Keywords

2.1 Introduction

Yeasts are found in all kinds of aquatic habitats, but in low numbers of less than 10 l−1 in open ocean and lake waters, in larger populations up to 500 l−1 near terrestrial influence of the shore and often over 1000 l−1 in eutrophic waters such as in aquatic ecotones. Open water habitats of oceans, bays, lakes, ponds, rivers, and streams have been the subject of many studies of yeast communities or guilds (Hagler and Ahearn 1987; Nagahama 2006; Kutty and Philip 2008). The yeast diversity in freshwater of worldwide lakes and rivers and in nearshore and offshore seawater is summarized in Chap. 1 of this book. However, natural aquatic ecotones with much higher nutrient levels in their waters have received little attention in studies of yeast ecology. Yeasts survive well in salt- and freshwater in pure culture but compete in mixed cultures (Ahearn et al. 1968; Hagler and Mendonça-Hagler 1979; Starmer and Lachance 2011). They survive so well that suspensions of pure cultures in distilled water are used as a method to maintain fungi, including yeasts, for prolonged periods (McGinnis et al. 1974). The tendency to be unicellular should favor growth of yeasts over filamentous fungi in aquatic habitats (Lachance and Starmer 1998). The prevalent species in some types of aquatic ecotones are compared in Table 2.1. Estuaries are a classic example of ecotone habitats as interphases between marine and freshwaters carrying nutrients and microbial populations from terrestrial to marine ecosystems (Odum 1993).

Table 2.1 Prevalent yeasts in aquatic ecotones
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Human-associated yeasts are frequent in most estuaries and can serve as pollution indicators (Hagler et al. 1986; Hagler 2006; Starmer and Lachance 2011). Artificial ponds for sewage treatment, recreational activities, and cultivation of aquatic life are dominated by species entering with sewage or fertilizers making them more the subject of industrial and public health microbiology and not included in this chapter. Wetland areas have relatively less volume of water and contain high amounts of degrading leaf litter and other organic materials compared with open waters (Fig. 2.1). This concentration of nutrients makes wetlands prime locations for feeding and breeding sites of many animals which can vector yeasts into the habitat. The shallow waters in these habitats are closely associated with organic and inorganic sediments supporting development of large fungal populations, including yeasts, in complex communities associated with the specialized flora and fauna of such regions (EPA 2016). Although these conditions favor diverse natural yeast communities, such habitats have not received much study. Sampling is complicated by uneven distribution in diverse microhabitats. Difficult access, unpleasant odors, mud, and insects are frequent factors. If close to human population centers, wetlands are often drained or contaminated by sewage. Smaller and somewhat ephemeral isolated volumes of water that are not wetlands, but have similar function in many terrestrial habitats, are phytotelmata (plant ponds). They are aquatic ecotones involving the plants and water but not soil. Although small and ephemeral relative to humans, they are large and permanent enough to support significant microbial populations. Examples are holes in tree trunks and cups formed by leaves and flowers. Bromeliad tanks are a good example and very important in diverse neotropical habitats especially in the forest canopy. These wetland habitats and phytotelmata have in common the degradation of large amounts of lignocellulose and other plant materials in shallow waters or water-saturated sediments. The plant species in such habitats are limited in diversity by exclusion of oxygen from the roots, but wetlands can support large and diverse microbial and animal populations. Hydrothermal vents in the oceans are another notable form of aquatic ecotone but fed by chemoautotrophic bacteria rather than degrading organic matter and not included in this chapter (Gadanho and Sampaio 2005; Nagahama 2006; Le Calvez et al. 2009). The aquatic surface films, sediment-water interface, and associated benthic organisms in all aquatic habitats are ecotones and have more concentrated nutrients than the water (Hagler and Ahearn 1987; Kachalkin 2014) and are part of more conventional marine and freshwater habitats. Diverse yeast populations are consistently present in waters, sediments, and biota of aquatic ecotones.

