Bijlage B Variety in the width of gametophyte branches as method of trait recognition. Introduction

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1 Bijlage B Variety in the width of gametophyte branches as method of trait recognition Introduction Haploid gametophyte cultures are the first stage in seaweed s lifecycle where actual morphological differences can be observed. Discovering and the eventual recognition of certain morphological traits in gametophytes can be very beneficial in the breeding industry. These morphological traits can be indicative for the health-, origin-, or growth characteristics of gametophytes. It could even lead to indications in the diploid sporophytes health, growth characteristics, and genetic diversity. Stable parameters are needed in order to find a good starting point in which morphological traits are recognized, archived, and compared. The most obvious parameter is the size of the gametophyte. This trait is easily observed and compared, but it lacks in terms of uniformity. Comparing sizes between populations is difficult because of the differences in branching, unlimited growth of the gametophyte, and colony forming within one population. In this study a start has been made in finding a stable parameter so that comparative studies can been done. The width of a gametophyte branch has been measured and compared between supposedly male and female specimen from Holland, Ireland and Norway in order to find such a stable parameter for intra- and interspecific comparisons. If this parameter is stable enough, and the genetic difference within one species is large enough, than differences should be observed. Clone lines are used as comparative material in order to see if there are not only inter specific differences between gametophytes from different geographical locations, but also to see if there are morphological differences within a gametophyte extraction from one mother plant. A great step can be made in the process of trait selection and selective cross breeding between species when there are differences found in morphology. Material & Methods Clone lines from Laminaria digitata & Sacharina latissima are used for comparative purposes. These clone lines are made from individual gametophytes that through time are cut manually into several multiple gametophyte clones. These gametophyte fragments are then grown until they have a large enough size for another cutting step. This will increase the amount of genetically identical gametophytes exponentially and will eventually result in large enough batches to do measurements on. The width of a gametophyte branch is measured using a Image focus that is integrated in the microscope under a magnification of 400*. Every clone line will be photographed 10 times and width measurements shall be taken for statistical purposes. SPSS 20 shall be used for statistical analysis, using the t-test and boxplots to show significance and trends.

2 In order to get accurate width measurements in gametophyte branches some clear rules are set to reduce the error to the minimum. First of all the measurements need to be taken perpendicular from the cell border. Any difference in degrees shall influence the length substantially. Secondly the length of the broadest part of the cell is measured. The broadest part of the cell is easy to recognize, reducing the error in measuring. Thirdly, a distinction needs to be made between regular branch cells and anomalies that more often than not are broader.

3 Results Figuur 1: boxplot comparing the branch width of gametophyte individuals from different species (Sacharina latissima, Laminaria digitata) and from different origins (Norway, Netherlands, Ireland). The x-axis depicts the branch width (µm) and the y-axis depicts the different individuals. Different colors are used for easy recognition ( SANO, red; SAIR, orange; SANL, yellow; LDNL, blue; LDNO, green; LDIR, purple) Figuur 2: boxplot comparing the branch width of gametophyte individuals from different species (Sacharina latissima, Laminaria digitata) and from different origins (Norway, Netherlands, Ireland). The x-axis depicts the branch width (µm) and the y-axis depicts the different species and sex. Different colors are used for easy recognition (SA-male, red; SA-female, green; LD-male, orange; LD-female, blue).

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5 Figuur 3: Gametophyte branch width comparison between different Sacherina latissima (SA) individuals from Ireland (IR). The x-axis depicts the branch width (µm) and the y-axis depicts the different individuals. Colors are used to differentiate between males ( red) and potentially female (orange). Figuur 4; Gametophyte branch width comparison between different Sacherina latissima (SA) individuals from Norway(NO). The x-axis depicts the branch width (µm) and the y-axis depicts the different individuals. Colors are used to differentiate between males (red) and female (blue).

