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Germination responses to salt stress of two intertidal populations of the perennial glasswort Sarcocornia ambigua
Accepted Manuscript Title: Germination responses to salt stress of two intertidal populations of the perennial glasswort Sarcocornia ambigua Author: Ricardo F. Freitas C´esar S.B. Costa PII: DOI: Reference:
S0304-3770(14)00055-2 http://dx.doi.org/doi:10.1016/j.aquabot.2014.04.002 AQBOT 2663
To appear in:
Aquatic Botany
Received date: Revised date: Accepted date:
22-7-2013 31-3-2014 3-4-2014
Please cite this article as: Freitas, R.F., Costa, C.S.B.,Germination responses to salt stress of two intertidal populations of the perennial glasswort Sarcocornia ambigua, Aquatic Botany (2014), http://dx.doi.org/10.1016/j.aquabot.2014.04.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Freitas&Costa Highlights (for review)
Highlights Sarcocornia ambigua is a widespread halophyte of the Atlantic coast of South America. Higher germination (81–84%) after 22 days occurred at salinities 0 and 5 g NaCl L-1.
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Osmotically induced inhibition of germination is reversible by salt relief.
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S. ambigua seeds were able to germinate (3%) even in 45 g NaCl L-1.
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*Freitas&Costa manuscript
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Germination responses to salt stress of two intertidal populations
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of the perennial glasswort Sarcocornia ambigua
3 Ricardo F. Freitas1*, César S.B. Costa2
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(1)
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Federal do Rio Grande (FURG), Av. Itália, km 8, Bairro Carreiros, 96203-900, Rio Grande,
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Brazil.
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(2)
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Programa de Pós-Graduação em Aqüicultura, Instituto de Oceanografia, Universidade
Laboratório de Biotecnologia de Halófitas, Instituto de Oceanografia, Universidade Federal
do Rio Grande (FURG), Av. Itália, km 8, Bairro Carreiros, CEP 96203-900, Rio Grande,
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Brazil.
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* Corresponding author: Tel.: +55 53 3233 6534. E-mail address: [email protected]
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(R.F. Freitas).
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Abstract - Sarcocornia ambigua is a perennial halophyte of the Atlantic coast of South
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America. The species occurs in salt marshes and mangrove swamps and is able to grow under
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hypersaline conditions. S. ambigua seeds were collected from two intertidal populations
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located inside and at the entrance of the Patos Lagoon estuary (RS, Brazil). The seeds were
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germinated by incubation for 22 days in 0, 5, 15, 30 and 45 g NaCl L-1 solutions at 20-30 °C
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and with a 12:12 h photoperiod. There were no significant differences in germination between
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S. ambigua populations. High average germination (81-84%) occurred at low salinities (0 and
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5 g NaCl L-1) and decreased at salinities of 15 g NaCl L-1 (41-46%) and above. Seeds were
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able to germinate (3%) even in 45 g NaCl L-1. Both populations of S. ambigua demonstrated
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an inhibition of germination under saline conditions. This inhibition was reversible through
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2 removal of the salt stress. The percentage of unviable seeds increased from 4% to 11-18% as
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the salinity increased from 5 to 15-40 g NaCl L-1. The germinative responses to salinity of S.
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ambigua are typical of an extreme halophyte. The predominant outflow tendency of the
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waters in the study area and the choked morphology of Patos Lagoon favour the flux of seeds
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between marshes in the middle estuary and at the narrow estuarine inlet, preventing local
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differentiation of S. ambigua populations.
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Keywords: Sarcocornia ambigua, halophyte, population differentiation, germination.
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34 1. Introduction
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Several species of the genus Sarcocornia (Chenopodiaceae) are considered ‘extreme
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halophytes’ based on their ability to thrive in seawater-flooded (35g NaCl L-1) and hypersaline
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soils (Redondo-Goméz et al., 2004; Davy et al., 2006; Ventura et al., 2011; Ventura and Sagi,
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2013). Native populations of Sarcocornia species occur in salt marshes, mangrove swamps
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and salt deserts. Population differentiation occurs primarily due to spatial heterogeneity of
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salinity and periodic flooding stresses (Redondo-Goméz et al., 2004; Davy et al., 2006),
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which are the major determining factors for germination success and plant establishment
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(Ungar, 1995).
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Along the South American Atlantic coast, Sarcocornia ambigua (Michx.) Alonso and
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Crespo (syn. Salicornia gaudichaudiana Moq.) is the most widely distributed perennial
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species of the genus, occurring in salt marshes and hypersaline mangrove areas from
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Venezuela to the Valdés Peninsula in Argentina (Costa, 2006; Isaach et al., 2006; Alonso and
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Crespo, 2008). In the marshes of the south-western Atlantic coast, S. ambigua colonizes
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physically stressful middle and upper marshes, and field experimental manipulation (Alberti
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3 et al., 2008) has shown that it facilitates the recruitment of the South American cordgrass
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Spartina densiflora, which co-dominates these marshes. More than 740 km² of the coast
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between southern Brazil (32º S) and the Valdés Peninsula (43º S) are covered by S. ambigua-
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dominated salt marshes (Isaach et al., 2006), which support large populations of polychaetes,
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pelecypods, and crustaceans. These organisms are subject to predation by oystercatchers,
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gulls, and terns (Costa et al., 2003, 2009; Alberti et al., 2008).
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S. ambigua propagates through seeds and vegetative growth (Leite et al., 2007; Alonso
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and Crespo, 2008). This species is very widespread, it occurs in places with high variability in
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salinity, both in time and space, which could lead to selective specialization or plasticity
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(Ungar, 1995; Gul et al., 2013). The control of germination in mature seeds may occur
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through phytohormones or the mechanical structures of the coat, producing innate dormancy
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(Baskin and Baskin, 2001; Cardoso, 2009). In coastal environments and certain arid regions,
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quiescent seeds may show a delay or inhibition of germination due to soil salinisation (Pujol
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et al., 2000; Gul et al., 2013), caused by a low osmotic potential and the toxic effect of certain
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elements, such as sodium and chlorine, at high concentrations. In sensitive species, the salt
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content of the sediment may produce a partial or even complete loss of seed viability (Khan,
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2003; Vicente et al., 2007; Gul et al., 2013). In contrast, certain halophytes may be stimulated
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to germinate by a combination of saline stress and a subsequent dilution of the salinity
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(‘saline stress relief’); this osmotic shock apparently denatures certain phytohormones that are
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considered inducers of innate dormancy (Ungar, 1995; Baskin and Baskin, 2001; Gul et al.,
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2013). For example, both S. fruticosa, which inhabits hypersaline high marshes in Spain, and
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S. perennis, which grows in regularly flooded low marshes of this country, showed decreases
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in the final germination percentage as salinity increased. However, the seeds of the former
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species showed accelerated germination after relief from prolonged salinity exposure, whereas
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such acceleration was absent in the seeds of the latter species (Redondo-Goméz et al., 2004).
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4 Along the estuary of Patos Lagoon, located in the state of Rio Grande do Sul in
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southern Brazil (32°S, 52°W), S. ambigua has a disjunct distribution and is extremely
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abundant in certain salt marshes (Costa, 2011). Patos Lagoon is the world’s largest choked
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lagoon, with a surface area of 10,000 km², and connects with the sea through a single 700 m
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wide inlet. The circulation of the Patos Lagoon estuary is driven primarily by winds and
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freshwater runoff, but the discharge of the principal tributary rivers is strong during the late
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winter and early spring, with low to moderate runoff during the summer and autumn (Costa et
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al., 2003; Möller et al., 2009; Garcia, 2011). During the seasonal flood period, only very
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strong southerly winds can drive sea water into the lagoon and reverse the seaward flow, and
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the water in most of the estuary can remain oligohaline or even fresh for several months
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(Costa et al., 2003; Möller et al., 2009). This seasonal pattern may exert selective pressure on
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local S. ambigua populations; in particular, both saline inhibition of the germination of the
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seeds dispersed during the autumn and acceleration of germination accompanying the relief
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from salt stress occurring in the spring would be expected.
