15.5 Anaerobic fungi and the rise of the ruminants
We point out above Schultz & Brady’s (2008) statement that agriculture is a specialised symbiosis that has evolved in the four animal groups: ants, termites, bark beetles, and humans. So far we have dealt with ants, termites and bark beetles, and though we do not intend to deal at any length with human agriculture most of the rest of our topics do have some relevance to farms and agriculture. In this section we will expand on the symbiotic association between chytrid fungi and ruminant mammals. This is crucial to human agriculture because the diet of grazing farm animals, consisting predominantly of plant structural carbohydrates such as lignocellulose, cellulose and hemicellulose, can only be digested because the animals have evolved to rely on symbiotic microorganisms in their alimentary tracts to hydrolyse these compounds under the anaerobic conditions that prevail in the gastrointestinal tract.
Ruminants are well adapted to achieve maximum digestion of the otherwise indigestible fibrous components of plant foods within the rumen. The most characteristic behaviour pattern of all ruminants is the regurgitation, rechewing and reswallowing of partially fermented food from the foregut, which is termed rumination. Foregut fermenting mammals also produce two enzymes, stomach lysozyme and pancreatic ribonuclease which accompany and are adaptations to this mode of digestion. The microbial community of the ruminant gastrointestinal tract contains the full range of microbes: bacteria and bacteriophage, archaea, ciliate protozoa, and anaerobic fungi; and all these at characteristically high species diversity and population densities, and exhibiting complex interactions (Mackie, 2002).
We will concentrate on just one component of this complex community: the anaerobic chytrid fungi. These fungi are not exclusively found in the rumen of the animals, but throughout the entire digestive tract. Further, anaerobic fungi have also been recovered from the faeces of ruminants, suggesting that the fungi have the ability to enter a resistant stage enabling them to survive desiccation and the oxygenated atmosphere.
Through to the middle of the twentieth century it was commonly assumed that all fungi required oxygen to survive, which lead to the view that the microbial population of the rumen consisted primarily of anaerobic bacteria and flagellate protozoa. Indeed, in his 1949 book Chemical Activities of Fungi Jackson W. Foster made one of those decisive statementsthat hindsight proves so decisively wrong that it is repeated regularly:
‘the major metabolic differences between molds and bacteria is that there are no anaerobic molds, either obligate or facultative’ (Foster, 1949).
This view held until 1975, when the rumen ‘flagellate’, Neocallimastix frontalis was properly described as a Chytridiomycete fungus even though it was a strict anaerobe (Orpin, 1975).
Chytrids are an ancient group of true fungi (see Section 3.2 entitled The Chytrids in Chapter 3; CLICK HERE to view the page). They usually produce uniflagellate zoospores, but some of the obligately anaerobic chytrids produce bi- or multiflagellated zoospores. All chytrid flagella are of the whiplash type, lacking hairs or scales, and are located posteriorly on the zoospore. Chytrids are usually described as aquatic organisms, but in fact they are equally abundant in terrestrial environments, and have even been isolated from sand from arid canyons in Arizona, and from the permafrost in the Arctic.
Chytrids are an ancient group of true fungi (see Section 3.2). They usually produce uniflagellate zoospores, but some of the obligately anaerobic chytrids produce bi- or multiflagellated zoospores. All chytrid flagella are of the whiplash type, lacking hairs or scales, and are located posteriorly on the zoospore. Chytrids are usually described as aquatic organisms, but in fact they are equally abundant in terrestrial environments, and have even been isolated from sand from arid canyons in Arizona, and from the permafrost in the Arctic. Chytrids are saprotrophs or parasites and their real importance lies in their ecological role as decomposers able to digest complex polymers such as cellulose, hemicellulose, chitin, and keratin, as well as some of the most recalcitrant materials in the biological world such as lignin and sporopollenin (a complex, highly cross-linked polymer found in the outer wall layers of pollen grains and some fungal spores). As parasites, they exist on/in a wide range of hosts including algae, other fungi, plants, mosses, insects and invertebrates; the first chytrid parasite of a vertebrate, Batrachochytrium dendrobatidis, has been found parasitising and killing amphibians. Chytrids are found throughout the world, the majority (80%) in temperate regions of the world, although this relative abundance of chytrids is most likely due to biased collection from these regions; that is chytrids have been under-collected in tropical and polar regions (Shearer et al., 2007).