6 Figuur 5; Gametophyte branch width comparison between different Sacherina latissima (SA) individuals from Netherlands (NL). The x-axis depicts the branch width (µm) and the y-axis depicts the different individuals. Colors are used to differentiate between males (red) and female (blue). Figuur 6; Gametophyte branch width comparison between different Laminaria digitat (LD) individuals from the Netherlands (NL). The x-axis depicts the branch width (µm) and the y-axis depicts the different individuals. Colors are used to differentiate between males (red) and female (blue).

7 Figuur 7; Gametophyte branch width comparison between different Laminaria digitata (LD) individuals from Norway(NO). The x-axis depicts the branch width (µm) and the y-axis depicts the different individuals. Colors are used to differentiate between males (red) and female (blue). Figuur 8; Gametophyte branch width comparison between different Laminaria digitata (LD) individuals from Ireland (IR). The x-axis depicts the branch width (µm) and the y-axis depicts the different individuals. Colors are used to differentiate between males (red) and female (blue).

8 Tabel 1: Std. Deviation and Std.Error Mean of branch width differences between individuals from the same sex and origin. One-Sample Statistics N Mean Std. Deviation Std. Error Mean SANO-male 193 7, , , SAIR-male 248 6, , , SANL-male 284 6, , , SANO-female , , , SANL-female 71 9, , , LDNO-male 76 9, , , LDIR-male 50 9, , , LDNL-male 46 9, , , LDNO-female 69 17, , , LDIR-female 25 18, , , LDNL-female 23 13, , , Tabel 2: One sample Test of the gametophyte branch width with Mean values and 95% confidence interval of the difference within the same sex and origin. One-Sample Test Test Value = 0 t df Sig. (2-tailed) Mean Difference 95% Confidence Interval of the Difference Lower Upper SANO-male 59, ,000 7, , , SAIR-male 76, ,000 6, , , SANL-male 73, ,000 6, , , SANO-female 58, ,000 12, , , SANL-female 48,902 70,000 9, , , LDNO-male 54,891 75,000 9, , , LDIR-male 47,478 49,000 9, , , LDNL-male 39,078 45,000 9, , , LDNO-female 36,733 68,000 17, , , LDIR-female 49,408 24,000 18, , , LDNL-female 23,327 22,000 13, , ,801824

9 Morphologies Figuur 9: Healthy female gametophyte with dense brown coloration. (SANO-002) Figuur 10: Healthy female gametophyte with dense brown coloration. (SANO-004) Figuur 11; In red the possible male trademark lump (SANO-004). Figuur 12; In red the possible male trade mark lump (SANL-012). Figuur 13; short lumped morphology(sair-001) Figuur 14; Elongated thin morphology (SAIR-005)

10 Figuur 15: LDNL-003 female gametophyte (amplification 100*). Figuur 16: SANL-012 female gametophyte (amplification 100*).