Fig. 2.1
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The Sammamish River in Redmond Washington showing typical wetland vegetation with peat bog in the willows behind them

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2.2 Estuaries

Few studies have been made of pristine estuaries. The prevalent species from some examples with different levels of urban influence increasing from left to right are presented in Table 2.2. The yeast species most typical of estuaries are Candida spp. (especially Candida intermedia, Candida parapsilosis, and Candida tropicalis), Debaryomyces hansenii, Pichia kudriavzevii (Issatchenkia orientalis and Candida krusei), Meyerozyma (Candida) guilliermondii, Cutaneotrichosporon (Trichosporon) cutaneum, Papiliotrema (Cryptococcus) laurentii, and Rhodotorula spp. (especially Rhodotorula glutinis and Rhodotorula mucilaginosa). Fell et al. (1960) started a sequence of studies of yeasts in estuaries, most of them near major urban centers. Various studies have been made of the Tagus river estuary in Portugal since the 1960s (Taysi and van Uden 1964). Coelho et al. (2010) working in the same estuary, but with various cultivation methods and the identifications of cultures by rDNA gene sequences, estimated the influence from marine, terrestrial runoff, urban effluents, and resident populations from samples taken in a transect downstream from Lisbon. Some species were associated with different sources entering the estuary. Mey. guilliermondii, C. parapsilosis, and Clavispora lusitaniae made up most of the isolates at 37 °C, and the counts at this temperature had a high correlation with counts of the fecal indicator Escherichia coli. Hagler et al. (1986) also found a high correlation of 40 °C yeast counts, largely of P. kudriavzevii and C. tropicalis, with coliform counts in Rio de Janeiro. High-temperature yeast counts can serve as indicators of domestic sewage (Hagler 2006). Rh. mucilaginosa was found as the prevalent species by Coelho et al. (2010) and considered to be mostly from terrestrial runoff although this species is also common in seawater and lakes (Hagler and Ahearn 1987). Deb. hansenii was considered a marker of marine origin although it is a species associated also with diverse nonmarine habitats. Pollution from urban areas is an important factor in most estuaries, and human-associated yeasts were very evident in the heavily polluted estuary site in Guanabara Bay, Rio de Janeiro (Hagler and Mendonça-Hagler 1981), but much less present in the more pristine Suwannee river estuary (Lazarus and Koburger 1974) and Everglades (Ahearn et al. 1968). An interesting strategy was used to detect yeasts of the Tagus river estuary, Portugal, by Gadanho and Sampaio (2004). The PCR-DGGE method of Muyzer (1999) was applied to analyze two water samples directly from environmental DNA and DNA extracted after cultivation on solid and liquid enrichment media. This combined procedure increased the species richness assessed in the water samples, allowing the detection of species present in low abundance or less competitive in culture. Ascomycetous species were not detected by PCR-DGGE using environmental DNA, but were after cultivation of the samples on YM agar or broth. The prevalent species detected in this study were Deb. hansenii, Rh. mucilaginosa, and Vanrija longa (Cryptococcus longus). Sites with brackish water from ice melts and tidal action similar to an estuary were studied in the north of Russia where salinity in the Kandalaksha Gulf does not exceed 20 ppt. Kachalkin (2014) found in water, sand, silt, and a sponge in the intertidal zone some species typical of estuaries in warmer regions, but also various obligate psychrophilic species including a new species Glaciozyma litoralis. The largest yeast populations in these two sites were associated with healthy algae and over 10,000 CFU g−1 dominated by Metschnikowia zobellii, whereas yeast counts in water were about 100 l−1 and much lower than in other substrates of the intertidal zone.

Table 2.2 Estuarine yeasts present in more than five samples
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2.3 Mangroves

