Johana Hallett, Amy Moyer, and Jonathan Burgos. BIOTECH

Using cladistic analysis of twenty weighted, non-ordered characters, we examined twenty-four genera within the kingdom Fungi. We chose twenty genera belonging to the phyla Ascomycota and Lichenomycota and four outgroup genera from the phyla Zygomycota and Basidiomycota. By utilizing our researched characters, we were able to construct a phylogram from which a taxonomy was deduced. From our cladogram and phylogram, a single clade illustrates that the lichen fungi and ascomycetes should be placed in one single phylum. We concluded that the researched characters such as sexual spore type, photosynthesis, and saprophytic nature greatly support the unification of the phylum Lichenomycota and the phylum Ascomycota.

"At least one-quarter of all described fungi, an estimated 12,000 to 20,000 species, can enter symbiotic association with photosynthetic microbes such as algae to form lichens" (Margulis and Schwartz, 1998). The relationship between these lichen fungi and lichen algae has perplexed scientists for many years concerning their proper placement within the classification system. Holt (1992) and Desimone et al. (1996) place the lichen fungi into a separate phylum. Holt (1992) places the lichen into a form-phylum, Lichenomycota, and separates these fungi from the ascomycetes, which are placed in the phylum Ascomycota. Holt (1992) and Desimone et al. (1996) believe that the lichen fungi are unique in comparison with the other ascomycetes because of the general inability of these organisms to survive without the benefits of a symbiotic relationship with algae. However, Margulis and Schwartz (1998) and Blackwell et al. (1993) have chosen to place the lichen fungi into the phylum Ascomycota, because of the similar symbiotic relationship that can be formed between other ascomycetes and algae.

Many scientists believe that the relationship between lichen fungi and lichen algae evolved from a common need to survive (Margulis and Schwartz, 1998). The two organisms, fungi and algae, eventually became dependent upon each other for survival. Laboratory research has proven that the lichen components can rarely be grown individually. The dependency of lichen components upon one another caused confusion within the scientific community in regard to the identification of this organism. Scientists formally believed that lichen were a mass of inseparable organisms, but upon closer examination, the symbiotic relationship between the fungi and algae was discovered (Margulis and Schwartz, 1998; Bold et al., 1987).

Lichen are prevalent in a wide range of environments such as the Antarctic, the Arctic tundra, high mountains, and the tropics. Within the aforementioned environments, the lichen may be found growing on rock and tree bark (Bold et al., 1987; Hawksworth, 1984; Margulis and Schwartz, 1998). These organisms are also the first to invade newly exposed volcanic regions. The fungi component of lichen can be characterized by the presence of a particular fruiting body, such as an ascocarp or basidiocarp (Margulis and Schwartz, 1998; Griffin, 1981). However, this classification is problematic because of the different fruiting bodies of the lichen fungi and their free-living relatives. The majority of lichen fungi are ascomycetes and bear an ascocarp. Others classify lichen fungi by their external appearance of the thallus, which may be crusty, leafy, or bushy. Another feature of the lichen includes the similarities between the sexual reproduction to that of the ascomycetes (Margulis and Schwartz, 1998; Ross, 1979). The asexual reproduction of these lichen involves the production of conidia similar to those of the ascomycetes. Lichen produce compounds, such as lichenic acids and pigments, which are absent when the algae and fungi are independent of one another (Bold et al., 1987; Margulis and Schwartz, 1998). Chitin and glucan are the two main components of the cell wall. In addition to these characteristics, lichen fungi are the only fungi able to utilize photosynthesis.

