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Reproductive isolation in Chara aspera populations
Aquatic Botany 91 (2009) 224–230
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Reproductive isolation in Chara aspera populations Irmgard Blindow a,*, Nils Mo¨llmann a, Michael G. Boegle b, Manuela Schu¨tte a a b
Biological Station of Hiddensee, University of Greifswald, Biologenweg 15, D-18 565 Kloster, Germany Limnologische Station der Technischen Universita¨t Mu¨nchen, Hofmark 3, D-82 393 Iffeldorf, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 January 2009 Received in revised form 23 June 2009 Accepted 24 June 2009 Available online 1 July 2009
Possible reproductive isolation between freshwater and brackish water populations of the dioecious charophyte Chara aspera was studied by means of cross-fertilization experiments and AFLP (Amplified Fragment Length Polymorphism). Three Swedish freshwater populations and three (German and Swedish) Baltic Sea populations of C. aspera were sampled. Cross-fertilization experiments were performed in a full combination setup of all populations and with two different salinities (0 and 10 PSU). Both freshwater and brackish water females formed about 70% more gametangia at 0 than at 10 PSU. Male individuals collected from freshwater had higher fertility than brackish water males at both salinities. 57% of all gametangia of females from freshwater developed into oospores compared to only 8% of gametangia of brackish water females. 42% of all oospores were fertilized in crosses between habitats (freshwater–brackish water) compared to 36% in crosses within habitats, the difference was not significant. Oospore and bulbil germination was investigated using propagules from freshwater and brackish water populations and incubation salinities of 0, 5, 10 and 20 PSU. None of the oospores collected from brackish water germinated. Germination of oospores and bulbils from freshwater was higher at 0 and 5 PSU than at higher salinities. Only around 40% of bulbils from brackish water germinated at 20 PSU compared to around 70% at the other three salinities. Germination of all bulbils was delayed at 20 PSU compared to other salinities. Genetic similarities (Jaccard indices of AFLP data) were higher within than between populations, but comparisons within habitat (freshwater–freshwater and brackish water–brackish water) were not different from comparisons between habitats. Our results did not identify any reproductive isolation between freshwater and brackish water populations, but indicate low gene flow between the two habitats. Oospore and bulbil germination success were highest at salinities corresponding to the conditions of their original habitat, suggesting genetic adaptation to their environmental conditions and indicating that propagules transported from freshwater to brackish water or vice versa will hardly develop into fertile plants. Additionally, brackish water plants perform poorer in all aspects of sexual reproduction than freshwater plants. Possibly, successful dispersal of oospores is not subjected to high selective pressure within the Baltic Sea where new sites easily can be colonized by means of vegetative reproduction. We assume that these adaptations will favour speciation within C. aspera and support the idea of the geologically young Baltic Sea as a ‘‘cradle of plant evolution’’. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Salinity Baltic Sea Cross-fertilization Oospore Dispersal Evolution AFLP
1. Introduction The taxonomy of charophytes has been subject to several revisions and much debate. High variability within single taxa as well as small morphological differences among closely related taxa prevent the identification of clearly separated species, and different authors either combined similar taxa into the same
* Corresponding author. Tel.: +49 38300 50251; fax: +49 38300 60672. E-mail address: [email protected] (I. Blindow). 0304-3770/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2009.06.009
species (Wood, 1962, 1965) or segregated them into separate species (Migula, 1897; Krause, 1997). Beyond the species level, a number of subspecies, varieties and forms have been described (e.g., Migula, 1897). While determination keys discriminate between morphologically similar species such as Chara intermedia and C. baltica, other species are characterized by high intraspecific morphological variability (Krause, 1997; Boegle et al., 2007). Cross-fertilization experiments are one way to investigate if the different ‘‘species’’ described in monographs and determination keys, and if the characters used for determination, reflect genetic isolation among groups. Such experiments have mainly been
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148320 E), both situated in Scania, and Redensee Bay (548220 N, 128350 E) in the Darß-Zingster bodden chain, Germany. The four Scanian sites were sampled during mid June, Lake Storacksen and Redensee Bay during end August. After collection, the plants were transported to the laboratory in plastic bags and stored at 4 8C until further treatment (cross-fertilization experiments and genetic analyses) within a few days.
performed in different taxa within the genus of Chara and resulted into successful crosses between different ‘‘species’’ (Proctor, 1971a, 1975), but also the identification of reproductive isolation among populations belonging to the same ‘‘species’’ (Proctor, 1971b; Grant and Proctor, 1972; Croy, 1982). The criteria used for species determination are not always the same as the morphological features that are under selective pressure in the evolution of a species (Proctor, 1980). Genetic techniques are another method to compare morphologically separate taxa with genetically isolated groups. Among these techniques, AFLP has been suggested to be most suitable to identify polymorphism within and among closely related taxa (Garcia-Mas et al., 2000) and has been successfully applied to various charophyte taxa (Mannschreck, 2003; Mannschreck et al., 2002). Like cross-fertilization experiments, these investigations identified high genetic distances between morphologically similar plants (Boegle et al., 2007), but also small genetic differences between morphologically distinct taxa (O’Reilly et al., 2007). Chara aspera Willd. is distributed all over the northern hemisphere (Croy, 1982; Nielsen, 2003). It occurs in both freshwater and brackish water and inhabits a wide range of different habitats, both permanent and temporary (Hasslow, 1931; Olsen, 1944; Krause, 1997). Typical characters are dioecism, triplostichous cortex, acute stipulodes and single-celled, circular bulbils (Krause, 1997), but high morphological variability exists within the ‘‘species’’, mainly concerning the spine characters (Krause, 1997; Nielsen, 2003). Several taxa have been described on a subspecies level such as C. aspera f. aspera with long, acute and single spines, C. aspera f. subinermis without spines or with small papilliformous spines and C. aspera var. curta with spines in bunches. Reproductive isolation has been shown between individuals originating from different continents as well as between individuals collected from the same continent (Croy, 1982). A certain level of genetic isolation can therefore be assumed within this species. The wide salinity range of this species from about 0 to 15 PSU (Hasslow, 1931; Olsen, 1944; Blindow, 2000) further suggests specific adaptations of single populations to their environment. Considerable morphological differences have been described between freshwater and brackish water plants. While brackish water plants have a ‘‘stunted’’ growth and grow typically in single tussocks (Olsen, 1944), freshwater plants use to be thinner and often form a dense vegetation ‘‘mat’’ (Blindow, 1992; van den Berg et al., 1998). To investigate the level of possible speciation within this variable taxon, we studied ecophysiological (Blindow et al., 2003) as well as morphological characteristics (Blindow and Schu¨tte, 2007) of freshwater and Baltic Sea populations of C. aspera. The aim of this study was to identify possible reproductive isolation between these populations by means of cross-fertilization experiments, genetic analyses, and investigations of salinity effects on fertility, fertilization success and germination of oospores and bulbils.
Under a stereomicroscope, the plants were separated into sterile plants, which were planted into beakers for further use, and fertile plants which were used for cross-fertilization. For this experiment, five replicates were prepared for each of two different salinity treatments and each combination of male and female plants from the six sites including within-population crosses. In total, the setup should have corresponded to 5 2 6 6 = 360 beakers. Due to lack of fertile plants, however, no treatments with male plants from Lake Storacksen, and only some treatments with male plants from Redensee Bay could be prepared. Therefore, the setup consisted of only 280 beakers in total. Each replicate consisted of one 400 mL beaker containing autoclavated sediment (1 cm height) and GF/C-filtered water from one of the sites (Lake Krankesjo¨n) with (10 PSU treatment) or without (0 PSU treatment) addition of salts, respectively, corresponding to the composition of natural sea water (see Blindow et al., 2003). In each beaker, three fertile female plants (whole plants or several upper whirls) were planted and incubated indoors at room temperature. Plants collected from freshwater were gradually adapted to the 10 PSU treatments by incubating them at 5 PSU for several days, and then filling the beakers to a final concentration of 10 PSU. After 2 weeks, all plants were examined, and oospores which had eventually been formed were removed. Thereafter, three fertile male plants were added to the beaker. The cross-fertilization experiment was run outdoors. All beakers were covered with a commercial mosquito net which reduced the natural irradiance (around 60 mol m2 day1 on a sunny summer day) by 50%, a light intensity resulting in higher growth rates and higher fertility according to former results (Blindow et al., 2003). All beakers were checked regularly, and dead male plants eventually replaced. On 15 October, the beakers were taken indoors and incubated at 12:12 h L:D and about 10% of natural irradiance. At the end of October, all plants were removed. Male plants were checked for vitality and fertility (no. of antheridia). For all female plants, young oogonia, aborted oogonia and oospores were counted. Parthenogenesis has not been described for C. aspera, but to exclude this possibility, 30 additional beakers containing three female plants each collected from Ho¨llviken and Edenryd were incubated without male plants at 0 PSU (15 beakers) and 10 PSU (15 beakers), respectively. The plants were checked on 11 November for numbers of oogonia, aborted oogonia, and oospores.