Mangroves are a type of tropical swamp with influence of tidal action and marine water, and like salt marshes are typically found in estuaries where they show a high level of microbial diversity (Ghizelini et al. 2012; Pires et al. 2012; Fig. 2.2). The yeast counts in water entering the mangrove vegetation of the Coroa Grande mangrove in Sepetiba Bay, Rio de Janeiro, had a geometric mean of 340 l−1. Yeast counts were about 10 times higher in sediments than in water and 100 to 1000 times higher in the intestines of invertebrates living in this mangrove. Counts of filamentous fungi were typically one to two orders of magnitude higher than yeast counts in the same mangrove area (Araujo et al. 1995). The more frequent yeasts found in mangroves are presented in Table 2.3. Ahearn et al. (1968) made an extensive study of yeast communities in South Florida saline and freshwater habitats including the Everglades. More than 50 species were identified with the less advanced taxonomic methods of that time, and representative species were tested for survival as pure or mixed cultures in fresh- and seawater. Among them in all estuarine mangrove locations was Kluyveromyces aestuarii that had been described by Fell (1961) from a Miami Florida estuary. It has since been found in mangroves of Rio de Janeiro, Brazil (Araujo and Hagler 2011), in China (Chi et al. 2012), and in Thailand where it occurred with another very similar species, Kluyveromyces siamensis, described by Am-In et al. (2008). The consistent presence of K. aestuarii in this habitat and absence from others suggested its use as an indicator organism (Araujo and Hagler 2011). Urban beaches of mangrove sediments, but cleared of mangrove vegetation, had more human-associated yeasts including Candida glabrata, C. tropicalis, C. parapsilosis, P. kudriavzevii, and Mey. guilliermondii and absence of K. aestuarii (Hagler et al. 1982; Soares et al. 1997). The intestines of detritus feeding crabs and a filter-feeding shipworm (mollusk) in the Coroa Grande mangrove in Rio de Janeiro were all found to contain K. aestuarii. However, it was not found in predatory and leaf-feeding crabs or a mussel attached to tree roots under the sediment, and it was nearly absent from clams in the mud flat of the same location (Araujo et al. 1995). K. aestuarii appears to be endemic to mangroves and associated with the detritus, presumably from degrading mangrove leaves at the sediment surface. The yeasts common in open waters are present in mangroves. But, there are also a striking number of diverse species isolated at low frequency, making a total of over 130 species in the work included in this review, many of which were probable new species. Many new species have been described from mangroves. These include Candida sharkiensis, Candida rhizophoriensis, Sakaguchia (Rhodotorula) cladiensis, Rhodotorula evergladiensis, and Papiliotrema mangalensis (Cryptococcus mangaliensis) (Fell et al. 2011); Candida spencermartinsiae, Candida taylorii, and Ustilago (Pseudozyma) abaconensis (Statzell-Tallman et al. 2011); Candida chanthaburiensis, Candida kungkrabaensis, and Candida suratensis (Limtong and Yongmanitchai 2010); Candida thaimueangensis (Limtong et al. 2007); Geotrichum siamensis and Geotrichum phurueaensis (Kaewwichian et al. 2010); K. siamensis (Am-In et al. 2008); Kwoniella mangroviensis (Statzell-Tallman et al. 2008); Lachancea meyersii (Fell et al. 2004); Martiniozyma (Candida) asiatica (Limtong et al. 2010a); Rhodotorula paludigena (Rhodosporidium paludigenum—Fell and Tallman 1980); Saturnispora (Candida) siamensis (Boonmak et al. 2009); Candida phangngensis (Limtong et al. 2008a); Saturnispora (Candida) sanitii and Saturnispora (Candida) suwanaritii (Limtong et al. 2010b); Tetrapisispora arboricola (Ueda-Nishimura and Mikata 1999); and Torulaspora maleeae (Limtong et al. 2008b).

Fig. 2.2
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Mangrove at Mosqueiro, Aracaju, Sergipe, Brazil, on the margin of the estuary of Rio Vaza-Barris showing typical tree seedlings and aerial roots, but no smaller plants

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Table 2.3 Yeasts found in more than five samples from mangroves
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Recent studies on fungal diversity in mangroves of New Caledonia used 454-pyrosequencing method, in which DNA was extracted directly from the environmental samples. Sequences from four regions of rDNA (ITS1, ITS2, SSU V5, and SSU V7) were obtained for fungi from submerged and aerial parts of trees. Species richness values were dependent on the gene marker used, ranging from 271 to 1001 OTUs (operational taxonomic units) and with the larger values for ITS sequences. Ascomycetes were dominant with 82% of the sequence reads, whereas Basidiomycetes represented 3%, and 15% could not be assigned to known taxa (Arfi et al. 2012a). The fungal diversity associated with anoxic-sulfidic sediments in the same mangrove was assessed by 454-pyrosequencing using the ITS1 and ITS2 regions (Arfi et al. 2012b). Over a hundred distinct OTUs were detected mostly of filamentous fungi but included the yeasts Dipodascus australiensis, Galactomyces geotrichum, and a few reads of Malassezia sp. and Deb. hansenii.