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In addition to its seasonal hydrodynamics, the Patos Lagoon estuary shows a
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pronounced salt gradient between the head and mouth of the estuary (≈ 60 km; Coutinho and
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Seeliger, 1984; Garcia, 2011), and hypersaline sediments have periodically been reported
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during the dry summer-autumn only in salt marshes near the inlet of the estuary (Cordazzo et
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al., 2007; Costa, 2011). Mature specimens of S. ambigua growing in marshes near the inlet of
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the estuary possess distinctive shoots with reddish colouration, whereas the plants in the
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marshes of the middle estuary have dark green shoots (Costa et al., 2006). The reddish colour
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of Sarcocornia shoots can indicate the accumulation of anthocyanins and other phenolic
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compounds, which are antioxidant compounds used by these plants to tolerate stresses, e.g.,
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water deficits, high soil salinisation and a high incidence of ultraviolet radiation (Costa et al.,
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2006; Davy et al., 2006). Because disjunct populations of S. ambigua can be differentially
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5 affected by salt intrusion in the Patos Lagoon estuary, their seeds might show contrasting
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sensitivity and germination responses to salt stress. Salinity is a major determinant of the
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plant community structure of the local marshes, and population differences among aquatic
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plants have previously been demonstrated along the Patos Lagoon estuarine gradient. For
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example, the plant communities of the low intertidal zone of the salt marshes of Patos Lagoon
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are differentiated into those of the oligohaline region at the head of the estuary(<6 g NaCl L-1)
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and those of the mesohaline region in the central-low part of the estuary (>6 g NaCl L-1)
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(Costa, 2011). Koch and Seeliger (1988) have demonstrated that germination patterns differ
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between populations of the submerged phanerogam Ruppia maritima that grow in the middle
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of the estuary and at the mouth of the estuary.
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This study aimed to compare the effects of different salinity treatments and of saline
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stress relief on the germination of S. ambigua seeds from two native populations that inhabit
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distinct areas of the salt gradient along the estuary of Patos Lagoon. We used laboratory
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experiments to evaluate the hypothesis that seed germination in local populations of S.
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ambigua is inhibited by salt exposure and enhanced after decreases in salinity as an
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adjustment to the seasonal pattern of estuarine salinity, as well as the hypothesis that the seeds
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of S. ambigua populations subjected to contrasting salt exposure show differential salt
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tolerance.
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2. Material and Methods
2.1. Characteristics of the study populations
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The Patos Lagoon estuary has a warm temperate climate, with minimum and
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maximum average monthly temperatures of 13 °C and 25 °C, respectively. The salt marshes
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occupy 70 km2 of the estuary margins (Isaach et al., 2006). The studied populations of S.
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ambigua are located 15.4 km apart, on Pólvora Island (32°01′S, 52°06′W) and on the sand spit
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at ‘Barra’ (local name of the estuarine inlet; 32°09′S, 52°04′W). Pólvora Island is situated on the middle region of the estuary. Approximately 45 ha of
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this island consists of marshlands covered by dense vegetation, which is dominated by
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grasses. The local population of S. ambigua is distributed over mud flats in the middle zone of
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the salt marshes. This zone is flooded less than 25% of the time by oligo-mesohaline, with an
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annual average salinity of 10 g NaCl L-1 (Costa et al., 2003). The Barra salt marsh at the
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mouth of the estuary has an area of approximately 9 ha (Marangoni and Costa, 2009). Locally,
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S. ambigua occupies high intertidal ground that is periodically subjected to hyper-saline
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conditions. These areas flood only during spring tides and are strongly influenced by sea
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spray (Cordazzo et al., 2007). Coutinho and Seeliger (1984) have estimated that 90% of the
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surface water reported in the area of the Barra is meso-euhaline, ranging from 13 to 34 g NaCl
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L-1.
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2.2. Germination experiments
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S. ambigua seeds were collected in the marshes of Pólvora Island and Barra during
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autumn of 2010. In each population, 10-15 individual mature plants were collected; their
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ripped seeds were removed from the fertile segments and placed in a label paper bag. The
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seeds of each population were sub-sampled and the hundred seeds weight determined. After
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collection, the seeds were dried at room temperature for one week and stored at 5°C (cold pre-
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treatment) for 60–70 days in the laboratory. Previously, Lopes (2000) showed that
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approximately 50% of mature S. ambigua seeds are unable to germinate under the appropriate
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environmental conditions when freshly released from the mother plant during the autumn, and
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a 2-month cold pretreatment is required to terminate this innate dormancy. It is possible that
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this response results from increased levels of gibberellins in the seeds. This germination
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7 response is compatible with the low overwinter temperatures (daily minimum below 10 ºC)
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and the optimal temperature conditions observed in late spring-early summer. After cold
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storage, the seeds were washed with 5% hypochlorite for 10 min to reduce fungus infestation
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(Leite et al., 2007) and then rinsed with distilled water. Subsequently, the seeds were placed in
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autoclaved Petri dishes with filter paper dampened with 6 mL of saline solution of different
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concentrations: 0, 5, 15, 30 and 45 g NaCl L-1. All Petri dishes were wrapped (to prevent
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evaporation and change in salinity) and incubated for 22 days in a germination chamber with
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a thermoperiod of 30/20 °C and photoperiod (12 h light/12 h dark; 40 μmol photons m-2 s-1,
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400-700 nm) provided by cold white fluorescent light (Universal-L-Duramax GE). The
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occurrence of the maximum total germination of S. ambigua seeds under these thermal and
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photoperiod conditions has previously been described (Lopes, 2000; Leite et al., 2007). Four
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Petri dishes (replicates) were used for each salinity level, with 25 seeds in each dish. Petri
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dishes were observed every two days to monitor the number of germinating seeds. Seeds were
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considered to have germinated if the emergence of the radicle through the seed coat was
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visible.
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To determine the occurrence of the inhibition of germination due to salt stress and the
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loss of seed viability at high salinity due to ionic toxicity, the seeds that did not germinate
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after 22 days of incubation in salinities higher than zero were subjected to a procedure
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producing saline stress relief (Redondo-Gomez et al., 2004). Seeds were washed with distilled
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water and placed on Petri dishes with filter paper dampened with distilled water and incubated
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under the above-mentioned light and thermoperiod conditions for an additional 22 days. After
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this period, the viability of the seeds that remained ungerminated was determined under a
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stereomicroscope (8× magnification) after immersion in a 0.25% (m/v) tetrazolium solution
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(2,3,5-triphenyltetrazolium chloride) at 30 °C for 24 h in the dark (Moore, 1985). This
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procedure was also employed for seeds that were initially incubated in zero salinity and that
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did not germinate, thus, indicating the germinability of the seed lots. A two-way ANOVA was used to test differences in the percentages of final
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germination and loss of viability among populations (P) and salinity levels (S), followed by a
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Bonferroni comparison test at a 5% significance level. Both germination and viability
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percentage data were previously transformed into angular values (arcsin √x/100) to satisfy the
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assumptions of normality and homoscedasticity (Zar, 2010). In the test of the effect of saline
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stress relief, the initial number of seeds varied among the Petri dishes (these seeds were the
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ungerminated seeds resulting from the test of the effect of salinity). For each Petri dish, the
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percentage of final germination was calculated by cross multiplication of the final number of
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germinated seeds, with a value of 100% representing the initial number of seeds.
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Because the time to first germination (days) did not follow a normal distribution for
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any population, this variable was compared nonparametrically among the salinity levels to
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which each population was exposed. A Kruskal-Wallis test, followed by pairwise comparisons
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using a Mann-Whitney test (Zar, 2010), was used for this analysis. A Bonferroni correction (p
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= 0.05/10 = 0.005) was used to determine differences in statistical significance among 5 levels
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of salinity treatment. Three salinity levels (15, 30, and 45 g L-1 NaCl) did not show 50%
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germination (T50) after 22 days of incubation; hence, this parameter was not included in the
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statistical analysis.
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3. Results
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3.1. Effect of salinity on total germination and seed mass
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After 22 days of incubation, seeds from both Pólvora Island and the Barra showed
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higher average germination rates (81–84%) at salinity levels of 0 and 5 g NaCl L-1 then at
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9 higher salinities. All salinities ≥15 g NaCl L-1 resulted in a significant decrease in total seed
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germination (41-46%; FS = 128.6; p <0.001, Bonferroni test; Tables 1 and 2). Both
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populations showed the same pattern of germination response to the different salinities tested
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(FPxS= 0.72; p > 0.05). Even at salt concentration of 45 g NaCl L-1, i.e., the highest salinity
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showing average germination of S. ambigua seeds from Pólvora Island (0%) and the Barra
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(3%), respectively, no differences between the populations were found by the Bonferroni test
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(Table 2). However, the seeds of Barra weighed on average 0.10 ± 0.01 mg (± standard error),
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42% more than the seeds from Pólvora Island (0.07 ± 0.01 mg) (p < 0.01; Mann-Whitney
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test).
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3.2. Effect of salinity on the time of germination
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At salinity levels of 0 and 5 g NaCl L-1, germination was observed two days after
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incubation of the seeds (Fig. 1A-B), and T50 was achieved between the 4th and 6th days (Table
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2). High salinities induced a significant delay in the time to first germination in both
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populations (Kruskal–Wallis tests; p < 0.01); seeds from the Barra and Pólvora Island exposed
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to a salinity level of 30 g NaCl L-1 required an average of 9.5 ± 4.3 and 14.5 ± 4.3 days,
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respectively, to begin germination (Fig. 1A-B; Table 2).
