Methods for Sampling and Analyzing Wetland Fungi
Most fungi are terrestrial, but representatives of all major groups of fungi along with three groups of fungus-like organisms (water molds, slime molds and lichens), usually studied by mycologists, can be found in wetlands. The primary ecological role of the fungi and water molds in wetland habitats is to decompose dead plant material—both woody and herbaceous debris as well as dead bryophytes. Although sometimes present in wetlands, slime molds and lichens occur almost exclusively on emergent (dry) substrates. Because the vast majority of fungi and fungus-like organisms associated with wetlands are microscopic, efforts to document their distribution and patterns of occurrence often pose a real challenge to ecologists. This chapter reviews some of the more useful and effective methods that can be used to study these organisms in wetland habitats. These include collecting specimens directly in the field, isolating specimens from substrate samples placed in moist chamber cultures and obtaining specimens on various types of organic baits.
KeywordsFruiting Body Woody Debris Wetland Habitat Slime Mold Dead Plant Material
Fungi (singular: fungus) are a large, diverse and ecologically important group of eukaryotic organisms found in every ecosystem on earth. These organisms constitute a separate kingdom, distinct from both plants and animals, from which they appear to have diverged more than one billion years ago (Bruns 2006). With a few exceptions, the vegetative body of a fungus is made up of microscopic filaments called hyphae. The latter are usually extensively branched, have a cell wall consisting largely of chitin, and are either septate or aseptate (coenocytic), depending upon the group of fungi involved. Collectively, the system of hyphae making up a single fungus is referred to as a mycelium. Because they lack the photosynthetic pigments found in plants and algae, fungi have a heterotrophic mode of nutrition. In contrast to animals, which feed by ingestion of organic material, fungi obtain their nutrition by extracellular digestion that is facilitated by enzymes secreted from the hyphae; they then absorb the solubilized breakdown products (Webster and Weber 2007). Both sexual reproduction and asexual reproduction occur in fungi, although some species seem to have either lost the capability for sexual reproduction or do so only infrequently. Both types of reproduction generally involve the production of microscopic spores on or within some type of fruiting structure. The spores represent the primary means of dispersal, but once these reach a suitable substrate and germinate, the fungus can proliferate rapidly by means of hyphal growth and potentially colonize the entire substrate (Stephenson 2010).
In most traditional taxonomic treatments of the kingdom Fungi, five phyla have been accepted as being “true” fungi: Chytridiomycota, Zygomycota, Glomeromycota, Ascomycota, and Basidiomycota (Alexopoulos et al. 1996; Stephenson and Stempen 1994). In addition to these “true” fungi, there are three other groups of “fungus-like” organisms that have long been studied by mycologists. The first example is the group known as water molds, which have often been treated in the context of the true fungi as the phylum Oomycota although they actually belong to an entirely different kingdom (the Chromista). Water molds have a vegetative body consisting of hyphal-like filaments that superficially resemble the hyphae of fungi and they obtain their food in the same manner. However, water molds also possess a number of other features that indicate they are not closely related to fungi. The most important of these is that the cell wall contains cellulose-like compounds and not chitin. The slime molds, members of yet another kingdom (the Amoebozoa), are a second group of fungus-like organisms. Some slime molds produce fruiting structures similar to, albeit usually much smaller than, those of certain macrofungi. Other than this, slime molds share few other features in common with the true fungi. However, they have been traditionally studied along with fungi and are typically included in most mycology textbooks (Stephenson and Stempen 1994). The members of yet another group, the lichens, are more than just fungi. These organisms are fungi that have established a mutualistic symbiotic relationship with another organism (either an alga or cyanobacterium, or a combination of the two) that enables them to survive under conditions that could not be tolerated by the fungus alone. This “composite” organism is usually very different in appearance from what it would be with only the fungus present, and the fungal component might not be recognized as such (Stephenson 2010). Because some representatives of each of these three groups can be found in wetlands and the fact that mycologists have traditionally considered them along with the true fungi, they are considered herein.