Freshwater swamps and ponds near urban centers are mostly polluted showing wide fluctuations in yeast counts compared with pond and swamp waters of unpopulated regions. There were notable studies done by Ahearn et al. (1968) in the Everglades and by van Uden and Ahearn (1963) in a small unpolluted lake in Michigan that give an idea of what yeasts are present in shallow freshwaters of uninhabited and less populated areas. The human-associated yeasts present in many studies from near urban areas were not common in these waters where yeasts belonging to the former genera Cryptococcus and Rhodotorula (now classified in diverse basidiomycetous genera) were most common. Yeast counts in Everglades freshwater sites were largely in the 150–500 range and with some up to 1200 l−1 (Ahearn et al. 1968). Yeast counts in the sites with 0–9 ppt salinity were mostly in the 100–1000 l−1 range, whereas with salinity of 25 ppt, most counts were less than 100. The most common species were strictly oxidative with Pa. laurentii the most frequent. The principal difference from estuarine mangrove regions in the Everglades was the lack of K. aestuarii, lower frequency of Cystofilobasidium infirmominiatum, and much higher frequency of Sporobolomyces. The only ascomycetes frequent in the freshwater swamps were Deb. hansenii, P. kudriavzevii (C. krusei), Metschnikowia reukaufii, and Yamadazyma triangularis.

2.4 Salt Marshes

Salt marshes are found frequently in regions protected from the action of the surf in temperate waters of bays and estuaries. Yeasts were studied in a salt marsh in Louisiana in southern USA where a new species was prevalent, Scheffersomyces (Pichia) spartinae, and with concentrations as great as 9 × 107 cells g−1 associated with the plant culm of Spartina alterniflora, oyster grass, the dominant plant of the habitat. The prevalent yeasts in the sediment rhizosphere were species of the then polyphyletic genera Trichosporon, Rhodotorula, and Rhodosporidium and Kluyveromyces lactis, a species similar to K. aestuarii that is prevalent in mangroves (Ahearn et al. 1970; Meyers et al. 1975; Hagler and Ahearn 1987). The population of K. lactis was followed in 30 samples each of water and sediment using 2% galactose YNB agar with pH adjusted to 4.0 with lactic acid on which it formed deep rose- to maroon-colored colonies and was compared with the overall yeast population growing on YM agar. It was found to be consistently present as a significant portion of the total yeast population which was 10 to 100 times higher in sediments than in water (Meyers et al. 1971). A more recent study using cultivation-independent methods was unable to confirm these species in a salt marsh in Georgia, but such methods have not shown good detection of yeasts (Buchan et al. 2002). Dini-Andreote et al. (2016) reported a comprehensive study using a high-throughput sequencing method to access fungal community dynamics related to marine-terrestrial transition at a pristine salt marsh (Schiermonnikoog Island, The Netherlands). The natural sedimentation process on this island resulted in a chronosequence developed over a hundred years of terrestrial ecosystem succession. The majority of OTUs based on ITS region sequences were assigned to Ascomycota (66.8%), followed by Basidiomycota (4.3%) with Tremellomycetes yeasts mainly represented by species previously assigned to the former polyphyletic genus Cryptococcus found especially in the early succession stages of transition from marine to terrestrial habitats. Yeasts have important populations as part of the normal biota of salt marshes, but have not received much study in this habitat.