Although they are morphologically similar to other chytrids, differences are sufficient for the anaerobic chytrids to be placed in their own phylum, which is called Neocallimastigomycota (see the section entitled Neocallimastigomycota in Section 3.2 and the photographs of Neocallimastix on that page; CLICK HERE to view the page). They appear to be among the most primitive of all the fungal phyla; the Neocallimastigomycota being the earliest diverging lineage (so the lineage originated long, long before its current hosts, ruminant herbivores, appeared on the geological scene) as a sister group to the rest of the Chytridiomycota.
Six genera have been described among rumen fungi, on the basis of number of flagella and sporangial characters, and all are placed in the Order Neocallimastigales. No sexual stage is known. Anaerobic chytrids may be monocentric (having one centre of growth, producing either a single or several sporangia) or polycentric (having several centres of growth), and the sporangia may have filamentous or bulbous rhizoids and produce multiflagellate or uniflagellate zoospores. The six genera described are: Neocallimastix, Piromyces, Caecomyces, Anaeromyces, Orpinomyces and Cyllamyces (Ho & Barr, 1995, Ozkose et al., 2001). Neocallimastix frontalis was the original isolate from the domestic cow and Piromyces spp. have been isolated from horses and elephants.
Rumen chytrids are the primary invaders of freshly ingested plant material in the rumen and, overall, the rumen chytrid biomass can amount to about 20% of the total rumen microbial biomass (Rezaeian, Beakes & Parker, 2004a & b). Zoospores alight on plant fragments and encyst, forming a thallus with a well-developed rhizoidal system that penetrates the plant material to extract energy by fermentation of its carbohydrate and other polymers within the animal’s rumen and intestine. The nucleus of the zoospore is retained in the cyst as it develops into a sporangium, which is cut off from the anucleate rhizomycelium by a septum. Protoplasm in the sporangium is cleaved into uninucleate zoospore initials; eventually, zoospores (with up to 16 flagella per zoospore in Neocallimastix frontalis) are formed in the sporangium and eventually released from the apex of the sporangium into the surrounding fluid. N. frontalis is obligately anaerobic and grows on fragments of grass in the rumen of cattle, sheep (see illustrations in Rezaeian et al., 2004a & b) and other herbivorous animals including water buffalo, goat and deer.
When grown in culture N. frontalis forms an extensive rhizomycelium. The fungal mode of growth is what makes the role of the chytrids so crucial. The filamentous rhizoids extend into the plant material secreting the array of enzymes needed to degrade cellulose (animals do not produce their own cellulose-degrading enzymes) and other polymers of the fragments of plant material that make up the herbivore’s food.
The rumen is a dynamic habitat, nutrient rich and oxygen poor. The pH is continuously modified by the host’s diet, the metabolic activity of the resident microorganisms and by the tissues of the host. Anaerobic fungi are deficient in mitochondria, and so unable to produce energy by aerobic respiration. Instead, they possess hydrogenosomes that allow a mixed acid fermentation of carbohydrate to be carried out. As a result of mixed acid fermentation hexose and pentose sugars are converted to formate, acetate, lactate, and ethanol which the organelle converts to energy in the form of ATP, CO2 and hydrogen by producing pyruvate oxidoreductase and hydrogenase.
Subsequently, methanogenic bacteria convert the excess H2 into methane, which is expelled from both ends of the animal. The fungi also produce a wide range of digestive enzymes, giving them broad substrate specificity, and enabling the fungi to transform the core structural polymers of plant cell walls into a variety of simple oligosaccharides, disaccharides, monosaccharides, amino acids, fatty acids, etc., which enter mixed acid fermentation to create energy resources, and other aspects of anabolism that contribute to cell growth, reproduction and population growth. Microbial growth is eventually passed onto the host’s stomach where digestion by the animal makes it available as a source of nutrients and energy (Trinci et al., 1994; van der Giezen, 2002).