The ability to recognize a particular fungus and then to assign it to the proper taxonomic group is, with rare exceptions, dependent upon features of the spores and fruiting structures produced by the fungus in question. Although the fruiting structure is macroscopic in some members of the Ascomycota and many members of the Basidiomycota, the vast majority of fungi produce spores on or within fruiting structures that are too small to be observed in any kind of detail without the use of a microscope. The Chytridiomycota and water molds produce motile, flagellated spores (called zoospores), but this type of spore is not found in any of the other groups. Even for those forms that produce macroscopic fruiting structures (usually referred to as fruiting bodies), the occurrence of the fungus itself in a wetland is often not immediately apparent when these structures are not present. This is because the vegetative body of most fungi is limited in extent, highly dispersed, or more or less completely immersed within a particular substrate. As such, surveys for these organisms are more likely to involve isolation from samples collected in nature and brought into the laboratory for analysis than by direct detection in the field (Stephenson 2010).
The total number of species of fungi and fungus-like organisms found on the Earth is not known, but it almost certainly exceeds one million and some estimates are appreciably higher (Hawksworth 2001). Since no more than about 100,000 species of fungi have been described to date, it follows that there are tremendous numbers of fungi yet to be discovered. It seems likely that many of these are associated with ecosystems (e.g., tropical forests) that only recently have been the focus of relatively intensive mycological studies. However, even in temperate regions of the world there are certain types of habitats that remain understudied, and among these are wetlands.
3.2 Fungi and Fungus-Like Organisms Found in Wetlands
All of the major groups of fungi and fungus-like organisms occur in wetlands, but some groups are much better represented than others, and particular examples are associated with only certain kinds of wetland situations. The primary role of the true fungi in all types of ecosystems in which they occur is to decompose dead plant material, but some fungi attack and live on or within other living plants, animals, or even other fungi. Fungi that decompose dead plant material are called saprotrophs, whereas those that feed on living hosts are called parasites if the host is harmed, but not killed and pathogens if their presence produces a condition (disease) that has the potential of resulting in the death of the host. The distinction between parasite and pathogen is not necessarily absolute, and a parasite may become a pathogen over time or under a different set of circumstances (Stephenson 2010). In addition, as discussed in more detail elsewhere in this chapter, fungi also form beneficial mutualistic associations with many vascular plants (e.g., mycorrhizae in roots and endophytes in other tissues) and certain types of algae and cyanobacteria.
3.2.2 Water Molds
The Glomeromycota, also known as the arbuscular mycorrhizal fungi, were once considered to be part of the Zygomycota. Members of this group form intimate relationships with vascular plants in which some of the fungal hyphae making up the organism live inside cells of the root, forming what is referred to as an endomycorrhizal association. The species of fungi involved in endomycorrhizal associations cannot survive without their plant host. Both the plant and the fungus benefit from the association. These fungi do not produce fruiting bodies and occur as non-septate hyphae growing inside root cells. Members of this group are not known to reproduce sexually, but they produce exceedingly large, multinucleate spores that often exceed 80 μm in diameter. Identification is based largely upon diagnostic features of these spores.
3.2.7 Slime Molds and Lichens
3.3 Ecological Roles of Fungi in Wetland Habitats
The primary ecological role of fungi in wetland habitats is to decompose dead plant material—both woody and herbaceous debris as well as bryophytes in those wetlands in which these organisms are present. As a group, fungi have the capability to decompose an incredible diversity of organic substrates, although a particular species may be limited to one or a few types of substrates. For example, fungi that decompose woody debris are not the same species that decompose either herbaceous debris or bryophytes. In some instances, a single species of fungus is confined to an even more restricted range of substrates. For example, some fungi decompose only the woody debris of conifers, while others are restricted to woody debris from angiosperms.
Decomposition of all types of woody debris is primarily accomplished by various ascomycetes and basidiomycetes. All of these fungi possess the enzymes required to degrade cellulose, but there are far fewer species (mostly basidiomycetes along with only a few ascomycetes) that have the capability of decomposing lignin (Hudson 1991).
Taxa of wood-decomposing fungi are often assigned to two categories on the basis of whether or not they can degrade both cellulose and lignin or just cellulose alone. The members of the first category (the so-called “white-rot” fungi) have the enzymes necessary to degrade both cellulose and lignin more or less simultaneously. The residual material that is left behind has a somewhat fibrous appearance and is very pale in color, looking as if it had been bleached. In contrast, wood degraded by members of the second group (the so-called “brown-rot” fungi) is brown in color and tends to be broken up into somewhat cuboidal fragments that quickly disintegrate into a powdery brown residue. In the both instances, the structural integrity of the wood is lost. Common and widespread examples of white-rot fungi are Trametes versicolor and Daldinia concentrica, whereas Fomitopsis pinicola and Laetiporus sulphureus are among the better known brown-rot fungi. The term “soft-rot” is sometimes applied to situations in which only the outermost layers of wood are subject to decay. Soft rots occur only in wood that has an unusually high level of moisture, which is often the case for woody substrates in wetlands. Most of the fungi involved are ascomycetes, with species of Chaetomium among the most common and best known. Only the cellulose of the wood is degraded by soft rot fungi (Stephenson 2010).