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For the two populations of S. ambigua, the overall germinability of seeds in the
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experimental conditions was 83% (estimated at zero salinity), and 7.5% of the seeds remained
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dormant (Fig. 2A). This average percentage of dormant seeds showed no significant
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difference (p > 0.05) among the salinity levels (FS = 1.25) and populations (FP = 0.77),
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ranging from 3 ± 1% (Barra, 5 g NaCl L-1) to 11 ± 2% (Pólvora Island, 15 g NaCl L-1) (Fig.
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2A).
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3.3. Effect of saline stress relief on seed viability and germination
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10 The temporal pattern of germination after saline stress relief at salinity levels of 30
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and 45 g NaCl L-1 was similar to that in the seeds initially incubated at zero salinity, in which
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germination was initiated after 1–2 days and T50 was attained between the 3rd and 4th days
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(Fig. 2B-C). The saline relief responses (germination from 76–81% of the seeds) at these two
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salinity levels showed no significant differences (FS = 1.05; p > 0.05), and no differences were
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detected between populations (FP = 0.08; p > 0.05).
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The optimal condition for germination was determined as that with a salt
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concentration of 5 g NaCl L-1, at which a high percentage of embryos emerged (Fig. 2A). The
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tetrazolium test showed 4% of unviable seeds incubated in 5 g NaCl L-1 and values 2–3 times
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significantly higher (11–18%; FS = 4.37; p < 0.01) when incubated at salinity concentrations
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within a range of 15-45 g NaCl L-1 (Table 2; Fig. 2A). In addition, 8% (from Pólvora Island)
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to 11% (from the Barra) of the seeds incubated at 0 g NaCl L-1 did not germinate, and these
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percentages of dormant seeds did not vary between populations (FP = 0.47; p > 0.05).
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4. Discussion
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4.1. General pattern of germination response to increasing salinity The germination responses of S. ambigua to salinity was similar to that of true
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halophytes (Ungar, 1995; Gul et al., 2013), with a maximum germination percentage under
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low salinities (0-5 g NaCl L-1) and a marked reduction in the percentage of seed germination
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at salinities higher than 15 g NaCl L-1. Nevertheless, seeds were able to germinate at 45 g
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NaCl L-1. The same pattern of response to salinity has been shown in extreme halophytes such
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as Salicornia dolichostachya, S. brachystachya (Huiskes et al., 1985), Sarcocornia fruticosa,
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S. perennis (Redondo-Goméz et al., 2004) and Arthrocnemum macrostachyum (Vicente et al.,
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2007). Glycophytes also show decreases in germination with increasing salt concentration
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11 (Khan, 2003; Cardoso, 2009), but in ‘Carioca bean’ (Phaseolus vulgaris), for example, the
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seeds did not germinate in salinities higher than 200 mol m-3 (≈ 8.4 g NaCl L-1; compared to
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germination in 0 g NaCl L-1 = 91%; Dantas et al., 2007). Facultative halophytes, such as the
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grass Hordeum jubatum (Ungar, 1974) from moderately saline soils and the sub-bush Cakile
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maritima from embryo dunes affected by high tides (Megdiche et al., 2007), showed
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maximum germination at approximately 0-5 g NaCl L-1, strong germination inhibition at
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salinities greater than 10 g NaCl L-1 and no germination at salinities ≥ 20 g NaCl L-1. As
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shown in the Results section, the germination response of S. ambigua identifies it as one of
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the most salt tolerant species cited in the literature.
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The halophytic characteristics of S. ambigua were clearly demonstrated by the
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germinative response of the seeds to extreme salinities. In a recent review of the germination
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strategies of several plants living in different saline habitats, Gul et al. (2013) showed that
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plants capable of germinating in salinities higher than that of seawater (even if less than 10%
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of the seeds germinated) were among the most tolerant halophytes. The mean germination
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rate of S. ambigua from Barra (7%) at a salt concentration of 30 g NaCl L-1 was two times the
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observed results in experiments conducted using seeds of the halophytes Suaeda fruticosa
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(Khan and Ungar, 1996) and Sarcocornia fruticosa (Redondo-Gomez et al., 2004) (both ≈
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3%). Small percentages of germination at salinities greater than or equal to 40 g NaCl L-1, as
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shown by the seeds of S. ambigua from the Barra, have been observed only in extreme
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halophytes, such as Salicornia stricta, S. ramosissima (Ungar, 1995), Sarcocornia fruticosa
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(Redondo-Goméz et al., 2004) and Haloxylon salicornicum (El-Keblawy and Al-Shamsi,
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2008).
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4.2. Salt tolerance of seeds and the effects of saline stress relief on germination
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12 The seed viability loss of 11–18% observed in S. ambigua exposed to mid-high
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salinities (including hypersaline) might be considered low relative to the high stress level to
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which the seeds were subjected. Redondo-Goméz et al. (2004) reported a significant increase
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in the average proportion of unviable seeds in three species of halophytes (Sarcocornia
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perennis, Sarcocornia fruticosa, and Sarcocornia perennis x Sarcocornia fruticosa), from 7–
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12% to 24–34%, between incubation salinities of 0 and 40 g NaCl L-1. Facultative halophytes,
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which are dominant in habitats marginal to salt marshes, exhibit high losses in seed viability
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when exposed to high salinities. For example, the seeds of Myrsine parvifolia, a bush that
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colonises the high intertidal zone of salt marshes in southern Brazil, showed a 20–30%
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viability loss when incubated for a month at salinities from 20 to 30 g NaCl L-1 (Ribeiro,
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2010). According to Ungar (1995), the ability of seeds to remain viable for long periods under
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saline stress represents an important attribute of halophytes, distinguishing them from
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glycophytes and allowing the exploration of soils with low water potential. No information
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was available on the longevity of seeds of S. ambigua on saline soils, but high seed viability is
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still observed in cold stored protoplasm after 15 years (C.S.B. Costa, pers. comm.).
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Most ungerminated seeds of S. ambigua initially exposed to high salinities (30 and 45
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g NaCl L-1) were quiescent, and germinated after saline stress relief. Consequently, both
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populations of S. ambigua showed osmotic inhibition of non-dormant seeds. Similar results
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have been observed in seeds of three Sarcocornia species from Mediterranean salt marshes.
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The germination of these seeds was partially inhibited by incubation at salinity levels from 20
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to 60 g NaCl L-1 and the seeds germinated after salt stress relief (Redondo-Goméz et al.,
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2004). Pujol et al. (2000) dismissed ionic intoxication as the main mechanism of germination
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inhibition in halophytes of south-western Spain after achieving reversibility of this inhibition
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at high salinities, with saline relief occurring in seeds incubated at low osmotic potentials.
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Seeds of other halophytic species inhabiting subtropical and temperate salt marshes, including
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13 the genera Atriplex, Salicornia, Limonium, Salsola, Crithmum, and Sporobolus, did not
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germinate at high salinities, but recovered more than 50% of their ability to germinate after
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they were removed from saline stress (Ungar, 1995; Khan and Ungar, 1996; Gul et al., 2013).
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Saline inhibition of germination is an efficient mechanism for the spatiotemporal distribution
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of germination of halophytes, allowing seeds to initiate germination when (and where) low
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salinity may favour the survival of seedlings (Khan and Ungar, 1997; Pujol et al., 2000;
305
Baskin and Baskin, 2001). Thus, quiescent seeds of S. ambigua may rest in the soil seed bank
306
for a long time, and their germination can be triggered during periods of reduced salinity. The
307
results presented above support the hypothesis that S. ambigua can explore the seasonal
308
pattern of estuarine salinity at Patos Lagoon, where highly saline waters prevail during the
309
summer-autumn drought and oligohaline waters are prevalent in late winter and early spring
310
(Costa et al., 2003; Möller et al., 2009; Garcia, 2011). The seeds of S. ambigua then find
311
optimal temperatures and saline conditions for germination in the late spring and early
312
summer.
