitner, Oliver
Whaley y Marthita; a Martin Gardner y Sabina Knees, por su generosidad y constante
apoyo; a Gunnar Ovstebo y Tom Christian por sus valiosas enseñanzas en el Jardín
Botánico de Edimburgo.
A la Corporación Nacional Forestal (CONAF), por los permisos otorgados para llevar a cabo
las colectas en terreno, en especial al personal del Parque Nacional Pan de Azúcar y
Parque Nacional Llanos de Challe. A Milsa Tapia, administradora del parque Puquén en
Los Molles, por permitir el acceso para colectar semillas. Al Centro UC del Desierto de
Atacama, por facilitar la visita a la Estación Experimental Atacama UC en el oasis de niebla
de Alto Patache, y por tantos años de amistad y trabajo en el desierto, sobre todo a Camilo
del Río, Juan Pablo Astaburuaga, Eric Parra, Nicolás Zanetta, Javiera Machuca, don
Horacio Larraín, Pilar Cereceda, Ana María Errrázuriz, Pablo Osses, Juan Luis García,
Margot Lagos, Cristina Vidal y Alicia Guentulle.
A mis compañeros del Laboratorio de Semillas, en especial a Carolina Ayala, Francisca
Subiabre, Paola Guilleminot, Elisa Cabrera, Inés Vilches, Patricia Caroca, Jennifer Soto,
por la constante motivación para trabajar y pasarlo bien al mismo tiempo. A Pedro Espinoza,
por su ayuda con los cortes histológicos; a Fabiola Orrego, por el apoyo en propagación
vegetativa; a Ignacia Erices, por su disposición y ayuda con los experimentos; a Pablo
Espinoza, por facilitar los instrumentos. A todos los que me acompañaron en algún
momento a terreno (Sofía Vio, Miguel Gómez, Elisa Cabrera, Benjamín Moreno, Daniela
Richaud, Nicolás Jiménez, Camila Di Domenico, Ester Vargas, Felipe Lobos, Constanza
Gazmuri, Eduardo Contreras, don Horacio Larraín, Carolina Ayala, María Ignacia Cuadra,
Laura Bascuñan y Sergio Ibañez). También a Marcelo Rosas y equipo del Banco de
Semillas - INIA Vicuña por invitarme a participar en una campaña de colecta por
Antofagasta. Al equipo de la DIP que siempre facilitó la vida en el doctorado: Susan Arenas,
Sandra Osorio, Ximena Ortega, Claudia González, Marcela Pérez, María José Bravo,
Wendy Wong y Marlene Rosales. Al equipo del Jardín Botánico Chagual (Fernanda Bustos,
María Victoria Legassa, Gonzalo Flores, Daniela Escobar y Natalia Smith), por su ejemplo
de compromiso y por recibir la colección de varias semillas de Nolana. A Pablo Morales por
generar el protocolo de propagación in vitro para Nolana intonsa, y felizmente permitirnos
donar esas plantas al JBCH.
A todos mis amigos queridos, que siempre me animan a seguir en este rumbo. A mi papá,
Pedro, mi mamá, Mariana, y mis hermanos Mariana, Florencia y Pedro, por guiarme
también con su ejemplo de pasión y dedicación. A mis sobrinitos Bautista y Jacinta: todo
esto es para ustedes. A mi abuela Erica por su genuino interés en lo que hago. Al Benja,
no solo por construirme tamices de semillas y ayudarme con definiciones de la tesis, sino
también por estar conmigo alegremente construyendo la vida. A mis dos florcitas que
quedaron en el camino.
Por último, mis respetos a todos los que han trabajado por la protección y mejor
comprensión de la vida sembrada en el desierto: vida latente, esperando las condiciones
favorables para germinar.
Chapter I. General introduction
The environmental conditions that seeds require to germinate in diverse plant populations
determine the main limits for their establishment at different times and habitats (Bansal and
Sen, 1981). The existence of a variety of complex mechanisms that regulate the germination
of seeds proves that they are ecophysiological adaptations, that is, differential physiological
responses of organisms according to the environment in which they live and the prevailing
environmental factors. These adaptations increase the survival potential of the species and
have been shaped in the normal pattern of evolution (Bansal and Sen, 1981).
Deserts are found on planet Earth north and south of the Equator, forming two belts of arid
ecosystems with species specifically adapted to those areas. Despite being regions of very
rigorous climates and often unfavorable conditions for life, together they occupy a quarter of
the surface of the Earth and provide important services to support life on the planet and
resources for human populations (UNEP, 2006).
The objective of this work is to understand the germination strategies and dormancy
mechanisms presented by plants in desert areas, in particular species of the genus Nolana
(Solanaceae), which would allow them to ensure their continuity in extreme and
unpredictable environments, especially the Atacama Desert in northern Chile.
1.1 Definition of desert
According to Shmida (1985), land deserts can be separated into three categories in terms
of the amount of rainfall they receive:
•
•
•
Semi-deserts: rainfall between 150 and 300 or 400 mm per year.
True deserts: less than 120 mm of annual rainfall.
Extreme deserts: less than 70 mm of annual rainfall.
Arid ecosystems usually combine true and extreme deserts, while a semi-arid ecosystem
corresponds to a semi-desert (Shmida, 1985). The limits set between one category and
another are not arbitrary; it is generally known that these tree formations are not found in
regions where the amount of annual rainfall is less than 400 mm. The 120 mm mark the
difference between steppe (semi-desert) and desert vegetation, which differ significantly in
coverage density; in steppes, coverage is around 20 to 50%, while in deserts, it is less than
10%. As for the 70 mm of rainfall, they would establish the difference between “diffuse”
vegetation and “contracted”, or restricted to only certain places (Shmida, 1985).
The arid and semi-arid zones, apart from being distinguished by the low amount of annual
rainfall, are also differentiated by the season or seasons in which rainfall occurs (Griffiths,
1972, cited in Gutterman, 1994). The more extreme the desert, the more unpredictable are
the low amounts and the distribution of rainfall, as well as the beginning and length of the
rainy season or seasons (Gutterman, 1994).
It is interesting to note that, although a desert is considered “extreme” if it presents an
average of less than 70 mm of annual rainfall, there are deserts where rainfall is much lower.
In the coastal Atacama Desert, the annual average rainfall is between 0.2 and 2 mm; in the
last century, not a drop of water fell in 60% of the years (Cereceda, 2012). The Atacama
Desert is considered the most arid in the world; this means that the hydrological balance
(balance between the contributions of water to the system by rainfall, and evaporation /
evapotranspiration outflows) is the most negative when compared to that of other deserts
(Weischet, 1975).
1.1.1 The coastal Atacama Desert in Chile
The Atacama and Peruvian Desert forms a nearly continuous, hyper-arid belt for around
3,500 km along the south-western coast of South America (7°-29°S; Dillon et al., 2009). In
Chile, the Atacama Desert occupies about 180,000 km2 of the national territory; here, it
extends from the Arica and Parinacota region (18°34'S, 69°27'W) to the north of the Atacama
region (26°10'S, 70°00'W), but sometimes the semi-desert area is also considered, which
includes the south of the Atacama region and the Coquimbo and Valparaíso regions
(Cereceda, 2012). According to Rundel et al. (1991), and from a floristic point of view, the
southern limit of the Atacama Desert should correspond to La Serena (29° 55´S). Inland,
there is an area that corresponds to the absolute desert, which almost completely lacks plant
life, except in some sectors with presence of groundwater (Luebert and Pliscoff, 2006).
The climate in the coastal desert of Chile is moderate in terms of temperature, which in
general is quite stable along the latitudinal range along the coast (Rundel et al., 1997). The
atmospheric humidity is high (annual average of 65-80%), and annual rainfall ranges from
less than 1 mm in the northernmost weather stations to around 80 mm in La Serena (Fig. 1,
Table 1). Rains generally occur during the southern winter (from May to September), but
some sectors also receive summer rains (Schultz et al., 2011).
400
350
Rainfall (mm)
300
250
200
150
100
50
0
Arica
Iquique
Calama
Antofagasta
La Serena
Valparaíso
Fig. 1. Amounts of annual rainfall (mm) in a normal year (average of 30 years between 1981 and
2010) in different cities of Chile. Source: Own elaboration based on data from the Chilean
Meteorological Office.
Table 1. Normal annual amounts in different coastal cities in northern and central Chile, some of
which are included in the Atacama Desert. Average temperature calculated based on data from 1971–
2000. Source: Chilean Meteorological Office, Integrated Territorial Information System (ITIS) and
Schultz et al., 2011.
Annual rainfall
(mm)
Arica
0,5
Iquique
0,6
Antofagasta
1,7
Copiapó
18,5
La Serena
78,5
Valparaíso
372,5
City
Mean temperature
(°C)
18,9
18,4
16,7
15,2
13,7
14,0
In the northernmost area (north of 21°30'S) of the coastal desert, vegetation is concentrated
in small patches in isolated sites that receive the influence of fog, usually on slopes with
south or southwest exposure (Schultz et al., 2011). These small patches are commonly
known as “fog oases” (Ellenberg, 1959, cited in Rundel et al., 1991) or “lomas vegetation”
(Rundel et al., 1997). Continuing south, up to around 26°S, vegetation increases but remains
dispersed. From there towards 30°S, the fog oases extend in a relatively closed formation
along the windward slope of the coastal mountain range (Rundel et al., 2007, cited in Schultz
et al., 2011).
The high frequency and intensity of the fog is a decisive ecological factor, to which the
existence and maintenance of fog oases is attributed. It acts in different ways: by protecting
plants from direct solar radiation and reducing evapotranspiration rates, maintaining low
temperatures and high atmospheric humidity, and also by providing water to certain taxa
adapted to the collection of fog water (Schultz et al., 2011).
The regeneration and replacement of the annual and perennial seed bank, however, is the
consequence of sporadic rains (Gutiérrez et al., 2000; Dillon and Rundel, 1990, cited in
Schultz et al., 2011). In years of heavy rains and high temperatures, associated to ENSO
(El Niño Southern Oscillation) years, propagule density in the soil seed bank increases 5 to
10 times, compared to normal years (Gutiérrez et al., 2000). This phenomenon occurs
approximately every 3.5 to 7 years (Dillon, 2005).
An increasing number of evidences indicate a decline in the oasis vegetation, especially in
the northernmost part of the coastal desert, which manifests in the loss of vitality of the
plants, withdrawal of individual populations, and probably also loss of species (Rundel et al.
1997; Muñoz-Schick et al. 2001; Pinto et al. 2001; Egaña et al., 2004; Pinto and Luebert,
2009; Schultz et al., 2011). Schultz et al. (2011), in their study of fog oases in Chile and Peru
between 2005 and 2009, conclude that this decline would be the product of recent changes
in the climate: less abundant and less frequent rainfall, and greater sunstroke caused by
less cloudiness in the area. They also mention local pollution as an additional factor in
vegetation degradation.
Since projections indicate that rainfall will continue to decline in the arid and semi-arid zone
of northern Chile towards the end of the 21st century (while temperatures may increase on
average) (Santibañez et al., 2017), the opportunity for an improvement in the conditions of
development of these plant formations is scarce, and on the contrary, they are expected to
continue receding (Schultz et al., 2011). Therefore, it is necessary to find ways to preserve
these ecosystems and species; an alternative is ex situ cultivation in Botanic Gardens, and
storage in seed banks (León-Lobos et al., 2012). Therefore, it is necessary to understand
the dormancy mechanisms and germination requirements presented by the stored seed
collections and their cultivation.
1.2 Germination in desert environments
Plants that grow in desert environments have a set of strategies that allow them to continue
existing in these complex habitats (Gutterman, 1994). The more extreme the desert, and the
more unpredictable and scarce the annual rainfall, the more important the complementary
adaptations and survival strategies during the different stages of the life cycle of a plant,
particularly for annual species (Gutterman, 2002).
Obviously, the availability of water, through rainfall, is the main limiting factor for the
germination, growth and productivity of plants in arid ecosystems (Noy-Meir, 1973; Baskin
and Baskin, 1998). There is a high variability in terms of quantity and temporality of rainfall
in desert ecosystems, which means that when a habitat is favourable for the germination of
seeds, and in particular for the completion of the life cycle of annual species, is unpredictable
(Baskin et al., 1993).
Gutterman (1994, 2000, 2002) has described two main types of dispersal and germination
strategies for annual species, based on observations in the Negev and Judea desert: the
“escape” strategy for seed dispersal and "opportunism" in germination; and "cautious"
dispersion and germination. Several species show combinations of these two extreme
strategies (Gutterman, 1994), demonstrating the high variability that exists in the effort to
secure the future of new generations of annual plants. For example, in a two-year study by
Loria and Noy-Meir (1980) on the demography of six species (Schismus arabicus,
Spergularia diandra, Erodium bryoniifolium, Gymnarrhena micrantha, Carrichtera annua,
Filago desertorum) in the Negev desert, he found that there were no two species identical
or even similar in terms of population distribution and behaviour: they all showed different
patterns of germination, dispersion, survival and seed production (size and shape), in time
and space (Loria and Noy-Meir, 1980, cited in Gutterman, 1994). Schwienbacher et al.
(2011) also studied the dormancy types present in 28 species of alpine plants (Austria), and
concluded that species in this alpine zone had different germination requirements, that is,
there was no "common" strategy or mechanism to all of them.
1.3 Dormancy mechanisms and their ecological importance
Seeds, the plant structures with the greatest capacity to withstand extreme environmental
conditions, eventually give way to seedlings, which are the most sensitive. For this reason,
it is very relevant for the survival of the species that fractions of the total seed population
germinate at the right time and place, especially for those that inhabit more extreme deserts
(Gutterman, 1994).
Dormancy can be defined as "an innate property of the seed that defines the environmental
conditions that must be met before the seed can germinate" (Finch-Savage and LeubnerMetzger, 2006). It is a quantitative genetic character that involves several genes and exhibits
continuous phenotypic variation (Baskin and Baskin, 2004). The induction of dormancy is
controlled both genetically and by the environment. Genetic control can be associated with
both the genotype of the embryo and of the mother plant (Contreras and Rojas, 2010); the
dormancy state is influenced by the environment in which the seed develops and matures
(maternal environment), but also by the external environment when dispersed, so it can
change over time (Finch-Savage and Leubner-Metzger, 2006). To break dormancy, and
depending on its type and level, a seed must undergo certain environmental factors for
minimum periods of time, which induce metabolic and structural changes within the seed
and eventually lead to germination (Bewley et al., 2013).
The dormancy of seeds in environments with high temporal variability, such as deserts, is
often described as a bet-hedging strategy, or bet diversification (Eberhart and Tielbörger,
2012). Not all mature seeds produced by a plant germinate immediately after dispersion, or
in the following season; in many cases, as a survival strategy for the species, a significant
amount of seeds remains dormant in the soil, for a period that can last for years or even
decades, forming what is known as a seed bank (Fenner, 1985, cited in Aguado et al., 2012).
There are essentially two types of soil seed banks: transitory, and persistent or permanent.
The first are those in which all seeds germinate or lose viability during the same year of
production; in the latter, a variable fraction of seeds germinates during the first year and the
rest remains viable for several more years (Aguado et al., 2012). Persistent banks are
considered one of the main survival strategies for desert plants, particularly for annual or
ephemeral species (Baskin and Baskin, 1998; Figueroa et al., 2004; Facelli et al., 2005).
According to Baskin and Baskin (1998, 2004), seed dormancy can be classified into: a)
physiological dormancy, which in turn has three levels of depth (non-deep, intermediate and
deep); b) morphological dormancy; c) morphophysiological dormancy (with several levels);
d) physical dormancy; and e) combined dormancy (physical and physiological). Table 3
summarizes the main characteristics of each type of dormancy.
Baskin and Baskin (1998), in a general review on the scientific knowledge about dormancy,
found that the percentage of species with seeds that have some type of dormancy, increased
from 40% in tropical rain forests to 84% in hot deserts (Gutterman, 2002). The most common
type of dormancy would be physiological, then physical.
Table 3. Types of dormancy, description and treatments to break dormancy, according to Baskin
and Baskin (1998, 2004).
Type of dormancy
Non-dormant (ND)
Physiological
dormancy (PD)
Morphological
dormancy (MD)
Description
A non-dormant seed is able to germinate at a specific time if the
environmental conditions are favourable; otherwise, it is said to be
dormant.
It is an inhibitory physiological mechanism from the embryo that prevents
the emergence of the radicle. The structures surrounding the embryo
(endosperm, testa, fruit walls) may also have a role in preventing
germination. There are three levels of physiological dormancy depth: non
deep, intermediate and deep; the first is the most common form of
dormancy, and it occurs both in gymnosperms and angiosperms. Non deep
PD is broken with relatively short periods of cold stratification (days to
weeks); intermediate PD needs longer periods of cold stratification
(months), and deep PD requires cold stratification for even longer periods.
The exogenous application of gibberellins could replace stratification in the
case of several species; in the case of deep PD, embryo removal would be
necessary for this application to take effect.
Embryos are differentiated (it is possible to distinguish radicle and
cotyledons), but not fully developed. For germination to take place, the
embryo needs time to complete its development. Hence, it is the
morphological characteristics of the embryo that prevent germination.
It is a combination of morphological and physiological dormancy, that is,
underdeveloped embryos that also have physiological dormancy. Two
things must happen so that a seed with MPD can germinate: (1) the embryo
Morphophysiological must grow to a critical and specific size (depending on the species); and
(2) physiological dormancy must be overcome. The key is to determine
(MPD)
what environmental condition each event can promote; for some species,
the same condition promotes embryo growth and dormancy rupture; in
others, specific conditions are required.
Physical (PY)
The impermeability of the testa (or walls of the fruit) to water is the main
reason why germination does not occur. All areas where water could enter
(micropyle, hilum, chalazal area) become impermeable. To break this type
of dormancy, the following methods are cited: cold stratification,
mechanical scarification, acid treatments, hot water (Ren and Tao, 2004)
and temperature treatments (Baskin and Baskin, 1998).
Combinada (CD)
An embryo with physiological dormancy of the non-deep type is added to
an impermeable testa. Germination does not occur until both types of
dormancy have been overcome.
There are several studies in which dormancy patterns have been investigated for different
populations of related species. Meyer and Monsen (1991) studied the mature seeds of 15
populations of mountain sagebrush (Artemisia tridentata spp. Vaseyana), which were
located in an altitudinal gradient. They were interested in finding out if it was possible to
predict germination responses in seeds from populations of different environments. They
found that in general, the seeds of more risky environments germinated more incompletely
and more slowly than those of less risky environments, that is, they were more dormant.
This variation in the germination response of collections from different environments could
be representing “germination ecotypes”, although a genetic study was not done; their results
suggest that, for this species, the variation increased the probability of seedling survival in
each habitat within a wide range (Meyer and Monsen, 1991).
Seed germination patterns of 135 populations (28 species and 13 sections) of the genus
Penstemon (Scrophulariaceae) were examined in the laboratory by Meyer et al. (1995). The
environments in which these seeds were found ranged from warm desert to alpine tundra.
Most species had dormant seeds at the time of dispersal and required a wet stratification
period to germinate. The response to stratification was related to the probable duration of
cold at the collection site; thus, populations of environments with severe winters produced
seeds with prolonged stratification requirements, while populations of environments with
mild winters produced seeds with short stratification requirements. The seeds that came
from intermediate altitude habitats, presented intermediate cold requirements, but there was
a considerable fraction that did not respond to any stratification period. Species with a wide
distribution in terms of habitats showed contrasting germination patterns among populations,
with a wide range of habitat-specific mechanisms. In fact, habitat-related patterns were
observed (similar mechanisms for the same habitat in seeds of different sections). The
common garden experiments showed a strong genetic basis for differences in germination,
among populations and also among individuals in a population. The Penstemon genus thus
shows that the multiple lineages within the genus have adapted and evolved in different
habitats with different dormancy mechanisms, specific to those environments (Meyer et al.,
1995).
Baskin and Baskin (2014) compile other studies that also investigate species with variation
in level/depth of dormancy in terms of their populations. For example, Japanese populations
of Cardamine hirsuta (Brassicaceae) had stronger dormancy (less germination) than those
from Europe (Kudoh et al., 2007, cited in Baskin and Baskin, 2014). Depth of dormancy in
16 ecotypes of Hordeum spontaneum (Poaceae) from Israel ranged from low in mesic sites
to high in dry sites (Yan et al., 2008, cited in Baskin and Baskin, 2014). In the case of
Biscutella didyma (Brassicaceae), dormancy increased with an increase in aridity and
decreased with an increase in soil moisture (Lampei and Tielborger, 2010, cited in Baskin
and Baskin, 2014).
There is little information on germination requirements or dormancy mechanisms in Chilean
desert species. Figueroa et al. (2004) mention that physical dormancy would be important
in species of the genus Prosopis (Fabaceae) and other legumes such as Caesalpinia,
Balsamocarpum, Adesmia and Cassia, and eventually also in Nolana (Solanaceae).
Physiological dormancy has been reported in Atriplex (Chenopodiaceae), Solanum
(Solanaceae) and Rhodophiala (Myostemma; Amaryllidaceae). For their part, Jara et al.
(2006) studied the behavior of six endemic and native herbaceous species of northern Chile
(Cistanthe salsoloides (Montiaceae), Leucocoryne purpurea (Amaryllidaceae), Pasithea
coerulea (Asphodelaceae), Placea amoena (Amaryllidaceae), Schizanthus litoralis
(Solanaceae) and Trichopetalum plumosum (Asparagaceae). The trials determined that for
all the species studied, the defined factors (hydration time, lighting, temperature and
scarification) and their interactions were highly significant, and that in fact the seeds of the
species had similar germination thresholds (Jara et al., 2006). The most important
conclusion of this study is that the combinations of factors with which the highest percentage
of seed germination was achieved, coincide with the environmental conditions of the sites
from where the species are native (Jara et al., 2006).
Given the scenario of decline of vegetation in the Chilean coastal desert, it is relevant to
increase the knowledge regarding germination requirements and dormancy mechanisms
that species present, and to study in particular whether there are patterns that would allow
prediction of their levels or depths of dormancy and therefore, their responses to germination
experiments.
1.3 The genus Nolana
Nolana is the fifth genus with more species in the Solanaceae family, after Solanum,
Lycianthes, Cestrum and Nicotiana (D’Arcy, 1991, cited in Dillon, 2005). It is composed of
89 species, of which 49 are distributed in Chile and 43 in Peru; four species have been
registered in both countries and one in the Galapagos Islands (Dillon et al., 2007a). Around
70 are considered endemic to oasis formations in Chile and Peru; 13 other species occur in
higher altitude habitats (> 1,000 m) and one of them, N. paradoxa, has an extensive
distribution from the center to the south of coastal Chile (33° S - 42° 30’S) (Dillon, 2005).