Leaves and other non-woody plant parts (e.g., fruits and seeds) represent an entirely different type of substrate than wood or bark. Considerable diversity exists for the leaves of plants, and what might be termed a “typical leaf” from a common and widespread angiosperm is markedly different from the needle-like leaf of many conifers. Moreover, angiosperm leaves vary from relatively “soft” and readily decomposed examples to those that are rather “tough” and fairly resistant to decomposition. The former would include the leaves of virtually all herbaceous plants, whereas the leaves of such plants as Rhododendron maximum and Kalmia latifolia would represent the latter category. Spores of fungi can reach a leaf while it is still attached, and the extent to which the leaf serves as a “spore trap” is related to such factors as its size, position on the tree, surface (smooth or hairy) and whether or not the spore lands when the leaf is moist. Studies have shown that numerous spores are already present on older leaves prior to leaf-fall. Once a leaf falls from a plant, it can be invaded by soil-inhabiting fungi or, if the leaf becomes submerged, various aquatic fungi. The fruiting bodies of certain basidiomycetes (e.g., species of Marasimus and Collybia) often occur in abundance on dead leaves, which simply reflects their biological role as litter-inhabiting saprotrophs. The fungi associated with fruits and seeds are not necessarily the same ones found on leaves. In some cases, a particular species may be restricted to the substrate represented by a certain type of fruit or seed (Stephenson 2010). Two examples are Mycena luteopallens, which occurs only on the husks of hickory (Carya) nuts and walnuts (Juglans), and Strobilurus conigenoides, a species found only on old fruits of magnolia (Magnolia).
An individual living plant is a complex and spatially diverse structure that represents a habitat that supports a diverse assemblage of fungi. Some of these fungi (termed epiphytes) colonize the surfaces of living leaves and stems, but others (termed endophytes) occupy internal tissues. Many of these (called foliar endophytes) occur inside leaves, whereas others are associated with stem tissues. It has been increasingly apparent that even a healthy plant has an assemblage of endophytes present, and there is considerable evidence that this situation is beneficial to the plant itself. For example, certain endophytes may increase the tolerance of the plant to the effects of temperature extremes and drought situations. Endophytes have been described as having biologically active secondary metabolites that serve to protect the plant against herbivory (Clay 1990) or have antibacterial or antifungal activity (Fisher et al. 1984). In return, the endophyte receives photosynthates from the plant. Many endophytic fungi are transmitted from one generation to the next by inoculating the seeds produced by the host plant. Interestingly, when the host plant dies, the endophytic fungi already present apparently play a role in the early stages of decomposition prior to the appearance of other species of fungi more typically associated with this process in the particular habitat where the plant occurs (Gessner et al. 2007; Van Ryckegem et al. 2007). Studies specifically directed towards the endophytes of wetland plants appear to be lacking, but these fungi, most of which are ascomycetes, have received considerable study in some groups of plants (e.g., grasses) that are not uncommon in some types of wetlands.
Some fungi form a symbiotic relationship with the roots of trees and other plants. This relationship, which is called a mycorrhizal association, is mutually beneficial to both the plant and the fungus. The fungus enables the plant to take up nutrients that would otherwise be unavailable, and the plant provides nutrition for the fungus. The majority of plants on Earth are involved in these associations. In some instances, the mycorrhizal association is so essential to the plant that the latter would not survive without its fungal partner. There are two fundamentally different types of mycorrhizal associations—ectomycorrhizal (usually involving a basidiomycete) and endomycorrhizal (most often involving a member of the Glomeromycota). In the former, the fungus produces a covering of hyphae (called a sheath or mantle) around the outside of smaller rootlets of the host plant. Other hyphae invade the cortex of the rootlet but do not disrupt the individual cells. In endomycorrhizal associations, no sheath is formed and hyphae of the fungus actually invade cells of the cortex of the rootlet. Perhaps 80 % of all vascular plants form mycorrhizal associations with fungi. Endomycorrhizal associations are predominant, but many of the trees (e.g., oak [Quercus], beech [Fagus], willow [Salix], spruce [Picea] and fir [Abies]) that are important in temperate and boreal regions of the Northern Hemisphere are ectomycorrhizal. However, the fungi that form ectomycorrhizal associations do not normally survive in areas that are permanently flooded (Bauer et al. 2003; Jurgensen et al. 1997), which limits their occurrence in wetlands. Moreover, saturated soil conditions, such as those characteristic of wetlands, also have been reported to restrict the growth of endomycorrhizal fungi, most likely as a result of low oxygen levels (Slankis 1973). Nevertheless, endomycorrhizal fungi are known to be present in many types of wetlands (Hoewyk et al. 2001; Rickerl et al. 1994) and certainly play important roles in the growth of numerous species of wetland plants (Dunham et al. 2003).