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4.3. Population differences in salt tolerance of seeds
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The Barra and Pólvora Island marshes are exposed to flooding waters with different
316
salinities (Coutinho and Seeliger 1984; Costa et al., 2003; Cordazzo et al., 2007), and mature
317
plants of S. ambigua show distinctive shoot colouration related to the accumulation of anti-
318
stress phenolic compounds (Costa et al., 2006). Our results also showed that plants from
319
Barra and Pólvora Island showed significant differences in seed size. These differences may
320
be related to genetic factors (e.g., Salicornia bigelovii; Zerai et al., 2010) and/or phenotypic
321
responses to environmental factors (Cordazzo, 1994). Other than this contrast, there were no
322
significant differences between S. ambigua populations in the germination response to
323
increasing salinity, in saline stress relief responses or in losses of seed viability due to saline
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Page 14 of 26
14 324
stress. Thus, populations of S. ambigua thriving in localities covered by euhaline and meso-
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oligohaline waters in the estuary of the Patos Lagoon did not show differences in the salt
326
tolerance of their seeds. Elsewhere, persistent temporal differences in salt stress have been shown to produce
328
differences between the salt tolerance of the seeds of different Sarcocornia species (Redondo-
329
Goméz et al., 2004). The lack of differentiation in salt tolerance between S. ambigua
330
populations could be explained by the particular hydrodynamics of the choked lagoon and by
331
the maintenance of genetic flow between marshes in the middle estuary and at the narrow
332
estuarine inlet. The circulation of Patos Lagoon is driven primarily by winds and freshwater
333
runoff (Costa et al., 2003; Möller et al., 2009; Garcia, 2011), typically with an average feature
334
is a residual seaward flow of 2,400 m³ s-1 (Möller et al., 2009). Because Sarcocornia seeds are
335
distributed by tides and currents (Davy et al., 2006), the predominant outflow of the water and
336
the morphology of Patos Lagoon might favour seed dispersal from the marshes of the middle
337
estuary toward the inlet marshes. This flux of seeds between marshes could prevent local
338
differentiation in the genetic structure of populations within the estuary, even in the presence
339
of a persistent horizontal gradient of salinity.
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342 343 344 345
Acknowledgements
This research was supported by the Brazilian National Research Council (CNPq)
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under grants n. 574600/2008-6 and 573884/2008-0 (INCTSAL).
References
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Alonso, M.A., Crespo, M.B., 2008. Taxonomic and nomenclatural notes on South American
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Dantas, B.F., Ribeiro, L.S., Aragão, C.A., 2007. Germination, initial growth and cotyledon
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E.M., Luque, T., Figueroa, M.E., 2006. Biological Flora of the British Isles: Sarcocornia
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perennis (Miller) A. J. Scott. Journal of Ecology 94, 1035-1048.
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El-Keblawy, A., Al-Shamsi, N., 2008. Salinity, temperature and light on seed germination of
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Haloxylon salicornicum, a common perennial shrub of the Arabian deserts. Seed Science and
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Garcia, C.A.E., 2011. Hydrological characteristics. In: Seeliger, U., Odebrecht, C., Castello,
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Atlantic, 2.ed., Springer Verlag, New York, pp.18-20
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Gul, B., Ansari, R., Flowers, T.J., Khan, M.A., 2013. Germination strategies of halophyte
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seeds under salinity. Environmental and Experimental Botany 92, 4-18.
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Huiskes, A.H.L., Stienstra, A.W., Koutstaal, B.P., Markusse, M.M., Van Soelen, J., 1985.
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Iribarne, O.O., 2006. Distribution of salt marshes plant communities associated with
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environmental factors along a latitudinal gradient on the south-east Atlantic coast. Journal of
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Biogeography 33, 888-900.
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Khan, M.A., 2003. Halophyte seed germination: Success and Pitfalls. In: Hegazi, A.M., El-
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Shaer, H.M., El-Demerdashe, S., Guirgis, R.A., Abdel Salam Metwally, A., Hasan, F.A.,
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Centre, Cairo, pp.346-358.
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Khan, M.A., Ungar, I.A., 1996. Influence of salinity and temperature on the germination of
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Haloxylon recurvum. Annals of Botany 78, 547-551.
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Khan, M.A., Ungar, I.A., 1997. Germination responses of the subtropical annual halophyte
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Zygophyllum simplex. Seed Science & Technology 25, 83-91.
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Koch, E.W., Seeliger, U., 1988. Germination ecology of two Ruppia maritima L. populations
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in southern Brazil. Aquatic Botany 31, 321-327.
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Leite, M.S., Barros, F.J.A, Khoury, S.H., Bonilla, O.H., Costa, C.S.B., 2007. Cultivo de
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plântulas de Salicornia gaudichaudiana Moq. para uso em bioremediação junto a viveiros de
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criação de camarão. Revista Brasileira de Biociências 5, 297-299.
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Lopes, M.F., 2000. Germinação de Salicornia gaudichaudiana Moq. em diferentes condições
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de temperatura e salinidade. Undergraduate Thesis, Universidade Federal do Rio Grande, Rio
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Grande, Brazil.
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Marangoni, J.C., Costa, C.S.B., 2009. Natural and anthropogenic effects on salt marsh over
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five decades in the Patos Lagoon (southern Brazil). Brazilian Journal of Oceanography 57,
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18 Megdiche, W., Amor, N.D.A, Hessini, K., Ksouri, R., Zuily-Fodil, Y., Abdelly, C., 2007. Salt
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tolerance of the annual halophyte Cakile maritima as affected by the provenance and the
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developmental stage. Acta Physiologiae Plantarum 29, 375-384.
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Möller, O.O., Castello, J.P., Vaz, A.C., 2009. The effect of river discharge and winds on the
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interannual variability of the pink shrimp Farfantepenaeus paulensis production in Patos
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Lagoon. Estuaries and Coasts 32, 787-796.
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Moore, R.P., 1985. Handbook on tetrazolium testing. International Seed Testing Association,
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Zürich.
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Pujol, J., Calvo, J., Ramirez-Diaz, P., 2000. Recovery of germination from different osmotic
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conditions by four halophytes from southeastern Spain. Annals of Botany 85, 179-186.
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Redondo-Goméz, S., Rubio-Casal, A.E., Castillo, J.M., Luque, C.J., Alvarez, A.A., Luque, T.,
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Figueroa, M.E., 2004. Influences of salinity and light on germination of three Sarcocornia
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taxa with contrasted habitats. Aquatic Botany 78, 255-264.
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Ribeiro, J.N.S., 2010. Germinação de Myrsine parvifolia A.D.C. em diferentes condições de
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temperatura e salinidade. Undergraduate Thesis, Universidade Federal do Rio Grande, Rio
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Grande, Brazil.
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Ungar, I.A., 1974. The effect of salinity and temperature on seed germination and growth of
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Hordeum jubatum. Canadian Journal of Botany 52, 1357-1362.
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Ungar, I.A., 1995. Seed germination and seed-bank ecology in halophytes. In: Kigel, J.,
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Galili, G. (Eds.), Seed Development and Germination. Marcel Dekker Incorporated, New
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York, pp. 599-628.
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Ventura, Y., Sagi, M., 2013. Halophyte crop cultivation: The case for Salicornia and
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Sarcocornia. Environmental and Experimental Botany 92, 144-153.
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Ventura,Y., Wuddineh, W.A., Myrzabayeva, M., Alikulov, Z., Khozin-Goldberg, I., Shpigel,
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M., Samocha, T.M., Sagi, M., 2011. Effect of seawater concentration on the productivity and
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19 nutritional value of annual Salicornia and perennial Sarcocornia halophytes as leafy
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vegetable crops. Scientia Horticulturae 128, 189-196.
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Vicente, M.J., Conesa, E., Álvarez-Rogel, J., Franco, J.A., Martínez-Sánchez, J.J., 2007.
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Effects of various salts on the germination of three perennial salt marsh species. Aquatic
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Botany 87, 167-170.
450
Zar, J.H., 2010. Biostatistical Analysis, 5th ed. Pearson Prentice-Hall, Upper Saddle River,
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NJ, USA.
452
Zerai, D.B., Glenn, E.P., Chatervedi, R., Lu, Z., Mamood, A.N., Nelson, S.G., Ray, D.T.,
453
2010. Potential for improvement of Salicornia bigelovii through selective breeding.
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Ecological Engineering 36, 730-739.
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20 455
Figure Legends
456 Fig. 1: Cumulative germination of Sarcocornia ambigua seeds collected at (A) Barra and (B)
458
Pólvora Island during 22 days under five salinity treatments (n = averages of 4 Petri dishes,
459
standard errors not included for clarity (standard errors of total germination are shown in
460
Table 2).
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Fig. 2: (A) General response of Sarcocornia ambigua seeds (overall average results of Barra
463
and Pólvora Island populations) to salt exposure and salt stress relief. Percentages of unviable
464
seeds, seeds with persistent dormancy, seeds that germinated after 22 days in five salinity
465
treatments and seeds that germinated only after salt stress relief are shown. Cumulative
466
germination of Sarcocornia ambigua seeds collected at (B) Barra and (C) Pólvora Island after
467
salt stress relief from salinity treatments of 30 g and 45 g NaCl L (n = average ± standard
468
error for 4 Petri dishes).
-1
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Freitas&Costa Figure 1
Page 22 of 26
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Freitas&Costa Figure 2
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Freitas&Costa Table 1
1
Table 1: Analysis of Variance of total germination and percentage of unviable seeds of
2
Sarcocornia ambigua collected from Barra and Pólvora Island populations and exposed to
3
five salinity treatments for 22 days (n = 4). All values were arcsin(√x/100) transformed prior
4
to analysis.