According to Dillon et al. (2009), the genus Nolana is the only one found throughout the
entire range of hill formations.
In addition to contributing to the knowledge of the regeneration dynamics of a group of plants
representative of the Atacama Desert, there are several reasons that make the study of the
Nolana genus particularly interesting: a) its high degree of endemism and presence in
habitats with extreme conditions of aridity and salinity, which gives it a high conservation
value (Tu et al., 2008; Dillon et al., 2009); b) the abundance of species with high ornamental
potential due to their foliage and flowering characteristics, such as N. acuminata, N.
aplocaryoides, N. baccata, N. balsamiflua, N. carnosa, N. coelestis, N. crassulifolia, N.
elegans, N. filifolia, N. linearifolia, N. parviflora, N. pterocarpa, N. ramosissima, N. reichei,
N. rostrata, N. rupicola, N. salsoloides and N. sedifolia (Freyre et al., 2005; Riedemann et
al., 2006); c) the presence, in at least one species, of compounds with fungicidal activity in
fungi of agricultural importance (N. sedifolia; Vio et al., 2012); and d) its relation with species
of economic importance, such as potato, tomato, pepper and petunia, which makes it a
possible source of genes of interest for development of cultivars better adapted to aridity
and/or salinity conditions. Despite these reasons, the genus Nolana have generally been
poorly studied: there are still many species for which relevant aspects of their reproductive
biology and establishment strategies are unknown. This knowledge is essential for their
conservation and potential use.
Nolanas, or "suspiros," as they are commonly known, are annual, perennial or shrubby
herbaceous species (Tu et al., 2008). Leaf succulence is a characteristic feature of these
species (Dillon et al., 2007a); their corollas can be regular, irregular or slightly zigomorphic,
with wide variations in their shape, size and colour (Dillon et al., 2007a). The most frequent
colour is lavender or blue, then white; in addition, in populations of species with bluish
flowers, sometimes individuals with white flowers occur (Dillon et al., 2007a). These species
may exhibit allogamy (obligatory or not) (Freyre et al., 2005) or autogamy.
Until relatively recently, the genus Nolana was considered as belonging to the Nolanaceae
family; Johnston, who described several Nolana species, noted in 1936 that there was an
obvious relationship with the Solanaceae family, but also an important difference in terms of
the nature and structure of the fruit. It is currently included in the Solanaceae family, after
analyzes based on chloroplastidial DNA, which also suggested that the Nolana genus would
be deeply inserted in the Solanoideae subfamily (Olmstead and Palmer, 1992; Olmstead et
al., 2008), to which tomato and pepper also belong.
Nolana is considered a monophyletic genus and is distinguished by the 5-15 carpellar ovary
that develops fibrous fruits (Knapp, 2002) that correspond to schizocarps (indehiscent fruit
originated by a gynoecium of two or more concrescent carpels; Font Quer, 2000), which split
up into mericarps when ripe (Fig. 2). Therefore, in Nolana, dispersal units are not seeds but
mericarps, which can vary in number between species from 2 to 30 per schizocarp and from
laterally united and multi-seeded to completely free and single-seeded (Tago-Nakazawa and
Dillon, 1999; Knapp, 2002). The seeds in the mericarp are separated by parenchymal tissue,
so that each one remains in an independent “embryonic chamber” (Saunders, 1936).
Although variable in size, shape and degree of fusion between species, the mericarp is a
convincing synapomorphy for Nolana (Dillon et al., 2007a), an evolutionary novelty that
allows the identification of the members of the group.
Schizocarp
Mericarp
Fig. 2. General scheme of schizocarp and mericarp in Nolana (taken from Saunders, 1936).
As for germination experiments, little is known for Nolana species. However, Freyre et al.
(2005) and Douglas and Freyre (2006) mention that germination occurs when the
“germination plugs” are opened in the mericarps of eight Nolana species, including N.
adansonii, N. aticoana, N. humifusa, N. laxa, N ivaniana, N. plicata, N. elegans and N.
rupicola; and that the germination percentage increased when adding gibberellic acid, but
not with chemical treatments (H2SO4, KNO3, HNO3 and ethephon). Cabrera et al. (2015)
also obtained a higher germination percentage with scarification in a specific area of the
mericarp (funicular scar) and addition of gibberellic acid. After-ripening treatment, under
specific conditions of temperature and relative humidity, could also have an effect on
germination (Douglas and Freyre, 2006).
Hypothesis
Similarity between species is expected in species of Nolana in terms of type and level of
dormancy that their seeds present; the similarity would be greater in more closely related
species. In addition, there should be a relationship between the level of dormancy and the
range of distribution (or niche diversity) for the species of the genus, with species with a
wider distribution presenting a lower dormancy than species whose distribution is more
restricted.
Objectives
a. General objective
To characterize and relate the dormancy of seeds of twelve Nolana species (Solanaceae)
from different populations of the coastal desert of Atacama, Chile, according to habitat
diversity and phylogeny.
b. Specific objectives
1. To establish the germination requirements of twelve Chilean species of the genus
Nolana (Solanaceae) in different populations along the Atacama coastal desert.
2. To determine the existence of dormancy patterns in terms of habitat diversity and
phylogeny.
3. To examine the anatomy and morphology of fruits of twelve species of the genus
Nolana.
4. Characterize the different stages in the fruit development of a selected Nolana
species.
Species under study
A phylogenetic estimate for Nolana, using a combination of molecular markers, has been
reconstructed by Dillon et al. (2007, 2009) and Tu et al. (2008). This phylogeny supports the
monophyly of the genus and has identified several clades supported by geographic
distributions and morphological synapomorphies (Dillon et al., 2009). According to these
analyses, the genus is of Chilean origin, with two different introductions to Peru and
subsequent radiation (Dillon et al., 2009). Of the seven clades identified, four are confined
to Chile and three are mainly Peruvian with some presence in Chile (Fig. 3). A brief
description of each clade, and a detail of the twelve species that were the focus of this study,
will be given below.
Fig. 3. Biogeographic diversification of Nolana, modified from Dillon et al. (2009), showing the clade
of the 12 studied Nolana species. C, Chile; P, Peru; G, Islas Galápagos, Ecuador.
•
Clade G (Chile): N. aplocaryoides, N. crassulifolia, N. divaricata, N. linearifolia, N.
onoana, N. sedifolia.
Clade G has recovered a strictly Chilean group of 13 species represented by small to
moderate shrubs (traditionally called "Dolia") and annuals, all with highly reduced corollas,
often white or yellowish (Dillon et al., 2009) but some have bluish flowers.
-
N. aplocaryoides (Gaudich.) I.M. Johnst. (Fig. 4) is an annual herb, erect and
branched, with uniseriate glandular hairs (Mesa, 1981). Endemic to northern Chile,
it grows between the regions of Tarapacá and Atacama, in plains in full sun (Zuloaga
et al., 2008). Its flowers are small, with an infundibuliform tubular corolla (funnelshaped), white, lilac or light blue (Mesa, 1981; Riedemann et al., 2006).
Fig. 4. N. aplocaryoides. Habitat context and growth habit.
-
N. crassulifolia Poepp. (Fig. 5) is an endemic prostrate shrub that forms mats on
rocks near the sea. Abundant in the Valparaiso region, it is also found in Atacama
and Coquimbo regions (Zuloaga et al., 2008). Leaves can be spathulate or slender
and linear, 15-25 mm long; pedicels are normally 3-8 mm long (Johnston, 1936),
corollas white.
Fig. 5. N. crassulifolia. Habitat context and growth habit.
-
N. divaricata (Lindl.) I.M. Johnst. (Fig. 6) usually forms a dense succulent globose
bush 1-2.5 m tall and seems to be confined to the hills near the sea (Johnston, 1936),
in the regions between Antofagasta and Coquimbo (Zuloaga et al., 2008). Leaves
can be broadly spathulate or clavate, quite glabrous; corollas are usually purple
(Johnston, 1936).
Fig. 6. N. divaricata. Habitat context and growth habit.
-
N. linearifolia Phil. (Fig. 7) is a trailing, herbaceous annual plant, although it can also
behave as persistent. It is only found in the northern regions of Antofagasta and
Atacama, in Chile (Zuloaga et al., 2008). The delicate, bright blue tubular corollas of
this species have lobes deeply notched, so it appears to be 10-lobed (Johnston,
1936).
Fig. 7. N. linearifolia. Habitat context and growth habit.
-
N. onoana M.O. Dillon & Nakazawa (Fig. 8) is a robust annual tap-rooted herb to 1
m tall; stems erect to ascending, but decumbent when densely leafy. Corollas are
narrowly infundibuliform, lavender to light blue (Dillon et al., 2007b). Its distribution
covers only the Antofagasta region (Zuloaga et al., 2008).
Fig. 8. N. onoana. Habitat context and growth habit.
-
N. sedifolia Poepp. (Fig. 9) is an endemic perennial shrub or sub-shrub that can
measure up to 100 cm tall, with very small and succulent leaves, and small, axillary,
white and tubular corolla (Dillon et al., 2007; Mesa, 1981). It has an extensive
distribution between the regions of Tarapacá and Valparaíso (Zuloaga et al., 2008).
Fig. 9. N. sedifolia. Habitat context and growth habit.
•
Clade F (Peruvian and Chilean): N. intonsa
Clade F is comprised of 18 Peruvian species and one Chilean species (N. intonsa), which
shares strong morphological resemblance to southern Peruvian species, such as N. pallida
(Dillon et al., 2009).
-
N. intonsa I.M. Johnst (Fig. 10) is an annual or perennial plant, with pubescent leaves
and whitish, long hairs. The flowers are solitary and have a whitish, lanuginous calyx,
and an infundibuliform corolla 18 to 23 mm long, purple with a deep-purple throat
(Mesa et al., 1998). It is another Chilean endemic, only found in the Tarapacá region
(Zuloaga et al., 2008).
Fig. 10. N. intonsa. Habitat context and growth habit.
•
Clade C (Chilean): N. carnosa, N. rostrata
Clade C has recovered a monophyletic group comprised of 6 species traditionally
classified as Alona. These are all large-flowered shrubs confined to Chile (Dillon et al.,
2009).
-
N. carnosa (Lindl.) Miers ex Dunal (Fig. 11) is a compact, densely and ascendingly
branched bush with large fruit and flowers. Sessile leaves hug the stem, although
not completely. It is a plant of the sandy coastal plains (Johnston, 1936), growing
only in Chile in the Tarapacá region and Atacama region (Zuloaga et al., 2008).
Fig. 11. N. carnosa. Habitat context and growth habit.
-
N. rostrata (Lindl.) Miers ex Dunal (Fig. 12) is a very slender, loosely branched,
sprawling shrub. The sessile leaves also hug the stems but are less dense than in
N. carnosa; they can be short or long. Corolla is normally blue, but there is also an
albino form (Johnston, 1936). It has an extense distribution, only in Chile, between
Tarapacá and Coquimbo regions (Zuloaga et al., 2008).
Fig. 12. N. rostrata. Habitat context and growth habit.
•
Clade B (Chilean): N. paradoxa, N. jaffuelii.
Clade B, previously recognized as a segregate genus, Sorema, currently consists of two
sub-groups: N. pterocarpa-N. baccata-N. parviflora, which are erect annuals with small
flowers; and N. paradoxa-N. rupicola, which are rosette-forming, taprooted plants with larger
flowers (Dillon et al., 2009). These species are only present in Chile (Dillon et al., 2009).
-
N. jaffuelii I.M. Johnst (Fig. 13) is an annual, glabrescent (almost hairless) plant; with
succulent leaves, oblanceolate, adorned by small vesicles. The corolla of the flower
is infundibuliform, violet, 13-20 mm long; the calyx is 8-10 mm long (Johnston, 1936).
It has been reported for Chile and Peru; in Chile, it is found between Tarapacá and
Antofagasta regions (Zuloaga et al., 2008).
Fig. 13. N. jaffuelii. Habitat context and growth habit.
-
N. paradoxa Lindl. (Fig. 14) is an annual, succulent species with a deep fleshy taproot; its elongate stems are prostrate and freely and loosely branched; the corolla
doubles the calyx in length, and its colour is light blue with a white throat (Reiche,
1910). This is the sole member of the family south of the Valparaíso area; it ranges
along the whole coast of central Chile south to Chiloe, and grows exclusively on the
sandy sea-shore (Johnston, 1936).
Fig. 14. N. paradoxa. Habitat context and growth habit.
-
N. parviflora (Phil.) Phil. (Fig. 15) is a decumbent annual with small inconspicuous
white corollas (Johnston, 1936). Being an endemic, it is found in Chile between
Antofagasta and Atacama regions (Zuloaga et al., 2008).
Fig. 15. N. parviflora. Habitat context and growth habit.
Characterization of seed collecting sites
The collection sites (Fig. 16) for the twelve species studied correspond to:
-
Alto Patache Fog Oasis, Tarapacá region: the cliff reaches 800 masl. The
development of ephemeral vegetation begins at 250 m, and it is possible to observe
small shrubs and cacti over 600 m (Pinto and Luebert, 2009). In years when there
are rains (very rare), it is also possible to find plants in the inner plateau (Pliscoff et
al., 2017).
-
Antofagasta hills, Antofagasta region: located in close vicinity to the city of
Antofagasta and near La Chimba National Reserve, which presents a high diversity
of species in very specific spots, and is threatened by human activities. It is possible
to find here a great profusion of annual species that appear in rainy years.
-
Pan de Azúcar National Park, Atacama region: it is characterized by an
underdeveloped coastal plain, which gives rise to a coastal cliff with a maximum
height of 800 masl, dissected by numerous ravines. The flora of the park is the typical
predominant in the arid zones. In the sectors near the coast, there is a greater
diversity of vegetation, due to the presence of coastal fog (CULTAM, 2014).
-
Llanos de Challe National Park, Atacama region: main scenario of the blooming
desert phenomenon on the coast, there are more than 220 different species, of which
206 are native (CONAF, 2019).
-
Punta de Choros, Coquimbo region: located next to the Pingüino de Humboldt
National Reserve, it corresponds to a coastal sector with presence of shrubs growing
very close to the sea.
-
Los Molles, Valparaíso region: located in the transition zone between the dominant
sclerophyllous scrub in Coquimbo and the sclerophyllous forest characteristic of the
vegetation of central Chile (Lund and Teillier, 2012), it is considered a biodiversity
hotspot and hosts several species in threat categories (Lund and Teillier, 2012).
Although it is located to the south of the area considered part of the coastal desert,
it is in the Valparaíso region, which is the southern limit of distribution for some
Nolana species (N. crassulifolia, N. elegans and N. sedifolia).
Fig. 16. Distribution map of seed collection showing locations (in yellow) and reference cities (in red).
Map made using Google Earth.
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nuclear LEAFY second intron. Molecular Phylogenetics and Evolution 49: 561-573.
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(Ed). Nairobi, Kenya.
Vio-Michaelis, S., G. Apablaza-Hidalgo, M. Gómez-Ungidos, R. Peña-Vera, and G.
Montenegro-Rizzardini. 2012. Antifungal activity of three Chilean plant extracts on
Botrytis cinerea. Botanical Sciences 90: 1-5.
Weischet, W. 1975. Las condiciones climáticas del desierto de Atacama como desierto
extremo de la tierra. Norte Grande 1: 363-373.
Zuloaga, F.O., Morrone, O. & M.J. Belgrano. 2008. Catálogo de plantas vasculares del Cono
Sur (Argentina, sur de Brasil, Chile, Paraguay y Uruguay). Missouri Botanical Garden
Press.
St.
Louis,
Missouri,
USA.
3884
pp.
Disponible
en
http://www2.darwin.edu.ar/Proyectos/FloraArgentina/FA.asp
Chapter II. Characterization of seed dormancy of twelve Chilean species of
Nolana (Solanaceae) from the coastal Atacama Desert
Josefina Hepp1,2, Miguel Gómez1,2, Pedro León-Lobos3, Gloria Montenegro1,2, Samuel
Contreras1,2
1
Departamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería Forestal,
Pontificia Universidad Católica de Chile
2
Centro del Desierto de Atacama, Pontificia Universidad Católica de Chile
3
Centro Regional de Investigacion La Platina, Instituto de Investigaciones Agropecuarias,
INIA
Corresponding author. Tel.: +562 2354 4112
E-mail address: scontree@uc.cl (S. Contreras), pleon@inia.cl (P. Leon-Lobos)
Postal address: Facultad de Agronomía e Ingeniería Forestal, Vicuña Mackenna 4860,
Macul, Santiago, Chile.
Abstract
The genus Nolana (Solanaceae) comprises numerous species endemic to the coastal
Atacama Desert of Chile and Peru, of high potential ornamental and conservation value.
These species could face adverse environmental conditions in the future, so in order to
support conservation efforts, it is necessary to understand their germination requirements
and characterize their dormancy. Different treatments were performed on mericarps of 12
species of Nolana: control (intact seeds embedded in distilled water), scarification in
germination plug and distilled water, and scarification in germination plug and addition of
GA3 (500 ppm); their permeability to water was also tested. It was determined that the
species did not present physical dormancy, as had been previously reported, but rather
physiological dormancy. Germination results after treatments were not homogeneous
among all twelve species, indicating differences in their dormancy levels. Also, the important
role of the endosperm in the prevention of germination for the studied Nolana species was
highlighted. Regarding the relationship between the level of physiological dormancy
(expressed as the percentage of germination for the most successful treatment) and the
latitudinal distribution of the species or their phylogenetic closeness, it was determined that,
for the studied species, their proximity in terms of clades was more relevant than their
latitudinal distribution.
Keywords: mericarp, physiological dormancy, germination, endosperm
Introduction
Dormancy of seeds in environments with high temporal and spatial variability, such as
deserts, is often described as a bet-hedging strategy (Eberhart and Tielbörger, 2012). Not
all mature seeds produced by a plant germinate immediately after dispersal, or in the
following season; in many cases, as a survival strategy for the species, a significant amount
of seeds remains dormant in the soil, for a period that can last for years or even decades
(Aguado et al., 2012), waiting for favourable conditions for seedling establishment. The more
extreme the environmental conditions, the greater the percentage of plant species that
exhibit dormant seeds (Gutterman, 2002).
The Atacama Desert is considered the most arid in the world; the hydrological balance
(balance between the input of water to the system by precipitation, and outputs by
evaporation/evapotranspiration) is the most negative when compared with that of other
deserts (Weischet, 1975). Along the coast it has a temperate climate, with constant
presence of fog produced by cold currents in the Pacific Ocean (Shmida, 1985). The high
frequency and intensity of fog is a decisive ecological factor, to which the existence and
maintenance of vegetation formations known as "fog oases" (Ellenberg, 1959, cited in
Rundel et al., 1991) or "lomas vegetation" (Rundel et al., 1997) is attributed.
In the coastal desert of Chile and Peru, the Solanaceae family is represented by 18 genera
and 128 species and is the second family with better representation in the lomas vegetation
(Dillon, 2005). The genus Nolana L. ex L. f. (Solanaceae) currently comprises 90 species of
arid and semi-arid environments in Chile and Peru, of which 46 are exclusively in Chile and
another three are shared with Peru. They are annual or perennial herbaceous plants or small
shrubs with leaves with different degrees of succulence and flowers of different colors and
sizes, tubular and hermaphrodites (Dillon et al., 2007; Tu et al., 2008; Dillon et al., 2009).
Nolana is the only genus found in the whole range of lomas formations, where it stands out
as the most conspicuous floristic element (Tago-Nakazawa and Dillon, 1999). Some Nolana
species are important components of the blooming desert phenomenon, associated with
ENSO (El Niño Southern Oscillation) events of short periods of heavy rainfall and relatively
high temperatures (Dillon, 2005) that occur every 3 to 7 years, which lead to massive
blooming episodes that replenish soil seed banks for annual and perennial plants (TagoNakazawa and Dillon, 1999; Dillon, 2005). Several of these species have high ornamental
potential due to their foliage and flowering characteristics (Freyre et al., 2005; Riedemann
et al., 2006; Fig. 1), and also high conservation value (Tu et al., 2008, Dillon et al., 2009)
given that they are endemic to habitats with extreme conditions of aridity and salinity.
Dillon et al. (2009) identified several strongly supported clades within the Nolana genus, with
geographic and morphological fidelity, four of which are confined to Chile and three are
mainly Peruvian with some presence in Chile. The majority of the species grows
preferentially in the arid and semiarid zones of coastal Chile and Peru, although some
species are found in high altitude habitats (> 1,000 m a.s.l.) and a few of them, i.e. N.
paradoxa and N. sedifolia, have distributions that range over 1,000 km (Johnston, 1936).
The Solanaceae family has been included among the families that present physical
dormancy (Baskin et al., 2000); however, Baskin et al. (2000) have not yet found evidence
that representatives of this family present this type of dormancy. Physiological non-deep
dormancy, apparently induced by the endosperm and the testa, has been detected in
Solanaceae (Finch-Savage and Leubner-Metzger, 2006). For Nolana, Freyre et al. (2005)
and Douglas and Freyre (2006), working in Nolana paradoxa and N. aplocaryoides among
others, mention that germination occurs when opening a "funicular plug” or “germination
plug” in mericarps, and that the percentage of germination increases when adding gibberellic
acid, but not with chemical treatments (H2SO4, KNO3, HNO3 and ethephon). Cabrera et al.
(2015) also obtained a higher percentage of germination in N. jaffuelii with specific
scarification in the funicular scar area (which indicates the location of the germination plug),
and application of gibberellic acid, reporting for the studied species dormancy of the physical
and physiological type. For the other studied species, no references have been found
regarding their dormancy.
A growing body of evidence indicates a decline in the lomas vegetation or fog oases of the
coastal desert, which is manifested in loss of vitality of the plants, receding individual
populations, and probably also loss of species (Rundel et al. 1997; Muñoz-Schick et al.
2001; Pinto et al. 2001; Egaña et al., 2004; Pinto and Luebert, 2009; Schultz et al., 2011).
Since projections indicate that rainfall will continue decreasing in the arid and semi-arid area
of northern Chile by the end of the century, while temperatures could rise on average
(Santibañez et al., 2017), the opportunity for an improvement in the development of these
ecosystems is low, and conversely, they are expected to continue deteriorating (Schultz et
al., 2011). It is crucial to establish the germination requirements of the species, and their
possible dormancy mechanisms, so that its storage in seed banks and subsequent
propagation for in situ restoration or ex situ cultivation, can be successful and help preserve
them and the ecosystems in which they live (León-Lobos et al., 2012).