Phylum Ascomycota, as classified by Margulis and Schwartz (1998) and Blackwell et al. (1993), not only includes lichen but also yeast, blue-green mold, truffles and morels (Margulis and Schwartz, 1998; Bessy, 1953). The ascomycetes are similar to many of the lichen in that both contain ascocarps as their method of sexual reproduction, and their asexual reproduction utilizes conidia. Another similarity between these two phyla is the composition of the cell wall, which contains chitin and glucan. Ascomycetes and lichen fungi are also saprophitic, meaning these organisms absorb dead organic material from their surroundings. The prevalence of ascomycetes living symbiotically on plants similar to lichen fungi serves as yet another unifying trait. All of these characters unify the lichen and the ascomycetes, which is why Margulis and Schwartz (1998), Blackwell et al. (1993), and we have chosen to place these fungi in the same phylum, Ascomycota.

Organisms Examined-

We examined 20 different genera (Table 1) representing the phylum Ascomycota, and the form-phylum Lichenomycota from the taxonomy of Holt (1992). The four genera from the phyla Zygomycota (Rhizopus and Entomophthora) and Basidiomycota (Coprinus and Geastrum) were used as outgroups for further comparison. These genera were selected from a multitude of sources listed in Table 1 and were utilized in our cladistic analysis of the lichen fungi and ascomycetes.

Table 1. The table lists the 24 genera used in our cladistic analysis and their respective phyla taken from the taxonomies of Holt (1992) and Margulis and Schwartz (1998). The four outgroup genera are identified by an asterisk. The following sources were used in our research and the abbreviation Obs signifies the organisms that we observed: Bessy, 1953 (B); Bold et al., 1987 (Bo); Dix, 1995 (D); Griffin, 1981 (G); Hawksworth, 1984 (H); Holt, 1992 (Ho); Landecker, 1992 (L); Margulis and Schwartz, 1998 (M); Moore et. al, 1985 (Mo); Ross, 1979 (R).

Characters Examined-

We chose 20 characters and their relative states (Table 2) for our cladistic analysis of the 24 genera listed in Table 1. No character was ordered, but many were weighted. The characters we deemed unimportant in the comparison of genera were assigned a weight of zero. The trivial characters include the presence of hyphae, hymenium, and fruiting bodies. A weight of one was given to the intermediate characters, and the characters we considered most important were assigned a weight of two, such as type of sexual spore, photosynthesis, and the saprophitic nature of these organisms.

Table 2. This table displays a list of characters and their respective character states. The character states provide the data for our cladogram and phylogram represented in Figures 1 and 2. The unordered characters were weighted according to their relative importance. We used the following sources in our research: Bessy, 1953 (B); Bold et al., 1987 (Bo); Dix, 1995 (D); Griffin, 1981 (G); Hawksworth, 1984 (H); Holt, 1992 (Ho); Landecker, 1992 (L); Margulis and Schwartz, 1998 (M); Moore et. al, 1985 (Mo); Ross, 1979 (R).

The cladogram was constructed using MacClade 3.07 (Maddison and Maddison, 1992). Figure 1 is a cladogram illustrating the relationship between the phyla Ascomycota and Lichenomycota (Holt, 1992). The maximum tree length was 153 while the minimum tree length was 78. The lowest tree length was reached by the manipulation of the tree branches. Figure 2 represents our proposed phylogram showing the evolutionary trends of the researched genera.

Figure 1 represents a cladogram of our proposed taxonomy, which illustrates the addition of the form-phylum Lichenomycota into the phylum Ascomycota (Holt, 1992). The hypothetical minimum tree length is 78 and the maximum is 153. By the manipulation of the tree branches, our proposed cladogram has a tree length of 104. The first clade on our cladogram to the left contains the genera from two of our outgroups, Geastrum and Coprinus, which represent the phylum Basidiomycota. The second clade includes the phylum Zygomycota with the genera Entomophthora and Rhizopus. The largest clade consists of lichen and ascomycetes and represents our proposed phylum Ascomycota.

