These are defined here to include Ascomycetes (including anamophic states) that are small but visible with or without the use of low magnification. These fungi can be collected directly in the field. This is in contrast to the many other Ascomycetes that can only be included in diversity studies by culturing those occurring in the leaf litter or soil using selective media. The microfungi to be included in the Costa Rican inventory encompass a great diversity of taxa that serve a number of different ecological tasks from plant pathogens to decomposers of woody substrates. While these fungi will be collected in the field, many of them also need to be brought into culture to observe important morphological characters necessary for identification and for understanding their life histories. Cultures are also important to determine the ability of these fungi to produce useful chemical compounds. These cultures will be an important product of this work.

Byssosphaeria schierdermaryeriana

Protocols:

The techniques for describing and processing microfungi were provided to the parataxonomists by Fernando Fernández, Sandy Salas and Loengrin Umaña during the first advanced fungal course held in January 1999.

Three types of sampling protocols will be employed: A) random sampling, B) sampling of fixed sized plots, and C) sampling of a fixed number of downed logs.

A) same as macrofungi.

Byssosphaeria schierdermaryeriana
In order to observe microfungi a magnifying
glass is needed due to hteir size

B) sampling of fixed sized plots:

1. Microfungi plots, one-square-meter in size, will be established adjacent to the macrofungi subplots. Because much of the leaves and twigs occurring in the plots will be collected, different plots will need to be laid out each time. Sampling microfungi plots is very time and labor intensive. The actual number of plots sampled will vary depending on the diversity of substrate in the plots and the number of people sampling the plots. The goal is to carefully and quantitatively sample the microfungi growing on small substrata for species that are infrequent and/or rarely collected, not to cover a lot of area. Sampling will continue for one week.

All microfungi on twigs, branches and leaves occurring within the plot(s) will be collected, labeled and placed in appropriate bags for transport back to the field station at the end of the collecting day. The samples will then be sorted, examined under a stereomicroscope to determine if they are fertile, described, divided, and dried.

One parataxonomist would only be able to sample 2 or 3 one-square-meter subplots in a week. So, in July each parataxonomist would sample 2-3 subplots and then in October they would sample 2-3 different subplots.

2. The microfungi plots will be sampled twice a year, in the middle and end of the rainy season, in months that the parataxonomists are not sampling the transects for macrofungi (July and December).

C) sampling of a fixed number of downed logs:

Restricting quantitative sampling to the 0.1 ha plots would exclude most of the fungi found on larger pieces of wood as the frequency of these larger substrates within the plots would be very low. Therefore, this substrate must be sampled separately. The following protocols should capture a good percentage of the diversity, as well as provide quantitative data to enable some analysis of abundance and host and size class specificity. Logs will be chosen and marked at one site in each conservation area during 2000 and early 2001. Then, they will be sampled 4 times per year for 3 years (March, July, September and December) starting in year 2001. These will be the same logs used to sample wood-inhaviting macrofungi.

1. Logs to be included in the sampling will be:

Diameter more than 20 cm

Length more than 2 m

Lying on the ground

2. There will be approximately 30 logs in each of the following decay classes.

Class 1: Relatively newly fallen, usually with its bark on.

Class 2: Medium rotten, bark fallen off, and knife can penetrate 1-2 cm into the wood without undue pressure.

Class 3: Thoroughly rotten, knife can penetrate into the log without much pressure, the wood can be partly destroyed with the fingers. 3. Each log will be marked with a colored plastic band and given a number. A map will be made so that the collector will see that she/he has missed a log if the numbers jumps from 35 to 37 without any intervening log.

4. The genus or species of the host tree will be noted when possible.

Protocols for documenting and preserving fungi in the field:

1. Collection data for all specimens will be entered into a database on laptop computers. This database will be compatible with ATTA and will enable ready transfer to that web accessible system.

2. Microfungi will be examined under a stereomicroscope to determine if they are in good conditions. Portions of the material will be air dried and transported to INBio for culturing and further characterization. When possible, this transfer should be done at the end of the week spent collecting microfungi. The remaining portions will be warm-air dried and sealed in plastic bags for protection against breakage and high humidity that will cause mold growth before being transported to INBio.

"Microfungi are examined under a stereoscope to determine
if they are in good condition"

Protocols for culturing, identifying, and curating the collections:

Identification of microfungi is based primarily on micromorphological characters. Curators and technicians will use compound and stereo microscopes equipped with digital image capture systems to facilitate capturing images for documentation, rapidly sharing information with international collaborators through e-mail, publications and other products.

Plot based inventories generate an incredible number of specimens, and an accession policy is mandatory to prevent a herbarium from being overwhelmed (both in processing time and space) by redundant collections. In general, it is recommended that 2 or 3 good samples of each taxon from each site be retained.

A wide variety of techniques can be used to isolate the different groups of microfungi. Some of the most common techniques are: direct isolation, tissue culture, and spore discharges. There are various culture mediums that can be used to isolate microfungi.

The principal factor in the selecting the preservation method is whether the specimens are going be to maintained as dry material in the herbarium or as live cultures.

The cultures (stock) is kept in petri boxes for a short time, then agar disks with micelio are transfered to vials with distilled water at room temperature, or kept at chamber temperature of -80 C.

"Cultures in vials with distilled water"