The microorganisms within the rumen form both co-operative and competitive interactions, producing a complex ecosystem. Some of the interactions are purely competitive. Ciliate protozoa are a major component of the rumen microflora. Ciliates ingest fungal zoospores as well as bacteria, and their predation of the fungal population can reduce overall cellulolytic activity. Bacteria like Ruminococcus albus reduce the ability of Neocallimastix frontalis to digest the xylan of barley straw, maize stem and wheat straw, compared to fungal monocultures. The bacteria secrete extracellular factors that inhibit fungal xylanases and cellulases. Some species of fungi produce inhibitors effective against bacteria. These characteristics seem to be simply an expression of the competition between the organisms. However, the overall degradation of plant material is greater when the fungi and bacteria are working together than when they are working individually.
Some of the interactions extend the mutualisms; for example the methanogenic bacteria are the primary hydrogenotrophs in the rumen ecosystem and their activity enables the chytrids to work more efficiently. Even low levels of free hydrogen inhibit the action of hydrogenase, yet this enzyme is crucial to fungal metabolism, so accumulation of hydrogen results in a decrease in carbon flow and an increase in inhibitory products such as ethanol and lactate. The methanogenic bacteria use the hydrogen in the rumen, releasing the hydrogenase enzyme from inhibition. The result of methanogenic bacterial activity is an increase in carbon flow through the chytrids, and, incidentally, increased production of H2.
Consequently, the methanogenic bacteria and fungi are synergistic; together they carry out a more efficient fermentation process, consequently releasing a higher biomass yield from the food, generating a larger microbial community, and greater benefit to the host. So we now have a tripartite mutualism: mammal-chytrid-methanogen. If we add the farmer who manages the pasture land for his cattle, the butcher who prepares the meat, the restaurateur who turns the meat into a meal, and the diner who eats that meal, the range of mutual dependencies increase even further.
A newly born ruminant does not possess this microbial flora; instead it must acquire the anaerobic fungi, bacteria and protozoa that would normally inhabit a mature animal. This colonisation is achieved rapidly, before the rumen becomes functional. It is brought about by accidental exposure to faeces in its pasture that contain resistant stages of the chytrids; also, fungi are present throughout the alimentary canal of ruminants, including the mouth and throat, suggesting that saliva is a likely vehicle for inoculation through licking and grooming of the infants by their mother, and interactions with other juveniles. Air samples have also been found to contain several species of anaerobic fungi, suggesting the possibility that aerosols act as another route for transmission (especially likely in large herds or flocks of individuals such as occur in intensive farming).
CLICK HERE to visit a page giving a little further information about the metabolism of anaerobic chytrids
High-efficiency fermentation is achieved by larger mammals in two different ways.
- The first, termed hindgut fermentation occurs in non-ruminant herbivores, which possess an enlarged area of the hindgut, usually the caecum, where fermentation takes place long after the initial gastric digestion in the stomach.
- The second, referred to as foregut fermentation applies in ruminants; these large animals provide accommodation for their microbial partners in a stomach which is modified into four chambers. The three forestomachs, sometimes considered to be elaborations of the oesophagus though other authorities consider them derivatives of the stomach comprise: the rumen (by far the largest), reticulum and omasum. The true stomach or abomasum then follows; this is the only site in the digestive tract that produces acid and digestive enzymes (pepsin and rennin).
In the newborn calf, the abomasum makes up about 80% of the total stomach volume, while in the mature cow it amounts to only 10%. Total volume of the stomach in a dairy cow is about 130 l (human monogastric stomachs generally have a volume of about one litre) and in the cow these organs, collectively, occupy almost 75% of the abdominal cavity.