2. Materials and methods
2.3. Oospore and bulbil germination
2.1. Plant collection
Oospores were collected from the upper sediment layer (0– 10 cm) in two of the freshwater sites (Lakes Krankesjo¨n and Bo¨rringesjo¨n) and two of the brackish water sites (Ho¨llviken and Edenryd Bays) within C. aspera vegetation during June 2002. Bulbils were collected directly from the rhizoids of the plants between end of July and end of August 2002 within dense vegetation of C. aspera. Both oospores and bulbils were transported to the laboratory and stored for several weeks at 4 8C in the dark. For the germination experiments, 20 oospores or bulbils, respectively, were placed into 400 mL beakers containing 1 cm of autoclavated sediment and covered with another 1 cm of
Plants of C. aspera were collected from three freshwater and three brackish water sites between mid June and end of August of 2002. Lake Storacksen (608560 N, 158120 E), province of Dalarna (middle Sweden), is a calcium-rich, oligotrophic lake. Lake Krankesjo¨n (558420 N, 138290 E), province of Scania, south Sweden, is a calcium-rich, meso- to eutrophic lake. Lake Bo¨rringesjo¨n (558290 N, 138200 E), Scania, is highly eutrophic with algal blooms throughout the vegetation period. The brackish water sites were Ho¨llviken Bay (558250 N, 128560 E) and Edenryd Bay (568020 N,
2.2. Cross-fertilization
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autoclavated sediment. The beakers were filled with GF/C-filtered water with (5, 10 and 20 PSU treatments) or without (0 PSU treatment) addition of salts, respectively, corresponding to the composition of natural sea water (see Blindow et al., 2003). Both sediment and water were taken from Lake Krankesjo¨n. The beakers were incubated in the laboratory at room temperature and natural light. The oospore germination experiment started during August 2002, the bulbil germination experiments during September 2002. All beakers were checked six times during the experiments for germlings. Both experiments were terminated during March 2003.
2.5. Data analysis
2.4. Genetic analyses
The female plants which were incubated in absence of male plants (parthenogenesis experiment) formed in total 11 (0 PSU) and 18 (10 PSU) oogonia, all of which were aborted. Female plants collected from freshwater had more gametangia at the end of the experiment than females collected from brackish water, the difference, however, was not significant (p = 0.191, Mann–Whitney). Both freshwater and brackish water females formed more gametangia at 0 than at 10 PSU (p = 0.015 and 0.022, respectively, Mann–Whitney, Fig. 1A). Male individuals collected from freshwater were more fertile than individuals collected from brackish water (p = 0.005, Mann– Whitney). While males originating from freshwater had a somewhat higher fertility at 0 compared to 10 PSU, brackish water males had a higher fertility at 10 compared to 0 PSU (Fig. 1B). The differences between salinities were not significant, however (p = 0.31 and 0.25, respectively, Mann–Whitney). The numbers of oospores did not differ between beakers with fertile male plants and beakers where the male plants were not fertile any more at the end of the experiment (p = 0.75, Mann– Whitney), or beakers with male plants originating from freshwater and males originating from brackish water (p = 0.72, Mann– Whitney). The oospore numbers were higher in beakers with female plants originating from freshwater than in beakers with females collected from brackish water (p = 0.00004, Mann– Whitney), and higher in beakers incubated at 10 PSU compared to beakers incubated at 0 PSU (p = 0.035, Mann–Whitney, Fig. 2). Only 8% of all gametangia of female plants originating from brackish water were fertilized and developed into oospores compared to 57% in females originating from freshwater. As an overall mean, 36% of all female gametangia from withinhabitat crosses (brackish–brackish or freshwater–freshwater, respectively) developed into oospores compared to 42% for between-habitat crosses. The difference was not significant (p = 0.98, Mann–Whitney). The percentage of gametangia which developed into oospores was not correlated with the distances between sites (p = 0.71, linear regression, Fig. 3). None of the oospores collected from brackish water germinated. Germination of oospores collected from the freshwater sites Lakes Krankesjo¨n and Bo¨rringesjo¨n was significantly affected by salinity (p = 0.010, Kruskal–Wallis). Only few oospores germinated at 20 PSU (Fig. 4), and the germination success at this salinity was lower compared to 0 and 5 PSU, respectively (p < 0.05, Kruskal– Wallis Multiple Comparison Z-value Test). Germination percentages were lower at 10 PSU compared to 0 and 5 PSU in both lakes (Fig. 4), but the differences were not significant (p > 0.05, Kruskal– Wallis Multiple Comparison Z-value Test). The effects of both origin (freshwater–brackish water) and salinity on bulbil germination were significant as was the interaction effect of both factors (p < 0.05, two-way ANOVA). In bulbils collected from freshwater, germination success at 10 PSU was lower than at 0 PSU, and germination success at 20 PSU significantly lower than at the other three salinity levels (p < 0.05, Kruskal–Wallis Multiple Comparison Z-value Test). For 5, 10 and 20 PSU, germination success was lower compared to germination success of bulbils collected from brackish water and cultivated at
After morphologic separation and removal of epiphytes in an ultrasonic bath the charophytes were stored at 20 8C. Genomic DNA of each individual was isolated separately from 40 mg of freezedried, powdered plant material using the DNeasy Plant Mini Kit (Quiagen, Hilden, Germany). The quality and quantity of DNA was determined on 1% agarose gel electrophoresis on 50 V in 30 min. The AFLP procedure was performed according to Vos et al. (1995), with slight modifications suggested by Mannschreck et al. (2002), for detailed description see Boegle et al. (2007). The DNA was digested with EcoRI and MseI, and simultaneously adapters were ligated (for sequences of adapters and primers see Mannschreck et al., 2002). The digested ligated DNA was diluted according to estimated DNA content with 40–140 mL H2O for subsequent preamplification steps. For preamplification primers were used complementary to the Mse1 and EcoR1 adaptors, the recognition site and possessing one additional nucleotide at the 30 primer end (for primer description and preamplification protocol see Mannschreck et al., 2002). After preamplification the quality and quantity of 10 mL preselective amplification product was determined on 1% agarose gel. The remaining 10 mL product was diluted according to estimated preselective amplification product with 40–140 mL H2O. Selective amplification was carried out with EcoRI and MseI primers having three additional nucleotides. For fragment analysis, EcoRI primers with the selective nucleotides ACA or ACG were labelled with a 5-FAM or JOE fluorochrome respectively, the MseI primers were used unlabelled. Preparation of samples and polyacrylamid gel electrophoresis was based on Mannschreck et al. (2002) with the exception that instead of 2 mL only 1.8 mL of PCR product was used. Semi-automated AFLP fragment analysis was performed with GenotyperTM DNA fragment analysis software version 2.1 (Applied Biosystem, Weiterstadt, Germany). To combine the information for a sample, each primer pair was linked into a single file according to the following protocol. Due to differing intensities of the primer combinations used, scale factors and peak levels were optimised to the sum of signal. The height of each ACA/CGA and ACA/CGC signal was divided by 300,000, each ACG/CGA signal was divided by 250,000, and each ACG/CGC signal by 150,000. Peaks were labelled if rescaled height was >150 for ACA fragments between 100 and 460 base pairs (bp), >100 for ACG fragments between 100 and 460 bp, and >50 for fragments between 461 and 500 bp for all primer combinations. The range between 100 and 500 bp was divided into 267 segments, each being 1.5 bp wide. Within each segment, labelled peaks were scored as one and the absence of a labelled peak was counted as 0, thus generating a presence/ absence matrix. Presence/absence matrices of each primer pair were combined to a single file per sample. The AFLP procedure was carried out twice for each sample. In the data analysis, only individuals with both AFLP procedures resulting in pairwise samples within an unweighted pairgroup method using arithmetic averages (UPGMA) phenogram were used for further analyses (not shown).
All data analyses were performed using NCSS 2001 (Hintze, 2001). Nonparametric methods (Kruskal–Wallis and nonparametric post hoc tests) were applied when homoscedasticity of the data material was not achieved even after transformation. Twoway ANOVA was applied on log-transformed data followed by Tukey post hoc tests. 3. Results
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Fig. 1. (A) Female fertility (no. of gametangia beaker1) and (B) male fertility (% of beakers with fertile plants) of Chara aspera collected from freshwater (white columns) and C. aspera collected from brackish water (hatched columns) after incubation at 0 and 10 PSU, respectively, at the end of the experiment. Mean values and SE are given. In (B), each crossing combination (five beakers) was treated as one replicate. Different letters indicate significant (Kruskal–Wallis Multiple Comparison Z-value Test, p < 0.05) differences between groups.
the corresponding salinities (p < 0.05, Tukey–Kramer Multiple Comparison Test). There were no significant differences in germination success of bulbils collected from brackish water at 0, 5 and 10 PSU (p > 0.05, Tukey–Kramer Multiple Comparison Test), but germination success at 20 PSU was significantly lower compared to the other three salinities (p < 0.05, Tukey–Kramer Multiple Comparison Test; Fig. 5). Neither for bulbils collected from the three freshwater sites nor for bulbils collected from the three brackish water sites, there was a significant difference in germination success among the single sites (p > 0.05, Kruskal–Wallis). Apart from lower germination success, germination of bulbils collected from freshwater was delayed at higher salinities. While 41% of all bulbils that germinated at 0 PSU had germinated already after 19 days of incubation, only 16% had germinated at 5 PSU and 12% at 10 PSU. The first germination at 20 PSU was recorded after 89 days (Fig. 6A). For bulbils collected in brackish water, 33% of all bulbils that germinated at 0 PSU had germinated after 19 days of incubation compared to 56% at 5 PSU and 46% at 10 PSU. The first germination at 20 PSU was recorded after 35 days (Fig. 6B). Both origin (freshwater–brackish water) and culture salinity had a
Fig. 2. Cross-fertilization success (no. of oospores beaker1) for all combinations of male and female C. aspera at 0 PSU (white columns) and 10 PSU (dotted columns), respectively. Mean values and SE are given. FF = freshwater female freshwater male. FB = freshwater female brackish water male. BF = brackish water female brackish water male. BB = brackish water female brackish water male. Different letters indicate significant (Kruskal–Wallis Multiple Comparison Z-value Test, p < 0.05) differences between groups.