For certain species, it has been found that there is a relationship between dormancy level
and habitat (Penstemon spp., Scrophulariaceae, in Meyer et al., 1995); or between
dormancy level and phylogenetic closeness (several species in Dayrell et al., 2016).
However, for Nolana such relationships have not been established.
Therefore, the objective of this study was to characterize the type of dormancy of twelve
Nolana species present in the coastal desert of Chile, to determine their germination
requirements, and to evaluate if there is any relationship between level of dormancy and
geographical distribution or phylogeny of the species.
Materials and methods
2.1 Plant material
Mericarps of 12 species of Nolana (Fig. 1) were collected between 2015 and 2016 from
individual plants at maturity and in their dispersal phase at different locations (Table 1) and
kept in labelled paper bags. Whenever possible, mericarps were collected randomly within
the population from at least 50 different individuals, to obtain a representative sample for
each species (León-Lobos et al., 2003). Fruits were then kept in paper bags, partially dried
(they were put inside jars with equal weight of silica gel, for 2 to 3 days) and stored at 20°C
and 40% RH until used for analysis. Selection of species is the result of availability of suitable
fruits (i.e. mature mericarps, black/brown in colour and with dry or senescent calyx) in the
field, which explains the overrepresentation of certain clades.
Figure 1. Context, flowers and fruits of four Nolana species included in this study. A, B and C: N.
divaricata (clade G); D, E and F: N. intonsa (clade F); G, H and I: N. carnosa (clade C); J, K and L:
N. jaffuelii (clade B).
Table 1. Species of the genus Nolana selected in the study, latitudinal distribution in Chile, collection
site and clade (Flora del Conosur, 2014 and Enciclopedia de la Flora Chilena, 2014).
1
Species
Latitudinal
distribution*
Collection site
Nolana aplocaryoides
(Gaudich.) I.M. Johnst.
22° 4'S / 27° 4'S
Pan de Azúcar, Atacama
(26°08.786'S, 70°39.943'W)
Clade*
G
Llanos de Challe, Atacama
(28°11.086'S, 71°09.751'W)
2
26° 21.054'S / 33°
Nolana crassulifolia Poepp.
55.002'S
3
Nolana divaricata (Lindl.)
I.M. Johnst.
23° 30.582'S / 30° 54'S
4
Nolana linearifolia Phil.
Hills of Antofagasta,
21° 55.98'S / 26° 1.998'S Antofagasta (23°30.133'S,
70°23.069'W)
5
Nolana onoana M.O. Dillon
& Nakazawa
23° 12'S / 23° 51.738'S
6
Nolana sedifolia Poepp.
Punta de Choros, Coquimbo
(29°14.786'S, 71° 28.021'W)
Punta de Choros, Coquimbo
(29°14.601'S, 71°27.840'W)
Llanos de Challe, Atacama
(28°11.991'S, 71°09.524'W)
Hills of Antofagasta,
Antofagasta (23°29.903'S,
70°21.817'W)
Alto Patache fog oasis,
Tarapacá (20°49.542'S,
70°09.392'W)
19° 55.998'S / 33°
5.922'S
Los Molles, Valparaíso (32°
14.444'S, 71°31.133'W)
Nolana intonsa I.M. Johnst.
19° 55.998'S
25.002'S
8
Nolana rostrata (Lindl.)
Miers ex Dunal
18° 49.998'S / 30° 3'S
Llanos de Challe, Atacama
(28°06.806'S, 71°09.257'W)
9
Nolana carnosa (Lindl.)
Miers ex Dunal
26° 9'S / 28° 35.094'S
Llanos de Challe, Atacama
(28°02.612'S, 71°07.022'W)
10 Nolana jaffuelii I.M. Johnst.
20° 13.002'S / 26° 9'S
Alto Patache fog oasis,
Tarapacá (20°48.903'S, 70°
09.630'W)
11 Nolana paradoxa Lindl.
29° 54'S / 43° 21'S
Los Molles, Valparaíso
(32°14.433'S, 71°31.133'W)
27° 4.002'S / 28°
13.002'S
Hills of Antofagasta,
Antofagasta (23°30.133'S,
70°23.069'W)
12
Nolana parviflora (Phil.)
Phil.
/
21°
Alto Patache fog oasis,
Tarapacá (20°49.309'S,
70°09.366'W)
7
*Letters for clades according to Dillon et al., 2009; a brief description of clades is given in Figure 2.
F
C
B
Figure 2. Biogeographic diversification of Nolana, modified from Dillon et al. (2009), showing the clade
of the 12 studied Nolana species. C, Chile; P, Peru; G, Islas Galápagos, Ecuador.
Seed viability
A tetrazolium test with 2,3,5-triphenyl-2H-tetrazolium chloride (Tz) was performed on a
sample of 25 mericarps for each species, to evaluate the viability of seed lots. This was done
in a separate sample of seeds, and not in the same mericarps after finishing the germination
experiments, given the extension of the experiments and the possibility of fungal damage.
Mericarps were scarified (germination plug was removed) and stained with 1% Tz solution
for approximately 24 hours at 30° C. After that time, mericarps were cut in halves and
surfaces were observed using a magnifier. Only seeds that were completely stained red
(embryo and endosperm) were considered viable.
Seed imbibition
To evaluate if water was able to penetrate and reach the embryo in intact mericarps,
methylene blue (1gr/100 mL) was used. Twenty seeds of 12 species were left in metal
containers with enough dye to cover them (the largest were covered in half), and 5 were
evaluated after 3, 24, 48 and 196 hours (room temperature). They were washed with distilled
water and dried with paper towel, then allowed to air dry for 20 minutes at room temperature.
Finally, stained seed were sectioned using a scalpel and observed under a stereoscopic
magnifier Olympus SZ2-ILST (Olympus Corporation, Tokyo, Japan), making sure that the
cut allowed to see the plug and half the embryo (longitudinal cut).
Evaluation of germination and characterization of dormancy
Evaluation of germination for the twelve species was performed in three experiments.
Mericarps were placed in 9 cm diameter Petri dishes, over three layers of filter paper,
saturated in distilled water or a solution of gibberellic acid (GA 3). Plates were hermetically
sealed with Parafilm to prevent drying and were watered as needed. Physiological
germination (radicle emergence over 2 mm) was evaluated. Results were reported as total
germination percentage.
Experiment 1. Role of funicular plug on germination
In order to make a comparison regarding the type and level of dormancy between the
different species, the same germination experiments were carried out on all the studied
species. Germination tests were carried out during 2015 and 2016, using seeds from ten
Nolana species collected in the previous months. Seeds were randomly assigned to one of
the following treatments: (1) Control (Ct), intact mericarps imbibed in distilled water; (2)
scarification (Sc), removal of germination plug and imbibition in distilled water; (3) Sc and
imbibition in a solution of 500 ppm GA3 (Sc+GA3). A scalpel was used to dissect the pericarp
at the funicular scar area avoiding radicle damage, as this proved to be the most effective
treatment for the germination of mericarps of N. jaffuelii (Cabrera et al., 2015). Germination
was evaluated three days a week during 45 days in a chamber at constant 20ºC (40% RH),
in four replicates of 25 seeds each.
Experiment 2: Additional experiment (N. linearifolia): a single species was selected due to
the number of available mericarps, and the following germination tests were performed: (1)
Control, intact mericarps imbibed in distilled water; (2) 500 ppm GA 3, intact mericarps
imbibed in gibberellic acid at 500 ppm; (3) 1000 ppm GA3, intact mericarps imbibed in
gibberellic acid at 1000 ppm; (4) Plug scarification, removal of germination plug, and
imbibition in distilled water; (5) Plug scarification with partial removal of endosperm, imbibed
in distilled water; (6) Plug scarification + 500 ppm GA3, scarified mericarps with partial
removal of endosperm imbibed in gibberellic acid (500 ppm GA3); (7) 10/25, intact mericarps
at alternating temperatures of 10/25°C, imbibed in distilled water; (8) 10/25 + GA 3, intact
mericarps at alternating temperatures of 10/25°C, imbibed in gibberellic acid (500 ppm GA 3);
and (9) Cold stratification (5°C) of intact mericarps for 4 months. Germination was evaluated
three times a week in four replicates (25 seeds each) per treatment, during 45 days in a
chamber at constant 20ºC (unless otherwise noted).
Experiment 3. Role of endosperm on germination
Based on observations after Exp. 1 and the further tests on N. linearifolia, a third set of
germination tests were performed using mericarps from species that had high germination
percentage, and two additional species (N. aplocaryoides and N. paradoxa) that were
collected and included afterwards, in order to better understand the role of endosperm in the
germination of seeds. Three treatments were defined: (1) Control (Ct), intact mericarps
imbibed in distilled water; (2) scarification (Sc), removal of germination plug and imbibition
in distilled water, as done for Exp. 1; and (3), Sc and partial removal of endosperm, and
imbibition in distilled water (Sc+en). Germination was evaluated three times a week in four
replicates (25 seeds each) per treatment, during 45 days in a chamber at constant 20ºC.
The constant temperature of 20°C was chosen for several reasons: (1) it was necessary to
have the same set of experiments to apply to all species; (2) the moderate temperatures of
the Chilean coast, on average, are around 20°C for the months in which germination occurs
(Fick & Hijmans, 2017); and (3) the constant temperature of 20°C was effective in the
treatments performed by Cabrera et al. (2015).
Statistical analysis
The effects of treatments on germination percentages were analyzed statistically using the
general linear model (GLM) procedure of the SAS program (SAS Institute, Cary, NC, USA).
An analysis of variance (ANOVA) was used to determine the existence of significant
differences between treatments. When significant differences were detected (p< 0.05),
Least Significant Difference (LSD; α=0.05) test was used to detect significant differences in
the comparison between pairs of treatments. Before the analysis, germination percentages
were transformed to the arcsin of the square root of the fraction value, but untransformed
data are presented in the results and used for discussion.
Results and discussion
3.1 Methylene blue staining
In Nolana, dispersal units are fruits called mericarps, which can vary in number between
species from 2 to 30 per schizocarp and from laterally united and multi-seeded to completely
free and single-seeded (Tago-Nakazawa and Dillon, 1999; Knapp, 2002). Seeds within the
mericarp remain in independent chambers (Saunders, 1936) and are firmly embedded within
the fruit. To facilitate analysis, in this study we consider only one seed per mericarp, so
“seed” will be the unit in which results are expressed.
The testa of the seed, and also certain tissues of the fruit (such as the pericarp), can prevent
germination for a considerable period of time by being impervious to water and preventing
its passage to the embryo (Baskin et al., 2000; Finch-Savage and Leubner-Metzger, 2006).
The impermeability may be caused by one or more layers of palisade cells in the testa, or
by sclereids with lignified secondary walls present in fruit tissues (Bewley et al., 2013). A
thick layer of sclereids was identified by Cabrera et al. (2015) in Nolana jaffuelii mericarps;
however, when embedding intact mericarps of different Nolana species in methylene blue,
we found that the water was able to enter until reaching the embryo for all the mericarps and
species, usually within 48 hr since imbibition started (Fig. 3) and confirmed that the entrance
route of the water is the germination plug. Therefore, there would be no impermeability to
water and therefore, it does not correspond to physical dormancy, since this type of
dormancy is defined as impermeability of the fruit or seed to water (Baskin and Baskin, 2004,
2014).
Figure 3. Longitudinal sections of intact mericarps (with germination plug) of N. divaricata, N. jaffuelii,
N. linearifolia and N. sedifolia at 3 hours, 24 hours, 48 hours and 8 days after imbibition in methylene
blue. Abbreviations: em: embryo; gp: germination plug; ra, radicle; * indicates regions which have
imbibed.
3.2 Evaluation of germination and characterization of dormancy
Role of funicular plug on germination
Seeds with physiological dormancy (PD) are permeable to water and possess a
physiological inhibiting mechanism that prevents radicle emergence (Baskin and Baskin,
2014), which could also be associated to structures surrounding the embryo (endosperm,
testa or fruit walls) (Baskin and Baskin, 2004). In hot semideserts and deserts, PD is the
most common type of seed dormancy for shrubs, perennial succulents, herbaceous
perennials and annuals (Baskin and Baskin, 2014). Three levels of PD (non-deep,
intermediate and deep) have been distinguished according to strength of the physiological
inhibiting mechanism, response to gibberellic acid and dormancy-breaking requirements
(Baskin and Baskin, 2014).
Our results indicate that seeds of the studied species present physiological dormancy,
following the definition by Baskin and Baskin (2004, 2014). Germination results after the first
experiment (expressed in different germination percentages in response to treatments)
indicate significant differences on percentages between control, scarification (removal of
germination plug) and scarification+GA3 treatments (p value <.0001) for each of the species,
indicating that the germination plug indeed plays a role on germination of Nolana species.
Therefore, it was confirmed that the best treatment for all species corresponds to a cut in
the germination plug and the addition of gibberellic acid. The only exception was N. intonsa,
which did not present germination with any of the treatments applied (Table 2).
Species also showed varying levels/degrees of depth in terms of their seed dormancy. This
has also been reported by other studies on seeds of species belonging to one same genus
(Van Assche et al., 2002 in Rumex (Polygonaceae); Barreto et al., 2016 in Stachytarpheta
(Verbenaceae), Giorni et al., 2018 in Xyris (Xyridaceae)). In our case, differences among
species ranged from 0 to 62% (Table 2). Species that in general had higher germination
percentages were those belonging to Clade G, then those of Clade B, and finally those of
Clade C. Species of Clade F (N. intonsa) did not register germination.
It is important to take into account the seed viability data (Table 2) given by the tetrazolium
test to correct some germination percentages. In general, the percentages remain relatively
stable for the species, except for N. crassulifolia, which increases significantly, as for N.
divaricata and N. sedifolia.
Table 2. Germination percentages of mericarps from 13 accessions of 10 Nolana species in response
to three treatments: (1) Control (Ct), intact mericarps imbibed in distilled water; (2) scarification (Sc),
removal of germination plug and imbibition in distilled water; (3) scarification and imbibition in a
solution of 500 ppm GA3 (Sc+ GA3). Percentage of seed viability was determined by tetrazolium test
(1%) of 25 mericarps per species.
Species
Clade
N. crassulifolia
G
N. crassulifolia*
G
N. divaricata
G
N. divaricata*
G
N. linearifolia
G
N. onoana
G
N. sedifolia
N. sedifolia*
N. intonsa
G
G
F
N. carnosa
C
N. rostrata
C
N. jaffuelii
B
N. parviflora
B
Collection site
Punta de
Choros
Llanos de
Challe
Llanos de
Challe
Punta de
Choros
Antofagasta
hills
Antofagasta
hills
Los Molles
Alto Patache
Alto Patache
Llanos de
Challe
Llanos de
Challe
Alto Patache
Antofagasta
hills
Ct1 (%)
Sc1 (%)
Sc+GA31
(%)
Viable
seeds
(%)
p value2
16 b
16 b
34 a
60
0.0683
1b
40 a
62 a
65
0.0038
0b
23 a
35 a
50
<0.0001
0b
4b
37 a
91
0.0004
3b
3b
55 a
96
<0.0001
15 b
25 b
54 a
75
0.0012
0b
0c
0
24 a
25 b
0
30 a
53 a
0
38
61
40
0.0008
<0.0001
0.4053
0b
2 ab
13 a
96
0.0457
0b
0b
17 a
76
<0.0001
0c
5b
27 a
88
<0.0001
0b
19 a
25 a
84
0.0011
1
For each species and collections, values with different letters are significantly different according to
a LSD test (α= 0.05)
2
P value from an analysis of variance.
* For N. crassulifolia, N. divaricata and N. sedifolia, data from two different populations are presented.
It was not possible to ascribe a level of physiological dormancy to each species, since the
experiments did not focus on determining the level of each but rather establishing
comparisons between species. However, it is possible to associate a less deep level of PD
in the case of some species which showed germination after imbibition in distilled water
(control treatment), such as N. onoana and N. crassulifolia; others with a more intermediate
level of PD (N. divaricata, N. linearifolia, N. sedifolia, N. jaffuelii, N. parviflora), which showed
germination after scarification and the addition of gibberellic acid; and finally, some that
hardly germinate with any of the treatments, which would correspond to a deeper dormancy
(N. carnosa, N. intonsa, N. rostrata) (Baskin and Baskin, 2004). Further experiments, taking
into account varying periods of cold stratification, need to be performed in order to stablish
more accurate degrees or levels of PD.
To add to the analysis, for those species for which it was possible to do so (N. crassulifolia,
N. divaricata and N. sedifolia), germination data from different populations was compared
for the same species (Table 2). Results showed differences between populations in the case
of N. crassulifolia (Punta de Choros and Llanos de Challe) and N. divaricata (Punta de
Choros and Llanos de Challe), but not for N. sedifolia, even when populations where very
far apart (Alto Patache and Los Molles). It would therefore seem that the dormancy trait in
two of these species is modified by the environment in which they grow, but this conclusion
could not be extended to all species, since for N. sedifolia, dormancy is determined more by
genotype than by the environment.
3.3 Additional experiments with Nolana linearifolia
The additional experiments carried out with N. linearifolia allowed us to confirm the type of
treatments that are most successful (Fig. 4), which related to scarification at the funicular
scar area, independent of the addition of gibberellic acid. In several other treatments,
mericarps were left intact (control, addition of different concentrations of GA3, cold
stratification and alternate temperatures) as a way to simulate what would happen in nature
and to understand how the germination plug is released; however, they yielded very low
results (<10%).
Fig. 4. Cumulative germination of Nolana linearifolia mericarps after eight treatments (T): (1) Control:
intact mericarps imbibed in distilled water; (2) 500 ppm GA3: intact mericarps imbibed in gibberellic
acid at 500 ppm; (3) 1000 ppm GA3: intact mericarps imbibed in gibberellic acid at 1000 ppm; (4) Plug
scarification, i.e., removal of germination plug, imbibed in distilled water; (5) Plug scarification with
partial removal of endosperm, imbibed in distilled water; (6) Plug scarification + 500 ppm GA3:
scarified mericarps with partial removal of endosperm imbibed in gibberellic acid (500 ppm GA3); (7)
10/25: intact mericarps at alternating temperatures of 10/25°C, imbibed in distilled water; (8) 10/25 +
GA3: intact mericarps at alternating temperatures of 10/25°C, imbibed in gibberellic acid (500 ppm
GA3); and (9) Cold stratification (5°C) of intact mericarps for 4 months. Data used is an average ±
standard error of four replicates.
3.4 Role of endosperm on germination
The PD may be caused by structures that cover the embryo, including endosperm (or
perisperm), seed coats and indehiscent fruit walls, among others, which can restrict radicle
emergence, especially in freshly matured seeds (Baskin and Baskin, 2014). In seeds of type
species of the family Solanaceae (pepper, tomato, tobacco), the diploid embryo is
surrounded by two layers: the endosperm (triploid) and the testa, of maternal origin (mostly
dead cells). The micropylar endosperm (of several layers of cells), which covers the tip of
the radicle, has been identified as a limiting factor for germination in species of the
Solanaceae family, as was reported in Datura (Sánchez et al., 1990) and Solanum (Groot
and Karssen, 1987). This micropylar endosperm acts as a mechanical barrier for
germination and participates in the regulation of the restriction of the embryo, by affecting
the balance of hormones (ABA / gibberellins) and sensitivity to them (Finch-Savage and
Leubner-Metzger, 2006).
After the experiments carried out for N. linearifolia, and having finished the first set of
experiments in the other species, a different set of treatments was tested to elucidate the
role of the endosperm in Nolana germination; this was only done for species that had high
germination percentages after removal of germination plug and addition of gibberellic acid
(Exp. 1), the majority of which belong to clade G. Results indicate that there was an evident
relationship between the partial removal of the endosperm and germination of these species
(Table 4), comparable to the addition of gibberellic acid. The extraction of the germination
plug, therefore, although it is necessary because it is the point from which the radicle
emerges, is not as decisive as the removal of the layer immediately below; that is, the
endosperm.
Table 4. Germination percentages of mericarps from 6 Nolana species in response to three
treatments: (1) Control (Ct), intact mericarps imbibed in distilled water; (2) scarification (Sc), removal
of germination plug and imbibition in distilled water; (3) scarification and partial removal of endosperm
(Sc+en) and imbibition in distilled water. Percentage of seed viability was determined by tetrazolium
test (1%) of 25 mericarps per species.
Viable
Collection
Sc+en*
Species
Clade
Ct1 (%)
Sc (%)
seeds p value2
site
(%)
(%)
Pan de
N. aplocaryoides G
0c
23 b
63 a
<0.0001
Azúcar
96
N. crassulifolia
G
6c
22 b
55 a
48
<0.0001
0c
23 b
40 a
84
<0.0001
G
Los Molles
Llanos de
Challe
Antofagasta
hills
N. divaricata
G
N. linearifolia
3b
3b
54 a
96
<0.0001
N. paradoxa
B
Los Molles
2b
16 b
54 a
84
0.0088
N. sedifolia
G
Los Molles
1c
22 b
40 a
60
<0.0001
1
For each column, values with different letters are significantly different according to a LSD test (α=
0.05)
2
P value from an analysis of variance.
* Scarification plus partial removal of endosperm
The weakening of this barrier seems to be a prerequisite for the protrusion of the radicle
during germination in the species that present it (Finch-Savage and Leubner-Metzger,
2006), particularly in the micropylar portion (Sánchez et al., 1990). While gibberellins
promote weakening -which may be due to enzymes that the same tissue synthesizes-, ABA
would be inhibiting it (Finch-Savage and Leubner-Metzger, 2006). The removal of the
endosperm and testa layers opposite the radicle tip may also assist this process to permit
radicle protrusion (Groot and Karssen, 1987).
3.5 Relationship between dormancy and geographical distribution of species and clades
Similar traits and life cycles are expected for species within a genus; different germination
requirements of the species may reflect specific adaptations to the occupied habitat (Van
Assche et al., 2002). Barreto et al. (2016) studied the viability and germination of seeds of
12 species of Stachytarpheta (Verbenaceae), and their results showed differences in
germination among species that appeared to be related to their natural habitats and
geographic distributions. Conversely, Giorni et al. (2018) found that, for the Xyris species
they studied (Xyridaceae), the effects of temperature on seed germination did not explain
the patterns of geographic distribution nor the endemism seen among the species
examined. In an extensive study on species from campo rupestre grasslands in Brazil,
Dayrell et al. (2016) found that phylogeny was better correlated than ecology with seed
dormancy categories, indicating that geological and climatic history had a more important
role in driving seed dormancy than contemporary factors (Dayrell et al., 2016).
When germination results of the studied Nolana species (corrected by the percentage of
seed viability) were correlated with the latitudinal distribution of each species (Fig. 5), no
evident relationship was obtained. Some species with a very limited distribution had a high
percentage of germination (N. onoana), whereas those with a wide latitudinal distribution
(i.e. N. rostrata), contrary to what was expected, had lower germination percentages.