Total Germination (%) df
MS
F
Sig.
MS
F
Sig.
Salinity
4
2.15
128.57
**
0.06
4.35
*
Population
1
0.02
1.41
NS
0.01
Sal X Pop
4
0.01
0.72
NS
0.01
Residual
30
0.02
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Variables
NS
0.32
NS
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0.47
0.02
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*p <0.01; ** p <0.001; NS = not significant (p >0.05).
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Unviable seeds (%)
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Page 24 of 26
Freitas&Costa Table 2
1
Table 2: Average values (± standard error) of total germination, number of days to first
2
germination, number of days to reach 50% germination (T50%) and percentage of unviable
3
seeds of Sarcocornia ambigua collected from Barra and Pólvora Island and exposed to five
4
salinity treatments for 22 days (n = 4). ND = Not detectable.
Total
First
T50%
Unviable
(g NaCl L-1)
Germination
Germination
(days)
Seeds (%)
(%)*
(days)#
0
84.0 ± 5.0 a
2.0 ± 0.0 A
4
8.0 ± 3.3 ab
5
82.0 ± 4.0 a
2.5 ± 0.5 A
4
4.0 ± 1.9 a
15
41.0 ± 8.7 b
3.5 ± 0.5 AB
ND
13.0 ± 3.5 bc
30
7.0 ± 0.1 c
9.5 ± 4.3 B
ND
11.0 ± 2.9 bc
ND
13.0 ± 1.2 bc
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Barra
3.0 ± 0.1 cd 17.5 ± 2.9 BC
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Salinities
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Population
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Pólvora Island
82.0 ± 3.5 a
2,0 ± 0,0 A
6
11.0 ± 3.5 abc
5
81.0 ± 4.4 a
3,0 ± 0.6 A
6
4.0 ± 1.9 a
15
46.0 ± 9.3 b
3,5 ± 0.5 AB
ND
15.0 ± 4.8 bc
30
2.0 ± 1.2 cd 14,5 ± 4.3 BC
ND
11.0 ± 5.1 abc
45
0.0 ± 0.0 d
ND
18.0 ± 3.0 c
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0
22.0 ± 0.0 C
6
* Different lowercase letters (column) show significant differences between the averages
7
(p < 0.05), according to Bonferroni multiple comparisons test.
Page 25 of 26
#Variable ranges from 0 to 22, with 22 days values indicating that no germination had
9
occurred during incubation period. Variable not normal and different capital letters
10
(column) indicate significant differences between averages within each population (a
11
Bonferroni correction was used yielding a critical p-value of 0.005), according to the
12
Mann-Whitney test.
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Page 26 of 26
S0304-3770(14)00055-2 http://dx.doi.org/doi:10.1016/j.aquabot.2014.04.002 AQBOT 2663
To appear in:
Aquatic Botany
Received date: Revised date: Accepted date:
22-7-2013 31-3-2014 3-4-2014
Please cite this article as: Freitas, R.F., Costa, C.S.B.,Germination responses to salt stress of two intertidal populations of the perennial glasswort Sarcocornia ambigua, Aquatic Botany (2014), http://dx.doi.org/10.1016/j.aquabot.2014.04.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Freitas&Costa Highlights (for review)
Highlights Sarcocornia ambigua is a widespread halophyte of the Atlantic coast of South America. Higher germination (81–84%) after 22 days occurred at salinities 0 and 5 g NaCl L-1.
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Osmotically induced inhibition of germination is reversible by salt relief.
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S. ambigua seeds were able to germinate (3%) even in 45 g NaCl L-1.
Page 1 of 26
*Freitas&Costa manuscript
1 1
Germination responses to salt stress of two intertidal populations
2
of the perennial glasswort Sarcocornia ambigua
3 Ricardo F. Freitas1*, César S.B. Costa2
4
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5 6
(1)
7
Federal do Rio Grande (FURG), Av. Itália, km 8, Bairro Carreiros, 96203-900, Rio Grande,
8
Brazil.
9
(2)
us
cr
Programa de Pós-Graduação em Aqüicultura, Instituto de Oceanografia, Universidade
Laboratório de Biotecnologia de Halófitas, Instituto de Oceanografia, Universidade Federal
do Rio Grande (FURG), Av. Itália, km 8, Bairro Carreiros, CEP 96203-900, Rio Grande,
11
Brazil.
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* Corresponding author: Tel.: +55 53 3233 6534. E-mail address: [email protected]
14
(R.F. Freitas).
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15
Abstract - Sarcocornia ambigua is a perennial halophyte of the Atlantic coast of South
17
America. The species occurs in salt marshes and mangrove swamps and is able to grow under
18
hypersaline conditions. S. ambigua seeds were collected from two intertidal populations
19
located inside and at the entrance of the Patos Lagoon estuary (RS, Brazil). The seeds were
20
germinated by incubation for 22 days in 0, 5, 15, 30 and 45 g NaCl L-1 solutions at 20-30 °C
21
and with a 12:12 h photoperiod. There were no significant differences in germination between
22
S. ambigua populations. High average germination (81-84%) occurred at low salinities (0 and
23
5 g NaCl L-1) and decreased at salinities of 15 g NaCl L-1 (41-46%) and above. Seeds were
24
able to germinate (3%) even in 45 g NaCl L-1. Both populations of S. ambigua demonstrated
25
an inhibition of germination under saline conditions. This inhibition was reversible through
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Page 2 of 26
2 removal of the salt stress. The percentage of unviable seeds increased from 4% to 11-18% as
27
the salinity increased from 5 to 15-40 g NaCl L-1. The germinative responses to salinity of S.
28
ambigua are typical of an extreme halophyte. The predominant outflow tendency of the
29
waters in the study area and the choked morphology of Patos Lagoon favour the flux of seeds
30
between marshes in the middle estuary and at the narrow estuarine inlet, preventing local
31
differentiation of S. ambigua populations.
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Keywords: Sarcocornia ambigua, halophyte, population differentiation, germination.
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33
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32
34 1. Introduction
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35 36
Several species of the genus Sarcocornia (Chenopodiaceae) are considered ‘extreme
38
halophytes’ based on their ability to thrive in seawater-flooded (35g NaCl L-1) and hypersaline
39
soils (Redondo-Goméz et al., 2004; Davy et al., 2006; Ventura et al., 2011; Ventura and Sagi,
40
2013). Native populations of Sarcocornia species occur in salt marshes, mangrove swamps
41
and salt deserts. Population differentiation occurs primarily due to spatial heterogeneity of
42
salinity and periodic flooding stresses (Redondo-Goméz et al., 2004; Davy et al., 2006),
43
which are the major determining factors for germination success and plant establishment
44
(Ungar, 1995).
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37
Along the South American Atlantic coast, Sarcocornia ambigua (Michx.) Alonso and
46
Crespo (syn. Salicornia gaudichaudiana Moq.) is the most widely distributed perennial
47
species of the genus, occurring in salt marshes and hypersaline mangrove areas from
48
Venezuela to the Valdés Peninsula in Argentina (Costa, 2006; Isaach et al., 2006; Alonso and
49
Crespo, 2008). In the marshes of the south-western Atlantic coast, S. ambigua colonizes
50
physically stressful middle and upper marshes, and field experimental manipulation (Alberti
Page 3 of 26
3 et al., 2008) has shown that it facilitates the recruitment of the South American cordgrass
52
Spartina densiflora, which co-dominates these marshes. More than 740 km² of the coast
53
between southern Brazil (32º S) and the Valdés Peninsula (43º S) are covered by S. ambigua-
54
dominated salt marshes (Isaach et al., 2006), which support large populations of polychaetes,
55
pelecypods, and crustaceans. These organisms are subject to predation by oystercatchers,
56
gulls, and terns (Costa et al., 2003, 2009; Alberti et al., 2008).
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S. ambigua propagates through seeds and vegetative growth (Leite et al., 2007; Alonso
58
and Crespo, 2008). This species is very widespread, it occurs in places with high variability in
59
salinity, both in time and space, which could lead to selective specialization or plasticity
60
(Ungar, 1995; Gul et al., 2013). The control of germination in mature seeds may occur
61
through phytohormones or the mechanical structures of the coat, producing innate dormancy
62
(Baskin and Baskin, 2001; Cardoso, 2009). In coastal environments and certain arid regions,
63
quiescent seeds may show a delay or inhibition of germination due to soil salinisation (Pujol
64
et al., 2000; Gul et al., 2013), caused by a low osmotic potential and the toxic effect of certain
65
elements, such as sodium and chlorine, at high concentrations. In sensitive species, the salt
66
content of the sediment may produce a partial or even complete loss of seed viability (Khan,
67
2003; Vicente et al., 2007; Gul et al., 2013). In contrast, certain halophytes may be stimulated
68
to germinate by a combination of saline stress and a subsequent dilution of the salinity
69
(‘saline stress relief’); this osmotic shock apparently denatures certain phytohormones that are
70
considered inducers of innate dormancy (Ungar, 1995; Baskin and Baskin, 2001; Gul et al.,
71
2013). For example, both S. fruticosa, which inhabits hypersaline high marshes in Spain, and
72
S. perennis, which grows in regularly flooded low marshes of this country, showed decreases
73
in the final germination percentage as salinity increased. However, the seeds of the former
74
species showed accelerated germination after relief from prolonged salinity exposure, whereas
75
such acceleration was absent in the seeds of the latter species (Redondo-Goméz et al., 2004).