Fig. 3. Oospore formation (% of gametangia, filled squares) and Jaccard similarities of AFLP-data (open squares) depending on geographic distances among sites.
significant effect on the germination percentage (of total germination) during day 19, and also the interaction effect between both factors was significant (p < 0.05, two-way ANOVA). Germination percentages (of total germination) during day 19 were lower for bulbils incubated at 20 PSU compared with bulbils
Fig. 4. Oospore germination (%) for C. aspera collected from L. Krankesjo¨n (Kra) and L. Bo¨rringesjo¨n (Bo¨r) at different salinities. Mean values and SE are given.
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Fig. 5. Bulbil germination (%) for C. aspera collected at different sites and salinities. Site abbreviations: Kra = Lake Krankesjo¨n, Bo¨r = Lake Bo¨rringesjo¨n, Sto = Lake Storacksen, Ho¨l = Ho¨llviken Bay, Red = Redensee Bay. Mean values and SE are given.
incubated at the other three salinities, and were lower for bulbils collected from freshwater sites incubated at 5 and 10 PSU than for bulbils collected from brackish water sites incubated at corresponding salinities (p < 0.05, Tukey–Kramer Multiple Comparison Test). Genetic similarities (Jaccard indices of AFLP-data) were higher within populations than between populations (p = 0.0014, twotailed t-test). There was no significant difference in Jaccard similarities between comparisons within habitats (freshwater– freshwater and brackish water–brackish water) and comparisons between habitats (freshwater–brackish water; p = 0.1694, twotailed t-test) (Table 1). Jaccard similarities were not correlated with the geographic distances between sites (p = 0.33, Fig. 3). 4. Discussion Despite of the fact that the brackish environment of the Baltic Sea has existed for only around 11,000 years, i.e., a very short time period from a geologic and evolutionary point of view, and despite the rapid salinity changes which have taken place within this period, a number of algal species which have colonized the Baltic Sea from either freshwater or marine habitats have already developed varieties different from their original populations. In some cases, these varieties show morphological or other physiological differences compared to their original populations, and in every case, they adapted to the environmental conditions of the Baltic Sea and are characterized by salinity optima which are within the brackish range and different from the original populations (Russell, 1985a,b, 1988, 1994; Thomas et al., 1990; Ba¨ck et al., 1991; Rietema, 1991, 1993, 1995; Kalvas and Kautsky, 1993). The Baltic Sea therefore has been assumed to be a spot of rapid evolution (Russell and Thomas, 1988).
C. aspera fits into this pattern. While morphological differences between freshwater and brackish water populations which have been described by Olsen (1944) can be explained by salinity effects rather than genetic differences (Blindow and Schu¨tte, 2007), our results suggest that C. aspera has developed a specific ecotype in the Baltic Sea which is adapted to the salinities of this environment. Even after longer adaptation periods, plants collected from the Baltic Sea have salinity optima at 5 to 10 PSU, freshwater plants at 0 to 5 PSU. This was found for photosynthesis, weight gain, and overall fertility (Blindow et al., 2003) as well as bulbil germination and (freshwater plants) oospore germination (this study). Apart from low bulbil germination success, the germination is delayed at salinities higher than the salinity range of the habitat where the plants were collected from. Waterfowl is assumed to be an important vector for gene flow among sites in charophytes as well as other submerged macrophytes, and sexual propagules of a number of submerged plants including charophytes tolerate gut passages or show even enhanced germination success after gut passage (Proctor, 1962; Clausen et al., 2002; Figuerola and Green, 2002). The amount of this transport is rather unknown, though high genetic within-site variabilities and low genetic between-site differences among waterfowl migration routes suggest that this transport may be substantial (Mader et al., 1998). Wigeon and pintail are two common duck species which switch between brackish water and freshwater habitats during migration and feed on submerged plants in brackish water (Clausen et al., 2002). In south Sweden, tufted ducks perform diurnal migration between freshwater and brackish water habitats (L. Nilsson, Department of Animal Ecology, University of Lund, personal communication). Therefore, it cannot be excluded that substantial oospore transport occurs between freshwater and brackish water. Nevertheless, we assume that gene flow between freshwater and brackish water is low in C. aspera. Oospores transported from freshwater to brackish water have low germination success. The few oospores which may manage to germinate will develop into plants that are not adapted to the salinity conditions of the Baltic Sea (Blindow et al., 2003), and will therefore be subject to strong inter- as well as intraspecific competition. Even if these plants grow up, low fertility and low bulbil germination success combined with a retarded bulbil germination will result in low reproduction success. The same ‘‘misfortune’’ is assumed to happen to oospores transported from brackish water to freshwater. Transport of oospores from brackish water to freshwater may be further reduced by an assumed overall lower sexual reproduction success of brackish water populations. In our experiment, substantially fewer oospores were formed by brackish water females as a result of lower male fertility, somewhat lower female fertility and a lower percentage of oogonia which developed into oospores, independent of the origin and fertility of the male partner and the experimental salinity condition. Though C. aspera germlings developed from oospores were found both in Ho¨llviken and Edenryd Bays (own observations), no oospores collected from brackish water sites germinated in opposite to oospores collected
Table 1 Genetic similarities (Jaccard indices) of Chara aspera from different sites according to AFLP-data. F = freshwater. B = brackish water. Within-population similarities are given in bold numbers. Jaccard indices
Freshwater Krankesjo¨n
Freshwater Bo¨rringesjo¨n
Freshwater Storacksen
Brackish Ho¨llviken
Brackish Redensee
F: Krankesjo¨n F: Bo¨rringesjo¨n F: Storacksen B: Ho¨llviken B: Redensee
35.8
23.1 62.8
22.5 27.1 35.4
23.6 39.5 27.7 49.3
24.6 34.7 26.6 38.0 52.6
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Fig. 6. Development of germination over time for bulbils collected from freshwater sites (A) and brackish water sites (B) incubated at different salinities. Percentage values of all bulbils that had germinated 173 days after the start of the experiment are given.
from freshwater. Again, this was independent of incubation salinity. These results indicate that high rates of sexual reproduction may have lost their adaptive value in the permanent habitat of the Baltic Sea where new sites easily can be colonized by bulbils or green plant parts. Though hibernation by means of oospores gives competitive advantage in shallow water before other submerged plants after severe ice winters (Idestam-Almqvist, 2000), oospore formation may be less important in this brackish environment, and allocation to bulbils may instead be more favourable. In freshwater, C. aspera is a typical pioneer plant characterized by rich oospore production and fast colonization of new habitats (Krause, 1997), including temporary habitats where oospores are more important for survival than in permanent habitats (Casanova, 1997). Also the wide geographic distribution range of this species, astonishing considering its dioecious nature (Proctor, 1980), indicates its high dispersal potential. In opposite to the observed ecophysiological differences between freshwater and brackish water plants (Blindow et al., 2003 and this study) neither our AFLP data nor our cross-
fertilization experiments show any genetic separation among these populations. Parthenogenesis has not been reported for C. aspera (Krause, 1997; Nielsen, 2003), nor was it found in our experiments. Before male and female plants were put together, the female plants had been incubated alone for 2 weeks to make sure that oogonia had not been fertilized before, i.e., a far longer time period than the 4–5 days assumed as sufficient by Grant and Proctor (1972). All oospores formed during our experiments are therefore assumed to originate from cross-fertilization. This lack of genetic isolation may be explained by the fact that not only the Baltic Sea has evolved rather recently, but also that the freshwater sites studied by us have been accessible for colonization first after the last glacial period. Young age of all sites and/or long transport distances of waterfowl (Claussen, 2002) may further explain the lack of distance-dependency in both genetic similarity and crossfertilization success. In opposite to our results, Croy (1982) found reproductive isolation in C. aspera collected from different freshwater sites in Spain, France, the Netherlands and the U.K., i.e., regions that were ice-free during the last glacial period.