Taxonomically, a ruminant is a mammal of the order Artiodactyla (even-toed ungulates); the anatomical features just described are exemplified by cattle, sheep, goats, giraffes, bison, yaks, water buffalo, deer, wildebeest, and various antelopes. All of these are placed in the suborder Ruminantia. Other animals, also generally called ruminants, have slightly different forestomach anatomy; camels, llamas, alpacas, vicunas have a reduced omasum and are occasionally referred to as pseudoruminants or as having ‘three stomachs’ rather than four. These are placed in the suborder Tylopoda. This drastic adaptation of the alimentary canal is the ‘evolutionary investment’ that the animals have made in this mutualism. It provides the microbes with a steady supply of freshly-cropped plant material and a warm safe habitat in which they can digest the supplied food matter; in return the animal gets a high-efficiency plant cell wall digester.
Piromyces and Caecomyces have been isolated from the horse and donkey (both of which are in the genus Equus in the order Perissodactyla or odd-toed ungulates), and Indian elephant (order Proboscidea). These animals are examples of the non-ruminant herbivores in which hindgut fermentation occurs. Hindgut fermentation provides the host with sources of energy and a range of nutrients that the microbes extract and make available from the digested plant materials, but because it takes place downstream of the stomach the lack of subsequent digestive processes means that the benefit is limited.
Hindgut fermentation is effectively a ‘downstream recovery’ process; it offers an evolutionary advantage to the animal by scavenging some of the nutritional value from the food that would otherwise be lost. But it is a relatively low-efficiency system: for example, elephants spend 16 hours a day collecting plants for food (about half is grasses, and they browse for other leaves, shoots, roots, fruits, etc.), but 60% of that food leaves the elephant’s body undigested. In comparison, the ruminant strategy offers a high efficiency nutrient extraction process since fermentation occurs for an extended period of time because of rumination, and because the products of fermentation enter the stomach where the digestive fluids of the host animal can digest both fermented plant materials and the extremely large populations of microbes.
The symbiotic relationship between Artiodactyla and chytrids enabled the animals to incorporate difficult-to-digest grasses into their diet, and the efficiency of ruminant digestion together with expansion of the grasslands gave the Artiodactyls the opportunity to become the dominant terrestrial herbivores throughout the world in the most recent epochs. However, the story starts long before the evolution of grasses. It is interesting that in fungal phylogenies the Neocallimastigomycota emerge as the earliest diverging lineage of the chytrid fungi (James et al., 2006). We take this to mean that these fungi have existed on Earth, presumably as saprotrophs in anaerobic niches like muds and stagnant pools, since before herbivorous animals of any sort emerged.
Fossilised flagellated fungi have been reported from the Precambrian, but the identification of the material is disputed. On the other hand, chytrids are “…probably the most common microbial element…” (Taylor et al., 2006) in the Devonian Rhynie Chert, which is 400 million years old. Several arthropod groups, including mites and collembolans are also well represented in the Rhynie Chert (Taylor et al., 2006) even though it is best known for its plant communities.
Consequently, this excellently preserved fossil record demonstrates that the chytrids and other fungal classes, and their associations with plants and microarthropods of the day were well established by about the middle of the Paleozoic era (Fig. 6 in Chapter 2).
From that time onwards the fungi were abundant, so any browsing animal that feasted on the community of plants represented in the Rhynie Chert would have got a mouthful of saprotrophic microfungi along with their salad. The first true herbivores were most probably fruit and seed eaters (frugivores) because the starch, protein and fats stored in fruits and seeds can be more easily digested than the plant fibres in foliage. It is argued that the evolution of large size was a prerequisite for the exploitation of leaves because of the need for a long residence time in the gut for fermentation to extract sufficient nutrients from foliage and herbage (Mackie, 2002).
In the Cretaceous (about 100 million years ago) dinosaurs probably occupied the herbivorous niche although they were still browsers; true grazing animals appeared much later in the Miocene (around 20 million years ago; Fig. 6 in Chapter 2) with the radiation of grassland-forming grasses of the plant family Poaceae (but see below).