Fig. 5. Correlation between germination percentage of 19 accessions of 12 species of Nolana for the
best treatment (removal of germination plug and imbibition in a solution of 500 ppm GA 3, or removal
of germination plug and partial removal of endosperm) and latitudinal distribution, expressed as the
difference between the northernmost and the southernmost latitudinal distribution point (decimal
degrees). R = 0,439 (p value = 0,060).
Regarding the relation between the level of physiological dormancy (expressed inversely as
the percentage of germination for the most successful treatment, i.e. scarification + GA 3)
and phylogeny, it could be determined that, for the studied species, their proximity in terms
of clades (Fig. 2) was more relevant than their latitudinal distribution. Species that belong to
Clade G showed, as a whole, higher percentages of germination (i.e. least deep dormancy)
for the most successful treatment than the species of the other clades, but Clade B also had
high germination percentages (Fig. 6).
Fig. 6. Germination percentage of Nolana species for the Sc+GA3 treatment (removal of germination
plug and imbibition in giberrellic acid), grouped into clades B, C, F, G. The number of
species/populations included for each clade is noted. Data used is an average ± standard error of
four replicates.
The species that showed higher germination percentages were N. crassulifolia (clade G), a
prostrate shrub that forms mats or draping rocks near the sea (Johnston, 1936), with a
distribution between 26° and 33°5´S; and N. aplocaryoides (clade G), an erect and branched
annual (Mesa, 1981), extending from Antofagasta to Atacama (22° to 27°4´S). Both have
wide distributions, but in different environments, since N. crassulifolia reaches to central
Chile (semi-desert), while N. aplocaryoides is only found in the desert. N. onoana (clade G),
however, which also presented a relatively high germination percentage, is only found at a
few locations in Antofagasta region. A particular case is N. paradoxa (clade B), with the most
extensive distribution from the center to the south of coastal Chile (33°S - 42°30'S), where
the climate is temperate humid; N. paradoxa had high germination percentages, but not
higher than the other species mentioned.
Clade B includes two subclades: rosette-forming, taprooted plants with larger flowers, and
erect annuals with slightly smaller flowers (Dillon et al., 2009), while Clade G is composed
of small to moderate shrubs and annuals, all with highly reduced corollas, often white or
yellowish (Dillon et al., 2009). These two clades have similar germination results, although
their growth habits are very different, and their mericarps (see Hepp et al., in press) are also
highly dissimilar. Altogether, this last clade (G) was found to have the higher germination
percentage, i.e. the least deep physiological dormancy. We would have expected a higher
level of dormancy in species with annual habits, since they depend more on seedling survival
and/or formation of a seed bank for persistence (Dayrell et al., 2016; Venable, 2007), which
was partly the case of N. paradoxa; but as mentioned before, its germination percentage
was not higher than those of most Clade G species. Dayrell et al. (2016) also found no
significant correlations between seed dormancy and life history traits, such as growth habit.
Although other studies have also found no evidence of a correlation between dormancy and
geographic distribution (Giorni et al, 2018; Dayrell et al., 2016), for Nolana it remains to be
studied if species that occupy a greater diversity of ecological niches present a greater
plasticity in their dormancy levels (lower levels of PD). Our analysis in this case focused on
the variation in conditions that may exist between different latitudes along the coast but did
not include the habitat or niche occupation for each of the species. Further studies are
needed to determine if there is such a pattern.
The highest germination percentage reached by the species studied was > 60%, and some
of them reached much lower percentages, even 0 for the most successful treatment in other
species; in fact, for most species there was almost no germination when the germination
plug was not removed (control treatment), that is, when local conditions were simulated.
This would indicate a very "cautious" germination strategy, as suggested by Gutterman
(1995), with higher thresholds of response to rainfall to begin germination, or deeper levels
of dormancy. It is important to consider that some of these species inhabit places where
rainfall occurs every 15 years or more, as is the case of N. intonsa, N. jaffuelii and N. sedifolia
in the fog oasis of Alto Patache in the North of Chile (Orellana et al., 2017; Pliscoff et al.,
2017).
Conclusions
Our results indicate that seeds of the studied species present physiological dormancy with
varying levels/degrees of depth. However, they do not present physical dormancy, as has
been reported previously, since water was able to enter the mericarp and reach the embryo
in all the species studied. Furthermore, the role of the endosperm in the prevention of
germination for the Nolana species studied is emphasized; the weakening (or removal) of
this barrier facilitates germination in the species that present it, particularly in the micropylar
portion. For the species studied, the results show a relationship between the percentage of
germination (or level of dormancy) of the species and their proximity in terms of clades, but
not with respect to their latitudinal distribution. Hence, our hypothesis of a greater similarity
in terms of type and level of dormancy among more closely related species was
corroborated; but we found no evidence of a relationship between level of dormancy and
range of distribution (latitudinal) for the species of the genus.
Acknowledgements
All species were collected with permission and support of the National Forest Commission
(CONAF Pan de Azúcar, CONAF Llanos de Challe), the Agricultural Research Institute
(INIA) and owners/administrators of private parks (Punta de Choros, Puquén Los Molles)
and research stations (Alto Patache Fog Oasis, managed by Universidad Católica de Chile).
CONICYT Scholarship (21130176) to Josefina Hepp made this study possible.
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Chapter III. Current and future patterns of distribution of Nolana species
(Solanaceae) from the coast of Chile and their relation to seed dormancy
Patricio Pliscoff1,2, Josefina Hepp2,3, Miguel Gómez2,3, Pedro León-Lobos4, Gloria
Montenegro2,3, Samuel Contreras2,3
1
Departamento de Ecología, Facultad de Ciencias Biológicas, Pontificia Universidad
Católica de Chile
2
Centro UC del Desierto de Atacama, Pontificia Universidad Católica de Chile
3
Departamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería Forestal,
Pontificia Universidad Católica de Chile
4
Centro Regional de Investigacion La Platina, Instituto de Investigaciones Agropecuarias,
INIA
Corresponding author. Tel.: +562 2354 2610
E-mail address: pliscoff@uc.cl (P. Pliscoff)
Postal address: Facultad de Ciencias Biológicas, Avda. Libertador Bernardo O'Higgins
340, Santiago, Chile.
Abstract
The genus Nolana (Solanaceae) comprises numerous species endemic to the arid and
semi-arid environments in the coastal area of Chile and Peru. Seed dormancy of the
physiological type with varying levels of depth has been reported for certain Nolana species.
The objective of this study is to establish the current and future distribution patterns of the
genus for Chilean species, to analyse if there is a relation between distribution (expressed
as niche breadth) and level of seed dormancy of a set of eight species, and how that may
influence survival of species under future climate change scenarios. Results for the current
distribution pattern confirm the two reported centres of diversity of Nolana, in Arequipa
(Peru) and between Antofagasta and Atacama (Chile); in the future, a contraction towards
the coast is projected. As for the change the eight studied species will present in future
climatic scenarios, there is a diversity of responses according to the species. Species that
have lower dormancy values are also those whose distribution range will remain stable or
expand, and conversely, distribution range will contract in the case of species with high
dormancy values. We found no evidence of a relation between distribution and dormancy;
that is, dormancy level is not explained by niche breadth.
Keywords: niche breadth, Atacama desert, climate change, mericarp
Introduction
The genus Nolana L. ex L. f. (Solanaceae) currently comprises 90 species of annual or
perennial herbaceous plants or small shrubs with leaves with different degrees of
succulence and flowers of different colors and sizes (Dillon et al., 2007; Tu et al., 2008;
Dillon et al., 2009). The majority of species grows preferentially in the arid and semiarid
zones of coastal Chile and Peru, although some species are found in high altitude habitats
(> 1,000 m a.s.l.) and a few of them, i.e. N. paradoxa and N. sedifolia, have distributions that
range over 1,000 km (Johnston, 1936). Dillon et al. (2009) have identified several strongly
supported clades within the Nolana genus, with geographic and morphological fidelity, four
of which are confined to Chile and three that are mainly Peruvian with some presence in
Chile.
According to Rundel et al. (1991) and Schultz et al. (2011), the coastal desert of Atacama
extends between 18°S and 30°S. The climate here is moderate in terms of temperature,
which in general is quite stable in the latitudinal range along the coast (average temperature
of 15-18° C from Valparaíso to Tarapacá; Fick & Hijmans, 2017). Atmospheric humidity is
high (annual average of 65-80%), and annual rainfall ranges from less than 1 mm in the
northernmost stations to around 80 mm in La Serena (Schultz et al., 2011; Chilean
Meteorological Office, Integrated Territorial Information System (SIIT)). In the area north of
21°30'S, the vegetation is concentrated in small patches in isolated sites that receive fog,
usually on slopes with south or southwest exposure (Schultz et al., 2011); the high frequency
and intensity of fog is a decisive ecological factor, to which is attributed the existence and
maintenance of these vegetation formations known as "fog oases" (Rundel et al., 1991) or
"Lomas vegetation" (Rundel et al., 1997). Continuing south, up to around 26°S, vegetation
increases but remains dispersed. From there towards 30°S, the fog oases extend in a
relatively closed formation along the windward slope of the coastal mountain range (Rundel
et al., 2007, cited in Schultz et al., 2011).
In environments with high temporal variability, such as deserts, seed dormancy is often
described as a bet-hedging strategy (Eberhart and Tielbörger, 2012). Not all mature seeds
produced by a plant germinate immediately after dispersal, or in the following season; in
many cases, as a strategy of survival, a significant amount of seeds remains dormant in the
soil, for a period that can last for years or even decades, forming what is known as a seed
bank (Aguado et al., 2012).
Dormancy can be defined as "an innate property of the seed that defines the environmental
conditions in which the seed is capable of germination" (Finch-Savage and LeubnerMetzger, 2006), and its induction is controlled both at the genetic level and by the
environment. Genetic control can be associated with both the embryo genotype and that of
the mother plant (Contreras and Rojas, 2010); the state of dormancy is influenced by the
environment in which the seed develops (maternal environment), but also by the external
environment when dispersed, so it can change over time (Finch-Savage and LeubnerMetzger, 2006). According to Baskin and Baskin (1998, 2004), there are five types of seed
dormancy, the most frequent being physiological dormancy (PD), which has three levels of
depth (not deep, intermediate and deep). The dormancy status of a batch of seeds can be
established based on embryo morphology, the permeability of the seed coat to water, and
the capacity of fresh seeds to germinate within one month (Baskin and Baskin, 2004;
Schwienbacher et al., 2011).
In a study on seed dormancy of 12 Nolana species, Hepp et al. (in preparation) found that
seeds presented PD with varying levels of depth, emphasizing the role of the micropylar
portion of the endosperm in the prevention of germination for the Nolana species studied.
Freyre et al. (2005) and Douglas and Freyre (2006) also reported PD for another nine Nolana
species, both from Chile and Peru. This property would allow the species to defer
germination in time; to this is added the fibrous hard fruits called mericarps that Nolana
species present, a character that is unique in the Solanaceae family (Knapp, 2002, Dillon et
al., 2009). The pericarp of mericarps is constituted by sclereids, which give strength and
rigidity to the fruit (Cabrera et al., 2015) and may provide protection to the embryo, as it
would allow the mericarps to remain relatively intact in the soil seed bank for many years
(Hepp et al., 2019, in press).
Although other studies have found no evidence of a correlation between dormancy and
geographic distribution (Giorni et al, 2018; Dayrell et al., 2016), in Nolana this has not been
studied. Hepp et al. (in preparation) focused in the latitudinal variation (expressed as the
difference between the northernmost and southernmost latitudinal distribution point in
decimal degrees), which showed no pattern, but did not include the habitat or niche
occupation for each of the species. The presence of the genus in the whole range of “lomas
formations”, where it stands out as the most conspicuous floristic element (Tago-Nakazawa
and Dillon, 1999), makes it interesting for this type of study.
The objective of this study was to establish the current and future distribution pattern of the
genus for Chilean species, and to determine which variables define their presence. A second
objective was to analyse if there is a relationship between distribution pattern and the level
of dormancy presented by seeds of a set of species. Also, if such a relationship exists, given
the projections of less rainfall and increasing temperatures in the arid and semi-arid area of
northern Chile by 2050 (Santibañez et al., 2017), how will it change in the future?
Materials and methods
Species ocurrences database
A presence occurrences database of 1,637 unique records was compiled for 45 species of
the genus Nolana (50% of total number of known species) of Chile and Peru (Fig. 1); only
species with at least 10 unique occurrences were selected for modelling. The complete list
of species and number of occurrences by database is presented in Annex 1. The source of
species occurrences where the following Herbaria: SGO (National Museum of Natural
History, Santiago, Chile); CONC (Botany Department, Universidad de Concepción,
Concepción, Chile); Global Biodiversity Information Facility (GBIF) and Michael Dillon
personal database.
Fig. 1. Study area with all ocurrences included for the Nolana genus.
Environmental variables
Current climatic surfaces (1950-2000) for the study area were obtained from an improved
climatic surface dataset presented in Pliscoff et al. (2014) and available for download in the
Spatial Ecology Group webpage (http://www.unil.ch/ecospat/home/menuguid/tools-data/data.html). Future projections for each monthly climatic variable were performed
according to the delta method (Ramirez-Villegas and Jarvis, 2010) using current climatic
surfaces modelled as the baseline; for future values, the most extreme climate change
scenarios of the IPCC Fifth assessment report (RCP 8.5) were selected and the Global
Circulation Models (GCM) scenario Hadgem2_ES at 2070-2100 time period. Future GCM
scenarios were downloaded from CIAT-GCM (Centro Internacional de Agricultura Tropical-
Global Climate Model http://ccafs-climate.org/). Future monthly climatic surfaces were used
to derive 19 bioclimatic variables (Nix, 1986; Busby, 1991), which are widely used in species
distribution modelling (Synes and Osborne, 2011), bioclimatic dataset are available for
download for each GCM for the year 2080 (RCP 8.5) in the Spatial Ecology Group webpage.
A variable selection step was applied in which pairwise Pearson correlations were calculated
between all variables, and in cases when correlation values were greater than 0.8
(Correlation matrix are presented in Annex 2), one of the two variables was excluded (Elith
et al., 2010) in order to avoid strongly correlated variables in the set of predictors (Kozak
and Wiens, 2006). This assessment resulted in five variables:
-
Annual Mean Temperature (BIO1)
Mean Diurnal Range (BIO2)
Annual Precipitation (BIO12)
Precipitation Seasonality (BIO15)
Precipitation of the Coldest Quarter (BIO19)
In addition, a cloud cover variable was added to the final bioclimatic dataset for the
characteristics of coastal desert environment where Nolana species inhabit. The Global 1km cloud cover dataset was used (Wilson and Jetz, 2016) which presents a monthly cloud
cover data derived from MODIS satellite images. The annual mean cloud cover extracted in
the study area extent was also used. Final six variables (five bioclimate plus cloud cover)
were used to calibrate the current models and project them to future conditions.
Species distribution modelling
MaxEnt software (Phillips et al., 2016) was used to define suitability areas (a habitat's
potential to support a particular species) of Nolana species under current and future
conditions. MaxEnt looks for the species distribution suitability of probabilities closest to
uniform (maximum entropy), constrained to the fact that feature values match their empirical
average (Phillips and Dudík, 2004), MaxEnt performs very well with relatively small sample
sizes of presence-only data (Elith et al., 2006; Wisz et al., 2008). Also, many studies have
found strong support for high temporal transferability of MaxEnt models (Rapacciuolo et al.,
2012). MaxEnt was ran using default parameters and the logistic output as an estimate of
environmental suitability of species was used. Presence records of each species were
randomly split into two subsets: 20% for training and 80% for testing. The accuracy of
models was evaluated with the area under the receiver operating characteristic curve (AUC)
(Lobo et al., 2008). After modelling the current distribution of Nolana species, the
environmental suitability of species was projected in one future climate scenario
(HadGem2_ES rcp 8.5 for 2070-2100 period). MaxEnt predictions were transformed into
binary outputs using the 10% percentile of training presences. Future changes in suitability
were evaluated using SDMtools (Brown, 2014) under ArcGis 10.6 software (ESRI 2017).
For each species, expansion, contraction and stability were calculated under future
scenarios, and also direction of change following centroids comparison between current and
future models.
Germination tests
In order to stablish the relation between habitat suitability and seed dormancy of a set of
species, the results of germination experiments and seed viability reported in Hepp et al. (in
preparation) were used for 10 species of Nolana (Table 1, Fig. 2). These 10 species belong
to 3 clades within the genus, as described in Dillon et al. (2009) and can be summarized as
follows:
-
Clade G has recovered a strictly Chilean group of 13 species represented by small
to moderate shrubs (traditionally called "Dolia") and annuals, all with highly reduced
corollas, often white or yellowish (Dillon et al., 2009) but some have bluish flowers.
-
Clade C has recovered a monophyletic group comprised of 6 species traditionally
classified as Alona. These are all large-flowered shrubs confined to Chile (Dillon et
al., 2009).
-
Clade B, previously recognized as a segregate genus, Sorema, currently consists of
two sub-groups: N. pterocarpa-N. baccata-N. parviflora, which are erect annuals with
small flowers; and N. paradoxa-N. rupicola, which are rosette-forming, taprooted
plants with larger flowers (Dillon et al., 2009). These species are only present in Chile
(Dillon et al., 2009).
Experiments considered for this study correspond to the most successful treatment for all
species, based on Cabrera et al. (2015) and Hepp et al. (in preparation): removal of
germination plug and addition of gibberellic acid (GA3 500 ppm); or removal of germination
plug and partial removal of endosperm. Germination tests were carried out during 2015 and
2016, using fruits from Nolana species collected in the previous months. Germination was
evaluated three days a week during 45 days in a chamber at constant 20ºC and light (40%
RH), in four replicates of 25 seeds each. Physiological germination (radicle emergence over
2 mm) was evaluated. Results were reported as total germination percentage.
Table 1. Selected species of the genus Nolana, collection site, results of germination experiments (Ct
and BT; Hepp et al., in preparation), and clade (based in Dillon et al., 2009). Treatments are as
follows: (Ct) control, intact mericarps imbibed in distilled water; (BT) best treatment, scarification
(removal of germination plug) and imbibition in a solution of 500 ppm GA3. Percentage of seed viability
was determined by tetrazolium test (1%) of 25 mericarps per species.
Collection site
Nolana aplocaryoides
(Gaudich.) I.M. Johnst.
Pan de Azúcar, Atacama
(26°08.786'S, 70°39.943'W)
0
63
96
Llanos de Challe, Atacama
(28°11.086'S, 71°09.751'W)
1
62
65
Punta de Choros, Coquimbo
(29°14.786'S, 71° 28.021'W)
16
34
60
Nolana crassulifolia
Poepp.
Ct (%)
Viable
BT (%) seeds
(%)
Species
Clade*
G
Punta de Choros, Coquimbo
(29°14.601'S, 71°27.840'W)
0
37
91
Llanos de Challe, Atacama
(28°11.991'S, 71°09.524'W)
0
35
50
Hills of Antofagasta,
Antofagasta (23°30.133'S,
70°23.069'W)
3
55
96
Alto Patache fog oasis,
Tarapacá (20°49.542'S,
70°09.392'W)
0
53
61
Los Molles, Valparaíso (32°
14.444'S, 71°31.133'W)
0
30
38
Nolana rostrata (Lindl.)
Miers ex Dunal
Llanos de Challe, Atacama
(28°06.806'S, 71°09.257'W)
0
17
76
Nolana carnosa (Lindl.)
Miers ex Dunal
Llanos de Challe, Atacama
(28°02.612'S, 71°07.022'W)
0
13
96
Nolana jaffuelii I.M.
Johnst.
Alto Patache fog oasis,
Tarapacá (20°48.903'S, 70°
09.630'W)
0
27
88
Nolana paradoxa Lindl.
Los Molles, Valparaíso
(32°14.433'S, 71°31.133'W)
2
54
84
Nolana parviflora (Phil.)
Phil.
Hills of Antofagasta,
Antofagasta (23°30.133'S,
70°23.069'W)
0
25
84
Nolana divaricata (Lindl.)
I.M. Johnst.
Nolana linearifolia Phil.
Nolana sedifolia Poepp.
C
B
Figure 2. Context, flowers and fruits of four Nolana species included in this study. A, B and C: N.
aplocaryoides (clade G); D, E and F: N. sedifolia (clade G); G, H and I: N. paradoxa (clade B); J, K
and L: N. rostrata (clade C).
Dormancy Index (DI)
With the germination results of the experiments described above, a dormancy index was
calculated. This Dormancy index was modified from Offord et al. (2004); percentage of
germinated seeds for the best treatment (BT; Hepp et al., in preparation) and control
treatment (Ct) were both included. Viability of seeds was tested using tetrazolium (2,3,5-
triphenyl-2H-tetrazolium chloride, or Tz) on a sample of 25 fruits for each species (staining
for 24 hours at 30° C, 1% Tz solution, all fruits without germination plug).
A first step was to calculate the initial Dormancy Index (DIi) taking into account only the
percentage of seeds germinated in the best treatment, as not all treatments were equally
successful, and the percentage of viable seeds. The percentage of seeds germinated in Ct
was then incorporated on a second step, resulting on a final Dormancy Index (DIf).
The higher the value of the Index, the more likely that the seed lot was dormant:
DIi= 1-(seed germinated in BT %/ viable seeds %)
DIf=(DIi*100)-(seed germinated in Ct %/DIi)
Niche overlap and breadth
To compare the potential distribution between Nolana species modelled, we
calculated niche overlap and breadth indexes. The niche overlap Schoener´s Index shows
the similarity between the distributions of Nolana niche models where low values indicate
low overlap and values near 1 reflects a high overlap between potential distribution modeled.
Also, Levin's niche breadth index was calculated, using inverse concentration and
uncertainty values to express distribution breadth, values close to 1 indicate broader niche.
All calculations were done using the ENMTools 1.3 package in R.
The maximum adequacy value from the location point (collection site) for each species
(probability of presence) was extracted.
Results and discussion
Current and future distribution
After the analysis of all occurrences for Nolana, information for the genus was updated:
there is a total of 90 species, 49 of which are present in Chile and 43 are found in Peru; both
countries only share three species (N. adansonii, N. gracillima and N. lycioides). N. jaffuelii,
which was previously reported for Peru, has no modern records and therefore is considered
a Chilean endemic. Dillon et al. (2009) mention there appears to be a barrier to dispersal
along the coast between 18° and 20°S (Dillon et al., 2009), between Tacna (Peru) and the
southern limit of the Arica region (Chile).