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Like in other aquatic plants (Santamaria, 2002; Kaplan and Stepanek, 2003; Uehara et al., 2006), genetic similarities are high within sites compared to similarities among sites which indicates low gene flow among populations or clonal growth as the dominant mode of reproduction within sites (Santamaria, 2002). C. aspera may reproduce vegetatively both by bulbils and by fragmentation (Nielsen, 2003). Low gene flow among populations and especially between freshwater and brackish water should result in increasing genetic differentiation. As this differentiation has an adaptive component, local adaptation should make successful establishment of less adapted immigrants unlikely and further reduce the gene flow between these populations (Ouborg et al., 1999). This scenario of a speciation process seems to fit to C. aspera. Acknowledgements We thank Peter Hansson for diving assistance in Lake Stroacksen. The study was financially supported by the German Research Foundation (DF-G, project BL 559/3). References Ba¨ck, S., Collins, J.C., Russell, G., 1991. Aspects of the reproductive biology of Fucus vesiculosus from the coast of SW Finland. Ophelia 34, 129–141. Blindow, I., 1992. Decline of charophytes during eutrophication: comparison with angiosperms. Freshwater Biol. 28, 9–14. Blindow, I., 2000. Distribution of charophytes along the Swedish coast in relation to salinity and eutrophication. Int. Rev. Hydrobiol. 85, 707–717. Blindow, I., Dietrich, J., Mo¨llmann, N., Schubert, H., 2003. Growth, photosynthesis and fertility of Chara aspera under different light and salinity conditions. Aquat. Bot. 76, 213–234. Blindow, I., Schu¨tte, M., 2007. Density and morphology of Chara aspera under different light and salinity conditions. Hydrobiologia 584, 69–76. Boegle, M.G., Schneider, S., Mannschreck, B., Melzer, A., 2007. Differentiation of Chara intermedia and C. baltica compared to C. hispida based on morphology and amplified fragment length polymorphism. Hydrobiologia 586, 155–166. Casanova, M.T., 1997. Oospore variation in three species of Chara (Charales, Chlorophyta). Phycologia 36, 274–280. Clausen, P., Nolet, B.A., Fox, A.D., Klaassen, M., 2002. Long-distance endozoochorous dispersal of submerged macrophyte seeds by migratory waterbirds in northern Europe—a critical review of possibilities and limitations. Acta Oecol. 23, 191–203. Croy, C.D., 1982. Chara aspera (Charophyta). Breeding pattern in the northern hemisphere. Phycologia 21, 243–246. Figuerola, J., Green, A.J., 2002. Dispersal of aquatic organisms by waterbirds: a review of past research and priorities for future studies. Freshwater Biol. 47, 483–494. Garcia-Mas, J., Oliver, M., Go´mez-Paniagua, H., De Vicente, M.C., 2000. Comparing AFLP, RAPD and RFLP markers for measuring genetic diversity in melon. Theor. Appl. Genet. 101, 860–864. Grant, M.C., Proctor, V.W., 1972. Chara vulgaris and C. contraria: patterns of reproductive isolation for two cosmopolitan species complexes. Evolution 26, 267– 281. Hasslow, O.J., 1931. Sveriges charace´er. Botaniska Notiser, pp. 63–136. Hintze, J., 2001. NCSS and PASS. Number Cruncher Statistical Systems. Kaysville, Utah. www.NCSS.com. Idestam-Almqvist, J., 2000. Dynamics of submersed aquatic vegetation on shallow soft bottoms in the Baltic Sea. J. Veg. Sci. 11, 425–432. Kalvas, A., Kautsky, L., 1993. Geographical variation in Fucus vesiculosus morphology in the Baltic and North Sea. Eur. J. Phycol. 28, 85–91. Kaplan, Z., Stepanek, J., 2003. Genetic variation within and between populations of Potamogeton pusillus agg. Plant Syst. Evol. 239, 95–112.
Krause, W., 1997. Charales (Chlorophyceae). In: Ettl, H., Ga¨rtner, G., Heynig, H., Mollenhauer, D. (Eds.), Su¨ßwasserflora von Mitteleuropa, vol. 18. 202 pp. Mader, E., van Vierssen, W., Schwenk, K., 1998. Clonal diversity in the submerged macrophyte Potamogeton pectinatus L. inferred from nuclear and cytoplasmic variation. Aquat. Bot. 62, 147–160. Mannschreck, B., Fink, T., Melzer, A., 2002. Biosystematics of selected Chara species (Charophyta) using amplified fragment length polymorphism. Phycologia 41, 657–666. Mannschreck, B., 2003. Genetische und morphologische Differenzierung ausgewa¨hlter Arten der Gattung Chara. Ph.D. Thesis. Technische Universita¨t Mu¨nchen, Germany. Shaker Verlag. Aachen. Migula, W., 1897. Die Characeen. Rabenhorst’s Kryptogamen Flora von Deutsch¨ sterreich und der Schweiz. Band 5. Schweitzerbarth, 765 pp. 149 Abb. land, O Nielsen, R., 2003. Chara aspera. In: Schubert, H., Blindow, I. (Eds.), Charophytes of the Baltic Sea. The Baltic Marine Biologists Publication No. 19. Gantner Verlag, Ruggell, pp. 42–52. Olsen, S., 1944. Danish Charophyta. Det Kongelige Danske Videnskabernes Selskab. Biologiske Skrifter, Bind III, Nr. 1. Copenhagen. O’Reilly, C.L., Cowan, R.S., Hawkins, J.A., 2007. Amplified fragment length polymorphism genetic fingerprinting challenges the taxonomic status of the nearendemic Chara curta Nolte ex. Ku¨tz. (Characeae). Bot. J. Linn. Soc. 155, 467–476. Ouborg, N.J., Piquot, Y., van Groenendael, J.M., 1999. Population genetics, molecular markers and the study of dispersal in plants. J. Ecol. 87, 551–568. Proctor, V.W., 1962. Viability of Chara oospores taken from migratory birds. Ecology 43, 528–529. Proctor, V.W., 1971a. Taxonomic significance of monoecism and dioecism in the genus Chara. Phycologia 10, 299–308. Proctor, V.W., 1971b. Chara globularis Thuillier (=C. fragilis Desvaux): breeding patterns within a cosmopolitan complex. Limnol. Oceanogr. 16, 422–436. Proctor, V.W., 1975. The nature of charophyte species. Phycologia 14, 97–113. Proctor, V.W., 1980. Historical biogeography of Chara (Charophyta): an appraisal of the Braun-Wood classification plus a falsifiable alternative for future consideration. J. Phycol. 16, 218–233. Rietema, H., 1991. Evidence for ecotypic divergence between Phycodrus rubens populations from the Baltic Sea and North Sea. Bot. Mar. 34, 375–381. Rietema, H., 1993. Ecotypic differences between Baltic and North Sea populations of Delesseria sanguinea and Membranoptera alata. Bot. Mar. 36, 15–21. Rietema, H., 1995. Ecoclinal variation in Rhodomela confervoides along a salinity gradient in the North Sea and Baltic Sea. Bot. Mar. 38, 475–479. Russell, G., 1985a. Some anatomical and physiological differences in Chorda filum from coastal waters of Finland and Great Britain. J. Mar. Biol. Assoc. UK 65, 343– 349. Russell, G., 1985b. Recent evolutionary changes in the algae of the Baltic Sea. Br. Phycol. J. 20, 87–104. Russell, G., 1988. The seaweed flora of a young and semi-enclosed sea: the Baltic. Salinity as a possible agent of flora divergence. Helgola¨nder Meeresuntersuchungen 42, 243–250. Russell, G., 1994. A variant of Pilayalla littoralis (Algae, Fucophyceae). Ann. Bot. Fenn. 31, 127–138. Russell, G., Thomas, D.N., 1988. The Baltic Sea: a cradle of plant evolution? Plants Today 1988 (May–June), 77–82. Santamaria, L., 2002. Why are most aquatic plants widely distributed? Dispersal, clonal growth and small-scale heterogeneity in a stressful environment. Acta Oecol. 23, 137–154. Thomas, D.N., Collins, J.C., Russell, G., 1990. Interpopulation differences in the salt tolerance of two Cladophora species. Estuar. Coast. Shelf S. 30, 201–206. Uehara, K., Tanaka, N., Momohara, A., Zhou, Z.-K., 2006. Genetic diversity of an endangered aquatic plant, Potamogeton lucens subspecies sinicus. Aquat. Bot. 85, 350–354. van den Berg, M.S., Coops, H., Simons, J., de Keizer, A., 1998. Competition between Chara aspera and Potamogeton pectinatus as a function of temperature and light. Aquat. Bot. 60, 241–250. Vos, P., Hogers, R., Blecker, M., Reijans, M., Van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., Zabeau, M., 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23, 4407–4414. Wood, R.D., 1962. New combinations and taxa in the revision of Characeae. Taxon 11, 7–25. Wood, R.D., 1965. Monograph of the Characeae. In: Wood, R.D., Imahori, K. (Eds.), A Revision of the Characeae, vol. 1. J. Cramer, Weinheim.