Figure 2 represents our proposed phylogram of the newly formed phylum Ascomycota. The phylogram divides into two major clades with our outgroups represented to the left of the phylogram. The genera from the outgroup phylum Basidiomycota, Geastrum and Coprinus, appear at the base of the phylogram. The genera from the phylum Zygomycota, Entomophthora and Rhizopus, are present in the first small clade. The large clade located to the right side of the phylogram represents our proposed phylum Ascomycota, which includes the lichen and ascomycetes. The second group is divided into two classes: Ascomycetes and Lichenomycetes. The class Ascomycetes has one order, Ascomycales, which is the first division of our phylogram. It includes genera such as Peziza, Hysterium, Neurospora, Saccharomycetes. The class Lichenomycetes has two orders: Ascolichales and Basidiolichales. The order Ascolichales is represented by the second and third divisions of our phylogram and includes Lepraria, Calicium,Cladonia, and Dermatina. The order Basidiolichales is represented by the last division and includes Omphalina, Cora and Pyrenula.

Figure 1. Our cladogram showing the phylum Lynchenomycota in the phylum Ascomycota and our four outgroups. The actual tree length is 104, with a minimum tree length of 78 and a maximum of 153. The cladogram has one distinct clade of ascomycetes and lichen and two clades for the outgroup phyla Zygomycota and Basidiomycota. The largest clade represents our proposed phylum Ascomycota. The cladogram was constructed with the use of 20 non-ordered, weighted characters, the 24 genera listed in Tables 1 and 2 and MacClade 3.07 (Maddison and Maddison, 1992).

Figure 2. Our phylogram showing the evolutionary relationship of our proposed phylum Ascomycota based on the cladogram shown in Figure 1. The actual tree length is 104, with a minimum tree length of 78 and a maximum of 153. The phylogram displays two distinct clades and two outgroups from phylum Basidiomycota are located along the base. The first small clade represents the other two outgroups from the phylum Zygomycota. The largest clade represents the ascomycetes and lichen in our proposed phylum Ascomycota. The phylogram was constructed with the use of MacClade 3.07 (Maddison and Maddison, 1992). The scale located to the right of the phylogram represents the number of evolutionary changes.

The goal of this cladistic analysis was to study the relationship between the lichen fungi and ascomycetes, and to arrive at a unification of the two groups. Margulis and Schwartz (1998) and Blackwell et al. (1993) place the lichen into the phylum Ascomycota, while Holt (1992) and Desimone et al. (1996) choose to separate the ascomycetes from the lichen, placing the lichen into their own form-phyla, Lichenomycota (Holt, 1992). Using the information gathered from our phylogram and cladogram (Figure 1 and 2), we have decided to unite the ascomycetes and lichen into one phylum Ascomycota, as Margulis and Schwartz (1998) and Blackwell et al. (1993) have done.

According to Figure 2, there were two main lines of evolution, with our outgroup genera, Geastrum and Coprinus, from the phylum Basidiomycota being unresolved. The first clade represents the two outgroup genera, Entomophthora and Rhizopus, from the phylum Zygomycota. The larger clade represents our proposed phylum Ascomycota. At the right base of the phylogram, the genera Elsinoe is found, which is unresolved. The first grouping of the large clade are the ascomycetes, containing genera such as Peziza, Hysterium, Neurospora, Taphrina, and Saccharomyces. The clade further divides into two groups of lichen bearing ascocarps such as Lepraria, Opegrapha, Calicium, Cladonia, and Parmelia. The final division includes the lichen bearing basidiocarps such as Omphalina, Cora, and Pyrenula, which are the most derived genera of our proposed phylum Ascomycota. Within our proposed phylum, Pyrenula is the most derived genera.

Our phylogram suggests that the ancestral lichen was closely related to the ascomycetes, and as they evolved, they formed a symbiotic relationship with algae. The lichen fungi and lichen algae formed this relationship as a means of survival, and eventually they became dependent on each other. Our phylogram supports this evolutionary trend by illustrating that a non-symbiotic ascomycetes resembles the ancestral lichen fungi. As our phylogram continues, the ability of some ascomycetes to have symbiotic relationships with algae is evolved, and eventually the lichen fungi enter into an inseparable symbiotic relationship with algae.