The various types of ecological associations that exist for fungi and plants in wetland habitats are most apparent for vascular plants, although bryophytes also must be considered in those instances in which at least some representatives of the group are present. Although both chytrids and water molds commonly colonize dead parts of vascular plants that are introduced into the aquatic habitats where they occur and these organisms are easily isolated in laboratory culture from this type of material (e.g., plant pollen is a standard “bait” for chytrids), they are ecologically more important for their roles as parasites of phytoplankton (chytrids) and primary colonizers of the bodies of aquatic invertebrate animals (water molds). Chytrids are often surprisingly abundant on filamentous algae and diatoms, and some species are known to severely deplete local populations of their algal hosts (Webster and Weber 2007). Water molds, including species belonging to the common and widespread genera Saprolegnia and Achlya, quickly colonize the bodies of aquatic insects (Dick 1970) and other invertebrates. In addition, some species of both chytrids and water molds are parasites of larger animals found in aquatic habitats, including crayfish and fish. In some instances, their ecological impact can be considerable.
3.4 Wetlands as a General Habitat for Fungi
Fungi are primarily terrestrial organisms, but numerous species can be found in wetlands. Some of these are aquatic forms while others are restricted to substrates that are above water level. For the most part, fungi are aerobic organisms, and they do not thrive in situations where an oxygen deficit develops. Interestingly, some otherwise terrestrial fungi are able to survive in aquatic habitats, but they do not complete their life cycle, at least what is known of it, in water. By definition, truly aquatic forms normally do complete their entire life cycle in the aquatic habitats in which they are found. For the majority of these, their whole evolutionary history seems to have been in water (Hudson 1991). They possess motile zoospores as the primary unit of dispersal and the latter function only in an environment with water present. As already noted, both chytrids and the fungus-like water molds share this feature.
More fungi are associated with forested wetlands (pocosins, swamps, bottomland hardwood communities, glades and bogs) than non-forested freshwater and saltwater marshes. This is a direct result of the greater substrate heterogeneity in wetland situations with trees and other woody plants present. In other words, the wider the range of available substrates, the larger the number of fungi likely to be present. Few fungi can tolerate salt water, which limits their occurrence in saltwater marshes. In acidic mountain bogs, the low pH represents another limiting factor for fungi, either directly on the fungi themselves or indirectly as a result of the constraints it places on the other organisms that cannot survive under conditions of low pH. Such wetlands often contain rare species of plants and animals (Grafton and Eye 1982). The same situation is likely to be true for fungi, but the fungi of acidic mountain bogs are understudied.
Regardless of the type of wetland being considered, the numbers and types of fungi present are determined largely by the type and nature of the substrates available, the relative abundance of those substrates and their condition (i.e., whether submerged or not, living or dead, and the stage of decomposition when the latter is the case). For example, the fungi involved in the decomposition of coarse woody debris are generally not the same as those associated with twigs derived from the same plant, and the fungi found on submerged woody debris are different from those that occur on the same type of woody debris located above the water level. Moreover, as already noted, the assemblages of fungi that decompose woody debris from conifers—which are important in some mountain bogs—and angiosperm trees are often quite different.
3.5 Fungi and Fungus-Like Organisms Associated with the Different Microhabitats Found in Wetlands
The composition and structure of the vegetation and thus the potential range of plant hosts available and the ultimate sources of the input of dead plant material vary considerably for different types of wetlands (Ellis and Chester 1989; Richardson and Gibbons 1993; Sharitz and Mitsch 1993). For example, Rentch and Anderson (2006) listed more than 1,700 species of vascular plants for wetland and riparian habitats in West Virginia. Approximately 38 % of these were plants that usually to almost always occur in such habitats. Obviously, only a smaller subset of this total would be found in a particular habitat, but even then the number of species present can be impressive. For example, Brown (1982) recorded 281 species for a swamp in western Maryland.