In terms of the current distribution of Nolana species, as mentioned in Dillon et al. (2007),
there are two concentration areas or centres of greater species diversity: one in the southern
Peruvian Department of Arequipa (14°38’S / 70°50’W to 17°17’S / 75°05’W) where 28
species are recorded (Rosado, 2019), and another in the northern Chile from Antofagasta
(21°26’S / 69°18’W to 25°56’S / 69°37’W) to Atacama region (25°56’S / 69°37’W to 29°24’S
/ 70°20’W, with 31 species (Fig. 3A).
In the future, a contraction towards the coast is projected (Fig. 3B), although both centres of
diversity are maintained (highlighted in boxes, Fig. 3A and 3B); in addition, there would be
an increase in the number of Nolana species that would be distributed in these areas.
Direction of change (Fig. 4) indicates the loss of species in certain areas of higer altitude,
outside of the areas of greater concentration of species. The calculated expansion,
contraction and stability for all species under future scenarios is found in Annex 3.
Fig 3. Current (A) and future (B) distribution of Nolana species in Chile and Peru. B: Environmental
suitability of species was projected in one future climate scenario (HadGem2_ES rcp 8.5 for 20702100 period).
Fig. 4. Direction of change following centroids comparison between current and future models.
Species distribution modelling
The variable that best defines the presence for most species is annual precipitation (Annex
4). In the case of N. crassulifolia, a species with distribution between Atacama (26° 21.054'S)
and Valparaíso (33° 55.002'S), it is the precipitation seasonality; and in the case of N.
paradoxa, with a very extensive and fundamentally coastal distribution, it is the mean diurnal
temperature range.
In terms of their spatial distribution, the species that most coincide, both in terms of overlap
(how their habitat suitability overlap in space) and similarity (how similar they are in shape
of habitat suitability), are N. rostrata (clade C) and N. divaricata (clade G). These are two
similar species in terms of growth habit, although they belong to two different clades. N.
divaricata usually forms a dense succulent globose bush 1-2.5 m tall and seems to be
confined to the hills near the sea (Johnston, 1936), in the regions between Antofagasta and
Coquimbo (Zuloaga et al., 2008). N. rostrata is a very slender, loosely branched, sprawling
shrub with an extense distribution, only in Chile, between Tarapacá and Coquimbo regions
(Zuloaga et al., 2008).
N. aplocaryoides and N. sedifolia also have high values for both indexes; in this case, these
are two species with different forms of growth, but corresponding to the same clade (G). N.
sedifolia is an endemic perennial shrub or sub-shrub that can measure up to 100 cm tall; it
has an extensive distribution between the regions of Tarapacá and Valparaíso (Zuloaga et
al., 2008). N. aplocaryoides is an annual herb, erect and branched; endemic to northern
Chile, it grows between the regions of Tarapacá and Atacama, in plains in full sun (Zuloaga
et al., 2008).
Table 2. Overlap Index. The numbers highlighted in bold correspond to high values.
Species
N. sedifolia (Nsed)
N. aplocaryoides (Nap)
N. carnosa (Ncar)
N. crassulifolia (Ncra)
N. divaricata (Ndiv)
N. linearifolia (Nlin)
N. paradoxa (Npa)
N. rostrata (Nro)
Nsed Nap Ncar Ncra
0.621 0.342 0.378
0.276 0.233
0.391
Ndiv
0.536
0.519
0.544
0.350
Nlin
0.473
0.607
0.135
0.105
0.271
Npa
0.177
0.093
0.116
0.389
0.132
0.038
Nro
0.343
0.283
0.591
0.275
0.645
0.121
0.093
Table 3. Similarity Index. The numbers highlighted in bold correspond to high values.
Species
N. sedifolia (Nsed)
N. aplocaryoides (Nap)
N. carnosa (Ncar)
N. crassulifolia (Ncra)
N. divaricata (Ndiv)
N. linearifolia (Nlin)
N. paradoxa (Npa)
N. rostrata (Nro)
Nsed Nap Ncar Ncra
0.878 0.643 0.688
0.568 0.509
0.613
Ndiv
0.811
0.787
0.824
0.632
Nlin
0.780
0.878
0.355
0.315
0.525
Npa
0.338
0.252
0.235
0.610
0.263
0.159
Nro
0.618
0.551
0.837
0.515
0.883
0.305
0.189
As for the change that the eight species will present in future climatic scenarios, there is a
diversity of responses according to the species (Fig. 5). Some will remain stable and will
even increase their range (N. crassulifolia, N. divaricata, N. sedifolia), but others will be more
affected and their range will contract (for example, N. aplocaryoides, N. carnosa, N.
linearifolia, N. paradoxa, N. rostrata; see also Annex 4).
Fig 5. Spatial patterns of change under future climate scenarios for 8 species: N. aplocaryoides, N.
carnosa, N. crassulifolia, N. divaricata, N. linearifolia, N. paradoxa, N. rostrata and N. sedifolia.
Habitat suitability of species was projected in one future climate scenario (HadGem2_ES rcp 8.5 for
2070-2100 period).
Relation between distribution and dormancy
According to previous studies by Hepp et al. (in preparation), species of clade G present
the less deep seed dormancy, adding to N. paradoxa (clade B); species belonging to clade
C are the most dormant. Table 4 presents the dormancy index (initial and final) for the 8
species studied plus another two (N. jaffuelii and N. parviflora) that could not be modelled,
where this information is confirmed.
Through species distribution modelling, we found that the potential areas of N.
aplocaryoides, N. carnosa, N. linearifolia, N. paradoxa and N. rostrata would be reduced
under future global climate change scenarios. N. carnosa and N. rostrata have the higher
dormancy values of all studied species, while the rest present low dormancy values (Table
4).
Among the species that will remain stable or even increase their range of distribution is N.
crassulifolia, which is the one with the lowest dormancy value. N. sedifolia also has a low
value, while for N. divaricata the value is intermediate (Table 4).
Plasticity in the seed regeneration trait may counteract the projected narrowing of the
species distribution range, as mentioned in Peng et al. (2019) for Saussurea (Asteraceae)
species; however, in this case, the species that have lower dormancy values are also those
whose distribution range will not be reduced, according to the models used in this study;
and conversely, distribution range will contract in the case of species with high values for
the dormancy index.
Table 4. Dormancy Index (initial, DIi, and final, DIf) for 10 species of Nolana, ordered by lower to
higher dormancy level. HS corresponds to the highest suitability value for each collection point.
Species
Clade
DIi
DIf
HS
N. crassulifolia
G
0.433
6.41
0.930851
N. paradoxa
B
0.357 30.11
0.852613
N. sedifolia
G
0.333 30.33
0.977771
N. aplocaryoides G
0.344 34.38
0.988483
N. linearifolia
G
0.438 36.89
0.723844
N. divaricata
G
0.593 59.34
0.995523
N. jaffuelii
B
0.693 69.32 Not modelled
N. parviflora
B
0.702 70.24 Not modelled
N. rostrata
C
0.776 77.63
0.877566
N. carnosa
C
0.865 86.46
0.983326
To add to the analysis, Table 5 presents the niche breadth, expressed as the inverse
concentration for each species. It can be observed that N. sedifolia is the species with
greater niche breadth; not N. paradoxa. This last species stands out for having a wide
distribution between 29°S and 43°S, the only species of the genus that reaches this latitude,
but inhabits mainly on the coast, very close to the sea (Fig. 3, N. paradoxa). N. sedifolia also
has a very wide distribution (between 19°S and 33°S) and is also capable of inhabiting inland
environments (Fig. 3, N. sedifolia). The species with the lowest niche breadth would be N.
carnosa.
Table 5. Niche breadth expressed as inverse concentration for each of the 8 modelled Nolana
species.
Species
Inverse
concentration
Uncertainty
Nolana aplocaryoides
Nolana carnosa
Nolana crassulifolia
Nolana divaricata
Nolana linearifolia
Nolana paradoxa
Nolana rostrata
Nolana sedifolia
0.8101277
0.7468854
0.7496458
0.8188687
0.7820484
0.7887218
0.7894775
0.8601784
0.03394854
0.0135538
0.01372035
0.03815467
0.02815799
0.02333149
0.02724888
0.07315427
However, seed dormancy was not correlated with niche breadth (Fig. 6); that is, species with
a greater niche amplitude, capable of occupying a greater variety of habitats, is not
necessarily the one with the lowest dormancy index. At the same time, species with high
dormancy rates also have intermediate levels of niche occupation.
100
Dormancy Index (final)
90
80
70
60
50
40
30
20
10
0
0,74
0,76
0,78
0,8
0,82
0,84
0,86
0,88
Niche breadth
Fig. 6. Correlation between germination percentage and niche breadth for eight species of the Nolana
genus. The value of R2 for the trendline is given. R = -0,142 (p value = 0,736).
In an extensive study on species from campo rupestre grasslands in Brazil, Dayrell et al.
(2016) found that phylogeny was better correlated than ecology with seed dormancy
categories, indicating that geological and climatic history had a more important role in driving
seed dormancy than contemporary factors (Dayrell et al., 2016). This may also be the case
for the genus Nolana; Hepp et al. (in preparation) reported that the proximity of Nolana
species in terms of clades seemed to be more relevant than their latitudinal distribution
(expressed as the difference between the northernmost and the southernmost latitudinal
distribution point).
Clade C species (N. carnosa and N. rostrata) should have priority in conservation programs,
since they have the higher dormancy levels and a contraction of their potential habitat is
expected in the future. In particular, N. carnosa also has a low niche breadth. Seeds of both
species should be candidates for long-term storage in Seed Banks. On the other hand, live
collections of these plants should be kept in botanical gardens, in order to have more
material for propagation and research activities. In general, clade G species are of less
concern, given the wide niche they present and their low dormancy levels; however, some
populations of these species are more susceptible to the threats of climate change,
especially in the northernmost locations. In the case of N. paradoxa of clade B, although its
extensive distribution will contract significantly in the future, it is a species with high
germination levels (low dormancy value), whose detached leafy stems readily root and it it
also has floating mericarps that can be dispersed over long distances (Johnston, 1936).
Conclusions
The current pattern of distribution for the genus Nolana, that confirms two centres of species
diversity, could be established; in the future, there will be a contraction towards the coast,
but some species will keep their distribution area stable, and some will even expand.
However, it is worrisome that the species whose seeds have higher values according to the
dormancy index, are those that have a smaller niche breadth and also, whose distribution
will contract in the future. Special efforts should be directed to include these species in an
integrated conservation programme, including both in situ and ex situ initiatives.
On the other hand, no evidence was found of a relationship between the niche breadth of
the species and their dormancy level; this means that the response to germination
experiments (or level of physiological dormancy) cannot be predicted according to the
distribution presented by the species. It is likely that the dormancy trait has a greater genetic
influence than the maternal environment (environment under which the seed develops).
Acknowledgments
CONICYT Scholarship 21130176 to JH made this study possible. To Gloria Rojas and
Vanezza Morales for their support in the work at the SGO Herbarium. To Michael Dillon for
his generous help with determination of species and also for providing his personal database
for locations of Nolana species.
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Annexes
1. Complete list of species and number of occurrences.
Species
Nolana acuminata
Nolana adansonii
Nolana albescens
Nolana aplocaryoides
Nolana baccata
Nolana balsamiflua
Nolana carnosa
Nolana cerrateana
Nolana chancoana
Nolana clivicola
Nolana crassulifolia
Nolana coelestis
Nolana confinis
Nolana diffusa
Nolana divaricata
Nolana elegans
Nolana filifolia
Nolana flaccida
Nolana glauca
Nolana gracillima
Nolana humifusa
Nolana incana
Nolana inconspicua
Nolana inflata
Nolana leptophylla
Nolana linearifolia
Nolana lycioides
Nolana mollis
Nolana pallida
Nolana pallidula
Nolana paradoxa
Nolana parviflora
Nolana patula
Nolana peruviana
Nolana ramosissima
Nolana rostrata
Nolana rupicola
Nolana salsoloides
Nolana sedifolia
Records
75
13
52
65
65
10
22
10
11
18
62
35
11
20
44
57
73
32
13
10
18
48
18
12
107
31
11
39
10
10
45
17
16
93
42
70
64
62
52
Nolana spathulata
Nolana sphaerophylla
Nolana stenophylla
Nolana tarapacana
Nolana villosa
Nolana werdermannii
20
18
26
18
68
11
2. Correlation matrix for all bioclimatic variables plus cloud cover.
BIO1
BIO2
BIO3
BIO4
BIO5
BIO6
BIO7
BIO8
BIO9
BIO10
BIO11
BIO12
BIO13
BIO14
BIO15
BIO16
BIO17
BIO18
BIO19
NUBES
BIO1 BIO2
BIO3
BIO4
1 0,03368 0,38064 -0,32051
1 0,24463 0,27566
1 -0,81377
1
BIO5
BIO6
0,88574 0,93168
0,32209 -0,25221
0,12431 0,44866
0,10113 -0,52284
1 0,70341
1
BIO7
BIO8
-0,15291
0,9237
0,73869 0,16719
-0,44873 0,32028
0,8282 -0,20171
0,29568 0,86072
-0,47102 0,79247
1 0,00313
1
BIO9
BIO10
BIO11
0,85449 0,94914 0,96623
-0,16167 0,11549 -0,05264
0,41193 0,12848 0,55171
-0,45443 -0,00801 -0,5525
0,67918 0,96883 0,75235
0,90106 0,81541 0,96436
-0,36811 0,10647 -0,36239
0,61202 0,90284 0,86296
1
0,7585 0,88019
1 0,83776
1
BIO12
0,46528
-0,46274
0,32515
-0,55077
0,19134
0,6356
-0,61676
0,35593
0,52102
0,31513
0,56174
1
BIO13
0,51678
-0,34546
0,41716
-0,58386
0,25134
0,65388
-0,56688
0,4051
0,56713
0,35563
0,61428
0,96361
1
BIO14
0,07361
-0,57428
0,00418
-0,28559
-0,13693
0,27366
-0,53774
0,02206
0,13958
-0,0096
0,14597
0,79488
0,63637
1
BIO15
0,04401
0,45117
0,40922
-0,22401
0,02524
-0,05414
0,1041
0,10961
-0,02145
-0,04098
0,09063
-0,16974
0,00535
-0,42806
1
BIO16
0,51967
-0,35312
0,41661
-0,58722
0,25273
0,65967
-0,57294
0,40713
0,57103
0,35797
0,61783
0,969
0,99795
0,64341
-0,00696
1
BIO17
0,08144
-0,58073
0,00886
-0,2964
-0,13141
0,2854
-0,54666
0,02204
0,1577
-0,00465
0,15606
0,81567
0,6614
0,99565
-0,42409
0,66893
1
BIO18
0,5385
-0,27239
0,37329
-0,48402
0,28186
0,60814
-0,46753
0,53934
0,41944
0,40144
0,59852
0,84034
0,8353
0,63783
-0,02037
0,83554
0,64079
1
BIO19
0,03563
-0,46381
-0,00763
-0,23951
-0,08925
0,23615
-0,42815
-0,11374
0,26178
-0,02729
0,10816
0,69713
0,65311
0,65069
-0,21466
0,65395
0,68328
0,29444
1
NUBES
0,10232
-0,6427
0,03828
-0,37306
-0,15124
0,30064
-0,59175
-0,00958
0,2084
-0,00984
0,1943
0,61247
0,55147
0,53536
-0,4288
0,55714
0,54987
0,48435
0,41955
1
3. Expansion, stability and contraction under future scenarios for all Nolana modelled species.
Bold number correspond to the 8 species for which germination data is available.
Species
Nolana acuminata
Nolana adansonii
Nolana albescens
Nolana aplocaryoides
Nolana baccata
Nolana balsamiflua
Nolana carnosa
Nolana cerrateana
Nolana chancoana
Nolana clivicola
Nolana coelestis
Nolana confinis
Nolana crassulifolia
Nolana diffusa
Nolana divaricata
Nolana elegans
Nolana filifolia
Nolana flaccida
Nolana glauca
Nolana gracilima
Nolana humifusa
Nolana incana
Nolana inconspicua
Nolana inflata
Nolana leptophylla
Nolana linearifolia
Nolana lycioides
Nolana mollis
Nolana pallida
Nolana pallidula
Nolana paradoxa
Nolana parviflora
Nolana patula
Nolana peruviana
Nolana ramosissima
Nolana rostrata
Nolana rupicola
Nolana salsoloides
Expansion
12819
8062
6227
10707
5114
1505
7172
2253
1896
2629
4846
20653
6692
5724
17456
6326
8629
1983
11846
15413
371
4314
7049
1214
11986
2726
13712
4287
2405
9808
446
35169
13008
3988
3346
7226
3860
7471
Stability
27526
26631
5910
26819
8665
8678
1837
15215
7148
13748
4658
76132
9475
9918
41539
20441
14234
30192
4723
96298
7034
4533
14483
1743
36307
14974
78799
4809
11686
104386
10465
35674
6391
11653
1988
17071
8867
21511
Contraction
1861
4251
13456
15344
11438
8457
6729
2831
2625
10439
6066
16603
2175
8370
4471
4166
9360
168
354
59339
11133
2006
7071
681
9277
9485
23169
4854
4579
27693
39547
9837
75
11131
2127
13670
496
3349
Nolana sedifolia
Nolana spathuata
Nolana sphaerophylla
Nolana stenophylla
Nolana tarapacana
Nolana villosa
Nolana werdernannii
22502
2669
1620
10336
16855
12792
26893
76609
5792
3535
14167
47871
22584
13968
10788
8005
469
11962
80320
8860
2133
75
4. Final six variables (five bioclimate plus cloud cover) used for current and future models.
Species
Nolana aplocaryoides
Nolana carnosa
Nolana crassulifolia
Nolana divaricata
Nolana linearifolia
Nolana paradoxa
Nolana rostrata
Nolana sedifolia
Annual
Mean
Temp
0.5
8.3
2.2
1.4
0.1
10.6
1.8
8.1
Mean
Diurnal
Range
29.6
10.2
30.8
15.8
25.8
47.9
0.2
38.3
Pp of
Annual Pp
Coldest
Pp
Seasonality
Quarter
56.9
33.3
1
48
71.1
0
32.3
41.8
1.3
28.5
45.4
18.6
0
10.1
25.1
3.6
9.4
18.1
16.6
15
1.8
17.1
24
5.5
Cloud
Cover
2.3
1.6
4.1
1.2
1.1
14.3
16.7
2.6
76
Chapter IV. Comparative fruit anatomy and morphology of twelve species of
Nolana (Solanaceae) of the coastal Atacama Desert, Chile
Josefina Hepp1,2, Miguel Gómez1,2, Pedro León-Lobos3, Gloria Montenegro1,2, Samuel Contreras1,2
1
Departamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería Forestal, Pontificia
Universidad Católica de Chile
2
Centro del Desierto de Atacama, Pontificia Universidad Católica de Chile
3
Centro Regional de Investigación La Platina, Instituto de Investigaciones Agropecuarias (INIA)
Corresponding author. Tel.: +562 2354 4145
E-mail address: mgomezu@uc.cl
Postal address: Facultad de Agronomía e Ingeniería Forestal, Vicuña Mackenna 4860, Macul,
Santiago, Chile.
Abstract
The genus Nolana (Solanaceae) comprises numerous species endemic to the arid and
semi-arid environments in the coastal area of Chile and Peru and is distinguished within the
Solanaceae family by the penta-carpelar ovary that develops fibrous fruits called mericarps.
The objective of this study was to assess the anatomical and morphological differences or
similarities among mericarps in 12 Chilean species of Nolana in order to contribute to the
identification of species and examine possible phylogenetic relationships. Cross sections
were stained using safranin and fast green and prepared for microscopic examination.
Mericarps were also analysed using a scanning electron microscope and a stereoscopic
magnifier. Three different tissues were identified: an external one (exocarp), corresponding
to a mono-stratified epidermis; a middle one (mesocarp), of variable thickness, occupying
the bulk of the pericarp and the one that presents more variation between species; and an
inner one (endocarp), constituted by one layer of very dense sclereids, which was not always
easily distinguishable. It was not possible to determine a clear relationship between the
anatomy and morphology of the mericarps and the position of the species within clades of
the genus. However, trends were observed, and the new information generated could be of
use for the identification of species. The relationship between mericarp structure, and the
seed germination and survival of these species is also discussed.
Keywords: mericarp, mesocarp, seed, arid environments
Introduction
Species of the genus Nolana L. ex L. f. (Solanaceae-Nolaneae) are mainly restricted to the
Atacama and Peruvian Deserts along the western coast of South America, in Chile and
Peru. There are 90 species in total, of which 49 are present in Chile; 46 are endemic to this
country and just three species are shared with Peru. Around 70 species are endemic to the
lomas formations of both countries, generally located in the coastal Atacama and Peruvian
desert; the other 13 occur in habitats of higher altitude (> 1,000 masl) (Dillon, 2005), and
two of them, N. paradoxa and N. sedifolia, have extensive distributions along the coast of
Chile (over 1,500 and 1,400 km, respectively). According to Dillon et al. (2009), Nolana is
the only genus found throughout the entire range of lomas formations, and it stands out as
the most conspicuous floristic element there at the species level. Although the taxonomy of
this genus at species level is still not completely resolved, as new species continue to be
77
discovered (Hepp and Dillon, 2018) and doubts persist regarding the identity of many others
(Dillon, pers. com.), a phylogenetic estimate for Nolana, using a combination of molecular
markers, has been reconstructed by Dillon et al. (2007, 2009) and Tu et al. (2008). This
phylogeny supports the monophyly of the genus and has identified several clades supported
by geographic distributions and morphological synapomorphies (Dillon et al., 2009).
According to these analyses the genus is of Chilean origin, with two different introductions
to Peru and subsequent radiation (Dillon et al., 2009). Of the seven clades identified, four
are confined to Chile and three are mainly Peruvian with some presence in Chile.
Nolana species have been acknowledged to have high ornamental potential due to their
succulent foliage and flowering characteristics (Freyre et al., 2005; Riedemann et al., 2006),
but there are other reasons which make the study of this genus particularly interesting. It
has a high degree of endemism and presence in habitats with extreme conditions of aridity
and salinity, which gives it a high conservation value (Tu et al., 2008, Dillon et al., 2009);
also, some species present compounds with fungicidal activity in fungi of agricultural
importance (N. sedifolia, Vio-Michaelis et al., 2012). Additionally, the genus belongs to the
Solanaceae family and to the Solanoideae sub-family (Olmstead et al., 2008), where tomato
and pepper also belong. This makes it a possible source of genes of interest for the
development of cultivars that are better adapted to conditions of aridity and/or salinity; these
intergeneric hybridizations have been studied and produced in other families such as
Brassicaceae (Kaneko and Woo Bang, 2014). However, Nolana species have been poorly
studied and there is limited information regarding their ecology, reproductive biology and
establishment strategies, knowledge that is essential for their conservation and potential use
(but see Freyre et al., 2005; Douglas and Freyre, 2010; Jewell et al., 2012; Cabrera et al.,
2015).
