Phenotypic variation in sexually and asexually recruited individuals of the Baltic Sea endemic macroalga Fucus radicans: in the field and after growth in a common-garden.

BACKGROUND: Most species of brown macroalgae recruit exclusively sexually. However, Fucus radicans, a dominant species in the northern Baltic Sea, recruits new attached thalli both sexually and asexually. The level of asexual recruitment varies among populations from complete sexual recruitment to almost (> 90%) monoclonal populations. If phenotypic traits have substantial inherited variation, low levels of sexual activity will decrease population variation in these traits, which may affect function and resilience of the species. We assessed the level of inherited variation in nine phenotypic traits by comparing variation within and among three monoclonal groups and one group of unique multilocus genotypes (MLGs) sampled in the wild.
RESULTS: Of the nine phenotypic traits, recovery after freezing, recovery after desiccation, and phlorotannin content showed substantial inherited variation, that is, phenotypic variation in these traits were to a large extend genetically determined. In contrast, variation in six other phenotypic traits (growth rate, palatability to isopod grazers, thallus width, distance between dichotomies, water content after desiccation and photochemical yield under ambient conditions) did not show significant signals of genetic variation at the power of analyses used in the study. Averaged over all nine traits, phenotypic variation within monoclonal groups was only 68% of the variation within the group of different MLGs showing that genotype diversity does affect the overall level of phenotypic variation in this species.
CONCLUSIONS: Our result indicates that, in general, phenotypic diversity in populations of Fucus radicans increases with increased multilocus genotype (MLG) diversity, but effects are specific for individual traits. In the light of Fucus radicans being a foundation species of the northern Baltic Sea, we propose that increased MLG diversity (leading to increased trait variation) will promote ecosystem function and resilience in areas where F. radicans is common, but this suggestion needs experimental support.

Background

In most ecosystems, increased species richness tends to support ecosystem function [1-3], but in ecosystems dominated by one or a few foundation species - such as seagrass meadows, seaweed belts, and stands of forest trees - species diversity may be less important while instead phenotypic diversity within the foundation species is likely to affect properties of the ecosystem as a whole, such as function and resilience [4]. For example, a population of a habitat-forming species in which the individuals are phenotypically different will create a more complex habitat than one in which phenotypes are all alike, and most likely the more complex habitat will attract more associated species [5]. Many foundation species both in terrestrial and aquatic habitats form new individuals by either sexual or asexual reproduction, and populations formed by one or a few different clones will provide less phenotypic variation than multi-clonal populations, whenever phenotypic traits are largely explained by genetic variation.

Experimental studies with plant species of mixed sexual and asexual recruitment have shown that, for example, primary production, carbon storage, nutritional load of surrounding soils and diversity of associated species increase whenever genotype diversity of populations increases [6]. Indeed, effects are sometimes comparable in magnitude to effects of increased species diversity [7]. As for species biodiversity, the link between genotype diversity and ecosystem function is suggested to be either a sampling effect, or a complementarity effect, or a combination of both. A sampling effect is the consequence of adding more genotypes, and thereby increase the likelihood that more profitable traits are included in the population [8,9]. However, increased genotype diversity may also improve performance of a population through complementarities among genotypes, that is, individuals of different genotypes facilitate performance of each other [10,11].

In marine ecosystems, studies that manipulate MLG diversity in experimental plots have been performed in seagrass (Zostera marina) meadows. Such experiments have shown that meadows of high MLG diversity (many different clones) better resist periods of intense grazing, grazing from specific grazers, extreme weather conditions, and have higher productivity and biomass of associated invertebrate fauna than meadows of low MLG diversity (few clones) [4,12,13]. The positive correlation between MLG diversity and ecosystem performance in Zostera marina have been attributed to both sampling and complementarity effects [4,13].