Many of the fungal parasites or pathogens associated with living plants are rather host specific, sometimes to the point of being restricted to a single species or genus. For example, this is usually the case for two groups of basidiomycetes, the rust fungi and the smut fungi. Members of the latter group are particularly common on grasses and sedges, both of which are common wetland plants. Saprotrophic microfungi associated with living plants tend to be much less host specific, with the same species potentially occurring on a wide range of different hosts. Conceivably, the greater the biodiversity of potential host species in a particular wetland, the greater the biodiversity of fungi associated with these hosts. The majority of saprotrophic microfungi found on living plants are ascomycetes.
Once the plant is no longer alive, it represents a source of material that is subject to decomposition, and the nature of the material itself is the main factor determining just what fungi are involved. Ascomycetes are the most important group for both submerged and emergent nonwoody substrates as well as submerged woody substrates, whereas basidiomycetes also play an important role for emergent woody substrates. Zygomycetes commonly occur on nonwoody substrates in moist microhabitats such as at the margin of a shallow pool, chytrids and water molds can colonize submerged nonwoody substrates, and both slime molds and lichens are sometimes found on emergent woody substrates. Slime molds occasionally occur on emergent nonwoody substrates but are more likely to be found on both nonwoody and woody substrates in moist microhabitats. As mentioned earlier, neither slime molds nor lichens are decomposers. However, the vegetative stage in the slime mold life cycle feeds primarily upon bacteria, which are abundant in all types of dead plant material except for acidic bogs, where the low pH is a limiting factor.
Although they receive much less attention than the fungi that decompose wood or litter, there is a group of fungi which decompose bryophytes. In those wetlands where bryophytes are abundant, such as in many mountain bogs dominated by Sphagnum mosses, it would be a mistake to dismiss the ecological importance of these bryophilous (“moss-loving”) fungi. Thormann and Rice (2007) summarized the literature on the fungi associated with peatlands and reported that 601 species of fungi have been identified from such habitats. It has been proposed that fungi are the dominant microbial decomposers (even more so than bacteria) in relatively acidic situations such as Sphagnum-dominated bogs (Andersen et al. 2006). For example, 55 species of fungi were reported to be associated with Sphagnum fuscum from one bog sampled in Alberta, Canada (Thormann et al. 2001). The group of bryophilous fungi includes a number of species of basidiomycetes, ascomycetes, and zygomycetes, but most examples produce fruiting bodies that are relatively small and thus not easy to spot in the field. Among the more common bryophilous fungi are species in the genus Galerina, whose fruiting bodies often occur in small clusters on mats of bryophytes (Fig. 3.5). The bryophilous fungi are another group that has received relatively little study.
The yeasts are among the most common of all fungi in nature. Both the ascomycetes and basidiomycetes include taxa that are considered as yeasts, but the best known yeasts are ascomycetes. By definition, yeasts are fungi that are unicellular and reproduce by budding. Budding is a method of asexual reproduction, and some yeasts appear to have lost the capability for sexual reproduction. However, many other yeasts do reproduce sexually, although sometimes only rarely. Yeasts are exceedingly common on the surfaces of living plants, and they also can be found in soil and water. However, relatively few studies have been directed towards the occurrence of yeasts in wetland habitats, although there is little doubt that these organisms are associated with every available type of substrate present. Thormann et al. (2007) summarized the information available on yeasts in peatlands and indicated that 75 species had been reported as associated with this habitat. It appears that these yeasts play an important role in the initial decomposition of plant debris by feeding upon the simple polymers that leach out of dead and dying plants.
3.6 General Methods/Techniques Used to Sample the Various Groups of Fungi
The vast majority of fungi are microscopic. Although some basidiomycetes and a few ascomycetes produce macroscopic fruiting structures, these structures tend to be rather ephemeral in nature. Only the fungus-like lichens are characterized by vegetative structures that persist for a considerable period of time. As such, sampling for fungi is a real challenge, and the methods and/or techniques used for one group are rarely appropriate for other groups. Prior to initiating any field survey for the fruiting structures of ascomycetes, basidiomycetes, slime molds or lichens, an investigator should conduct some preliminary background work on the system to be studied. This would involve such things as assessing the types and relative abundance of the each of the various substrates present along with determining the most appropriate conditions (e.g., immediately after a period of rainy weather) for carrying out field surveys. Having some knowledge of what to look for, where to look, and how to actually collect any specimens that turn up as a result of the collecting effort are absolutely essential.