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Page 4 of 26
4 Along the estuary of Patos Lagoon, located in the state of Rio Grande do Sul in
77
southern Brazil (32°S, 52°W), S. ambigua has a disjunct distribution and is extremely
78
abundant in certain salt marshes (Costa, 2011). Patos Lagoon is the world’s largest choked
79
lagoon, with a surface area of 10,000 km², and connects with the sea through a single 700 m
80
wide inlet. The circulation of the Patos Lagoon estuary is driven primarily by winds and
81
freshwater runoff, but the discharge of the principal tributary rivers is strong during the late
82
winter and early spring, with low to moderate runoff during the summer and autumn (Costa et
83
al., 2003; Möller et al., 2009; Garcia, 2011). During the seasonal flood period, only very
84
strong southerly winds can drive sea water into the lagoon and reverse the seaward flow, and
85
the water in most of the estuary can remain oligohaline or even fresh for several months
86
(Costa et al., 2003; Möller et al., 2009). This seasonal pattern may exert selective pressure on
87
local S. ambigua populations; in particular, both saline inhibition of the germination of the
88
seeds dispersed during the autumn and acceleration of germination accompanying the relief
89
from salt stress occurring in the spring would be expected.
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In addition to its seasonal hydrodynamics, the Patos Lagoon estuary shows a
91
pronounced salt gradient between the head and mouth of the estuary (≈ 60 km; Coutinho and
92
Seeliger, 1984; Garcia, 2011), and hypersaline sediments have periodically been reported
93
during the dry summer-autumn only in salt marshes near the inlet of the estuary (Cordazzo et
94
al., 2007; Costa, 2011). Mature specimens of S. ambigua growing in marshes near the inlet of
95
the estuary possess distinctive shoots with reddish colouration, whereas the plants in the
96
marshes of the middle estuary have dark green shoots (Costa et al., 2006). The reddish colour
97
of Sarcocornia shoots can indicate the accumulation of anthocyanins and other phenolic
98
compounds, which are antioxidant compounds used by these plants to tolerate stresses, e.g.,
99
water deficits, high soil salinisation and a high incidence of ultraviolet radiation (Costa et al.,
100
2006; Davy et al., 2006). Because disjunct populations of S. ambigua can be differentially
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Page 5 of 26
5 affected by salt intrusion in the Patos Lagoon estuary, their seeds might show contrasting
102
sensitivity and germination responses to salt stress. Salinity is a major determinant of the
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plant community structure of the local marshes, and population differences among aquatic
104
plants have previously been demonstrated along the Patos Lagoon estuarine gradient. For
105
example, the plant communities of the low intertidal zone of the salt marshes of Patos Lagoon
106
are differentiated into those of the oligohaline region at the head of the estuary(<6 g NaCl L-1)
107
and those of the mesohaline region in the central-low part of the estuary (>6 g NaCl L-1)
108
(Costa, 2011). Koch and Seeliger (1988) have demonstrated that germination patterns differ
109
between populations of the submerged phanerogam Ruppia maritima that grow in the middle
110
of the estuary and at the mouth of the estuary.
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This study aimed to compare the effects of different salinity treatments and of saline
112
stress relief on the germination of S. ambigua seeds from two native populations that inhabit
113
distinct areas of the salt gradient along the estuary of Patos Lagoon. We used laboratory
114
experiments to evaluate the hypothesis that seed germination in local populations of S.
115
ambigua is inhibited by salt exposure and enhanced after decreases in salinity as an
116
adjustment to the seasonal pattern of estuarine salinity, as well as the hypothesis that the seeds
117
of S. ambigua populations subjected to contrasting salt exposure show differential salt
118
tolerance.
120 121 122
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2. Material and Methods
2.1. Characteristics of the study populations
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The Patos Lagoon estuary has a warm temperate climate, with minimum and
124
maximum average monthly temperatures of 13 °C and 25 °C, respectively. The salt marshes
125
occupy 70 km2 of the estuary margins (Isaach et al., 2006). The studied populations of S.
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6 126
ambigua are located 15.4 km apart, on Pólvora Island (32°01′S, 52°06′W) and on the sand spit
127
at ‘Barra’ (local name of the estuarine inlet; 32°09′S, 52°04′W). Pólvora Island is situated on the middle region of the estuary. Approximately 45 ha of
129
this island consists of marshlands covered by dense vegetation, which is dominated by
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grasses. The local population of S. ambigua is distributed over mud flats in the middle zone of
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the salt marshes. This zone is flooded less than 25% of the time by oligo-mesohaline, with an
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annual average salinity of 10 g NaCl L-1 (Costa et al., 2003). The Barra salt marsh at the
133
mouth of the estuary has an area of approximately 9 ha (Marangoni and Costa, 2009). Locally,
134
S. ambigua occupies high intertidal ground that is periodically subjected to hyper-saline
135
conditions. These areas flood only during spring tides and are strongly influenced by sea
136
spray (Cordazzo et al., 2007). Coutinho and Seeliger (1984) have estimated that 90% of the
137
surface water reported in the area of the Barra is meso-euhaline, ranging from 13 to 34 g NaCl
138
L-1.
139 140
2.2. Germination experiments
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S. ambigua seeds were collected in the marshes of Pólvora Island and Barra during
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autumn of 2010. In each population, 10-15 individual mature plants were collected; their
143
ripped seeds were removed from the fertile segments and placed in a label paper bag. The
144
seeds of each population were sub-sampled and the hundred seeds weight determined. After
145
collection, the seeds were dried at room temperature for one week and stored at 5°C (cold pre-
146
treatment) for 60–70 days in the laboratory. Previously, Lopes (2000) showed that
147
approximately 50% of mature S. ambigua seeds are unable to germinate under the appropriate
148
environmental conditions when freshly released from the mother plant during the autumn, and
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a 2-month cold pretreatment is required to terminate this innate dormancy. It is possible that
150
this response results from increased levels of gibberellins in the seeds. This germination
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Page 7 of 26
7 response is compatible with the low overwinter temperatures (daily minimum below 10 ºC)
152
and the optimal temperature conditions observed in late spring-early summer. After cold
153
storage, the seeds were washed with 5% hypochlorite for 10 min to reduce fungus infestation
154
(Leite et al., 2007) and then rinsed with distilled water. Subsequently, the seeds were placed in
155
autoclaved Petri dishes with filter paper dampened with 6 mL of saline solution of different
156
concentrations: 0, 5, 15, 30 and 45 g NaCl L-1. All Petri dishes were wrapped (to prevent
157
evaporation and change in salinity) and incubated for 22 days in a germination chamber with
158
a thermoperiod of 30/20 °C and photoperiod (12 h light/12 h dark; 40 μmol photons m-2 s-1,
159
400-700 nm) provided by cold white fluorescent light (Universal-L-Duramax GE). The
160
occurrence of the maximum total germination of S. ambigua seeds under these thermal and
161
photoperiod conditions has previously been described (Lopes, 2000; Leite et al., 2007). Four
162
Petri dishes (replicates) were used for each salinity level, with 25 seeds in each dish. Petri
163
dishes were observed every two days to monitor the number of germinating seeds. Seeds were
164
considered to have germinated if the emergence of the radicle through the seed coat was
165
visible.