Another evolutionary trend is the ability to photosynthesize. Since the ancestral fungus is closely related to ascomycetes, a non-photosynthetic fungus, it is more likely that the ancestor was also non-photosynthetic. As these fungi evolved, the ability to photosynthesize was obtained, as can be seen by the increase in the number of changes from the ascomycetes to the lichen fungi.

The type of sexual spore is another evolutionary trend in our phylogram. The ancestral fungus most likely utilized an ascocarp for sexual reproduction, as does the ascomycetes. The next section in our phylogram contains the ascomycetes, which also bear an ascocarp most likely resembling the sexual spore of the ancestor. The next two divisions of lichen also bear ascocarp, but they are not identical to those of the ascomycetes. The last section includes the lichens, which have a basidiocarp.

Some of the researched characteristics have shown no evolutionary trends. These characters include saprophitic nature, cell organization, the presence of hyphae, and thallus.

According to Figure 1, there are three distinct clades in our cladogram. The first two clades represent our outgroup genera, Geastrum and Coprinus, from the phylum Basidiomycota and Entomophthora and Rhizopus from the phylum Zygomycota. The larger clade represents our proposed phylum Ascomycota, which includes the lichen and the ascomycetes. The clade moves from the lichen, located to the left, to the ascomycetes, located to the right. The lichen fungi begin with Pyrenula and extend to the genus Lepraria. The ascomycetes begin with Saccharomyces and lead to Elsinoe.

In reference to the character traits in Table 2 and the phylogram in Figure 2, the lichen are closely related to the ascomycetes in various aspects. A single, unifying character among the two phyla is their saprophitic nature. Both the lichen and the ascomycetes thrive by the absorption of dead organic material from their surroundings. This saprophitic nature is characterized by the secretion of enzymes that break down the organic material, which can later be absorbed by the fungus. This saphrophitic nature unifies the lichen and the ascomycetes into our newly proposed phylum Ascomycota.

Another unifying character is the presence of an ascocarp in the ascomycetes and in the majority of the lichens. The ascocarp serves as the means of sexual reproduction in which spores are formed. The only genera that do not follow this trend are the lichen bearing basidiocarps, such as Omphalina, Cora, and Pyrenula.

Photosynthesis is another unifying character, which creates our classes by separating the non-photosynthetic fungi from the photosynthetic fungi. This trend is not evident in the cladogram (Figure 1), but it is clearly illustrated in the phylogram (Figure 2). The non-photosynthetic fungi, the ascomycetes are placed in the class Ascomycetes. This class has a single order Ascomycales. The photosynthetic fungi, or lichen, are placed in the class Lichenomycetes. This class is then divided into two orders: Ascolichales and Basidiolichales. The order Ascolichales includes all the lichen fungi that bear an ascocarp, while the order Basidiolichales includes the lichen fungi bearing a basidiocarp, as illustrated in Table 3.

As our proposed taxonomy illustrates (Table 3), we chose to unify the lichen fungi and ascomycetes into one phylum, Ascomycota. Margulis and Schwartz (1998) and Blackwell et al. (1993) support such unification, because of the aforementioned similarities between these two groups. Holt (1992) and Desimone et al. (1996) feel that the lichen fungi are unique in comparison with the other ascomycetes because of the general inability of these organisms to survive without the benefits of a symbiotic relationship with algae.

Table 3: Our proposed taxonomy uniting the lichen fungi with the ascomycetes and the taxonomy of Holt (1992).

Phylum Basidiomycota

Geastrum, Coprinus

Phylum Zygomycota

Entomophthora, Rhyzopus

Phylum Ascomycota

Class Ascomycetes

Order Ascomycales

Class Lichenomycetes

Order Ascolichales

Order Basidiolichales

Omphalina, Cora, Pyrenula

Phylum Ascomycota

Form-Phylum Lichenomycota

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