Temperature and rainfall are the major factors that determine when macrofungi produce fruiting bodies in nature. The latter is relatively less important in wetlands, where moist conditions are not necessarily dependent solely upon periods of rainfall. Nevertheless, the occurrence of fruiting bodies is affected by the amount of moisture in the soil or the substrate within which the mycelium of the fungus occurs, which can range from saturated to dry, depending upon the type of wetland and the time of year. If the only objective is to determine what species occur in a particular area of wetland, then obtaining good baseline data may involve intensive collecting every 1–2 weeks throughout the fruiting season in order to maximize the number of species collected. It is widely known that a particular species of fungus may not produce fruiting bodies every year. This underscores the need for long-term (at least several years) studies, which may not be practical in every situation, to document most of the species for the area being investigated. In many instances, especially when surveying larger areas, the opportunistic sampling protocol described by Cannon and Sutton (2004) is appropriate. This method simply involves walking through the entire area in a random fashion to maximize the probability of finding fruiting bodies of most of the species present at the time sampling is being carried. Otherwise, a plot can be delimited and the sampling effort confined to just the area within the plot. This allows the calculation of abundance measures for the fruiting bodies of the various macrofungi present. Moreover, the use of a plot is especially appropriate when the fruiting bodies are so small as to be easily overlooked and thus require a more intensive examination (often on the hands and knees) to detect. This is often the case for some of the fruiting bodies of the species of Galerina that are associated with mats of bryophytes or the small cup fungi that are found on the surface of moist soil. Since the advent of digital photography, it is often standard practice to document field-collected fruiting bodies of macrofungi with images, which can be invaluable in making identifications (e.g., when comparing a particular fruiting body with the illustrations available in most field guides and taxonomic monographs).
Except for those few basidiomycetes and ascomycetes that produce fruiting bodies that are tough, leathery or woody, which is case for certain species (e.g., most polypores, some corticoid fungi and many members of the Xylariaceae) that occur on decaying wood, field-collected fruiting bodies should be dried to preserve many of the features considered when making an identification. Prior to drying, it is exceedingly useful to prepare a spore print (which reveals the color of the spores in mass) for those fungi (mostly agarics and boletes) for which this is possible. This simply involves placing the cap (or a section of the cap) on a piece of paper, covering this under a cup, bowl or other container in order to maintain moist conditions, and allowing it to remain in place for several hours (usually overnight). Spores that fall from the cap produce the spore print. Afterwards, the fruiting bodies should be dried by placing them on some sort of drying apparatus. Commercial food dehydrators are usually the best option. Larger fruiting bodies should be split in half from top to bottom before being placed on the dryer. This practice speeds the drying process, prevents the interior of the fruiting body from decaying and stops the feeding activities of any insect larvae that might be present (Lodge et al. 2004). Once dry, fruiting bodies can be placed in small paper boxes, paper bags or plastic bags for permanent storage.
As mentioned in the introductory information provided for chytrids and water molds, the most common method of sampling for this group of organisms involves the use of baits, either directly in the field or under laboratory conditions. The former involves placing suitable baits in the water, allowing them to remain in place for several days, and returning the baits to the laboratory. In order to confine the items used as baits, they are usually placed in some sort of trap (e.g., a small-mesh wire cage or perforated plastic basket), which is suspended in the water by a piece of nylon cord. Some of the more commonly used baits include boiled hemp (Cannabis) seeds, dead insects, pieces of fruits (e.g., pear or apple), hair, and small pieces of cellophane (Bruns 2006; Stevens 1974). The length of time the baits are left in place will depend upon conditions, especially the temperature of the water. However, a period ranging from several days to a week is usually sufficient during most of the year. In winter, it may be best to recollect the baits after a somewhat longer period of 10 days to 2 weeks. Once the baits have been collected from the water, they are transported to the laboratory and kept under cool conditions (no more than 20–25 °C) until examined for the presence of both chytrids and water molds. Conversely, it is possible to collect water molds directly in the field by examining substrates upon which they are likely to be present. Examples include floating or submerged small animals (e.g., insects) surrounded by an obvious halo of white threads. Water molds are usually easy to recognize from their coarse, stiff and radiating hyphal-like structures. Chytrids are too small to be spotted in the field but can be found when filaments of algae are collected from the water, brought back to the laboratory and examined under a compound microscope.
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