11 Discussion Variety in gametophyte width Looking at the results a distinct difference can be observed in gametophyte branch width between Sacharina latissima and Laminaria digitata (Fig. 1 & Fig. 2). Gametophytes from Laminaria are overall bigger than the same sex gametophytes from Sacharina (Fig. 2). There is also another major difference between the two species and that is variety in gametophyte widths within same sex/origin. Laminaria has distinct size differences within male or female gametophytes with the same origin (table 1 & 2). These differences are less clear within Sacharina and this can have two possible reasons. First, the genetic difference between Sacharina individuals from different locations could be less than within their Laminaria counterpart. Secondly, the larger size in gametophyte width within Laminaria could give more room for variation. This seems more plausible since there is a systematically higher Std.deviation found within larger female gametophytes branch widths than their smaller male gametophyte counterparts. There is one exception and that goes for the male SA gametophytes from Norway. But taking the amount of samples (n) into account the resulting std. Error Mean returns to be smaller than the std. Error Means of female gametophytes. Other observations can be made interspecifically. There are distinct size differences between the same species from different regions. A very clear observation is the large size of Norwegian female Sacharina gametophytes compared to the Dutch or Irish counterpart. Even within Laminaria Digitata the same observation can be made although the interregional size distribution is more widely dispersed. Male Laminaria digitata gametophytes are furthermore also slightly bigger when they come from Norway. A very clear observation is the systematically smaller size of male gametophytes compared to their female counterpart. This male/female size difference is already a well-known characteristic that goes for Laminaria as well as Sacharina. The female/male width ratio is furthermore bigger for Laminaria digitata then for Sacharina latissima. No clear male/female size difference could be found within Irish Sacharina latissima individuals. This could be because of thinner female branches. More plausible is the possibility that only male gametophytes are cloned in the process of the last months. Morphology Observations could also be made concerning the male and female morphology of SA as well as LD. These observations can give insight to the functioning of different parts of the gametophyte, but could also be helpful in trait recognition in early stages of the seaweeds life cycle. First of all there is a certain distinct type of branch ending within SA male gametophytes (encircled in red; Fig. 11 & Fig. 12). This broadening of cells at the end of some branches is very distinct and found a lot in male Sacharina gametophytes from Ireland, Norway, and Holland. Female gametophytes have round endings just like male gametophytes but the distal cells are not bigger than more proximal cells. Furthermore these male gametophyte nubs have pointy tips and resemble ancient arrows points. Second is the strange observation of the branch length between not only different individuals but also within one and the same clone line (Fig. 13 & 14). Two different morphotypes can be distinguished, in one hand the very elongated thinner branches and in the second the shorter thicker branches. Gametophytes of the latter morphotype usually also have more lumped appearances since the

12 individual cells are more rounded. The strange thing is that it only has little influence on the width of the gametophyte. But concerning the study between branch widths a remark should be placed when the gametophytes are very short and lumped. This could influence the data, resulting in false information. It seems that the shorter and more lumped the gametophytes are the less healthy they become. This is everything but statistically clear but this trend seems to be pointing to this direction looking at the observations that were made on the clone lines through time. These gametophyte morphotypes seem however to change little through time. Clones that were short and lumped in the beginning are still short and lumped. Small changes are however observed the other way around. Within elongated clone-lines there are colonies formed that start to be shorter and more lumped. This is also one of the reasons that health is proposed as a factor that influences these morphological differences. Future work The results are very promising considering the variety in branch width found. There is a clear variety in branch width intra-specifically but also inter-specifically. Creating more clone lines through time results in more data and thus getting closer to a standard per species from the different locations. Exceptionally broad or thin individuals shall be easier to recognize with increasing datasets. The broadening of these cells influences the results substantially, thus creating false information. A clear measuring protocol needs to be made in order to reduce the error in measuring the width of the gametophytes. This protocol is partially proposed in the material en methods of this research. Gametophyte morphology studies can be highly beneficial for early recognition of health, growth and reproduction. It is thus thinkable to look further into morphological traits within gametophytes. New morphological traits could be cell content, color, chloroplast density, or fluorescence measurements. Less likely characteristics could be the amount of branching, lump formation, or growth rate through longer periods of time. One very important morphological characteristic is the branch length of the gametophyte. The length of gametophyte branches are however more difficult to parameterize because of the hooks and corners in the branches. It seems however that the branch length is none the less indicative for the health of the gametophyte and thus important to research in the future.

13 - Differences in size o Male female o Origin o Interspecific o SAIR no difference male female Females are found to be thicker in normal cultures. This indicates on male only clone lines from Ireland. o Standard deviation bigger in females Standard deviation is mostly bigger in females except for SANO but the standard error which takes the sample size (n) into account makes it uniformly bigger within females Dutch female smaller than Noregian gametophyte - Morphology differences o Elongated v short branches Thicker cells in short branches Influenced the results Seemingly healthier gametophytes when elongated Changes trough time? SA as well as LD o Male v female Thicker nub on branch end o Sacharina V Laminaria o Chloroplast amount and internal distribution - Other observations o Spermatozoids in culture o