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To determine the occurrence of the inhibition of germination due to salt stress and the
167
loss of seed viability at high salinity due to ionic toxicity, the seeds that did not germinate
168
after 22 days of incubation in salinities higher than zero were subjected to a procedure
169
producing saline stress relief (Redondo-Gomez et al., 2004). Seeds were washed with distilled
170
water and placed on Petri dishes with filter paper dampened with distilled water and incubated
171
under the above-mentioned light and thermoperiod conditions for an additional 22 days. After
172
this period, the viability of the seeds that remained ungerminated was determined under a
173
stereomicroscope (8× magnification) after immersion in a 0.25% (m/v) tetrazolium solution
174
(2,3,5-triphenyltetrazolium chloride) at 30 °C for 24 h in the dark (Moore, 1985). This
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Page 8 of 26
8 175
procedure was also employed for seeds that were initially incubated in zero salinity and that
176
did not germinate, thus, indicating the germinability of the seed lots. A two-way ANOVA was used to test differences in the percentages of final
178
germination and loss of viability among populations (P) and salinity levels (S), followed by a
179
Bonferroni comparison test at a 5% significance level. Both germination and viability
180
percentage data were previously transformed into angular values (arcsin √x/100) to satisfy the
181
assumptions of normality and homoscedasticity (Zar, 2010). In the test of the effect of saline
182
stress relief, the initial number of seeds varied among the Petri dishes (these seeds were the
183
ungerminated seeds resulting from the test of the effect of salinity). For each Petri dish, the
184
percentage of final germination was calculated by cross multiplication of the final number of
185
germinated seeds, with a value of 100% representing the initial number of seeds.
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Because the time to first germination (days) did not follow a normal distribution for
187
any population, this variable was compared nonparametrically among the salinity levels to
188
which each population was exposed. A Kruskal-Wallis test, followed by pairwise comparisons
189
using a Mann-Whitney test (Zar, 2010), was used for this analysis. A Bonferroni correction (p
190
= 0.05/10 = 0.005) was used to determine differences in statistical significance among 5 levels
191
of salinity treatment. Three salinity levels (15, 30, and 45 g L-1 NaCl) did not show 50%
192
germination (T50) after 22 days of incubation; hence, this parameter was not included in the
193
statistical analysis.
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3. Results
196 197
3.1. Effect of salinity on total germination and seed mass
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After 22 days of incubation, seeds from both Pólvora Island and the Barra showed
199
higher average germination rates (81–84%) at salinity levels of 0 and 5 g NaCl L-1 then at
Page 9 of 26
9 higher salinities. All salinities ≥15 g NaCl L-1 resulted in a significant decrease in total seed
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germination (41-46%; FS = 128.6; p <0.001, Bonferroni test; Tables 1 and 2). Both
202
populations showed the same pattern of germination response to the different salinities tested
203
(FPxS= 0.72; p > 0.05). Even at salt concentration of 45 g NaCl L-1, i.e., the highest salinity
204
showing average germination of S. ambigua seeds from Pólvora Island (0%) and the Barra
205
(3%), respectively, no differences between the populations were found by the Bonferroni test
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(Table 2). However, the seeds of Barra weighed on average 0.10 ± 0.01 mg (± standard error),
207
42% more than the seeds from Pólvora Island (0.07 ± 0.01 mg) (p < 0.01; Mann-Whitney
208
test).
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3.2. Effect of salinity on the time of germination
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At salinity levels of 0 and 5 g NaCl L-1, germination was observed two days after
212
incubation of the seeds (Fig. 1A-B), and T50 was achieved between the 4th and 6th days (Table
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2). High salinities induced a significant delay in the time to first germination in both
214
populations (Kruskal–Wallis tests; p < 0.01); seeds from the Barra and Pólvora Island exposed
215
to a salinity level of 30 g NaCl L-1 required an average of 9.5 ± 4.3 and 14.5 ± 4.3 days,
216
respectively, to begin germination (Fig. 1A-B; Table 2).
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For the two populations of S. ambigua, the overall germinability of seeds in the
218
experimental conditions was 83% (estimated at zero salinity), and 7.5% of the seeds remained
219
dormant (Fig. 2A). This average percentage of dormant seeds showed no significant
220
difference (p > 0.05) among the salinity levels (FS = 1.25) and populations (FP = 0.77),
221
ranging from 3 ± 1% (Barra, 5 g NaCl L-1) to 11 ± 2% (Pólvora Island, 15 g NaCl L-1) (Fig.
222
2A).
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3.3. Effect of saline stress relief on seed viability and germination
Page 10 of 26
10 The temporal pattern of germination after saline stress relief at salinity levels of 30
226
and 45 g NaCl L-1 was similar to that in the seeds initially incubated at zero salinity, in which
227
germination was initiated after 1–2 days and T50 was attained between the 3rd and 4th days
228
(Fig. 2B-C). The saline relief responses (germination from 76–81% of the seeds) at these two
229
salinity levels showed no significant differences (FS = 1.05; p > 0.05), and no differences were
230
detected between populations (FP = 0.08; p > 0.05).
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The optimal condition for germination was determined as that with a salt
232
concentration of 5 g NaCl L-1, at which a high percentage of embryos emerged (Fig. 2A). The
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tetrazolium test showed 4% of unviable seeds incubated in 5 g NaCl L-1 and values 2–3 times
234
significantly higher (11–18%; FS = 4.37; p < 0.01) when incubated at salinity concentrations
235
within a range of 15-45 g NaCl L-1 (Table 2; Fig. 2A). In addition, 8% (from Pólvora Island)
236
to 11% (from the Barra) of the seeds incubated at 0 g NaCl L-1 did not germinate, and these
237
percentages of dormant seeds did not vary between populations (FP = 0.47; p > 0.05).
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240 241
4. Discussion
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4.1. General pattern of germination response to increasing salinity The germination responses of S. ambigua to salinity was similar to that of true
243
halophytes (Ungar, 1995; Gul et al., 2013), with a maximum germination percentage under
244
low salinities (0-5 g NaCl L-1) and a marked reduction in the percentage of seed germination
245
at salinities higher than 15 g NaCl L-1. Nevertheless, seeds were able to germinate at 45 g
246
NaCl L-1. The same pattern of response to salinity has been shown in extreme halophytes such
247
as Salicornia dolichostachya, S. brachystachya (Huiskes et al., 1985), Sarcocornia fruticosa,
248
S. perennis (Redondo-Goméz et al., 2004) and Arthrocnemum macrostachyum (Vicente et al.,
249
2007). Glycophytes also show decreases in germination with increasing salt concentration
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Page 11 of 26
11 (Khan, 2003; Cardoso, 2009), but in ‘Carioca bean’ (Phaseolus vulgaris), for example, the
251
seeds did not germinate in salinities higher than 200 mol m-3 (≈ 8.4 g NaCl L-1; compared to
252
germination in 0 g NaCl L-1 = 91%; Dantas et al., 2007). Facultative halophytes, such as the
253
grass Hordeum jubatum (Ungar, 1974) from moderately saline soils and the sub-bush Cakile
254
maritima from embryo dunes affected by high tides (Megdiche et al., 2007), showed
255
maximum germination at approximately 0-5 g NaCl L-1, strong germination inhibition at
256
salinities greater than 10 g NaCl L-1 and no germination at salinities ≥ 20 g NaCl L-1. As
257
shown in the Results section, the germination response of S. ambigua identifies it as one of
258
the most salt tolerant species cited in the literature.
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The halophytic characteristics of S. ambigua were clearly demonstrated by the
260
germinative response of the seeds to extreme salinities. In a recent review of the germination
261
strategies of several plants living in different saline habitats, Gul et al. (2013) showed that
262
plants capable of germinating in salinities higher than that of seawater (even if less than 10%
263
of the seeds germinated) were among the most tolerant halophytes. The mean germination
264
rate of S. ambigua from Barra (7%) at a salt concentration of 30 g NaCl L-1 was two times the
265
observed results in experiments conducted using seeds of the halophytes Suaeda fruticosa
266
(Khan and Ungar, 1996) and Sarcocornia fruticosa (Redondo-Gomez et al., 2004) (both ≈
267
3%). Small percentages of germination at salinities greater than or equal to 40 g NaCl L-1, as
268
shown by the seeds of S. ambigua from the Barra, have been observed only in extreme
269
halophytes, such as Salicornia stricta, S. ramosissima (Ungar, 1995), Sarcocornia fruticosa
270
(Redondo-Goméz et al., 2004) and Haloxylon salicornicum (El-Keblawy and Al-Shamsi,
271
2008).
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272 273
4.2. Salt tolerance of seeds and the effects of saline stress relief on germination
Page 12 of 26
12 The seed viability loss of 11–18% observed in S. ambigua exposed to mid-high
275
salinities (including hypersaline) might be considered low relative to the high stress level to
276
which the seeds were subjected. Redondo-Goméz et al. (2004) reported a significant increase
277
in the average proportion of unviable seeds in three species of halophytes (Sarcocornia
278
perennis, Sarcocornia fruticosa, and Sarcocornia perennis x Sarcocornia fruticosa), from 7–
279
12% to 24–34%, between incubation salinities of 0 and 40 g NaCl L-1. Facultative halophytes,
280
which are dominant in habitats marginal to salt marshes, exhibit high losses in seed viability
281
when exposed to high salinities. For example, the seeds of Myrsine parvifolia, a bush that
282
colonises the high intertidal zone of salt marshes in southern Brazil, showed a 20–30%
283
viability loss when incubated for a month at salinities from 20 to 30 g NaCl L-1 (Ribeiro,
284
2010). According to Ungar (1995), the ability of seeds to remain viable for long periods under
285
saline stress represents an important attribute of halophytes, distinguishing them from
286
glycophytes and allowing the exploration of soils with low water potential. No information
287
was available on the longevity of seeds of S. ambigua on saline soils, but high seed viability is
288
still observed in cold stored protoplasm after 15 years (C.S.B. Costa, pers. comm.).
