Traditionally diatoms have been identified and classified based on morphological characteristics (Round et al. 1990). However with the molecular techniques now also available it is important to establish the major clades of diatom phylogeny based on both morphological and molecular features (Evans et al. 2007, Alverson 2008). The 18S rDNA sequence is a highly conserved region and is used for basic reconstruction of diatom phylogeny. It provides insights into the evolution of diatoms and 18S rDNA sequences are available for most representatives of the diatoms (Medlin et al. 1996, Beszteri et al. 2001, Fox & Sorhannus 2003). Beszteri et al. (2001) characterised the phylogenetic structure within a clade of six diatoms belonging to the Naviculaceae in order to assess the monophyly using the 18S rDNA. Other studies focussed on intragenomic nucleotide variations among different Skeletonema species or the phylogenetic relationship among phytoplankton species which were distinguished by their 18S rDNA sequences (e.g.Sorhannus 2004, Alverson & Kolnick 2005).
In our study we used the 18S rRNA gene to determine the intraspecific variation of 26 selected Paralia sulcata strains over one year and to verify the phylogenetic relationship among the strains. Our analysis demonstrated that all strains displayed a high similarity (more than 99 %). After comparison with the reference sequence of the P. sulcata strain (EF 192995) (Rampen et al. 2007), all isolated strains were clearly identified as P. sulcata. Thus, all strains belonged to the same population of P. sulcata and showed a lower intraspecific variation regarding the 18S rRNA gene. This low differentiation indicated a homogenous distribution of the P. sulcata population in the water column at Helgoland Roads throughout the year.
ISSR amplification and genetic diversity of Paralia sulcata
In order to investigate and to infer the intraspecific phylogenetic relationship of phytoplankton species, molecular markers are an important tool. Thus, ISSR-PCR has been frequently used for differentiation of phytoplankton species within the last decade (Zietkiewicz et al. 1994, Bornet & Branchard 2001). In this study the ISSR fingerprinting method was used to examine the genetic diversity among 36 P. sulcata strains for the first time. The fragment patterns of all ISSR primers were highly variable and no primer has a similar fragment pattern within the P. sulcata strains.
GENETIC DIVERSITY OF PARALIA SULCATA
Three ISSR primers were not successful for all replicates. The most applicable primers seem to be (ATG)5 and (CCA)5 due to the wide range in fragment length and amplified fragment numbers. Indeed the primer (ATG)5 is described as the best primer for intraspecific interactions among phytoplankton species (Bornet et al. 2004).
Interestingly, all primers showed a higher number of total amplified fragments as within this population but the genetic differentiation among the populations was low (Zhang et al. 2006). 34 ISSR primers were used and PCR with eight of these primers exhibited highly polymorphic band pattern such as the (GACA)4 and the (CAA)5
primer which were also used in our study. In contrast to our study, Zhang et al. (2006) showed a lower range in amplified fragment lengths (from 200 to 1400 bp) which could indicate less abundant SSRs in the genome of C. polypinum compared to a higher complexity within the P. sulcata genome as shown in this study. Thus, the genome size, the complexity of the genome and the distribution of the SSRs as binding sites for the ISSR primers are important factors to determine the genetic diversity on P. sulcata.
In a study on genetic diversity on Arabidopsis thaliana it has been reported that ISSR markers are not suitable in comparison with CAPS (cleaved amplified polymorphic sequence) due to the uneven distribution of ISSR markers within the genome (Barth et al. 2002). Zietkiewicz et al. (1994) pointed out that the genome of A. thaliana is smaller than most other culture plants and therefore reflected a low number of observed amplified ISSR fragments due to a lower genome complexity.
ISSR-PCR shows high reproducibility and efficiency for study the intragenetic variability in phytoplankton species (Nagaoka & Ogihara 1997, Galvan et al. 2003, Bornet et al. 2004). In our study the reproducibility of all five ISSR primers was between 83 to 88 %. This was less than the observed results from a previous study by Charters et al. (1996) which exhibited a high reproducibility (100 %) of the ISSR-PCR with the primers in Brassica napus and B. rapa cultivars. However, similar to our results, the study of McGregor et al. (2000) displayed a low reproducibility of ISSRs
GENETIC DIVERSITY OF PARALIA SULCATA
with 87 % in 39 different potato cultivars. The authors postulated that competition for priming sites within the genome, which was also the possible reason for low reproducibility of RAPDs (Hallden et al. 1996) may also be the cause of the low reproducibility of ISSRs (McGregor et al. 2000). This problem with the lower reproducibility could limit the application of the ISSR primers as fingerprinting method for P. sulcata.
Due to the lower reproducibility of the replicates and to the missing data of some ISSR primers all statistical analyses were performed with the composition pattern of all five ISSR for 22 P. sulcata strains. The MDS analysis in combination with the factor
“simgroups” detected a clear separation of the P. sulcata population (Fig 3). Moreover the ANOSIM displayed significantly separated P. sulcata populations according to the ISSR patterns indicating high intraspecific diversity. The clear separation of the January to March strains (“simgroups” group d, c) from all other strains in the MDS plot for P. sulcata strains (“simgroups” group b) based on the Jaccard’s similarities (Figs 3) is notable. These results indicate that there is a high genetic variability within the P. sulcata population over one year. Unfortunately, the ISSR-PCR with three primers was not successfully in all P. sulcata strains and, especially the spring to summer strains were missing (April to June). Due to these missing data in the analysis we could not make ultimate conclusions for a shift in the P. sulcata population from winter (December to February) to a summer population (June to August).
However, we might explain our observed separation in the P. sulcata population with the ISSR primer fragment patterns as well as with the environmental parameters.
Helgoland Roads is a temperate marine system with a typical seasonal pattern. In general, spring to summer are characterised by warmer water temperatures and higher water transparency (Secchi depth) and autumn to winter by higher concentrations of nutrients due to recycling and resuspension processes in the water column (Wafar et al.
1983, Gebühr et al. 2009). As expected, this seasonal pattern of the environmental parameters was observed with the calculation of the Euclidean distance of the environmental parameters. The isolated P. sulcata strains were well correlated with the environmental parameters displayed by the BEST analysis and as well as with the changes in the environmental parameter due to the different seasons. The significant separation of the P. sulcata strain based on the factor “simgroups” indicates this well correlation with the environmental parameters and a more or less seasonal distribution of the isolates during the year. Thus, we can support our hypothesis of the existence of
GENETIC DIVERSITY OF PARALIA SULCATA
genetically variable P. sulcata populations at Helgoland Roads. This result pointed out that the North Sea provided a good adaptation of the P. sulcata population on its changing marine habitat.