It is likely that the success of Nolana species in these arid environments is due to the
development of anatomical characters such as foliar succulence, salt glands and trichomes
that capture moisture and restrict transpiration, and also to the development of fibrous hard
fruits, a character that is unique in the Solanaceae family (Knapp, 2002, Dillon et al., 2009).
These fruits correspond to schizocarps (indehiscent fruit originated by a gynoecium of two
or more concrescent carpels; Font Quer, 2000), which split up into mericarps when ripe.
Therefore, in Nolana, dispersal units are not seeds but mericarps, which can vary in number
between species from 2 to 30 per schizocarp and from laterally united and multi-seeded to
completely free and single-seeded (Tago-Nakazawa and Dillon, 1999; Knapp, 2002). The
pericarp is rich in sclereids, which give strength and rigidity to the fruit (Cabrera et al., 2015).
Although variable in size, shape and degree of fusion between species, the mericarp is
considered an autapomorphy of Nolana (Knapp, 2002).
The shape and structure of seeds or propagation units can provide important information to
identify species or make phylogenetic inferences in many plant families (Wada and Reed,
2008, Gontcharova et al., 2009, Wada and Reed, 2010, Camelia, 2011), although according
to Knapp (2002), this may sometimes be problematic since fruit type has proven to be highly
homoplasious. However, anatomical and morphological information provide valuable
information to help elucidate taxonomic uncertainties within a specific group (Liu et al., 2006;
Camelia, 2011). Additionally, when studying the structure of tissues it is possible to infer
78
their function (eg. resistance, defence against insects, etc.), as many studies have done
(Charini & Barboza, 2007; Gehan-Jayasuriya et al., 2007; Ruiz Sanchez et al., 2017).
The objectives of the present work are to identify morpho-anatomical differences across the
mericarps of 12 Chilean species of Nolana belonging to four different clades , in order to: 1)
better understand the relationship between fruit structure and the survival and germination
of the species, and 2) provide species specific characters useful for taxonomical purposes.
A greater similarity is expected in species that are closer according to clades, both in both
in morphology and anatomy of the mericarps.
Materials and methods
2.1 Plant material
Mericarps (dispersal units) of 12 species of Nolana were collected from individual plants
before dispersal at different locations in Chile (Table 1). Selection of species is the result of
availability of suitable fruits (i.e. mature mericarps, black/brown in colour and with dry or
senescent calyx) in the field (Fig. 1), which explains the overrepresentation of certain clades
(Fig. 2).
Collected mericarps were stored in an incubator at 20°C (18-24°C) and 40% RH (35-45%)
until evaluation. Identification of the species was possible with the help of Dr. Michael Dillon,
consulting herbarium specimens at the Natural History Museum of Santiago de Chile (SGO)
and using the available bibliography.
79
Figure 1. Flowers (above) and fruits (below) of six Nolana species included in this study. A and G: N.
crassulifolia (clade G); B and H: N. divaricate (clade G); C and I: N. sedifolia (clade G); D and J. N.
carnosa (clade C); E and K: N. intonsa (clade F); F and L: N. paradoxa (clade B).
Table 1. Coordinates of collection site and clade description of the 12 Nolana species of this study.
1
Species
Coordinates Clade* Description of clade*
Nolana aplocaryoides
(Gaudich.) I.M. Johnst.
26°08.786'S,
70°39.943'W
G
Clade G has recovered a strictly Chilean
group of 13 species represented by small
80
2
3
4
5
6
32°14.446´S,
71°31.177´W
29°14.601'S,
71°27.840'W
23°30.133'S,
70°23.069'W
Nolana onoana M.O.
23°29.903'S,
Dillon & Nakazawa
70°21.817'W
Nolana sedifolia Poepp. 20°49.542'S,
70°09.392'W
Nolana intonsa I.M.
Johnst.
20°49.309'S,
70°09.366'W
7
F
8
Nolana carnosa (Lindl.)
Miers ex Dunal
28°02.612'S,
71°07.022'W
9
Nolana rostrata (Lindl.)
Miers ex Dunal
28°06.806'S,
71°09.257'W
Nolana jaffuelii I.M.
10 Johnst.
Nolana paradoxa Lindl.
11
to moderate shrubs (traditionally called
"Dolia") and annuals, all with highly
reduced corollas, often white or yellowish
(Dillon et al., 2009) but some have bluish
flowers.
Nolana crassulifolia
Poepp.
Nolana divaricata
(Lindl.) I.M. Johnst.
Nolana linearifolia Phil.
C
20°48.903'S,
70°09.630'W
30°11.734'S,
71°25.377'W
Nolana parviflora (Phil.) 23°30.133'S,
70°23.069'W
12 Phil.
B
Clade F is comprised of 18 Peruvian
species and one Chilean species (N.
intonsa), which shares strong
morphological resemblance to southern
Peruvian species, such as N. pallida
(Dillon et al., 2009).
Clade C has recovered a monophyletic
group comprised of 6 species traditionally
classified as Alona. These are all largeflowered shrubs confined to Chile (Dillon
et al., 2009).
Clade B, previously recognized as a
segregate genus, Sorema, currently
consists of two sub-groups: N. pterocarpaN. baccata-N. parviflora, which are erect
annuals with small flowers; and N.
paradoxa-N. rupicola, which are rosetteforming, taprooted plants with larger
flowers (Dillon et al., 2009). These species
are only present in Chile (Dillon et al.,
2009).
*Letters for clades and description according to Dillon et al., 2009.
81
Figure 2. Biogeographic diversification of Nolana, modified from Dillon et al. (2009), showing the clade
of the 12 studied Nolana species. C, Chile; P, Peru; G, Islas Galápagos, Ecuador.
2.2 Stereoscopic magnifier
The colour, size, presence of dispersal structures, and external appearance of the
dispersion unit were recorded. A sample of each species was photographed using a
stereoscopic magnifier (Olympus SZ2-ILST).
2.3 Scanning electron microscope
Scanning electron microscopy (SEM) was used to examine the morphology of the fruits, the
surface of the funicular scar and the longitudinally sectioned mericarps. Samples of 12
species were mounted on aluminium SEM stubs and sputtercoated with a platinum-gold
alloy using a Quorum Sputter Coater Q150T. Mericarps were examined and photographed
using a HITACHI S-4700 SEM at an accelerating voltage of 2.0 kV and working distance of
12.0 mm. Images were saved as tagged image file format (TIFF).
2.4 Histological analysis
Five mericarps per species were randomly selected and fixed in formol acetic alcohol (FAA),
then dehydrated and preserved in paraffin in preparation for sectioning. Cross and
82
longitudinal histological sections were made through the ripe mericarps using a microtome
(to a thickness of 16 µm) and stained with safranin-fastgreen solution. Sections were
observed under a light microscope and photographed using a Moticam 5 camera and
measured with Motic Images Plus 2.0 ML.
Results and discussion
3.1 Fruit anatomy
In the anatomical analysis with SEM images, the presence of one to several seed or
embryonic chambers within mericarps, with evident differences between species, was
confirmed; species such as N. intonsa, N. linearifolia or N. rostrata have multiple embryonic
chambers, as opposed to N. aplocaryoides, N. jaffuelii or N. onoana, which usually bear one
to two embryonic chambers per mericarp (Fig. 3). According to Johnston (1936), the fruit of
Nolana would have evolved from a multiseminated berry in which the carpellar and placental
tissue have united and hardened forming a firm and lobed structure, morphologically a
schizocarp, divided at maturity into a variable number of mericarps (Saunders, 1936). These
mericarps are very particular since each embryo is divided into a separate embryonic
chamber, formed by parenchymal tissue (Bondeson, 1986).
Also evident in most of the mericarps is a slightly depressed, elliptical area which
corresponds to the funicular scar (Fig. 3A-3C; Fig. 3G-3I) and indicates the location of each
embryonic chamber (Fig. 3D-3F; Fig. 3J-3L). This structure was described in Bondeson
(1981) as a “germination plug”, since the protruding radicle pushes the plug out during
germination. It corresponds to the funicular tissue that goes through the pericarp; it can be
detached mechanically and appears to influence germination, as fruits scarified in this region
obtained higher percentages of germination than did intact fruits (Cabrera et al., 2015).
Since each plug separates independently, it is possible to assume that there would be more
than one opportunity for germination for those mericarps with more than one embryonic
chamber (Fig. 3J, 3K and 3L). No differences were observed between species in terms of
structure, size or position of the germination plug.
83
Figure 3. Scanning electron microscopy (SEM) images for mature (dispersed) mericarps of six Nolana
species: intact (A) and longitudinally cut (D) mericarps of N. aplocaryoides; intact (B) and
longitudinally cut (E) mericarps of N. onoana: intact (C) and longitudinally cut (F) mericarps of N.
jaffuelii; intact (G) and longitudinally cut (J) mericarps of N. intonsa; intact (H) and longitudinally cut
(K) mericarps of N. linearifolia; intact (I) and longitudinally cut (L) mericarps of N. rostrata. Arrows
indicate the location of funicular scar; circles enclose the structure that corresponds to the germination
plug. Bars indicate mm. abbreviations: emc, embryonic chamber.
The histological study performed on mericarps of these twelve Nolana species reveals the
presence of three layers. Following the definition of Richard (1819, quoted in Pabón-Mora
& Litt, 2011), we consider an external layer (exocarp), corresponding to a mono-stratified
epidermis whose cells contain vacuoles with tannins and a thin protective cuticle; an inner
84
layer, the endocarp, constituted by only one layer of very dense sclereids; and in the middle,
the mesocarp, of variable thickness, generally occupying the bulk of the pericarp, and the
one that presents more variation between species (Fig. 4).
Figure 4. Pericarp of three Nolana species at two magnifications (40x, left images; and 400x, right
images). A and B: N. crassulifolia (clade G); C and D: N. paradoxa (clade B); E and F: N. linearifolia
(clade G). Abbreviations: en, endosperm; end, endocarp; em, embryo; exo, exocarp; mes, mesocarp.
The exocarp is variable in terms of size of the cells, but it was generally presented as a
monostratified layer (Fig. 6 and 7, Table 2).
The mesocarp consists of two histologically differentiated zones, as has been described for
other Solanaceae species (Solanum spp.; Chiarini and Barboza, 2007). There is an internal
zone constituted by sclereids that can either be very dense, or present empty cells with
undulated but lignified walls, and an external one, formed by radially elongated cells with
suberized walls in some species (N. carnosa and N. jaffuelii; Fig. 5, Table 2), or by
parenchymatic cells that may be collapsed, in others (N. aplocaryoides, N. crassulifolia, N.
divaricata, N. intonsa, N. rostrata and N. sedifolia; Fig. 6 and 7, Table 2). The determination
of these two zones was possible due to the study of the fruit development of N. paradoxa in
different stages; in early stages, it was possible to visualize the origin of these two zones
85
within the mesocarp and to clearly distinguish the monolayer of the endocarp (Hepp et al.,
in preparation), although previous studies concluded that the lignified and extended portion
of the mericarp corresponded to the endocarp (Bondeson, 1986; Cabrera et al., 2015).
Figure 5. Pericarp of two Nolana species. A and B: N. carnosa (clade C); C: N. jaffuelii (clade B).
Abbreviations: exo, exocarp; mes1, internal zone of the mesocarp; mes2, external zone of the
mesocarp.
The several layers of sclereids of the mesocarp provide hardness and rigidity to the fruit and
would also allow the mericarps to remain relatively intact in the soil seed bank for many
years, despite the extreme conditions prevalent in the Atacama Desert. To this is added the
the external mesocarp with suberized cells in the case of N. carnosa and N. jaffuelii (Fig. 5),
which could help isolate the embryo from the external environment surrounding the fruit. An
additional protection may be given by the presence of chemical compounds in the mericarp
cover; for example, an antifungal activity on Botrytis cinereal was discovered for N. sedifolia
(Vio-Michaelis et al., 2012). The hard layer of sclereids, along with other compounds such
as tannins and cuticle present in the exocarp and suberized cells of the outer mesocarp,
may confer water impermeability to the mericarp, which could be related to physical
dormancy of the seeds, as was suggested by Cabrera et al. (2015).
86
Figure 6. Pericarp of six Nolana species: A: N. aplocaryoides (clade G); B: N. crassulifolia (clade G);
C: N. divaricata (clade G); D: N. intonsa (clade F); E: N. rostrata (clade C); F: N. sedifolia (clade G).
Abbreviations: exo, exocarp; mes1, internal zone of the mesocarp; mes2, external zone of the
mesocarp.
87
Figure 7. Pericarp of three Nolana species. A: N. linearifolia (clade G); B: N. onoana (clade G); C: N.
paradoxa (clade B). Abbreviations: en, endosperm; end, endocarp; exo, exocarp; mes1, internal zone
of the mesocarp; mes2, external zone of the mesocarp.
It is possible to classify the twelve Nolana species into three groups according to the
composition of the mesocarp: (1) those with two well differentiated zones, one external with
several layers of large cells with suberized walls and one internal of dense sclereids (Fig.
5), as in N. jaffuelii and N. carnosa; (2) those with a large (internal) zone of very dense
sclereids and an external layer that is collapsed or difficult to distinguish (Fig. 6), as in N.
aplocaryoides, N. crassulifolia, N. divaricata, N. intonsa, N. parviflora, N. rostrata and N.
sedifolia; and (3) those with a wide internal zone of large or medium sized cells, empty or
with large vacuoles (giving them a “porous” appearance), undulated lignified cell walls, and
a collapsed external layer that is difficult to distinguish (Fig. 7), as in N. linearifolia, N. onoana
and N. paradoxa (Table 2).
Table 2. Anatomical mericarp features of the 12 species of Nolana studied. Clades correspond to
phylogenetic study of Nolana by Dillon et al. (2007, 2009) and Tu et al. (2008).
Species
Clade
Mericarp
size
(sample)
Mesocarp
Exocarp
External zone
Internal zone
“Two-zones” group – Fig. 5
N. jaffuelii
N. carnosa
B
Unistrate,
isodiametric
1.8
mm
cells about
diameter
0.04 mm
diameter
1-4 layers of large elongated cells
with suberized walls, irregular
thickness in whole (0.13 thinnest
section - 0.43 mm thickest section)
Layer of irregular
thickness (0.12 thinnest
section - 0.48 mm
thickest section),
constituted by very
dense isodiametric
sclereids
C
Unistrate,
6
mm isodiametric
long, 5.5 cells, 0.05
mm wide mm average
width
1.2-1.7 mm width, several (8-10)
layers of large elongated cells with
suberized walls
0.5 mm average width,
layer of very dense
isodiametric sclereids
“Dense” group – Fig. 6
N.
G
aplocaryoides
0.03 mm average width, 1 apparent
1.5
mm Indistinct, thin
layer of isodiametric cells, with
diameter
unistrate layer
crushed appearance at maturity
0.15 to 0.45 mm width,
layer of very dense
isodiametric sclereids,
some intracellular
spaces noticeable
N.
crassulifolia
G
Unistrate,
2
mm isodiametric
long, 1.5 cells about
mm wide 0.07 mm
diameter
0.03 mm average width, 1 apparent
layer of isodiametric cells, with
crushed appearance at maturity
0.35 to 0.8 mm width,
layer of very dense
isodiametric sclereids
G
Unistrate,
isodiametric
2 mm long cells about
0.05 mm
diameter
0.04 mm average width, 1 apparent
layer of isodiametric cells, with
crushed appearance at maturity
0.5 mm average width,
layer of very dense
isodiametric sclereids,
some intracellular
spaces noticeable
N. divaricata
88
F
3.5
long
0.05 mm average width, 1 layer of
round or elongated cells with
suberized walls, with crushed
appearance at maturity
0.1-0.3 mm width (area
surrounding the
embryonic chambers),
very dense isodiametric
sclereids, some
intracellular spaces
noticeable
N. parviflora
B
0.04 mm average width, 1 layer of
Indistinct, thin
5 mm long
round or elongated cells with
unistrate layer
suberized walls
0.07 mm average width,
layer of very dense
isodiametric sclereids.
Slightly thicker at point
of union with wing
N. rostrata
C
6 mm long
G
Unistrate,
2
mm isodiametric
long, 1.5 cells about
mm wide 0.05 mm
diameter
N. intonsa
N. sedifolia
Unistrate,
isodiametric
mm
cells, 0.04
mm average
width
0.06 mm average width, 1 to 3
Indistinct, thin
layers of isodiametric cells, with
unistrate layer
crushed appearance at maturity
0.02 mm average width, 1 apparent
layer of isodiametric cells, with
crushed appearance at maturity
1 to 1.8 mm width, layer
of very dense
isodiametric sclereids
0.1 to 0.35 mm width,
layer of very dense
isodiametric sclereids
“Porous” group – Fig. 7
N. linearifolia
N. onoana
N. paradoxa
G
G
B
Unistrate,
isodiametric
2.3
mm
cells about
diameter
0.035 mm
diameter
0.025 mm average width, 1
apparent layer of isodiametric cells,
with crushed appearance at
maturity
0.02 mm average width, 1 apparent
1.5
mm Indistinct, thin
layer of isodiametric cells, with
diameter
unistrate layer
crushed appearance at maturity
2.6
long
Unistrate,
isodiametric
mm
or rectangular
cells, 0.02 mm
average width
1 to 3 layers of medium sized
elongated
cells,
crushed
appearance at maturity, irregular
thickness in whole (0.02 thinnest
section - 0.1 mm thickest section)
Medium sized,
apparently empty cells
with undulated but
sclerified walls (area
surrounding the
embryonic chambers:
0.15 thinnest section 0.4 mm thickest section)
Medium sized,
apparently empty cells
with undulated but
sclerified walls (0.11
thinnest section - 0.6
mm thickest section)
Medium to large, almost
empty
cells
with
undulated but sclerified
walls, forming a spongy
tissue in appearance
(0.11 thinnest section 0.85
mm
thickest
section)
It was not possible to identify an evident relationship between the anatomy of the mericarps
and the location of the species within the clades. However, a tendency was observed, since
most of the mericarps of species belonging to Clade G have an internal zone of the
mesocarp with very dense sclereids, and an external zone that is barely distinguishable,
very thin and with a crushed appearance. This feature was also presented by N. intonsa of
Clade F and N. rostrata of Clade C. Two species of Clade G also had an internal zone of
the mesocarp with large and empty cells, with undulated walls, which gave them a porous
89
or aerated appearance. This feature was more evident in N. paradoxa (Clade B) than in N.
linearifolia and N. onoana (both Clade G), as was also revealed in the SEM analysis (Fig.
3), where these last two species appear to have a pericarp very similar in density to the rest
of Clade G species (N. divaricata, N. crassulifolia, N. aplocaryoides and N sedifolia). Thus,
we can conclude that there is a gradient in terms of size and content of cells of the internal
zone of the mesocarp for the 12 species studied. Although this is not enough by itself to
identify species, it is a new tool which may assist identification, and that can be
complemented with other analyses for this purpose.
3.2 Fruit morphology
According to Dillon et al. (2009), although the mericarp is a unique character and
distinguishes Nolana from other members of the Solanaceae family, its morphology, and
their number and size, are of limited use in circumscribing internal groups. However, certain
trends can be observed.
Clade B is the only one that presents winged mericarps (Fig. 8), but just one of its two
subclades includes species that display this feature: the N. pterocarpa-N. baccata-N.
parviflora subgroup, which are erect annuals with small flowers and angular mericarps, at
times prolonged into wings (Dillon et al., 2009) (Fig. 8, Table 3). The other subclade is N.
paradoxa-N. rupicola, rosette-forming, taprooted plants with larger flowers, and more
spherical but still angular mericarps lacking prolonged wings (Dillon et al., 2009) (Fig. 8).
These winged mericarps could be a diagnostic character for this subclade (M. Dillon, pers.
com.), although in the case of N. jaffuelii, which has plants that are similar to those in this
first subclade, mericarps are rounded and angular, without wings (Fig. 8). All members of
Clade B present numerous mericarps (10-20, up to 30 per schizocarp), unlike members of
other clades as described by Dillon et al. (2009), which present smaller numbers (often 3 to
6, up to 8 mericarps). Species of this clade thus have a different dispersal strategy, releasing
multiple mericarps that accumulate under the mother plants, and are therefore important
components of the blooming desert phenomenon, at which time these species can be seen
blooming profusely in the coastal Atacama Desert and inland valleys.
90
Figure 8. Morphological features of mericarps of species that belong to Clade B, SEM and
stereoscopic magnifier images. A: N. paradoxa; B: N. parviflora (portion of mericarp); C: N. jaffuelii.
W: wing. Bar (right) indicates mm.
Mericarps of clade C are relatively large (Fig. 9, Table 3) compared to all other mericarps of
Nolana species, and in both N. carnosa and N. rostrata, they have a thin layer (exocarp plus
part of the mesocarp immediately below) that covers each mericarp completely or partially.
This layer can easily be detached and could be a distinguishing feature for members of this
clade. Their fruits are schizocarpic, rounded or merely 5-sulcate, composed of 5
multispermous sections which are broadly joined to one another laterally (Johnston, 1936).
91
Figure 9. Morphological features of mericarps of species that belong to Clade C, SEM and
stereoscopic magnifier images. A: N. carnosa, SEM image of portion of mericarp without cover, and
photograph of mericarp with and without thin covering layer; B: N. rostrata, SEM image of portion of
mericarp, and photograph of mericarp with and without thin covering layer. It is possible to see the
funicular scar in one region of the mericarp of N. rostrata.
N. intonsa is the only Chilean species belonging to clade F; its mericarps are easily
recognizable, shaped like a comma (Fig. 10, Table 3) with a rough surface of bright black
colour when freshly dispersed. N. intonsa presents a large number of embryonic chambers
within some of its mericarps (up to 8), the highest number for all studied species (Table 3).
92
Figure 10. Morphological features of mericarps of Nolana intonsa (Clade F), SEM and stereoscopic
magnifier images.
In clade G, mericarps of all its species have relatively different sizes, and differ in terms of
texture and gloss, but they are all rounded or spherical (Fig. 11) and usually bear 1 to 3
embryonic chambers, with exception of N. linearifolia which has larger mericarps and can
bear up to 5 chambers in one mericarp (Table 3). Johnston (1936) mentions that although
the species is aberrant in corolla, the fruit agrees closely with that prevailing among the
shrubby members of its genus (Clade G; Fig. 11, Table 3).