Contents lists available at ScienceDirect
Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Reproductive isolation in Chara aspera populations Irmgard Blindow a,*, Nils Mo¨llmann a, Michael G. Boegle b, Manuela Schu¨tte a a b
Biological Station of Hiddensee, University of Greifswald, Biologenweg 15, D-18 565 Kloster, Germany Limnologische Station der Technischen Universita¨t Mu¨nchen, Hofmark 3, D-82 393 Iffeldorf, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 January 2009 Received in revised form 23 June 2009 Accepted 24 June 2009 Available online 1 July 2009
Possible reproductive isolation between freshwater and brackish water populations of the dioecious charophyte Chara aspera was studied by means of cross-fertilization experiments and AFLP (Amplified Fragment Length Polymorphism). Three Swedish freshwater populations and three (German and Swedish) Baltic Sea populations of C. aspera were sampled. Cross-fertilization experiments were performed in a full combination setup of all populations and with two different salinities (0 and 10 PSU). Both freshwater and brackish water females formed about 70% more gametangia at 0 than at 10 PSU. Male individuals collected from freshwater had higher fertility than brackish water males at both salinities. 57% of all gametangia of females from freshwater developed into oospores compared to only 8% of gametangia of brackish water females. 42% of all oospores were fertilized in crosses between habitats (freshwater–brackish water) compared to 36% in crosses within habitats, the difference was not significant. Oospore and bulbil germination was investigated using propagules from freshwater and brackish water populations and incubation salinities of 0, 5, 10 and 20 PSU. None of the oospores collected from brackish water germinated. Germination of oospores and bulbils from freshwater was higher at 0 and 5 PSU than at higher salinities. Only around 40% of bulbils from brackish water germinated at 20 PSU compared to around 70% at the other three salinities. Germination of all bulbils was delayed at 20 PSU compared to other salinities. Genetic similarities (Jaccard indices of AFLP data) were higher within than between populations, but comparisons within habitat (freshwater–freshwater and brackish water–brackish water) were not different from comparisons between habitats. Our results did not identify any reproductive isolation between freshwater and brackish water populations, but indicate low gene flow between the two habitats. Oospore and bulbil germination success were highest at salinities corresponding to the conditions of their original habitat, suggesting genetic adaptation to their environmental conditions and indicating that propagules transported from freshwater to brackish water or vice versa will hardly develop into fertile plants. Additionally, brackish water plants perform poorer in all aspects of sexual reproduction than freshwater plants. Possibly, successful dispersal of oospores is not subjected to high selective pressure within the Baltic Sea where new sites easily can be colonized by means of vegetative reproduction. We assume that these adaptations will favour speciation within C. aspera and support the idea of the geologically young Baltic Sea as a ‘‘cradle of plant evolution’’. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Salinity Baltic Sea Cross-fertilization Oospore Dispersal Evolution AFLP
1. Introduction The taxonomy of charophytes has been subject to several revisions and much debate. High variability within single taxa as well as small morphological differences among closely related taxa prevent the identification of clearly separated species, and different authors either combined similar taxa into the same
* Corresponding author. Tel.: +49 38300 50251; fax: +49 38300 60672. E-mail address: [email protected] (I. Blindow). 0304-3770/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2009.06.009
species (Wood, 1962, 1965) or segregated them into separate species (Migula, 1897; Krause, 1997). Beyond the species level, a number of subspecies, varieties and forms have been described (e.g., Migula, 1897). While determination keys discriminate between morphologically similar species such as Chara intermedia and C. baltica, other species are characterized by high intraspecific morphological variability (Krause, 1997; Boegle et al., 2007). Cross-fertilization experiments are one way to investigate if the different ‘‘species’’ described in monographs and determination keys, and if the characters used for determination, reflect genetic isolation among groups. Such experiments have mainly been
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148320 E), both situated in Scania, and Redensee Bay (548220 N, 128350 E) in the Darß-Zingster bodden chain, Germany. The four Scanian sites were sampled during mid June, Lake Storacksen and Redensee Bay during end August. After collection, the plants were transported to the laboratory in plastic bags and stored at 4 8C until further treatment (cross-fertilization experiments and genetic analyses) within a few days.
performed in different taxa within the genus of Chara and resulted into successful crosses between different ‘‘species’’ (Proctor, 1971a, 1975), but also the identification of reproductive isolation among populations belonging to the same ‘‘species’’ (Proctor, 1971b; Grant and Proctor, 1972; Croy, 1982). The criteria used for species determination are not always the same as the morphological features that are under selective pressure in the evolution of a species (Proctor, 1980). Genetic techniques are another method to compare morphologically separate taxa with genetically isolated groups. Among these techniques, AFLP has been suggested to be most suitable to identify polymorphism within and among closely related taxa (Garcia-Mas et al., 2000) and has been successfully applied to various charophyte taxa (Mannschreck, 2003; Mannschreck et al., 2002). Like cross-fertilization experiments, these investigations identified high genetic distances between morphologically similar plants (Boegle et al., 2007), but also small genetic differences between morphologically distinct taxa (O’Reilly et al., 2007). Chara aspera Willd. is distributed all over the northern hemisphere (Croy, 1982; Nielsen, 2003). It occurs in both freshwater and brackish water and inhabits a wide range of different habitats, both permanent and temporary (Hasslow, 1931; Olsen, 1944; Krause, 1997). Typical characters are dioecism, triplostichous cortex, acute stipulodes and single-celled, circular bulbils (Krause, 1997), but high morphological variability exists within the ‘‘species’’, mainly concerning the spine characters (Krause, 1997; Nielsen, 2003). Several taxa have been described on a subspecies level such as C. aspera f. aspera with long, acute and single spines, C. aspera f. subinermis without spines or with small papilliformous spines and C. aspera var. curta with spines in bunches. Reproductive isolation has been shown between individuals originating from different continents as well as between individuals collected from the same continent (Croy, 1982). A certain level of genetic isolation can therefore be assumed within this species. The wide salinity range of this species from about 0 to 15 PSU (Hasslow, 1931; Olsen, 1944; Blindow, 2000) further suggests specific adaptations of single populations to their environment. Considerable morphological differences have been described between freshwater and brackish water plants. While brackish water plants have a ‘‘stunted’’ growth and grow typically in single tussocks (Olsen, 1944), freshwater plants use to be thinner and often form a dense vegetation ‘‘mat’’ (Blindow, 1992; van den Berg et al., 1998). To investigate the level of possible speciation within this variable taxon, we studied ecophysiological (Blindow et al., 2003) as well as morphological characteristics (Blindow and Schu¨tte, 2007) of freshwater and Baltic Sea populations of C. aspera. The aim of this study was to identify possible reproductive isolation between these populations by means of cross-fertilization experiments, genetic analyses, and investigations of salinity effects on fertility, fertilization success and germination of oospores and bulbils.
Under a stereomicroscope, the plants were separated into sterile plants, which were planted into beakers for further use, and fertile plants which were used for cross-fertilization. For this experiment, five replicates were prepared for each of two different salinity treatments and each combination of male and female plants from the six sites including within-population crosses. In total, the setup should have corresponded to 5 2 6 6 = 360 beakers. Due to lack of fertile plants, however, no treatments with male plants from Lake Storacksen, and only some treatments with male plants from Redensee Bay could be prepared. Therefore, the setup consisted of only 280 beakers in total. Each replicate consisted of one 400 mL beaker containing autoclavated sediment (1 cm height) and GF/C-filtered water from one of the sites (Lake Krankesjo¨n) with (10 PSU treatment) or without (0 PSU treatment) addition of salts, respectively, corresponding to the composition of natural sea water (see Blindow et al., 2003). In each beaker, three fertile female plants (whole plants or several upper whirls) were planted and incubated indoors at room temperature. Plants collected from freshwater were gradually adapted to the 10 PSU treatments by incubating them at 5 PSU for several days, and then filling the beakers to a final concentration of 10 PSU. After 2 weeks, all plants were examined, and oospores which had eventually been formed were removed. Thereafter, three fertile male plants were added to the beaker. The cross-fertilization experiment was run outdoors. All beakers were covered with a commercial mosquito net which reduced the natural irradiance (around 60 mol m2 day1 on a sunny summer day) by 50%, a light intensity resulting in higher growth rates and higher fertility according to former results (Blindow et al., 2003). All beakers were checked regularly, and dead male plants eventually replaced. On 15 October, the beakers were taken indoors and incubated at 12:12 h L:D and about 10% of natural irradiance. At the end of October, all plants were removed. Male plants were checked for vitality and fertility (no. of antheridia). For all female plants, young oogonia, aborted oogonia and oospores were counted. Parthenogenesis has not been described for C. aspera, but to exclude this possibility, 30 additional beakers containing three female plants each collected from Ho¨llviken and Edenryd were incubated without male plants at 0 PSU (15 beakers) and 10 PSU (15 beakers), respectively. The plants were checked on 11 November for numbers of oogonia, aborted oogonia, and oospores.
