Methodology for the study of freshwater fungi

During the study of freshwater fungi, it is necessary and important to maintain a uniform methodology to study the fungi from different habitats, and substrates. It is suggested that the methodology outlined below is followed.

Lignicolous fungi (fungi on woody substrates)

1. Site selection

Freshwater ascomycetes and anamorphic fungi can be found on decaying wood, grass and macrophytes in lakes, ponds, reservoirs, swamps, streams and rivers. In general, a good site for study the freshwater fungi would have a narrow channel and a high density of riparian vegetation along the banks and thus lots of dead woody material within. For the biodiversity study, independent streams with different types of riparian vegetation are suggested. For biogeographic studies, sites from different countries having different latitudinal gradients should be chosen.

2. Sampling

What to record in the field

When an ideal site has been selected, a field trip is needed to collect samples. A good record of all the parameters of the habitat should be made.
These should include where possible
• altitude,
• latitude and longitude (GPS),
• water temperature,
• pH of the water,
• dissolved oxygen content (DOC),
• running speed of water,
• description of the stream bottom
• description of the riparian vegetation.
Numerous photographs of the sampling site should be taken at the same time.
Normally we would go upstream of the access point to the river and collect samples by walking upstream to prevent the water becoming dirty.
It is important that collect samples that have been submerged for a long-time (at least 6 months). Tell tale sign are lack of bark, caddis fly larvae, erosion of the wood surface, wood becoming very hard. Be sure not to collect the newly falling down parts of the plant debris.
• Ideal number of samples = 50
• These should be collected from 100 metre stretch or longer.
• All the samples should be kept in moist environment and returned to the laboratory as soon as possible for further incubation and examination.

3. Incubation

Samples need to be incubated in a plastic bags or plastic boxed with moistened sterilized tissue paper inside for incubation. Incubated samples must be placed individually in separate containers or several containers with sufficient space to avoid one sample be colonized by fungi from the nearby substrate. The samples could be examined for the appearance of fungi within 7 days of incubation. While the best stage for checking the samples are from 7 to 30 days following incubation. Normally the samples should be examined prior to incubation and regularly for up to three months. Prolonged incubation for up to six months also induces sporulation of some taxa that do not appear initially.

4. Identification

• Samples should be observed under a dissecting microscope to examine the occurrence of fungi.
• Materials should be examined at 10× eye piece and 1.5-2× magnification = 15-20×
• Fruiting structures should be removed with a fine pair of forceps or needle, and a slide mount should be prepared in water.
• Structures could be observed under a compound microscope and the fungi are identified.
• A good description and detailed record need to be made for accurate identification and further diversity analysis.
• A well-organized sheet could be used for recording all the characters as well as all the measurements (see table 1).
• This kind of sheet or form should include all the important structures of the fungi will be observed. For example, freshwater ascomycetes are classified and identified based on ascomatal structure, ascomatal wall structure, presence of paraphyses/pseudoparaphyses, ascus morphology (e.g. shape, unitunicate/bitunicate, apical ring/ ocular chamber) , and ascospore characters (e.g. shape, dimension, septation, color, appendages). Anamorphic fungi are identified based on morphology of conidiophores and conidia (e.g. size, color, septation), and conidiogenesis.
• Photography – The pictures corresponding to the characters those have been recorded in the description sheet should be taken at the same time. A high quality digital camera is recommended, since lots of photos need to be taken for one character. All the photos should be detailed coded with your own reference number. Photoshop software could be used for further modification of the original photos, and also for make plates.

5. Data analysis

According to the biodiversity analysis, the following analyses should be performed after species identification. A good way to record fungi on samples is as follow (See Table 2)

• The number of species
• The species diversity
• The frequency of occurrence
• So, the number of species on each sample should be recorded as well.
• Species diversity of each sampling site can be calculated using Shannon-Wiener’s index.
• The frequency of occurrence can be calculated as following:
•
number of samples on which a given taxa occurred ×100％
total number of samples examined

6. Isolation

After description, single spore isolation can be performed immediately to obtain pure cultures of the fungi. The fresh specimens are the best material for this. Fungi sporulate directly on the substrata, and spores can be picked off and used for single spore isolation. The method provided by Choi et al. (1999) is quite commonly used. It involves making a spore suspension which is then pipetted onto the surface of agar and allowed to dry. The spores will usually germinate within 12-72 hours and newly emerged germ tubes can be seen using a stereomicroscope with illumination from below. Then the spore can be picked of on a minute piece of agar and transferred to a new plate. (see link to two papers)

7. Herbarium

After finish with the description and photography, the samples need to be dried and made into specimen for long-term preservation. Firstly, we should cut off the small piece of wood with fungi on it from the big sample, then this small piece of wood could be dried just under room temperature. The specimen also could be made by putting them into the heating chamber to fasten its drying procedure. Then the small box (as showed in pictures below) could be used to keep the specimen. A mark with detailed information about this specimen should be attached with the box.

Table 1 FUNGI DESCRIPTION SHEET Reference No.:
SAMPLE NO.__________ PLACE________________DATE_____/_______/_______