Fucus species are important foundation species of coastal waters, providing shelter and food for various invertebrate and fish species [14-16]. In the northern parts of the semi-enclosed Baltic Sea, two species of Fucus are present, F. vesiculosus L. and F. radicans Bergström & Kautsky [17,18] and here, in addition to sexual reproduction, individuals of both species have the capacity to form new attached thalli from adventitious branches that come loose from the thallus, are swept away by water motions, and later re-attach to the substratum by formation of rhizoids [19]. Asexual recruitment of attached thalli is hitherto not found outside the Baltic Sea, and the reason for this is not clear. A possible reason why it exists inside the Baltic Sea may be that zygote formation is partly impeded by the low ambient salinity in the northern part of the Baltic (< 4-5‰) [20,21]. However, asexual reproduction is overall much more common in F. radicans than in F. vesiculosus also in this area. In addition, populations of F. radicans living in similar salinities may differ a lot in the prevalence of asexual recruitment, ranging from populations strongly dominated by one clone to populations with only sexually recruited thalli [22]. A female clone, in particular, is exceptionally widespread and dominates many populations over much of the distribution of F. radicans.

The aim of the present study was to find out if phenotypic variation among thalli of F. radicans are to any substantial extent explained by genetic variation, or if most of observed trait variation in nature is non-genetic variation induced by variable environmental conditions. If there is substantial inherited phenotypic variation, we would expect that populations of low MLG diversity (few clones), for example, the nearly monoclonal populations found in some areas, will have lower capacity with respect to ecosystem function and resilience than populations of high MLG diversity (many clones present in the same population), as has been shown in Zostera marina and terrestrial plant species [6,13].

In this study we used thalli of F. radicans sampled in two areas about 30 km apart in the northern part of the Bothnian Sea (northern Baltic Sea). The multilocus genotype of each thalli was determined by microsatellite analysis and thalli were thereafter arranged into four groups; three monoclonal groups made up of the three most common MLGs in the samples, and one group composed of singletons, that is, unique MLGs found in single copies and presumably of sexual origin. We measured phenotypic variation within and among groups in nine phenotypic traits and found phenotypic variation in three of them showing evidence of strong inherited variation.

Results and discussion

Using 5 microsatellite loci we genotyped 147 thalli of F. radicans that were picked from separate holdfasts widely distributed over two sampling areas (approx. 200 m2 each and 30 km apart) in the northernmost part of the species' distribution. Among these thalli we found three clones that were represented by 14-93 thalli each and we selected 9 equally sized thalli per clone to make up our three monoclonal groups. In addition, we found 12 individuals of unique genotypes and selected 9 of these to represent a group of unique MLGs. In all individuals of all four groups we analysed phenotypic variation in thallus width and distance between dichotomies ("branches"), the two most important morphological traits used to separate F. radicans and F. vesiculosus in the field [17]. We furthermore measured phlorotannin concentrations and palatability to grazing from isopods, traits suggested to be important to local adaptation and to the geographic distribution of F. radicans [23]. These four traits were measured a few days after sampling, or for grazing, a few months later (late August), but on tissue grown before sampling, and hence likely included phenotypic variation that was already present in the field.

The total phenotypic variation we found in the traits measured without a common garden treatment were composed of two different parts (i) variation among thalli of the same MLG, and (ii) variation among MLGs. Variation among thalli and within MLGs must be environmentally induced and caused by thalli being grown in different microhabitats. Under the assumption that thalli of the same MLG were randomly distributed over the sampling area, differences among MLGs indicated inherited variation. The assumption of random distribution of individuals of the same MLG is supported by our observation that all the three common clones were present in both sampling areas, and that we in a detailed study of another site (of similarly size as the two sampled sites of the current study) found separate clones of F. radicans to be randomly distributed in space (Pereyra et al. pers. communication). For the traits measured at this stage, we found highly significant differences among MLGs in phlorotannin content while differences were non-significant in palatability to grazers, thallus width and distance between dichotomies (Table 1). This strongly suggests that variation in phlorotannin content is, to a large part, inherited.

Table 1

ANOVA statistics (P-values) indicating differentiation of traits.