2. Materials and methods
2.3. Oospore and bulbil germination
2.1. Plant collection
Oospores were collected from the upper sediment layer (0– 10 cm) in two of the freshwater sites (Lakes Krankesjo¨n and Bo¨rringesjo¨n) and two of the brackish water sites (Ho¨llviken and Edenryd Bays) within C. aspera vegetation during June 2002. Bulbils were collected directly from the rhizoids of the plants between end of July and end of August 2002 within dense vegetation of C. aspera. Both oospores and bulbils were transported to the laboratory and stored for several weeks at 4 8C in the dark. For the germination experiments, 20 oospores or bulbils, respectively, were placed into 400 mL beakers containing 1 cm of autoclavated sediment and covered with another 1 cm of
Plants of C. aspera were collected from three freshwater and three brackish water sites between mid June and end of August of 2002. Lake Storacksen (608560 N, 158120 E), province of Dalarna (middle Sweden), is a calcium-rich, oligotrophic lake. Lake Krankesjo¨n (558420 N, 138290 E), province of Scania, south Sweden, is a calcium-rich, meso- to eutrophic lake. Lake Bo¨rringesjo¨n (558290 N, 138200 E), Scania, is highly eutrophic with algal blooms throughout the vegetation period. The brackish water sites were Ho¨llviken Bay (558250 N, 128560 E) and Edenryd Bay (568020 N,
2.2. Cross-fertilization
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autoclavated sediment. The beakers were filled with GF/C-filtered water with (5, 10 and 20 PSU treatments) or without (0 PSU treatment) addition of salts, respectively, corresponding to the composition of natural sea water (see Blindow et al., 2003). Both sediment and water were taken from Lake Krankesjo¨n. The beakers were incubated in the laboratory at room temperature and natural light. The oospore germination experiment started during August 2002, the bulbil germination experiments during September 2002. All beakers were checked six times during the experiments for germlings. Both experiments were terminated during March 2003.
2.5. Data analysis
2.4. Genetic analyses
The female plants which were incubated in absence of male plants (parthenogenesis experiment) formed in total 11 (0 PSU) and 18 (10 PSU) oogonia, all of which were aborted. Female plants collected from freshwater had more gametangia at the end of the experiment than females collected from brackish water, the difference, however, was not significant (p = 0.191, Mann–Whitney). Both freshwater and brackish water females formed more gametangia at 0 than at 10 PSU (p = 0.015 and 0.022, respectively, Mann–Whitney, Fig. 1A). Male individuals collected from freshwater were more fertile than individuals collected from brackish water (p = 0.005, Mann– Whitney). While males originating from freshwater had a somewhat higher fertility at 0 compared to 10 PSU, brackish water males had a higher fertility at 10 compared to 0 PSU (Fig. 1B). The differences between salinities were not significant, however (p = 0.31 and 0.25, respectively, Mann–Whitney). The numbers of oospores did not differ between beakers with fertile male plants and beakers where the male plants were not fertile any more at the end of the experiment (p = 0.75, Mann– Whitney), or beakers with male plants originating from freshwater and males originating from brackish water (p = 0.72, Mann– Whitney). The oospore numbers were higher in beakers with female plants originating from freshwater than in beakers with females collected from brackish water (p = 0.00004, Mann– Whitney), and higher in beakers incubated at 10 PSU compared to beakers incubated at 0 PSU (p = 0.035, Mann–Whitney, Fig. 2). Only 8% of all gametangia of female plants originating from brackish water were fertilized and developed into oospores compared to 57% in females originating from freshwater. As an overall mean, 36% of all female gametangia from withinhabitat crosses (brackish–brackish or freshwater–freshwater, respectively) developed into oospores compared to 42% for between-habitat crosses. The difference was not significant (p = 0.98, Mann–Whitney). The percentage of gametangia which developed into oospores was not correlated with the distances between sites (p = 0.71, linear regression, Fig. 3). None of the oospores collected from brackish water germinated. Germination of oospores collected from the freshwater sites Lakes Krankesjo¨n and Bo¨rringesjo¨n was significantly affected by salinity (p = 0.010, Kruskal–Wallis). Only few oospores germinated at 20 PSU (Fig. 4), and the germination success at this salinity was lower compared to 0 and 5 PSU, respectively (p < 0.05, Kruskal– Wallis Multiple Comparison Z-value Test). Germination percentages were lower at 10 PSU compared to 0 and 5 PSU in both lakes (Fig. 4), but the differences were not significant (p > 0.05, Kruskal– Wallis Multiple Comparison Z-value Test). The effects of both origin (freshwater–brackish water) and salinity on bulbil germination were significant as was the interaction effect of both factors (p < 0.05, two-way ANOVA). In bulbils collected from freshwater, germination success at 10 PSU was lower than at 0 PSU, and germination success at 20 PSU significantly lower than at the other three salinity levels (p < 0.05, Kruskal–Wallis Multiple Comparison Z-value Test). For 5, 10 and 20 PSU, germination success was lower compared to germination success of bulbils collected from brackish water and cultivated at
After morphologic separation and removal of epiphytes in an ultrasonic bath the charophytes were stored at 20 8C. Genomic DNA of each individual was isolated separately from 40 mg of freezedried, powdered plant material using the DNeasy Plant Mini Kit (Quiagen, Hilden, Germany). The quality and quantity of DNA was determined on 1% agarose gel electrophoresis on 50 V in 30 min. The AFLP procedure was performed according to Vos et al. (1995), with slight modifications suggested by Mannschreck et al. (2002), for detailed description see Boegle et al. (2007). The DNA was digested with EcoRI and MseI, and simultaneously adapters were ligated (for sequences of adapters and primers see Mannschreck et al., 2002). The digested ligated DNA was diluted according to estimated DNA content with 40–140 mL H2O for subsequent preamplification steps. For preamplification primers were used complementary to the Mse1 and EcoR1 adaptors, the recognition site and possessing one additional nucleotide at the 30 primer end (for primer description and preamplification protocol see Mannschreck et al., 2002). After preamplification the quality and quantity of 10 mL preselective amplification product was determined on 1% agarose gel. The remaining 10 mL product was diluted according to estimated preselective amplification product with 40–140 mL H2O. Selective amplification was carried out with EcoRI and MseI primers having three additional nucleotides. For fragment analysis, EcoRI primers with the selective nucleotides ACA or ACG were labelled with a 5-FAM or JOE fluorochrome respectively, the MseI primers were used unlabelled. Preparation of samples and polyacrylamid gel electrophoresis was based on Mannschreck et al. (2002) with the exception that instead of 2 mL only 1.8 mL of PCR product was used. Semi-automated AFLP fragment analysis was performed with GenotyperTM DNA fragment analysis software version 2.1 (Applied Biosystem, Weiterstadt, Germany). To combine the information for a sample, each primer pair was linked into a single file according to the following protocol. Due to differing intensities of the primer combinations used, scale factors and peak levels were optimised to the sum of signal. The height of each ACA/CGA and ACA/CGC signal was divided by 300,000, each ACG/CGA signal was divided by 250,000, and each ACG/CGC signal by 150,000. Peaks were labelled if rescaled height was >150 for ACA fragments between 100 and 460 base pairs (bp), >100 for ACG fragments between 100 and 460 bp, and >50 for fragments between 461 and 500 bp for all primer combinations. The range between 100 and 500 bp was divided into 267 segments, each being 1.5 bp wide. Within each segment, labelled peaks were scored as one and the absence of a labelled peak was counted as 0, thus generating a presence/ absence matrix. Presence/absence matrices of each primer pair were combined to a single file per sample. The AFLP procedure was carried out twice for each sample. In the data analysis, only individuals with both AFLP procedures resulting in pairwise samples within an unweighted pairgroup method using arithmetic averages (UPGMA) phenogram were used for further analyses (not shown).
All data analyses were performed using NCSS 2001 (Hintze, 2001). Nonparametric methods (Kruskal–Wallis and nonparametric post hoc tests) were applied when homoscedasticity of the data material was not achieved even after transformation. Twoway ANOVA was applied on log-transformed data followed by Tukey post hoc tests. 3. Results
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Fig. 1. (A) Female fertility (no. of gametangia beaker1) and (B) male fertility (% of beakers with fertile plants) of Chara aspera collected from freshwater (white columns) and C. aspera collected from brackish water (hatched columns) after incubation at 0 and 10 PSU, respectively, at the end of the experiment. Mean values and SE are given. In (B), each crossing combination (five beakers) was treated as one replicate. Different letters indicate significant (Kruskal–Wallis Multiple Comparison Z-value Test, p < 0.05) differences between groups.
the corresponding salinities (p < 0.05, Tukey–Kramer Multiple Comparison Test). There were no significant differences in germination success of bulbils collected from brackish water at 0, 5 and 10 PSU (p > 0.05, Tukey–Kramer Multiple Comparison Test), but germination success at 20 PSU was significantly lower compared to the other three salinities (p < 0.05, Tukey–Kramer Multiple Comparison Test; Fig. 5). Neither for bulbils collected from the three freshwater sites nor for bulbils collected from the three brackish water sites, there was a significant difference in germination success among the single sites (p > 0.05, Kruskal–Wallis). Apart from lower germination success, germination of bulbils collected from freshwater was delayed at higher salinities. While 41% of all bulbils that germinated at 0 PSU had germinated already after 19 days of incubation, only 16% had germinated at 5 PSU and 12% at 10 PSU. The first germination at 20 PSU was recorded after 89 days (Fig. 6A). For bulbils collected in brackish water, 33% of all bulbils that germinated at 0 PSU had germinated after 19 days of incubation compared to 56% at 5 PSU and 46% at 10 PSU. The first germination at 20 PSU was recorded after 35 days (Fig. 6B). Both origin (freshwater–brackish water) and culture salinity had a
Fig. 2. Cross-fertilization success (no. of oospores beaker1) for all combinations of male and female C. aspera at 0 PSU (white columns) and 10 PSU (dotted columns), respectively. Mean values and SE are given. FF = freshwater female freshwater male. FB = freshwater female brackish water male. BF = brackish water female brackish water male. BB = brackish water female brackish water male. Different letters indicate significant (Kruskal–Wallis Multiple Comparison Z-value Test, p < 0.05) differences between groups.
