Bio 105 Lab 2: MARINE ALGAE “Algae” is a descriptive term with little scientific meaning regarding taxonomy (grouping and naming of organisms) and phylogeny (evolutionary history). This means that the common term “algae” encompasses a wide array of different marine and freshwater photosynthesizers from bacteria to giant kelp, and not just one branch of closely related species like “red algae” or “diatoms.” According to some estimates, over half of Earth’s primary productivity (plant and algal growth due to photosynthesis) occurs in the oceans – most of it by tiny unicellular algae. In this lab, you will have the opportunity to familiarize yourself with some of the major groups of algae, including cyanobacteria, dinoflagellates, diatoms, green algae, red algae and brown algae. The latter 3 groups include the seaweeds, which can be found growing nearshore, and you will learn to identify some common local species. To get an idea of the relationships amongst the 6 algal groups listed above, complete the following simplified phylogenetic tree with help of the diagram at the beginning of Chapter 6 or 7 in your text (Karleskint, 3rd ed.). The higher (more recent in time) the diverging point of two groups, the more closely they are related. Which of these groups contains the closest living relatives of land plants? _________________ Which of these groups is most distantly related to all the rest? ___________________

PART A: MICROSCOPIC ALGAE Station 1: CYANOBACTERIA Cyanobacteria a.k.a. blue-green bacteria a.k.a. blue-green “algae|” are, like all bacteria, prokaryotic (without a nuclear membrane surrounding their DNA). All other algae are eukaryotic (with a nuclear membrane). It is cyanobacteria who first evolved photosynthesis (using solar energy to convert CO2 and H2O into sugars, with O2 as a by-product). The main photosynthetic pigment in blue-green bacteria is the bluish-green chlorophyll a, with accessory pigments like the red/blue phycobilins. Pigments capture light energy at different wave lengths, making photosynthesis more efficient. The tiny unicellular photosynthetic bacteria in genus Prochlorococcus may collectively be the most abundant life form in the ocean and responsible for much of marine photosynthesis! Most cyanobacteria are unicellular, although some species can form long chains known as filamentous colonies. In great numbers, the tiny microscopic cyanobacteria are capable of forming formidable macroscopic structures: Stromatolites (see photo) are large mounds of cyanobacteria and other microbes matted together by mucilage and sediments. A few cyanobacteria are bioluminescent and live symbiotically with certain animal species, e.g. fish and squid, in organs called photophores (see photo). If Prochlorococcus is not available, look at some other representatives of cyanobacteria, including colonial forms like Oscillatoria and the closely related Anabaena/Nostoc. Use your microscope and wet mount skills to prepare and view the provided organisms. Like many colonial blue-greens, Nostoc colonies contain a round, non-photosynthetic heterocyst (cell specialized to produce and store nitrogen for the colony.) Nostoc can also form supercolonies of inter-twined filaments within a gelatinous mass, forming balls of up to 50 cm diameter! Check out the jar with some slightly smaller versions of Nostoc balls. Make a detailed, clearly labeled sketch of both cyanobacteria species provided. All your drawings should have all the components discussed in Lab 1, and should be specific enough to recognize organisms and their structures on a lab exam.

Station 2: DIATOMS Diatoms are the main component of the seas’ phytoplankton (assembly of small, drifting photosynthesizers). They use cholorophyll a and c. Beauty in Nature also occurs on the microscopic level, and many marine biologists would argue that diatoms are a case in point! See for yourself as you examine the prepared slides and photos of some examples of diatom art. There is great diversity amongst species with regard to the shapes and intricate patterns of their glassy (silica-containing) cell walls. The cell wall is divided into 2 valves which fit together like a miniature shoe box, protecting the cell within. Some species of diatoms also form chains. Provide a sketch of a variety of diatom species, using at least a couple of different slides available.

Next, make a wet mount of diatomaceous earth, containing fragments of fossilized diatoms. Provide a sketch of a variety of fragments, or – with any luck – a complete valve from millions of years ago.

Station 3: DINOFLAGELLATES Dinoflagellates, along with diatoms (s.a.), are a major component of the seas’ phytoplankton (assembly of small, drifting photosynthesizers). Like diatoms, dinoflagellates use cholorophyll a and c. The cell membranes of most algae within this group (e.g. Ceratium and the closely related genera Peridinium/ Glenodinium) are reinforced with cellulose, i.e. armored. However, some species are called naked dinoflagellates (e.g. Noctiluca), as they lack this reinforcement. Most dinoflagellates have 2 flagella, and are unicellular. Most are also globular/roundish in shape. A notable exception to the latter is the 3-pronged genus Ceratium. Some species are bioluminescent (check out the surfing video!). A common bioluminescent species is sea sparkle (Noctiluca scintillans). Another sub-group of dinoflagellates known collectively as zooxanthellae are important symbionts, notably of corals, providing their hosts with extra nutrients from photosynthesis in return for shelter and CO2 (see photo on display). Make a labeled sketch of the 4 dinoflagellate species (s.a.) available on prepared slides.