	No common garden treatment				Common garden treatment
Differentiation	Thallus width	Distance between dichotomies	Phlorotannin content	Palatability to grazers	Photochemical yield	Photochemical yield after desiccation	Photochemical yield after freezing	Water content after desiccation	Growth during 3 months
ANOVA clones	0.55	0.1	0.0001	0.49	0.08	0.059	0.002	0.99	0.36
Post-hoc (SNK)
Clone-0 vs Clone-1			P < 0.05				P < 0.05
Clone-0 vs Clone 4						0.05 <P < 0.10	P < 0.05
Clone-1 vs Clone 4			P < 0.05			0.05 <P < 0.10
Effect			Clone-1 higher			Clone-4 higher	Clone-0 lower

Variation of phenotypic trait values for nine different traits are compared among the 3 monoclonal groups and significant results further evaluated with SNK posthoc test. Trait values were used untransformed with low deviations from homogeneous variances. Significant values are indicated in bold

The initial measurements were followed by all thalli being cultivated in a common garden environment (large outdoor seawater tanks) starting in late May 2009. In September four additional phenotypic traits were measured; growth during 90 days, photochemical yield under normal conditions, and photochemical yield and water content after desiccation, and in January 2010 photochemical yield after freezing. These traits have physiological relevance and are likely to affect fitness of thalli under different environmental conditions. For these traits, all the measurements were performed on new tissue formed during the common garden treatment. As macroalgae do not possess a vascular system that connects different parts of the thallus, the metabolic status of old tissue is not easily transferred to new tissue [24]. Hence, it is most likely that for these traits environmental variation induced in the field was largely eliminated in the new grown tissue used in the measurements. Thus, in this case, differences among MLGs indicated genetic differences also if the assumption of random distribution of thallus of the same MLGs in the field would not hold. In this comparison we found a highly significant difference among MLGs in the photochemical yield after freezing, and MLGs were also nearly significantly different in photochemical yield after desiccation (Table 1). These differences show that variation in recovery from freezing stress, and desiccation stress, has a genetic component.

We also examined the variation within the group of unique MLGs and found these to be significantly larger than within the monoclonal groups for the same three phenotypic traits, and this observation strongly support the conclusion of inherited variation in resistance to desiccation, resistance to freezing and content of phlorotannins (Table 2, Figure 1). Furthermore, none of these traits were significantly correlated with each other, which tentatively suggest that they are inherited as fully independent traits (Table 3). A notable result was that only a few of the individuals contributed to the increased phenotypic variation in the group of unique MLGs (Figure 1). In addition, the MLGs that contributed the most to the deviation where different for the different traits with the exception of one MLG that were both highly resistant to freezing and to desiccation (Figure 1). The reason for the rather dramatic differences in trait values of individual MLGs is unclear but may perhaps be explained by relatively few quantitative trait loci (QTLs) or genomic regions being involved in these specific traits. While the results for the remaining traits did not indicate strong inherited variation, it is important to underline that sample sizes were too small to identify modest or low levels of inherited phenotypic variation.

Table 2

Cochran's test of homogeneous variances among experimental groups.

	No common garden treatment				Common garden treatment
Population	Thallus width	Distance between dichotomies	Phlorotannin content	Palatability to grazers	Photochemical yield	Photochemical yield after desiccation	Photochemical yield after freezing	Water content after desiccation	Growth after 3 months
Clone-0	0.40	24.33	104.98	0.01	21.30	31.18	19.02	1.41	7.98
Clone-1	0.16	4.89	77.44	0.01	16.89	59.06	28.89	9.56	11.24
Clone-4	0.38	22.52	55.06	0.01	21.45	119.31	35.24	19.64	6.21
Uniques	0.58	15.62	308.41	0.00	29.68	743.91	728.38	27.68	8.80
Cochran's C	0.38	0.36	0.56*	0.32	0.33	0.78*	0.90*	0.47	0.33

*Significant at P < 0.05, critical C = 0.52. Phenotypic trait variances are indicated for three monoclonal groups and one group of unique multilocus genotypes and compared with Cochran's C. Significant heterogeneity in variances are indicated with a C value in bold

Figure 1

Trait values for traits that showed significant inherited variation. Values for each individual thalli are indicated (separate bars) and groups are blue: clone-0, red: clone-1, green: clone 4 and black unique multilocus genotypes.

Table 3

Correlation among traits.

	N	Correlation coefficient	P
Growth rate vs Phlorotannin content	11	-0.53	0.05-0.1
Photochemical yield after freezing vs Thallus width	12	0.51	0.05-0.1
Photochemical yield after desiccation vs Water content after 2 h of desiccation	12	0.54	0.05-0.1

Correlation coefficients are shown for those phenotypic traits that showed marginally significant correlations (0.05

Correlation coefficients are shown for those phenotypic traits that showed marginally significant correlations (0.05