93
Figure 11. Morphological features of mericarps of species that belong to Clade G, SEM and
stereoscopic magnifying glass images. A: N. aplocaryoides; B: N. crassulifolia; C: N. divaricata; D: N.
linearifolia; E: N. sedifolia; F: N. onoana.
It is important to mention that for the recently described Nolana patachensis (Hepp and
Dillon, 2018), unique characteristics of its mericarps were determinant to identify it as a new
species for Chile. As the authors describe, its mericarps are elliptic or oval with finely bullate
surfaces, which distinguishes N. patachensis from its congeners in northern Chile.
Table 3. Morphological mericarp features of the 12 species of Nolana studied. Clades correspond to
phylogenetic study of Nolana by Dillon et al. (2007, 2009) and Tu et al. (2008).
Species
Clade
Number of
Mericarp size
mericarps
Mericarp shape
Number of
seeds per
mericarp
N. jaffuelii
B
numerous
(>15)
1-3 mm
diameter
angular
1(-2)
N. paradoxa
B
numerous
(>15-20)
3-6 mm long
rounded angles
(polyhedral)
1-2(-3)
2 types of mericarps,
1 (winged) to
major nutlets and
4-6 mm long, 2thinner, winged ones. 5 (only larger
3 mm wide
fruits)
Both laterally
compressed
N. parviflora
B
8-15
N. carnosa
C
4-5
N. rostrata
C
5-7-8
N. intonsa
F
7-8
N. aplocaryoides
G
3-6
N. crassulifolia
G
3-8
N. divaricata
G
(3)-5
N. linearifolia
G
3-6
N. onoana
G
6-7
N. sedifolia
G
3-8
5-8 mm long, 45 mm wide
4-6 mm
diameter
3.5 mm long 2.5 mm wide
1-2 mm
diameter
2-3 mm
diameter
2-3 mm
diameter
2-3 mm
diameter
2-3 diameter
irregular, globose,
extended
1 to 5
rounded
2-7
angular; unequal
1 to 8
spherical
1-2-3
rounded; unequal
1-2
globose; ovoid,
unequal
1-2
spherical
(1)2 to 5
ovoid
1(-2)
2-3 mm long, 1oval; unequal
2 mm wide
1-3
From: Dillon et al., 2007; Mesa et al., 1998; Mesa, 1981; Johnston, 1936; Reiche, 1910; and
personal observations
94
Conclusions
There are shared fruit characteristics among the twelve Chilean Nolana species studied, but
there is no clear pattern that would allow them to be identified based only on internal
anatomy of the mericarps. It was possible to classify the twelve species into 3 groups
according to the composition of the mesocarp, but this was not strictly related to clades. The
inner layer of the mesocarp, composed of dense sclereids, may provide protection to the
embryo, as it would allow the mericarps to remain relatively intact in the soil seed bank for
many years. Additionally, for species with mericarps containing more than one embryonic
chamber, there would be more than one opportunity for germination, since each plug is
detached independently. Therefore, the particular structure of the mericarps would be
relevant in the survival of these species in desert environments, both in terms of dispersion
and reproduction.
The identification of species can also be supported by the morphology or external structure
of the mericarps, since within each clade it is possible to find trends presented by their fruits,
particularly in terms of size and shape.
Acknowledgements
We thank Wolfgang Stuppy, who supported the internship at RBG Kew and allowed the use
of the scanning slectron microscope and other instruments. We also thank Michael Dillon
for his assistance with identification of species and helpful information throughout this study.
All species were collected with permission and support of the National Forest Commission
(CONAF Pan de Azúcar, CONAF Llanos de Challe), the Institute of Agricultural Research
(INIA) and owners/administrators of private parks (Punta de Choros, Puquén Los Molles)
and research stations (Alto Patache Fog Oasis, managed by Universidad Católica de Chile).
The CONICYT Scholarship (21130176) to Josefina Hepp made this study possible.
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Knapp, S. 2002. Tobacco to tomatoes: a phylogenetic perspective on fruit diversity in the
Solanaceae. Journal of Experimental Botany 53: 2001-2022.
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pour obtener le Titre de Doctoeur, 1981, 199 pp.
Mesa, A., Muñoz-Schick, M. & Pinto, R. Presencia de Nolana adansonii (Roemer y Schultes)
Johnst. y Nolana intonsa Johnst. (Nolanaceae) en el desierto costero de Iquique, Norte de
Chile. Noticiario mensual del Museo Nacional de Historia Natural, 1998, N° 333, p. 3-5.
96
Olmstead, R., Bohs, L., Abdel Migid, H., Santiago-Valentin, E., Garcia, V. & S.M. Collier.
2008. A molecular phylogeny of the Solanaceae. Taxon 57 (4): 1159-1181.
Reiche, K. 1910. Flora of Chile, 83. Familia Nolanaceae. 5: 410-435.
Riedemann, P., Aldunate, G. & Teillier, S. Flora nativa de valor ornamental: Zona Norte.
Santiago, Chile: Ediciones Chagual, 2006, 404 pp.
Saunders, E. 1936. On Certain Unique Features of the Gynoecium in Nolanaceae. The New
Phytologist 35(5): 423-431.
Tago-Nakazawa M, Dillon MO. 1999. Biogeografía y evolución en el clado Nolana
(Nolaneae-Solanaceae). Arnaldoa 6(2): 81±116.
Tu, T., Dillon, M., Sun, H. & Wen, J. Phylogeny of Nolana (Solanaceae) of the Atacama and
Peruvian deserts inferred from sequences of four plastid markers and the nuclear LEAFY
second intron. Molecular Phylogenetics and Evolution, 2008, N° 49, p. 561-573.
Vio-Michaelis, S., Apablaza-Hidalgo, G., Gómez-Ungidos, M., Peña-Vera, R. and
Montenegro-Rizzardini, G. 2012. Antifungal activity of three Chilean plant extracts on
Botrytis cinerea. Botanical Sciences 90 (2): 1-5.
Wada, S. and Reed, B.M. 2008. Morphological analysis of Rubus seed. Acta Horticulturae
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97
Chapter V. A new endemic species of Nolana (Solanaceae-Nolaneae) from
near Iquique, Chile
Arnaldoa 25 (2): 323-338, 2018
http://doi.org/10.22497/arnaldoa.252.25202
Josefina Hepp
Departamento de Ciencias Vegetales
Facultad de Agronomía e Ingeniería Forestal
&
Centro del Desierto de Atacama
Pontificia Universidad Católica de Chile, Santiago, Chile
(jnhepp@uc.cl)
Michael O. Dillon
The Field Museum, Integrative Research Center
1400 South Lake Shore Drive, Chicago, IL 60605-2496, USA
(mdillon@fieldmuseum.org)
Abstract
In connection with studies on the fog oasis or lomas formations at Alto Patache near Iquique,
Chile, the first author encountered several Nolana species, including one new to science
described here, N. patachensis J. Hepp & M.O. Dillon (Solanaceae-Nolaneae). The new
species is diagnosed, described, illustrated with photographs, and compared to nearest
geographic neighbors in northern Chile. To an aid in recognition, a key to Nolana species
reported from Región of Tarapacá is provided. Putative relationships between the various
Nolana species encountered at the type locality are discussed. Conservation efforts at the
type locality are highlighted, including its unique environmental conditions, biota, and
potential threats
Key Words: Nolana, Nolaneae, endemics, lomas formations, new species, Región of
Tarapacá, Chile, conservation, Solanaceae
Resumen
En relación a los estudios sobre el oasis de niebla o las formaciones de lomas en Alto
Patache cerca a Iquique, Chile; el primer autor recolectó varias especies de Nolana, incluida
una nueva especie para la ciencia que aquí se describe, N. patachensis J. Hepp & M.O.
Dillon (Solanaceae-Nolaneae). Además de la descripción, se realiza la diagnosis, se ilustra
con fotografías y se compara con las especies vecinas más cercanas del norte de Chile.
Para ayudar al reconocimiento, se proporciona una clave para las especies de Nolana
reportadas para la Región de Tarapacá. También se discuten las relaciones putativas entre
las diversas especies de Nolana encontradas en la localidad del tipo; así mismo, se resaltan
los esfuerzos de conservación en la localidad tipo, incluyendo sus condiciones ambientales
únicas, biota y amenazas potenciales.
98
Palabras clave: Nolana, Nolaneae, endémicas, formaciones de lomas, especie nueva,
Región de Tarapacá, Chile, conservación, Solanaceae
Introduction
Nolana L. ex L.f. (Solanaceae-Nolaneae) is a genus consisting of 90 species, including the
one described here (Dillon, 2005, 2016). With the addition of this species, it brings the total
number to 48 Chilean species, including 45 endemic species, and three species with disjunct
distributions ranging from southern Peru (i.e., Nolana adansonii (Roem. & Schult.) I.M.
Johnst., N. gracillima (I.M. Johnst.) I.M. Johnst., and N. lycioides I.M. Johnst.). Of this
number, 12-13 Nolana species have been verified from Región de Tarapacá (see Table 1).
In Chile, greatest species diversity in Nolana is confined to near-ocean localities termed
lomas formations, between 50--800 m elevation and along the foot of the Coastal Cordillera,
usually within 20 kms of the Pacific Ocean (Rundel et al., 1991; Dillon & Hoffmann, 1997).
Only a few species are distributed above 2000 m and/or at a distances of 50-500 kms inland
from the coast, e.g., Nolana leptophylla, N. sessiliflora.
Most Chilean species are narrow endemics, with small, restricted geographic ranges and
specific ecological requirements, but a few species have larger geographic distributions and
occupy several vegetation formations (Dillon, 2005).
Materials and Methods
Descriptions were made from dried herbarium specimens deposited in SGO. All acronyms
follow those in Index Herbariorum (http://sweetgum.nybg.org/science/ih/). Conservation
status was assigned using IUCN criteria (2017) combined with field observations and
geographic distribution based on herbarium specimens. Scanning electron microscopy
(SEM) was used to examine the structure of the mericarps. Samples were mounted on
aluminium SEM stubs and sputter coated with a platinum-gold alloy, using a Quorum Sputter
Coater Q150T. Mericarps were examined and photographed using a HITACHI S-4700 SEM
at an accelerating voltage of 2.0 kV and working distance of 12.0 mm. Images were saved
as TIFF (Tagged Image File Format). Sample were photographed using a stereoscopic
magnifying glass.
We utilize the "morphological cluster" concept in recognition of species in Nolana (see
Mallet, 1995), defined as "assemblages of individuals with morphological features in
common and separate from other assemblages by correlated morphological discontinuities
in a number of features". In addition to the diagnoses provided for the new species, specific
characters useful in recognition of species are detailed in the Key to Species of Región de
Tarapacá, Chile.
Taxonomic treatment
99
Nolana patachensis J.Hepp & M.O. Dillon, sp. nov.
Figure 1, 2, 3, 4, 5
TYPE: CHILE. Región de Tarapacá: Prov. Iquique, Alto Punta Patache, 20°49’S, 70°09’W,
1 Nov 1997, W. Sielfeld 32 (holotype: SGO-143057).
Diagnosis
Nolana patachensis is most similar to N. onoana, sharing similar leaf and floral morphology
(Fig. 7C); however, it differs from the latter species in its spreading, prostrate habit, and
erect, terete leaves. Further, its elliptic or oval mericarps with (1-)2-3(-4) seeds and finely
bullate surfaces (Fig. 5A) are essentially unique within its congeners in northern Chile. The
mericarps in N. patachensis also differ from mericarps found in N. onoana which are round
or spherical, with a finely reticulate or alveolate surfaces. Other annual, tap-rooted species,
e.g., N. aplocaryoides, have with much wider leaves with long, villous trichomes (Fig. 7A),
and round or spherical mericarps; and N. gracillima with glabrescent to pilose pubescence
leaves, shorter corollas (Fig. 7B), and pyriform mericarps.
Description
Taprooted annual herbs to 50 cm in diameter, 10-20 cm tall, basally-branched; stems
branched, prostrate to decumbent, to 15 cm long, densely stipitate-glandular. Leaves
alternate, sessile, the blades linear-oblong, 10-20 mm long, 3-4 mm wide, terete, erect or
perpendicular orientation, succulent, densely pubescence with stout, stipitate-glandular
trichomes, entire, apically rounded, the bases rounded. Inflorescences of solitary flowers
in upper leaf axils; pedicels cylindrical, densely pubescent, 2-7 (-10) mm long. Flowers 5merous; calyx narrowly campanulate, 5-7 mm wide at anthesis, densely covered with
stipitate-glandular trichomes, 5-lobed, the tube ca. 3-5 mm long, ca. 5 mm wide, the lobes
lanceolate, unequal, 2-3 mm long, ca. 1 mm wide, the apices obtuse or rounded; corollas
zygomorphic, infundibuliform, 18-24 mm long, 8-12 mm wide at anthesis, distally lavender
to light blue, the throat clear, externally and internally glabrous, the lobes obtuse; stamens
5, included, the filaments inserted on lower third of corolla, unequal, 3 long, 2 short, anthers
dithecal, purple, the thecae ca. 1.2 mm long, ca. 1 mm wide, glabrous; ovary glabrous, basal
nectary ca. 1 mm wide, the carpels 5, the style included, the stigma green. Fruits mericarps,
5, 1-seriate, oval to elliptic, black, 2-3 mm long, 1.5-2 mm wide, adaxial surfaces minutely
bullate; seeds (1-)2-3(-4) per mericarp.
Phenology: Annual, tap-rooted annual that responds to sufficient moisture for germination
and flowering during November, 1997, and more recently November-December, 2015.
Etymology: The species epithet is the latinisation of the geographic locality of the type
collection, Alto Patache fog oasis or lomas formation located in Región of Tarapacá of
northern Chile. As with many place names, the origins remain obscure, but it may have its
origins in Pukina, a language distantly related to Quechua. In any event, it is not associated
directly with either Quechua or Aymara. Further details on the locality are to be found below.
100
Distribution and ecology: Nolana patachensis has been recorded from two adjacent
localities in Región de Tarapacá (Fig. 6), Alto Patache fog oasis or lomas formation (20º48'S,
70º09'W) and Alto Punta Lobos (21º02'W, 70º09'W). Both of these locality exhibit southwest
oriented slopes that are constantly exposed to fog (Muñoz-Schick et al., 2001; Calderón et
al., 2010; Osses et al., 2017). Alto Patache fog oasis has been recognized as a priority area
for conservation and is currently protected in a concession granted by the Ministry of
National Assets for 25 years (since 2007) to Pontificia Universidad Católica de Chile,
through the Atacama Desert Center. Its isolation has provided conditions for development
of a particular biota. For example, it is the only known locality for Santessonia cervicornis
(Follmann) Follmann (an endemic Critically Endangered lichen species; Vargas et al., 2017)
and several other lichens common to the Atacama Desert. The vascular flora at Alto
Patache has been estimated at approximately 42 species of vascular plants (Pliscoff et al.,
2017), including one of the few populations of Alstroemeria lutea Muñoz-Schick (MuñozSchick 2000). The arthropod fauna includes two endemic Coleoptera, Scotobius
patachensis and Scotobius larraini (Sagredo et al., 2002), and two bees, Penapis
larraini (Hymenoptera: Halictidae: Rophitinae) (Packer, 2012) and Neofidelia submersa
(Hymenoptera: Apoidea: Megachilidae) (Dumesh & Packer, 2013). Lastly, this locality has
been implicated in the first record of a noctuid moth, Hemieuxoa polymorpha Forbes, for
Chile, when adults were collected at Alto Patache in 1999 (Angulo & Olivares, 2005). Alto
Punta Lobos is located approximately 25 kms south of Alto Patache, and like that formation,
it has a compliment of perhaps 20 endemics in a flora of ca. 40 species (Muñoz-Schick et
al., 2001).
Putative relationships: Nolana patachensis is distinctive among its congeners in Chile with
a combination of characters not encountered in any other described species. While DNA
results are not available for this species, overall morphological similarity suggests
relationships with Nolana onoana, a member of Clade G (Dillon et al., 2009).
Clade G is a strictly Chilean clade including Nolana aplocaryoides as the sister taxon to the
remainder of the clade (Dillon et al., 2009). This group is represented by small to large
shrubs and annuals, all with small leaves, (1-)10-20(-40) mm long and (1-)2-5(-7) mm wide,
all with smaller corollas when contrasted with those in Clade B (e.g., N. jaffuelii) and some
members with only white or yellowish corollas. The leaf pubescence is extremely variable
from glabrous to densely canescent with stellate or dendritic, or arachnoid, stipitate, or
simple trichomes, and rarely with superficial salt glands.
Conservation status: Critically Endangered (CR); overall distribution at two localities, each
with <10 km2 (CR) and perhaps <250 individuals. See IUCN (2017) for explanation of
measurements. The threats to these habitats are posed by ever-expanding human pressure
from mining and other industrial processes that can contaminate these fragile environments.
Efforts at studying, protecting and preserving the region are underway.
Additional specimens examined: CHILE. Región de Tarapacá: Prov. Iquique, Alto
Patache, E. Belmonte 97770 (CONC-143484), Alto Punta Patache, 20°49’W, 70°09’W, 800
m, 8 Nov 1997, R. Pinto s.n. (SGO-142975); Alto Punta Lobos, 21°02’S, 70°09’W, 800 m,
17 Jan 1998, R. Pinto s.n. (SGO-142976).
101
A series of Nolana collections from further south of the currently known range of N.
patachensis need to be evaluated in the light of recent discoveries; these are from Región
de Antofagasta, Prov. Tocopilla, camino a Mina Mantos de La Luna, M. Quezada & E. Ruiz
16 (CONC-121070), M. Quezada & E. Ruiz 17 (CONC-121207), and M. Quezada & E. Ruiz
19 (CONC-121076; SGO-127878). Another collection that should be scrutinized further;
Prov. Tocopilla, Quebrada Mamilla, F. Schlegel 7693 (CONC-115627).
Notes: When herbarium material of this plant was first encountered by MOD in 2009, they
had been determined as Nolana aplocaryoides, another tap-rooted annual species typically
recorded further to the south. That species has quite different leaves and pubescence, but
with similar corollas (Fig. 7A). At that time, MOD determined the sheets as N. gracillima, a
species originally described from southern Peru (Fig. 7B) but with populations reaching
northern Chile. However, upon examination of photographs of living plants with flowers
taken by JH on a field trip to Alto Patache in 2015, after the abundant August rains in the
sector (Figs. 2, 3), MOD recognized the plants as distinct, realized his error, and became
convinced that the taxon was new to science. While close to N. onoana (Fig. 7C) in its floral
morphology, that species has a very different growth habit and its leaves are more sulcate
on the abaxial surfaces, but most distinctive are the differences in the mericarps.
102
Key to Nolana species recorded from Región de Tarapacá, Chile.
1
2
3
-
4
-
5
-
Annual, tap-rooted herbs.
2
Perennial herbs, subshrubs or shrubs.
6
Leaves clearly petiolate, the bases auriculate, occasionally connate, the
blades cordiform, rarely reniform or elliptic, glabrous, the surfaces with
salt glands, attracting atmospheric moisture and causing the look and
feel of oil
Leaves, if petiolate, without auriculate bases nor connate, the blades
spathulate to oblanceolate, variously pubescent, but never oily
Basal leaves with entire blades, the flowering shoots with sessile oval to
ovate bracts subtending flowers, the corollas 30-40 mm wide; mericarps
more than 20, 3-seriate
Basal leaves lacking, the cauline leaves linear-lanceolate to linearspathulate, the corollas less than 10 mm wide; mericarps 2-5, 1-seriate
N. adansonii
Leaves linear-spathulate, the blades 7-30 mm long, apically acute, the
calyx 8-10 mm long, the mericarps 2-5, 2 largest, ca. 3 mm long.
Leaves linear to linear-lanceolate, the blades 8-25 mm long, apically
obtuse to rounded, the calyx 3-6 mm long, the mericarps typically 2-5,
the largest 1.5-2 mm long.
Leaves 8-25 mm long, finely pilose to glabrescent, the mericarps
pyriform, 1.5-2 mm long
Leaves 10-20 mm long, densely stipitate-glandular pubescent, the
mericarps oval to elliptic, 2-3 mm long.
N. tarapacana
3
N. jaffuelii
4
5
N. gracillima
N. patachensis
6
Leaves clavate to globular-obovate, the corollas 6-9 mm long
7
-
Leaves oblong to linear-lanceolate to linear-spathulate.
8
7
Leaves broadly clavate to globular-obovate, 5-10 mm long, densely
pubescent with stellate or dendritic trichomes, the corollas suburceolate,
white to yellowish.
Leaves clavate to globular, 1-5 mm long, densely pubescent with
arachnoid-tomentose pubescence, the corollas hypocrateriformis, white
or rarely bluish
Leaves narrowly linear-spathulate to linear-lanceolate, 20-30 mm long,
10-20 mm wide
Leaves 10 mm long or less, linear-oblong to oblong, 1-4 mm wide.
N. peruviana
Leaves narrowly linear-spathulate, conspicuously shaggy villous, the
corollas lavender with deep purple guides in the throat.
Leaves linear-lanceolate to linear-spathulate, 10-25 mm long, ca. 1 mm
wide, stipitate-glandular, the corollas blue, obvious guides absent.
Leaves linear-oblong, 4-5 mm long, ca. 1 mm wide, hispidulous with
capitate-glandular trichomes, 3-4(-5) mericarps
Leaves linear or oblong, 2-10 mm long, pubescence of elongate
trichomes, not of capitate-glandular trichomes.
Leaves oblong, 2-6 mm long, 0.7-1.5 mm wide, oblong, tomentose to
villous with flaccid-elongate trichomes, mericarps 5.
N. intonsa
_
8
9
10
11
-
Leaves linear, 10 mm long, 3-4 mm wide, pubescent with simple
trichomes, mericarps 2(-3).
N. sedifolia
9
10
N. lycioides
N. leptophylla
11
N. tocopillensis
N. foliosa
103
Acknowledgements
We thank the curators and staff at SGO and CONC for permitting examination and
photography of collections. Field studies were supported, in part, by grants to JH by
CONICYT – Beca Doctorado Nacional (21130176). CONAF (Corporación Nacional
Forestal) is thanked for the permits granted to carry out continuous field studies, and also
Estación Experimental Atacama UC - Oasis de Niebla de Alto Patache (Pablo Osses) for
facilitating the visit to the study site. JH would like to thank Eduardo Contreras and Dr.
Horacio Larraín, with whom she traveled in December 2015 to Alto Patache; Dr. Larraín and
Pilar Cereceda are specially remembered and JH would like to dedicate this species to them,
for their commitment to studies in the desert. Dr. Laurence Packer is acknowledged for help
with butterfly identification and appropriate literature. JH acknowledges the support of her
thesis committee, including Samuel Contreras, Miguel Gómez, Gloria Montenegro and
Pedro León. We thank Victor Quipuscoa (HSP) for critically reviewing the manuscript and
providing a digital image of Nolana gracillima (Fig. 7B).