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Most ungerminated seeds of S. ambigua initially exposed to high salinities (30 and 45
290
g NaCl L-1) were quiescent, and germinated after saline stress relief. Consequently, both
291
populations of S. ambigua showed osmotic inhibition of non-dormant seeds. Similar results
292
have been observed in seeds of three Sarcocornia species from Mediterranean salt marshes.
293
The germination of these seeds was partially inhibited by incubation at salinity levels from 20
294
to 60 g NaCl L-1 and the seeds germinated after salt stress relief (Redondo-Goméz et al.,
295
2004). Pujol et al. (2000) dismissed ionic intoxication as the main mechanism of germination
296
inhibition in halophytes of south-western Spain after achieving reversibility of this inhibition
297
at high salinities, with saline relief occurring in seeds incubated at low osmotic potentials.
298
Seeds of other halophytic species inhabiting subtropical and temperate salt marshes, including
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Page 13 of 26
13 the genera Atriplex, Salicornia, Limonium, Salsola, Crithmum, and Sporobolus, did not
300
germinate at high salinities, but recovered more than 50% of their ability to germinate after
301
they were removed from saline stress (Ungar, 1995; Khan and Ungar, 1996; Gul et al., 2013).
302
Saline inhibition of germination is an efficient mechanism for the spatiotemporal distribution
303
of germination of halophytes, allowing seeds to initiate germination when (and where) low
304
salinity may favour the survival of seedlings (Khan and Ungar, 1997; Pujol et al., 2000;
305
Baskin and Baskin, 2001). Thus, quiescent seeds of S. ambigua may rest in the soil seed bank
306
for a long time, and their germination can be triggered during periods of reduced salinity. The
307
results presented above support the hypothesis that S. ambigua can explore the seasonal
308
pattern of estuarine salinity at Patos Lagoon, where highly saline waters prevail during the
309
summer-autumn drought and oligohaline waters are prevalent in late winter and early spring
310
(Costa et al., 2003; Möller et al., 2009; Garcia, 2011). The seeds of S. ambigua then find
311
optimal temperatures and saline conditions for germination in the late spring and early
312
summer.
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313
4.3. Population differences in salt tolerance of seeds
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The Barra and Pólvora Island marshes are exposed to flooding waters with different
316
salinities (Coutinho and Seeliger 1984; Costa et al., 2003; Cordazzo et al., 2007), and mature
317
plants of S. ambigua show distinctive shoot colouration related to the accumulation of anti-
318
stress phenolic compounds (Costa et al., 2006). Our results also showed that plants from
319
Barra and Pólvora Island showed significant differences in seed size. These differences may
320
be related to genetic factors (e.g., Salicornia bigelovii; Zerai et al., 2010) and/or phenotypic
321
responses to environmental factors (Cordazzo, 1994). Other than this contrast, there were no
322
significant differences between S. ambigua populations in the germination response to
323
increasing salinity, in saline stress relief responses or in losses of seed viability due to saline
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Page 14 of 26
14 324
stress. Thus, populations of S. ambigua thriving in localities covered by euhaline and meso-
325
oligohaline waters in the estuary of the Patos Lagoon did not show differences in the salt
326
tolerance of their seeds. Elsewhere, persistent temporal differences in salt stress have been shown to produce
328
differences between the salt tolerance of the seeds of different Sarcocornia species (Redondo-
329
Goméz et al., 2004). The lack of differentiation in salt tolerance between S. ambigua
330
populations could be explained by the particular hydrodynamics of the choked lagoon and by
331
the maintenance of genetic flow between marshes in the middle estuary and at the narrow
332
estuarine inlet. The circulation of Patos Lagoon is driven primarily by winds and freshwater
333
runoff (Costa et al., 2003; Möller et al., 2009; Garcia, 2011), typically with an average feature
334
is a residual seaward flow of 2,400 m³ s-1 (Möller et al., 2009). Because Sarcocornia seeds are
335
distributed by tides and currents (Davy et al., 2006), the predominant outflow of the water and
336
the morphology of Patos Lagoon might favour seed dispersal from the marshes of the middle
337
estuary toward the inlet marshes. This flux of seeds between marshes could prevent local
338
differentiation in the genetic structure of populations within the estuary, even in the presence
339
of a persistent horizontal gradient of salinity.
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340
342 343 344 345
Acknowledgements
This research was supported by the Brazilian National Research Council (CNPq)
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under grants n. 574600/2008-6 and 573884/2008-0 (INCTSAL).
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20 455
Figure Legends
456 Fig. 1: Cumulative germination of Sarcocornia ambigua seeds collected at (A) Barra and (B)
458
Pólvora Island during 22 days under five salinity treatments (n = averages of 4 Petri dishes,
459
standard errors not included for clarity (standard errors of total germination are shown in
460
Table 2).
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cr
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Fig. 2: (A) General response of Sarcocornia ambigua seeds (overall average results of Barra
463
and Pólvora Island populations) to salt exposure and salt stress relief. Percentages of unviable
464
seeds, seeds with persistent dormancy, seeds that germinated after 22 days in five salinity
465
treatments and seeds that germinated only after salt stress relief are shown. Cumulative
466
germination of Sarcocornia ambigua seeds collected at (B) Barra and (C) Pólvora Island after
467
salt stress relief from salinity treatments of 30 g and 45 g NaCl L (n = average ± standard
468
error for 4 Petri dishes).
-1
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Freitas&Costa Figure 1
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Freitas&Costa Figure 2
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Freitas&Costa Table 1
1
Table 1: Analysis of Variance of total germination and percentage of unviable seeds of
2
Sarcocornia ambigua collected from Barra and Pólvora Island populations and exposed to
3
five salinity treatments for 22 days (n = 4). All values were arcsin(√x/100) transformed prior
4
to analysis.
Total Germination (%) df
MS
F
Sig.
MS
F
Sig.
Salinity
4
2.15
128.57
**
0.06
4.35
*
Population
1
0.02
1.41
NS
0.01
Sal X Pop
4
0.01
0.72
NS
0.01
Residual
30
0.02
us
cr
Variables
NS
0.32
NS
an
0.47
0.02
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*p <0.01; ** p <0.001; NS = not significant (p >0.05).
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Unviable seeds (%)
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Freitas&Costa Table 2
1
Table 2: Average values (± standard error) of total germination, number of days to first
2
germination, number of days to reach 50% germination (T50%) and percentage of unviable
3
seeds of Sarcocornia ambigua collected from Barra and Pólvora Island and exposed to five
4
salinity treatments for 22 days (n = 4). ND = Not detectable.
Total
First
T50%
Unviable
(g NaCl L-1)
Germination
Germination
(days)
Seeds (%)
(%)*
(days)#
0
84.0 ± 5.0 a
2.0 ± 0.0 A
4
8.0 ± 3.3 ab
5
82.0 ± 4.0 a
2.5 ± 0.5 A
4
4.0 ± 1.9 a
15
41.0 ± 8.7 b
3.5 ± 0.5 AB
ND
13.0 ± 3.5 bc
30
7.0 ± 0.1 c
9.5 ± 4.3 B
ND
11.0 ± 2.9 bc
ND
13.0 ± 1.2 bc
ed
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Barra
3.0 ± 0.1 cd 17.5 ± 2.9 BC
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45
cr
Salinities
us
Population
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Pólvora Island
82.0 ± 3.5 a
2,0 ± 0,0 A
6
11.0 ± 3.5 abc
5
81.0 ± 4.4 a
3,0 ± 0.6 A
6
4.0 ± 1.9 a
15
46.0 ± 9.3 b
3,5 ± 0.5 AB
ND
15.0 ± 4.8 bc
30
2.0 ± 1.2 cd 14,5 ± 4.3 BC
ND
11.0 ± 5.1 abc
45
0.0 ± 0.0 d
ND
18.0 ± 3.0 c
Ac
0
22.0 ± 0.0 C
6
* Different lowercase letters (column) show significant differences between the averages
7
(p < 0.05), according to Bonferroni multiple comparisons test.
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#Variable ranges from 0 to 22, with 22 days values indicating that no germination had
9
occurred during incubation period. Variable not normal and different capital letters
10
(column) indicate significant differences between averages within each population (a
11
Bonferroni correction was used yielding a critical p-value of 0.005), according to the
12
Mann-Whitney test.
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