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
]. 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 ). This strongly suggests that variation in phlorotannin content is, to a large part, inherited.
ANOVA statistics (P-values) indicating differentiation of traits.
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 ). 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 , Figure ). Furthermore, none of these traits were significantly correlated with each other, which tentatively suggest that they are inherited as fully independent traits (Table ). A notable result was that only a few of the individuals contributed to the increased phenotypic variation in the group of unique MLGs (Figure ). 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 ). 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.
Cochran's test of homogeneous variances among experimental groups.
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.
Studies in the sister species F. vesiculosus
have shown that the inherited part of the variation in growth rate is low while it is high in phlorotannin content [25
], and this is similar to what we found for F. radicans
. Phlorotannins provide a defence towards grazing, in particular by isopods, in F. vesiculosus
], and this seems most likely the case also in F. radicans
. We found only a weak and non-significant negative correlation (r = -0.02) between phlorotannin content and palatability to isopod grazers in the present study, but we did find an almost significant negative correlation with growth rate (Table ), suggesting phlorotannin production to be costly.
Isopod abundance is low in the Bothnian Sea compared to in the Baltic Proper [28
], and grazing pressure from isopods seems mostly low or absent in most areas where F. radicans
is present [18
]. In addition, phlorotannin production is known to be costly [29
], this study], and, thus there is probably a trade-off favouring a low phlorotannin production in areas of low grazing pressure. Indeed, in choice experiments, isopods graze substantially more on F. radicans
than on F. vesiculosus
], which suggests that phlorotannin content is generally lower in F. radicans
than in F. vesiculosus
. As we now show, there is genetic variation present in this trait in F. radicans
and hence selection may favour genotypes of high or low phlorotannin content depending on the local grazing pressure. A less likely alternative (owing to costs of production) is that the high phlorotannin production we found in some of the clones is a remnant from the more saline Littorina Sea period of the Baltic Sea a few thousand years ago, when isopods were likely more common in the Bothnian Sea than they are today. It has also been suggested that phlorotannins protect the thalli from UV radiation [30
] but this seems of less contemporary importance as F. radicans
in the Baltic lives permanently submerged.
Perhaps still more intriguing is the finding that populations of F. radicans
contain MLGs that fully recover from freezing dowo-15°C or desiccation during 3 h, while this species lives submerged and only very occasionally may be exposed to desiccation or freezing during periods of extreme low water in the Bothnian Bay. Indeed, it seems unlikely that selection favouring such characters would be more than very occasional. However, the ancestral population of F. radicans
, that is, the population of F. vesiculosus
that entered the Baltic Sea about 6000 years ago, and from which F. radicans
], came from intertidal habitats in the North Sea. Here, resistance to freezing and desiccation is critical to survival as most individuals live emerged during periods of low water. In fact, populations of F. vesiculosus
from the North Sea show much higher tolerance to desiccation than Baltic populations of this species [32
], and these differences seem largely inherited [33
]. The high tolerance of single MLGs of F. radicans
to desiccation and freezing may be what is left of a very common genotype in the ancestral gene pool entering the Baltic Sea. That this genotype is still present in low frequencies may be due to local and temporal positive selection during rare events of extreme low water level. Alternatively or in addition, there may be epistatic or pleiotrophic effects in combination with weak negative selection, or simply that genetic drift only very slowly replace near neutral variation in large populations.