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106
Annexes
Fig. 1. Nolana patachensis J. Hepp & M.O. Dillon. Holotype: Sielfeld 32 (SGO-143057).
Colección Museo Nacional de Historia Natural, Chile (SGO).
107
Fig. 2. Nolana patachensis habitats. A: Individuals of N. patachensis growing together with
N. jaffuelii, N. intonsa and Cristaria molinae in December 2015, at the hills and inner plateau
of Alto Patache oasis, which in dry years are devoid of plants. B: Prostrate growth habit of
N. patachensis.
108
Fig. 3. Nolana patachensis. A. Close-up of flowers showing light blue color on the edge of
the corolla. B. Lepidoptera (Family Hesperiidae - Skippers) pollinating the flowers available
in the oasis with rains, December 2015. It is possible to see the trichomes on the typical
erect leaves.
109
Fig. 4. Nolana patachensis mericarps. A. Abaxial view with stereoscopic magnifying glass
images, scale marks = 1 mm. B. Adaxial vew with stereoscopic magnifying glass images,
scale marks = 1 mm. C. X-ray photograph showing internal seed chambers within each
mericarps.
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Fig. 5. Nolana patachensis mericarp. A. SEM of adaxial mericarp surface. B. SEM of abaxial
mericarp surface, the arrows indicates the position of the funicular scars where the radicle
appears during germination.
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Fig. 6. Distribution of Nolana patachensis.
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Fig. 7. Nolana species confused with N. patachensis. A. N. aplocaryoides, close-up of leaves
and flowers. Voucher: Dillon 9055 (F). B. N. gracillima, close-up of flowering branch.
Voucher: Quipuscoa et al. 6728 (F). C. N. onoana, close-up of leaves and flowers. Voucher:
Dillon 9050 (F).
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Table 1.
Alphabetical list of Nolana species recorded from Región Tarapaca, Chile, distribution
and phylogenetic position as suggested by membership in clades (adapted from Dillon et
al. 2009).
Species
Distribution
Clade
1
N. adansonii (Roem. & Schult.) I.M. Johnst.
Chile-Peru
F
2
N. foliosa (Phil.) I.M. Johnst.
Chile
E
3
N. gracillima (I.M. Johnst.) I.M. Johnst.
Chile–Peru
E
4
N. intonsa I.M. Johnst.
Chile
F
5
N. jaffuelii I.M. Johnst.
Chile
B
6
N. leptophylla (Miers) I.M. Johnst.
Chile
G
7
N. lycioides I.M. Johnst.
Chile-Peru
D
8
N. patachensis J. Hepp & M.O. Dillon
Chile
G
9
N. peruviana (Gaudich.) I.M. Johnst.
Chile
G
10
N. sedifolia Poepp.
Chile
G
11
N. tarapacana (Phil.) I.M. Johnst.
Chile
E
12
N. tocopillensis (I.M. Johnst.) I.M. Johnst.
Chile
G
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Chapter VI. A study of fruit development in Nolana paradoxa (Solanaceae)
from the coast of Chile
Josefina Hepp1,2, Miguel Gómez1,2, Pedro León-Lobos3, Gloria Montenegro1,2, Samuel Contreras1,2
1
Departamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería Forestal, Pontificia
Universidad Católica de Chile
2
Centro del Desierto de Atacama, Pontificia Universidad Católica de Chile
3
Centro Regional de Investigación La Platina, Instituto de Investigaciones Agropecuarias (INIA)
Corresponding author. Tel.: +562 2354 4145
E-mail address: mgomezu@uc.cl
Postal address: Facultad de Agronomía e Ingeniería Forestal, Vicuña Mackenna 4860, Macul,
Santiago, Chile.
Abstract
Nolana paradoxa (Solanaceae) is the species with the greatest latitudinal distribution of the
genus, and one of the more extensively studied, in terms of flower morphology and fruit
development; however, aspects of its germination requirements and dormancy
mechanisms, and the role and formation of the funicular scar or germination plug, are still
unclear. The complete fruits (schizocarps) of N. paradoxa (Solanaceae) were analysed
structurally using light microscopy. The development of fruits, which were collected at 2, 5,
9, 12, 17, 25 and 35 days after flowering (DAF), was monitored, with particular focus on the
development of the funiculus and the formation of the germination plug. Ripe mericarps
(dispersal units) were also analysed using a Scanning Electron Microscope, a stereoscopic
magnifier and X-rays. In the anatomical analysis, it was possible to visualize the monolayer
of the endocarp and the origin of two zones within the mesocarp of the fruits. In addition, the
formation of the germination plug was observed, which sclerifies as mericarps mature; this
structure, however, is not impermeable to water.
Keywords: schizocarp, mericarp, funicular scar, germination plug
Introduction
The genus Nolana L.f. (Solanaceae), originally described by Linnaeus in 1762, derives its
name from the Latin word nola or small bell (Freyre et al., 2005). It currently comprises 90
species from Perú (40 endemic species) and Chile (46 endemic species), with both countries
sharing only three species; N. galapagensis is the only one endemic to the Galápagos
Islands (Dillon et al., 2009). The genus is considered monophyletic and is distinguished by
the fundamentally 5-carpellate ovary that develops sclerified fruits, a unique character in the
Solanaceae family (Knapp, 2002; Freyre et al., 2005). The complete fruit is a schizocarp
consisting of several uni or pluriseminate mericarps of different sizes (Bruno, 1994); when
the schizocarp is mature, it often splits up into several mericarps, the dispersal units.
Most Nolana species are found in the fog-dependant lomas formations in the coast of the
Atacama Desert, but some species occupy habitats at higher altitudes (> 1,000 masl). N.
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paradoxa Lindl. is the sole member of the family south of the Valparaiso area (Johnston,
1936), its distribution extending from Huasco (28°13´S) to the south of Chile, in Chiloé Island
(42°43´S). It is usually found in sandy sea-shore environments, from 0 to 100 masl. It is an
annual to perennial succulent herb with a fleshy taproot (Freyre et al., 2005), and the
elongate stems are prostrate and loosely branched (Johnston, 1936).
Nolana paradoxa was the second Nolana (after N. humifusa) to be introduced to European
gardens in the early 1820s, and today remains the most popular commercially available
Nolana, sold under the name of Chilean bellflower, with existing cultivars “Blue Bird” and
“Cliff Hanger Blue” (Freyre et al., 2005). For this reason, this species has been studied
extensively, with a focus on flower morphology and fruit development (Lindley, 1824; Payer,
1857; Saunders, 1936; di Fulvio, 1969; di Fulvio, 1971; Huber, 1980; Bondeson, 1986),
although it is not yet fully understood how the seeds break dormancy and germinate in their
natural habitat.
Many seeds, across several families, contain specialized structures that regulate the uptake
of water by closing the natural openings in the seed or fruit coat (Bewley et al., 2013) and
thus relate to physical dormancy by preventing water to reach the embryo. For example, a
chalazal cap or plug is found in members of the Malvaceae (Bewley et al., 2013; Baskin &
Baskin, 2015); an imbibition lid (gap adjacent to hilum) in Cannaceae (Baskin & Baskin,
2015); and a lens gap in Fabaceae (Gama-Arachchige et al., 2013). Before seeds can
germinate, these structures are ruptured, causing the seeds or fruits to become permeable
to water; they are called water-gaps or water-plugs (Gama-Arachchige et al., 2013; Baskin
& Baskin, 2015). A germination plug, constituted by remains of the funiculus (Saunders,
1936), has also been described for species of Nolana in the Solanaceae family (Bondeson,
1981 and 1986; Douglas and Freyre, 2006), and an impermeability to water, i.e. physical
dormancy, has been reported for the genus (Cabrera et al., 2015). However, the role this
plug plays in germination is not yet clearly established.
The objective of this study is to characterize the development of fruits of Nolana paradoxa,
particularly in relation to the germination plug of the mericarps, to better understand its role
in the germination and dormancy mechanisms of Nolana species.
Materials and methods
Plant material
Mericarps of Nolana paradoxa (Fig. 1) were collected in Guanaqueros (30°11.734'S /
71°25.377'W) and germinated according to Hepp et al. (2019, in preparation), removing the
germination plug and imbibing the fruits in gibberellic acid (500 ppm). The obtained
seedlings were transplanted to the greenhouse at the Facultad de Agronomía e Ingeniería
Forestal in Santiago, Chile, where they were watered twice a week and kept at ambient
temperature. The transplant was carried out in pots (14 cm diameter), with previous
disinfection with sodium hypochlorite (Clorox ©) at 5%. The substrate used was peat, sand
and perlite in 8:3:1.5 ratio, respectively. When flowers were open, they were hand-pollinated
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and marked, and then fruits in development (complete schizocarps) were harvested at 2, 5,
9, 12, 17, 25 and 35 days after flowering (DAF).
Fig. 1. Flowering plant (A) and developing fruits with persistent calyx (B) of Nolana paradoxa growing
at the sandy sea-shore in Guanaqueros, Coquimbo region, Chile.
Morphology of mericarps
The colour, size and external appearance of the mericarps (dispersal unit) were recorded.
A sample of fruits of N. paradoxa was photographed using a stereoscopic magnifier
Olympus SZ2-ILST (Olympus Corporation, Tokyo, Japan).
Fruits were also x-rayed using the Faxitron MX20 Digital X-ray (Faxitron Bioptics, LLC,
Arizona, USA). A sample of fruits were x-rayed and digital images of each sample were
recorded. Identification of full seeds and of cavities appearing empty in each fruit were made
based on differences in contrast within recorded images.
Scanning electron microscopy (SEM) was used to examine the structure of the fruit, the
surface of the funicular scar and the longitudinally sectioned mericarp. Samples of N.
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paradoxa were mounted on aluminium SEM stubs and sputtercoated with a platinum-gold
alloy, using a Quorum Sputter Coater Q150T (Quorum Technologies Ltd, East Sussex,
United Kingdom). Mericarps were examined and photographed using a HITACHI S-4700
SEM (Hitachi High-Technologies Corporation, Tokyo, Japan) at an accelerating voltage of
2.0 kV and working distance of 12.0 mm. Images were saved as Tagged Image File Format
(TIFF).
To evaluate if water was able to penetrate and reach the embryo in intact mericarps of N.
paradoxa, methylene blue (1gr/100 mL) was used. A sample of mericarps were left in metal
containers with enough dye to cover them and were evaluated after 48 hours (room
temperature). They were washed with distilled water and dried with paper towel, then
allowed to air dry for 20 minutes at room temperature. They were observed under magnifier
and sectioned using a scalpel (longitudinal cut).
Histological analysis
Two or three complete fruits (schizocarps) corresponding to each collection day (at 2, 5, 9,
12, 17, 25 and 35 DAF) were selected and fixed in formol acetic alcohol (FAA), then
dehydrated and preserved in paraffin to be sectioned off. Cross and longitudinal histological
sections were made through the schizocarp, using a microtome (to a thickness of 16 µm)
and safranin-fast green staining. Sections were observed under light microscope and
photographed.
Results and discussion
3.1 General structure of the mericarp
The dispersal unit in the case of N. paradoxa is a polyhedral fruit, violet to black in colour
(Johnston, 1936) called mericarp (Fig. 2A), usually 3-6 mm long (Johnston, 1936). Each
mericarp of N. paradoxa has 1 or 2 (even 3) seeds (Reiche, 1910) which are enclosed in a
separate chamber (Lindley, 1824). These embryonic chambers normally contain one
embryo but are sometimes empty (Fig. 2B).
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Fig. 2. Dispersal units (mericarps) of N. paradoxa, stereoscopic magnifier (A) and x-ray images (B).
Arrows indicate full embryonic chambers; circle encloses an empty embryonic chamber.
In the morphological analysis with SEM images, it is possible to distinguish a funicular scar
in the basal region of the mericarp (Fig. 3A and 3B), which corresponds to the area of union
with the floral receptacle (Saunders, 1936). This slightly depressed, elliptical area indicates
the location of a structure which has been termed a “germination plug” (Fig. 3C and 3D),
since the protruding radicle push the plug out during germination (Bondeson, 1981). Figure
4 shows in detail the union of the receptacle with individual mericarps, which altogether form
the complete fruit or schizocarp.
For the longitudinally cut mericarps, the intermediate layer corresponding to the mesocarp
stands out (Fig. 3C); it is composed of medium to large, almost empty cells with undulated
but lignified walls, forming an airy or porous tissue in appearance, as described in Hepp et
al. (2019, in press); not corky, as reported by Johnston (1936), since cell walls are not
suberized. For the majority of Nolana species, mericarps are passively distributed (Knapp,
2002); in the case of N. paradoxa, the porous pericarp might be an adaptation to floating.
Johnston (1936) even suggests that the wide distribution of this species may have been
119
aided in part to detached leafy stems (that readily root) washed by the waves and currents,
and also to the floating mericarps of the species.
Fig. 3. SEM images of mericarps of Nolana paradoxa. A: small funicular scar in the area of attachment
to the floral receptacle. B: Detail of funicular scar. C: Longitudinal section of mericarp. D: Detail of
embryo and germination plug. Abbreviations: co: cotyledons; fs: funicular scar; gp: germination plug;
mes: mesocarp.
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Fig. 4. Histological sections of the complete fruit of N. paradoxa. Longitudinal cut (A: 2 DDF, 40x) and
transversal cut (B: 2 DDF, 40x) of schizocarps showing the calyx (ca) and the floral receptacle (fr).
Arrows indicate the area of union of mericarps with floral receptacle.
3.2 Anatomy of the pericarp
By following the development of the fruits, it was possible to appreciate the formation of the
different layers of the pericarp. By the definition of Richard (1819, quoted in Pabón-Mora &
Litt, 2011), we consider a single-layered endocarp which is easily distinguishable in early
stages (Fig. 5A-5C). The outer layer or exocarp also corresponds to a mono layered
epidermis with cells that contain vacuoles with tannins and a thin protective cuticle, which
appear collapsed when mericarps are fully mature (Fig. 5D).
The tissue in between would correspond to the mesocarp, for which two histologically
differentiated zones can be visualized (Fig. 5A-5C): an internal one, which in the case of N.
121
paradoxa is constituted by sclereids, empty cells with undulated but lignified walls, and which
has been previously described as endocarp (Bondeson, 1986; Cabrera et al., 2015, working
with N. jaffuelii). The external one, formed by parenchymatic cells (Fig. 5B and 5C), is
reduced in size as mericarps mature (Fig. 5D).
Fig. 5. Layers of the pericarp of N. paradoxa. A: 2 DAF (40x). B: 12 DAF (10x). C: 17 DAF (400x). D:
Dispersed mature mericarp (400x). Abbreviations: end: endocarp; mes1: internal zone of the
mesocarp; mes2: external zone of the mesocarp; exo: exocarp.
3.3 Formation of the funicular plug
It can be observed that the seed is generally protected by the pericarp (Fig. 6), except for
the aperture or opening left by the invagination of the ovule and occupied by the funiculus
(Bondeson, 1986). The epidermis (or endocarp) appears as a layer of cells that cover the
inner wall of the embryonic chamber, the funiculus and the embryo (Bondeson, 1986).
As the mericarp matures, cells of a portion of the funiculus begin to lignify and sclereids are
formed (Fig. 6A-F). This sclerified funicular plug (Fig.7), or germination plug (Saunders,
1936) protects the opening after dispersal of the mericarps; then, when germination begins,
the pressing radicle will break the testa and push the funicular plug out (Bondeson, 1986).
122
Additionally, the presence of starch stored in the abscission zone (of the funiculus) was
observed (Fig. 7B), where the mericarp is attached to the floral receptacle. According to
Bondeson (1986), all cells that are to become endocarp cells accumulate starch; the cell
walls thicken and lignify.
Fig. 6. Detail of mericarps and developing embryo. The germination plug in formation is indicated with
an arrow. A: 2 DAF (100x). B: 9 DAF (40x). C: 12 DAF (40x). D: 17 DAF (40x). E: 25 DAF (40x). F:
35 DAF (100x). Abbreviations: em: embryo; fu: funiculus; pe: pericarp.
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Fig. 7. A. Funicular plug at 17 DAF (100x). B: Detail of sclereids of the funicular plug, 17 DAF (400x).
Abbreviations: em: embryo; fu: funiculus; gp: germination plug; re: receptacle; sc: sclereids; st: starch.
Baskin & Baskin (2014) and Gama-Arachchige et al. (2013) describing the water gaps or
water plugs in different species, identify three types of complexes based on the morphology
of openings and the anatomy of occluding strucures; type-III corresponds to a narrow-linear
or circular water-gap opening that is occluded by a plug-like structure formed by waterimpermeable sclerenchyma cells (Baskin & Baskin, 2014), which is similar to the structure
described in this study. However, the germination plug in Nolana is not impermeable to
water, as can be observed in Figure 8, since dye-tracking of intact mericarps of N. paradoxa
showed the imbibition route and water reaching the embryo within 48 hours. Therefore, N.
paradoxa does not present physical dormancy, according to the definition of impermeability
to water of the seed or fruit coat (Bewley et al., 2013; Baskin & Baskin, 2014).
124
Fig. 8. Stereoscopic image of longitudinal section of mericarp of N. paradoxa, imbibed in methylene
blue for 48 hrs. Abbreviations: dy: dye; em: embryo; en, endosperm; gp: germination plug; mes:
mesocarp; ra, radicle; * indicates regions which have imbibed.
Conclusions
The observation of the development of complete fruits (schizocarps) of N. paradoxa, allowed
the determination of three histologically different tissues: the monolayer of the endocarp, an
internal and an external tissue of the mesocarp, and an exocarp monolayer that appears
crushed at maturity. The tissues of the mesocarp develop differently, and one of them, the
external, is reduced in size as the mericarp matures. Therefore, at least for this species, it
is difficult to observe the external mesocarp when making cuts in fully mature, already
dispersed mericarps.
Within each mericarp, it was also possible to observe the development of a germination plug
that is constituted by a portion of the funiculus and which closes the opening of the
embryonic chamber; although it is sclerified, it is not impermeable to water, therefore it does
not constitute physical dormancy.
Acknowledgments
We thank Wolfgang Stuppy, who supported the internship at RBG Kew and allowed the use
of the Scanning Electron Microscope and other instruments. CONICYT Scholarship
21130176 to JH made this study possible.
References
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Dormancy and Germination. Elsevier, San Diego, USA. 2nd edition. 1586 pp.
Baskin, J.M., C.C. Baskin, and X. Li. 2000. Taxonomy, anatomy and evolution of physical
dormancy in seeds. Plant Species Biology 15: 139-152.
Bewley, J.D., K.J. Bradford, H.W.M. Hilhorst, and H. Nonogaki. 2013. Seeds: physiology of
development, germination and dormancy. 3rd Edition, Springer Science+Business Media,
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Bondeson, W. E. 1986. Gynoecial morphology and funicular germination plugs in the
Nolanaceae. Nordic Journal of Botany 6: 183-198. Copenhagen. ISSN 0107-055X.
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(Nolanaceae). Boletín de la Sociedad Argentina de Botánica 30 (1-2): 51-57.
Cabrera, E., Hepp, J., Gómez, M. & Contreras, S. 2015. Seed dormancy of Nolana jaffuelii
I.M.Johnst. (Solanaceae) in the coastal Atacama Desert. Flora 214: 17-23.
di Fulvio, T.E. 1969. Embriología de Nolana paradoxa (Nolanaceae). Kurtziana 5: 39-54.
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di Fulvio, T.E. 1971. Morfología floral de Nolana paradoxa (Nolanaceae), con especial
referencia a la organización del gineceo. Kurtziana 6: 41-51.
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(Solanaceae), a ubiquitous member of the Atacama and Peruvian Deserts along the western
coast of South America. Journal of Systematics and Evolution 47(5): 457-476.
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and characterization of ten new water gaps in seeds and fruits with physical dormancy and
classification of water-gap complexes. Annals of Botany 112: 69-84.
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VII. General conclusions
•
It was found that Nolana species do not have physical dormancy, as previously
reported, but physiological dormancy, with different levels of depth between the
twelve species studied. In this context, the importance of the endosperm as a
germination regulator in the studied species could be determined.
•
It was possible to germinate, and in fact seedlings were obtained for most of the
twelve species studied; the only one that did not show germination was N. intonsa,
for which a very deep dormancy was defined. The best treatment for all species
included the removal of the germination plug (scarification) and partial removal of
the endosperm or alternatively, removal of the germination plug and addition of
gibberellic acid.
•
It was possible to find a dormancy pattern for the species studied; the level of
dormancy was related to phylogeny, that is, to the clade to which the species
belong. Neither latitudinal distribution nor niche diversity was related to the level of
dormancy; it is therefore likely that the dormancy trait has a greater genetic
influence than the environment under which the seed develops (maternal
environment).
•
Under a climate change scenario in the future, the distribution pattern of the genus
Nolana will contract towards the coast, but some species will keep their distribution
area stable, and some will even expand. However, some species whose distribution
will contract, have high values according to the dormancy index (i.e. lower
percentages of germination). Special efforts should be directed to include these
species in an integrated conservation programme, including both in situ and ex situ
initiatives.
•
As for the anatomy, according to the histological sections of mericarps of all the
species studied, three different tissues were identified: an external one (exocarp),
corresponding to a mono-stratified epidermis; a middle one (mesocarp), of variable
thickness, occupying the bulk of the pericarp and the one that presents more
variation between species; and an inner one (endocarp), constituted by one layer of
very dense sclereids. It was determined that the thick layer composed of dense
sclereids, which had previously been described as endocarp, corresponds to the
inner layer of the mesocarp. This hard layer provides protection to the embryo, as it
would allow the mericarps to remain relatively intact in the soil seed bank for many
years.
•
It was not possible to determine a clear relation between the anatomy and
morphology of mericarps and the position of the species within clades of the genus.
However, trends were observed, and the new information generated could be of
use for the identification of species. In fact, it was possible to describe a new
species for the genus, Nolana patachensis from the fog oasis of Alto Patache, in
Tarapacá, based mainly on the morphological differences of mericarps between
127
otherwise very similar species. This means that by the end of this thesis, the genus
Nolana is composed of 90 species.
•
For a selected species, N. paradoxa, it was possible to observe the development of
the germination plug, constituted by a portion of the funiculus. As the mericarp
matures, this portion begins to lignify and sclereids are formed. The germination plug
protects the opening of the mericarp after dispersal of the mericarps; then, when
germination begins, the pressing radicle will break the seed cover and push the
funicular plug out.
128