Plant-eating mammals during the late Cretaceous and early Paleocene (say, 80 million years ago) were physically small frugivores; mammals did not become herbivores until the Middle Paleocene (60 million years ago). Herbivore browsers first appeared in the Middle Paleocene but they did not become major components of the fauna until the late Eocene (40 million years ago; Fig. 6 in Chapter 2).
It is envisaged that the earliest herbivores were large, ground dwelling mammals, reaching their dietary specialisation by evolution from large, ground dwelling frugivores or by a major size increase from small insectivorous ancestors (Mackie, 2002). It is also argued that hindgut fermentation must have developed first, with foregut fermentation emerging after this initial adaptation of the hindgut, and this seems to reflect the evolutionary appearance of Perissodactyls first, followed by Artiodactyls.
Grass-dominated ecosystems, including steppes, temperate grasslands, and tropical–subtropical savannas, play a central role in the modern world, occupying about 25% of the Earth’s land surface; these ecosystems evolved during the Cenozoic (Strömberg & Feranec, 2004). Grasses are thought to have initially evolved 60 million years ago (Jacobs, 2004); the first to appear used the C3 photosynthetic pathway, but the grasses that dominate the semi-arid savanna are the C4 grasses.
C3 photosynthesis is the typical photosynthetic pathway that most plants use; C4 plants can photosynthesise more efficiently in the higher temperatures and sunlight encountered by savanna grasses because they use water more effectively and have biochemical and anatomical adaptations to reduce photorespiration. There is a good argument for the evolution of the Artiodactyls being driven by the development and expansion of savanna and steppe grasslands in Africa and Eurasia (Cerling, 1992; Bobe & Behrensmeyer, 2004).
The appearance of grasslands in Africa and Eurasia during the Eocene epoch, and subsequent spread during the Miocene saw the Artiodactyls begin to dominate over the Perissodactyls. A credible hypothesis for the evolution of rumination in artiodactyls is that it represented a joint adaptation to increasing aridity of the local environment due to climatic cooling and drying.
The Eocene climate was humid and tropical, and is likely to have favoured browsers and frugivores (and hindgut fermentation). With the onset of the Oligocene, the climate became generally cooler and drier, and this trend persisted throughout the Tertiary. This increasing aridity, coupled with high sunlight exposure in the equatorial zone, would have favoured the C4 grasses, and as they became the dominant vegetation the emphasis in herbivore evolution would be to increase the efficiency of the fermentation of the more fibrous plant material. Selection pressure, in other words, against hindgut fermentation and in favour of foregut fermentation and rumination (Mackie, 2002; Bobe & Behrensmeyer, 2004).
The expansion of grassland at the expense of Miocene forests created conditions favourable for the evolution of Artiodactyla that could survive aridity and exploit grassland vegetation; changes in the environment drive major evolutionary events, and in this case major changes in bovid abundance and diversity were caused by dramatic climatic changes affecting the entire ecosystem (Bobe, 2006; Bobe & Eck, 2001; Franz-Odendaal, Lee-Thorp & Chinsamy, 2002). The artiodactyls became the most abundant and successful order of current and fossil herbivores, with about 190 species living today.
An added twist to the story is that the emergence of the genus Homo in the Pliocene of East Africa also appears to be broadly correlated in time with the advent of these same climatic changes and the introduction of the ecosystems they brought about (Bobe & Behrensmeyer, 2004). Grasslands currently represent 25% of the vegetation cover of planet Earth; and this family of plants (Poaceae) is the most important of all plant families to human economy as it includes our staple food cereal grains.
- The grasses owe their success to the environmental pressures plants faced during the evolution of the savanna grasslands of East Africa.
- The artiodactyls owe their success wholly to their symbiotic relationship with the rumen chytrids.
- Humans found their staple cereal foods among the Poaceae and their main food animals among the ruminants.
Updated December 17, 2016