Fig. 3. Oospore formation (% of gametangia, filled squares) and Jaccard similarities of AFLP-data (open squares) depending on geographic distances among sites.
significant effect on the germination percentage (of total germination) during day 19, and also the interaction effect between both factors was significant (p < 0.05, two-way ANOVA). Germination percentages (of total germination) during day 19 were lower for bulbils incubated at 20 PSU compared with bulbils
Fig. 4. Oospore germination (%) for C. aspera collected from L. Krankesjo¨n (Kra) and L. Bo¨rringesjo¨n (Bo¨r) at different salinities. Mean values and SE are given.
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Fig. 5. Bulbil germination (%) for C. aspera collected at different sites and salinities. Site abbreviations: Kra = Lake Krankesjo¨n, Bo¨r = Lake Bo¨rringesjo¨n, Sto = Lake Storacksen, Ho¨l = Ho¨llviken Bay, Red = Redensee Bay. Mean values and SE are given.
incubated at the other three salinities, and were lower for bulbils collected from freshwater sites incubated at 5 and 10 PSU than for bulbils collected from brackish water sites incubated at corresponding salinities (p < 0.05, Tukey–Kramer Multiple Comparison Test). Genetic similarities (Jaccard indices of AFLP-data) were higher within populations than between populations (p = 0.0014, twotailed t-test). There was no significant difference in Jaccard similarities between comparisons within habitats (freshwater– freshwater and brackish water–brackish water) and comparisons between habitats (freshwater–brackish water; p = 0.1694, twotailed t-test) (Table 1). Jaccard similarities were not correlated with the geographic distances between sites (p = 0.33, Fig. 3). 4. Discussion Despite of the fact that the brackish environment of the Baltic Sea has existed for only around 11,000 years, i.e., a very short time period from a geologic and evolutionary point of view, and despite the rapid salinity changes which have taken place within this period, a number of algal species which have colonized the Baltic Sea from either freshwater or marine habitats have already developed varieties different from their original populations. In some cases, these varieties show morphological or other physiological differences compared to their original populations, and in every case, they adapted to the environmental conditions of the Baltic Sea and are characterized by salinity optima which are within the brackish range and different from the original populations (Russell, 1985a,b, 1988, 1994; Thomas et al., 1990; Ba¨ck et al., 1991; Rietema, 1991, 1993, 1995; Kalvas and Kautsky, 1993). The Baltic Sea therefore has been assumed to be a spot of rapid evolution (Russell and Thomas, 1988).
C. aspera fits into this pattern. While morphological differences between freshwater and brackish water populations which have been described by Olsen (1944) can be explained by salinity effects rather than genetic differences (Blindow and Schu¨tte, 2007), our results suggest that C. aspera has developed a specific ecotype in the Baltic Sea which is adapted to the salinities of this environment. Even after longer adaptation periods, plants collected from the Baltic Sea have salinity optima at 5 to 10 PSU, freshwater plants at 0 to 5 PSU. This was found for photosynthesis, weight gain, and overall fertility (Blindow et al., 2003) as well as bulbil germination and (freshwater plants) oospore germination (this study). Apart from low bulbil germination success, the germination is delayed at salinities higher than the salinity range of the habitat where the plants were collected from. Waterfowl is assumed to be an important vector for gene flow among sites in charophytes as well as other submerged macrophytes, and sexual propagules of a number of submerged plants including charophytes tolerate gut passages or show even enhanced germination success after gut passage (Proctor, 1962; Clausen et al., 2002; Figuerola and Green, 2002). The amount of this transport is rather unknown, though high genetic within-site variabilities and low genetic between-site differences among waterfowl migration routes suggest that this transport may be substantial (Mader et al., 1998). Wigeon and pintail are two common duck species which switch between brackish water and freshwater habitats during migration and feed on submerged plants in brackish water (Clausen et al., 2002). In south Sweden, tufted ducks perform diurnal migration between freshwater and brackish water habitats (L. Nilsson, Department of Animal Ecology, University of Lund, personal communication). Therefore, it cannot be excluded that substantial oospore transport occurs between freshwater and brackish water. Nevertheless, we assume that gene flow between freshwater and brackish water is low in C. aspera. Oospores transported from freshwater to brackish water have low germination success. The few oospores which may manage to germinate will develop into plants that are not adapted to the salinity conditions of the Baltic Sea (Blindow et al., 2003), and will therefore be subject to strong inter- as well as intraspecific competition. Even if these plants grow up, low fertility and low bulbil germination success combined with a retarded bulbil germination will result in low reproduction success. The same ‘‘misfortune’’ is assumed to happen to oospores transported from brackish water to freshwater. Transport of oospores from brackish water to freshwater may be further reduced by an assumed overall lower sexual reproduction success of brackish water populations. In our experiment, substantially fewer oospores were formed by brackish water females as a result of lower male fertility, somewhat lower female fertility and a lower percentage of oogonia which developed into oospores, independent of the origin and fertility of the male partner and the experimental salinity condition. Though C. aspera germlings developed from oospores were found both in Ho¨llviken and Edenryd Bays (own observations), no oospores collected from brackish water sites germinated in opposite to oospores collected
Table 1 Genetic similarities (Jaccard indices) of Chara aspera from different sites according to AFLP-data. F = freshwater. B = brackish water. Within-population similarities are given in bold numbers. Jaccard indices
Freshwater Krankesjo¨n
Freshwater Bo¨rringesjo¨n
Freshwater Storacksen
Brackish Ho¨llviken
Brackish Redensee
F: Krankesjo¨n F: Bo¨rringesjo¨n F: Storacksen B: Ho¨llviken B: Redensee
35.8
23.1 62.8
22.5 27.1 35.4
23.6 39.5 27.7 49.3
24.6 34.7 26.6 38.0 52.6
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Fig. 6. Development of germination over time for bulbils collected from freshwater sites (A) and brackish water sites (B) incubated at different salinities. Percentage values of all bulbils that had germinated 173 days after the start of the experiment are given.
from freshwater. Again, this was independent of incubation salinity. These results indicate that high rates of sexual reproduction may have lost their adaptive value in the permanent habitat of the Baltic Sea where new sites easily can be colonized by bulbils or green plant parts. Though hibernation by means of oospores gives competitive advantage in shallow water before other submerged plants after severe ice winters (Idestam-Almqvist, 2000), oospore formation may be less important in this brackish environment, and allocation to bulbils may instead be more favourable. In freshwater, C. aspera is a typical pioneer plant characterized by rich oospore production and fast colonization of new habitats (Krause, 1997), including temporary habitats where oospores are more important for survival than in permanent habitats (Casanova, 1997). Also the wide geographic distribution range of this species, astonishing considering its dioecious nature (Proctor, 1980), indicates its high dispersal potential. In opposite to the observed ecophysiological differences between freshwater and brackish water plants (Blindow et al., 2003 and this study) neither our AFLP data nor our cross-
fertilization experiments show any genetic separation among these populations. Parthenogenesis has not been reported for C. aspera (Krause, 1997; Nielsen, 2003), nor was it found in our experiments. Before male and female plants were put together, the female plants had been incubated alone for 2 weeks to make sure that oogonia had not been fertilized before, i.e., a far longer time period than the 4–5 days assumed as sufficient by Grant and Proctor (1972). All oospores formed during our experiments are therefore assumed to originate from cross-fertilization. This lack of genetic isolation may be explained by the fact that not only the Baltic Sea has evolved rather recently, but also that the freshwater sites studied by us have been accessible for colonization first after the last glacial period. Young age of all sites and/or long transport distances of waterfowl (Claussen, 2002) may further explain the lack of distance-dependency in both genetic similarity and crossfertilization success. In opposite to our results, Croy (1982) found reproductive isolation in C. aspera collected from different freshwater sites in Spain, France, the Netherlands and the U.K., i.e., regions that were ice-free during the last glacial period.