PART B: MACRO-ALGAE “Seaweed” or “kelp” is a common name often given to large specimens of 3 diverse algal groups: Green Algae (phylum Chlorophyta), Red Algae (phylum Rhodophyta) and Brown Algae (phylum Phaeophyta). These phyla also contain microscopic forms, but we will focus here on the macroscopic representatives or macro-algae. While a seaweed’s outward coloration is often indicative of its taxonomic group, i.e. green, red or brown, there are many exceptions. Classification is based on the actual pigments (see Table 1) as well as storage products. Phylum Chlorophyta (Green Algae) Rhodophyta (Red Algae) Phaeophyta (Brown Algae)

Chlorophylls Accessory Pigments a, b

Carotenoids, e.g. β-carotene (orange-yellow)

a, d

Phycobilins incl. phycoerythrin (reddish), phycocyanin (bluish) Carotenoid: Fucoxanthin (brown)

a, c

Table 1. Pigments of the major macro-algae. The body of a seaweed is called thallus. The flattened parts of a thallus are referred to as blades or fronds, while a stem-like structure is known as stipe. The seaweed attaches to the surface by a holdfast (see Fig. 7.2 text). Unlike plants, such as sea grasses, algae have no vascular tissue and therefore, by definition, no true leaves, stems or roots. Seaweeds absorb water and minerals over their entire surfaces, eliminating any selective pressure for the evolution of internal transport vessels. Many seaweeds do, however, contain air bladders. What do you think, is the function of these? ______________________What is their selective advantage?____________________________ (hint: think photosynthesis!)

Go to the following stations to identify common local seaweed species in the 3 major phyla: Station 4: GREEN ALGAE Station 5: RED ALGAE (check out also examples of the red algal products agar and carrageenan) Station 6: BROWN ALGAE On unlined paper provided, make a sketch of each species designated. Be sure to include the name, phylum, approximate size, labels for any prominent thallus parts (s.a.) or special features, and perhaps a note on its shade of color – anything that would help you in identifying a particular species based on your labeled drawings and descriptive notes alone (ON THE BEACH OR ON A LAB EXAM!).

Station 7: CHROMATOGRAPHY Algal pigments play an important role in oceanic photosynthesis as well as in the phylogenetic history of algae. You can separate out individual pigments from algal or plant material by paper chromatography, resulting in a chromatogram showing bands of pigments identifiable by their color and position . Separation of the different pigments is possible because different pigments dissolved in a solvent adsorb to the surface of the paper differently. (Note: when one compound adsorbs to another, it adheres to the surface without forming a chemical bond.) The more polar the compound, the more strongly it is adsorbed to the paper, therefore more polar compounds remain near the bottom of the chromatography sheet, and less polar ones are carried further by the solvent as it moves up the sheet. In the following exercise, you will compare the pigments in cyanobacteria with those in green alga (Ulva sp.= sea lettuce) and plants (Zostera marina = eel grass). Take a piece of chromatography paper and draw a pencil line about 3 cm along the short edge of the paper. (Handle the paper only by its edges.) Place a small quantity of the green alga in a mortar. Add just enough acetone to make a thick paste, by grinding it thoroughly with the pestle. Let the particulate matter settle. If necessary, add only enough acetone to permit pipetting of the liquid extract onto the chromatography paper. Repeat for the eelgrass (cutting it up with small scissors beforehand). The bacterial extract has already been prepared for you. Using a small pipette (a different one for each specimen in order to avoid cross-contamination!), spot each extract in its own (labeled!) spot along the pencil line (Figure1). Be careful to make small spots. Let dry between applications but do about 25-50 spottings for best results. Your objective is to have a concentrated small circle of pigment on the line. Do not dry your spots under the light as both the light and heat will damage the pigments.

Up

down Pencil line cyanobacteria Ulva Zostera

Figure 1. Paper chromatogram

Place the chromatography paper in the graduated cylinder, being careful not to let the spots touch the liquid. The chromatography solvent is a mixture of 9 parts petroleum ether to 1 part acetone. Keep an eye on the chromatogram as it runs. It only takes a few minutes to separate out the pigments. Don’t let the solvent front run off the top of the paper. Sketch your chromatogram results (you can use Fig. 1 above) and label the resulting pigment bands. Include the solvent line (height to which the solvent traveled). When interpreting your chromatogram, keep in mind the following: in order of polarity, the sequence of pigments is chlorophyll b (yellowish-green), chlorophyll a (bluish-green), followed by carotenes (deep yellow) which will travel close to the solvent front. Check the presence of pigments in the table below: Pigment

Cyanobacteria

Ulva

Zostera

Carotene Chlorophyll a Chlorophyll b Compare the pigments of the 3 different photosynthesizers. Which 2 are the most similar? ______________ and _______________ Are they more closely related to each other than either is to the third? _____ While this is a very simplistic example, the study of pigments in bacteria, plants and the various algal groups can give a lot of insight into their evolutionary relationships.

Station 8: ALGAL ART The use of a plant press can be a handy skill for both science and art. Pick one of the delicate algae provided for this purpose and float it onto a piece of herbarium paper. Please use only paper of appropriate size, to prevent waste. With tweezers or probes, arrange the thallus into the desired position. Cover your specimen with a piece of jaycloth, then layer it as follows with the other class specimen into the plant press: cardboard, blotting paper, specimen on herbarium paper, jay cloth, blotting paper, cardboard etc. Your algal artwork should be dried and ready by next week’s lab and ready to use as a study aid and/or greeting card, book mark etc.