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Like in other aquatic plants (Santamaria, 2002; Kaplan and Stepanek, 2003; Uehara et al., 2006), genetic similarities are high within sites compared to similarities among sites which indicates low gene flow among populations or clonal growth as the dominant mode of reproduction within sites (Santamaria, 2002). C. aspera may reproduce vegetatively both by bulbils and by fragmentation (Nielsen, 2003). Low gene flow among populations and especially between freshwater and brackish water should result in increasing genetic differentiation. As this differentiation has an adaptive component, local adaptation should make successful establishment of less adapted immigrants unlikely and further reduce the gene flow between these populations (Ouborg et al., 1999). This scenario of a speciation process seems to fit to C. aspera. Acknowledgements We thank Peter Hansson for diving assistance in Lake Stroacksen. The study was financially supported by the German Research Foundation (DF-G, project BL 559/3). References Ba¨ck, S., Collins, J.C., Russell, G., 1991. Aspects of the reproductive biology of Fucus vesiculosus from the coast of SW Finland. Ophelia 34, 129–141. Blindow, I., 1992. Decline of charophytes during eutrophication: comparison with angiosperms. Freshwater Biol. 28, 9–14. Blindow, I., 2000. Distribution of charophytes along the Swedish coast in relation to salinity and eutrophication. Int. Rev. Hydrobiol. 85, 707–717. Blindow, I., Dietrich, J., Mo¨llmann, N., Schubert, H., 2003. Growth, photosynthesis and fertility of Chara aspera under different light and salinity conditions. Aquat. Bot. 76, 213–234. Blindow, I., Schu¨tte, M., 2007. Density and morphology of Chara aspera under different light and salinity conditions. Hydrobiologia 584, 69–76. Boegle, M.G., Schneider, S., Mannschreck, B., Melzer, A., 2007. Differentiation of Chara intermedia and C. baltica compared to C. hispida based on morphology and amplified fragment length polymorphism. Hydrobiologia 586, 155–166. Casanova, M.T., 1997. Oospore variation in three species of Chara (Charales, Chlorophyta). Phycologia 36, 274–280. Clausen, P., Nolet, B.A., Fox, A.D., Klaassen, M., 2002. Long-distance endozoochorous dispersal of submerged macrophyte seeds by migratory waterbirds in northern Europe—a critical review of possibilities and limitations. Acta Oecol. 23, 191–203. Croy, C.D., 1982. Chara aspera (Charophyta). Breeding pattern in the northern hemisphere. Phycologia 21, 243–246. Figuerola, J., Green, A.J., 2002. Dispersal of aquatic organisms by waterbirds: a review of past research and priorities for future studies. Freshwater Biol. 47, 483–494. Garcia-Mas, J., Oliver, M., Go´mez-Paniagua, H., De Vicente, M.C., 2000. Comparing AFLP, RAPD and RFLP markers for measuring genetic diversity in melon. Theor. Appl. Genet. 101, 860–864. Grant, M.C., Proctor, V.W., 1972. Chara vulgaris and C. contraria: patterns of reproductive isolation for two cosmopolitan species complexes. Evolution 26, 267– 281. Hasslow, O.J., 1931. Sveriges charace´er. Botaniska Notiser, pp. 63–136. Hintze, J., 2001. NCSS and PASS. Number Cruncher Statistical Systems. Kaysville, Utah. www.NCSS.com. Idestam-Almqvist, J., 2000. Dynamics of submersed aquatic vegetation on shallow soft bottoms in the Baltic Sea. J. Veg. Sci. 11, 425–432. Kalvas, A., Kautsky, L., 1993. Geographical variation in Fucus vesiculosus morphology in the Baltic and North Sea. Eur. J. Phycol. 28, 85–91. Kaplan, Z., Stepanek, J., 2003. Genetic variation within and between populations of Potamogeton pusillus agg. Plant Syst. Evol. 239, 95–112.
Krause, W., 1997. Charales (Chlorophyceae). In: Ettl, H., Ga¨rtner, G., Heynig, H., Mollenhauer, D. (Eds.), Su¨ßwasserflora von Mitteleuropa, vol. 18. 202 pp. Mader, E., van Vierssen, W., Schwenk, K., 1998. Clonal diversity in the submerged macrophyte Potamogeton pectinatus L. inferred from nuclear and cytoplasmic variation. Aquat. Bot. 62, 147–160. Mannschreck, B., Fink, T., Melzer, A., 2002. Biosystematics of selected Chara species (Charophyta) using amplified fragment length polymorphism. Phycologia 41, 657–666. Mannschreck, B., 2003. Genetische und morphologische Differenzierung ausgewa¨hlter Arten der Gattung Chara. Ph.D. Thesis. Technische Universita¨t Mu¨nchen, Germany. Shaker Verlag. Aachen. Migula, W., 1897. Die Characeen. Rabenhorst’s Kryptogamen Flora von Deutsch¨ sterreich und der Schweiz. Band 5. Schweitzerbarth, 765 pp. 149 Abb. land, O Nielsen, R., 2003. Chara aspera. In: Schubert, H., Blindow, I. (Eds.), Charophytes of the Baltic Sea. The Baltic Marine Biologists Publication No. 19. Gantner Verlag, Ruggell, pp. 42–52. Olsen, S., 1944. Danish Charophyta. Det Kongelige Danske Videnskabernes Selskab. Biologiske Skrifter, Bind III, Nr. 1. Copenhagen. O’Reilly, C.L., Cowan, R.S., Hawkins, J.A., 2007. Amplified fragment length polymorphism genetic fingerprinting challenges the taxonomic status of the nearendemic Chara curta Nolte ex. Ku¨tz. (Characeae). Bot. J. Linn. Soc. 155, 467–476. Ouborg, N.J., Piquot, Y., van Groenendael, J.M., 1999. Population genetics, molecular markers and the study of dispersal in plants. J. Ecol. 87, 551–568. Proctor, V.W., 1962. Viability of Chara oospores taken from migratory birds. Ecology 43, 528–529. Proctor, V.W., 1971a. Taxonomic significance of monoecism and dioecism in the genus Chara. Phycologia 10, 299–308. Proctor, V.W., 1971b. Chara globularis Thuillier (=C. fragilis Desvaux): breeding patterns within a cosmopolitan complex. Limnol. Oceanogr. 16, 422–436. Proctor, V.W., 1975. The nature of charophyte species. Phycologia 14, 97–113. Proctor, V.W., 1980. Historical biogeography of Chara (Charophyta): an appraisal of the Braun-Wood classification plus a falsifiable alternative for future consideration. J. Phycol. 16, 218–233. Rietema, H., 1991. Evidence for ecotypic divergence between Phycodrus rubens populations from the Baltic Sea and North Sea. Bot. Mar. 34, 375–381. Rietema, H., 1993. Ecotypic differences between Baltic and North Sea populations of Delesseria sanguinea and Membranoptera alata. Bot. Mar. 36, 15–21. Rietema, H., 1995. Ecoclinal variation in Rhodomela confervoides along a salinity gradient in the North Sea and Baltic Sea. Bot. Mar. 38, 475–479. Russell, G., 1985a. Some anatomical and physiological differences in Chorda filum from coastal waters of Finland and Great Britain. J. Mar. Biol. Assoc. UK 65, 343– 349. Russell, G., 1985b. Recent evolutionary changes in the algae of the Baltic Sea. Br. Phycol. J. 20, 87–104. Russell, G., 1988. The seaweed flora of a young and semi-enclosed sea: the Baltic. Salinity as a possible agent of flora divergence. Helgola¨nder Meeresuntersuchungen 42, 243–250. Russell, G., 1994. A variant of Pilayalla littoralis (Algae, Fucophyceae). Ann. Bot. Fenn. 31, 127–138. Russell, G., Thomas, D.N., 1988. The Baltic Sea: a cradle of plant evolution? Plants Today 1988 (May–June), 77–82. Santamaria, L., 2002. Why are most aquatic plants widely distributed? Dispersal, clonal growth and small-scale heterogeneity in a stressful environment. Acta Oecol. 23, 137–154. Thomas, D.N., Collins, J.C., Russell, G., 1990. Interpopulation differences in the salt tolerance of two Cladophora species. Estuar. Coast. Shelf S. 30, 201–206. Uehara, K., Tanaka, N., Momohara, A., Zhou, Z.-K., 2006. Genetic diversity of an endangered aquatic plant, Potamogeton lucens subspecies sinicus. Aquat. Bot. 85, 350–354. van den Berg, M.S., Coops, H., Simons, J., de Keizer, A., 1998. Competition between Chara aspera and Potamogeton pectinatus as a function of temperature and light. Aquat. Bot. 60, 241–250. Vos, P., Hogers, R., Blecker, M., Reijans, M., Van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., Zabeau, M., 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23, 4407–4414. Wood, R.D., 1962. New combinations and taxa in the revision of Characeae. Taxon 11, 7–25. Wood, R.D., 1965. Monograph of the Characeae. In: Wood, R.D., Imahori, K. (Eds.), A Revision of the Characeae, vol. 1. J. Cramer, Weinheim.