2.5 Bogs and Fens

Bogs and fens are the dominant wetlands of our planet and important as carbon sinks. Yeast species make up about 10% of all peatland fungi and probably use simple polymers leached from plant materials in the initial phases of decomposition (Thormann et al. 2007). Peat bogs and fens do contain yeasts, and basidiomycetous species tend to be more prevalent and increasingly so in colder climates. The yeast species found more than once in bogs and fens are noted in Table 2.4. Candida spp., Deb. hansenii, Rh. mucilaginosa, and Sporobolomyces roseus were most common, and Goffeauzyma (Cryptococcus) gilvescens dominated in the coldest regions. Kachalkin (2010) isolated psychrophilic yeasts Sterigmatosporidium polymorphum and Phenoliferia (Rhodotorula) psychrophenolica, and Aureobasidium pullulans var. subglaciale from the Sphagnum mosses and paludal vascular plants in a swamp region near Moscow. Broad assimilation spectrum fungal species, capable of utilization of organic acids and aromatic compounds, were prevalent in the moss-turf (Kurakov et al. 2008). Yeast populations were noted by Babjeva and Chernov (1995) to be lower in the litter complex at about 103 g−1 compared to the 105–106 g−1 found in tundra epiphyte complex. A study in Canada and Siberia yielded 12 identified and 8 unidentified probable new species from 34 isolates. Nadsonia starkeyi-henricii was included among them and is probably a peatland specialist (Thormann et al. 2007).

Table 2.4 Yeasts found in bogs and fens
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2.6 Phytotelmata

Phytotelmata are formed from rainwater collected and preserved in structures of some plants including many bromeliad species. Thousands of species of bromeliads are native to diverse tropical habitats of the Americas (Fig. 2.3). These are dynamic and complex microenvironments inhabited by communities of different organisms including endemic species (Benzing 1990; Whittman 2000; Lopez et al. 2009). The phytotelmata in bromeliad leaf rosettes are a major source of nutrients for these organisms and communities associated with them (Richardson et al. 2000). Animals including insects and small mammals, known to carry yeasts, can have mutualistic relationships with tank bromeliads (Abranches et al. 1997; Pagnocca et al. 2008; Duarte et al. 2016; Leroy et al. 2016). Leaves from the bromeliad itself are in contact with the tank water, but the tanks also collect the leaf litter falling into them from surrounding vegetation. Each plant has many partitions formed by the leaf rosette making it like a circular rack of enrichment cultures around the central tank, each with different conditions for yeast growth. The leaf litter and visiting animals vector diverse species to form metapopulations in an extensive matrix of natural aquatic microcosms. Heavy rains can flood the tanks and wash out the existing nutrients and microbial populations, and during prolonged dry periods, the tanks can dry out (Araujo et al. 1998; Araujo 1999; Garcia 2007). More than 112 yeast species have been found in phytotelmata, and the prevalent yeasts in them are presented in Table 2.5. Phytotelmata in direct sunlight have strong algal growth, rather than degradation of organic materials, as a principal source of organic nutrients for microbial growth. Their yeast community is dominated by basidiomycetous species, whereas shaded plants are also rich in ascomycetous species. The species Kazachstania bromeliacearum appears to be endemic to bromeliad phytotelmata, and Kazachstania rupicola has been found in rupestrian bromeliads. C. intermedia was frequent in the phytotelmata and was the dominant yeast in fruits of the bromelias Quesnelia quesneliana, Vriesea procera, and Aechmea nudicaulis. Deb. hansenii, its anamorph Candida famata, and similar species including Schwanniomyces occidentalis, Schwanniomyces polymorphus, Schwanniomyces vanrijiae and Mey. guilliermondii were prevalent especially in the shaded plants. An example of phytotelma other than bromeliad tanks is in the more ephemeral flower structures of the wild banana-like plant Heliconia velloziana. The 15 ascomycetous yeasts isolated from 14 phytotelmata of Hel. velloziana with more than one isolate were four cultures of Candida heliconiae, three each of Candida picinguabensis and Metschnikowia spp. and two each of Candida apis, Candida pseudointermedia, Candida restingae, Candida saopaulonensis, and Debaryomyces sp. (Ruivo 2005). The more common basidiomycetous species of the phylloplane were also among the prevalent phytotelmata yeasts (Fonseca and Inácio 2006). Phytotelmata are “in situ” enrichment cultures for yeasts making them a good natural source to tap the species richness of the phylloplane. Gomes et al. (2016) screened enzymes produced by yeasts from Vriesea minarum phytotelmata. These enzymes would allow them to participate in the degradation of plant and animal materials falling into the tanks. Phytotelmata yeasts have been the source of various new species: Carlosrosaea (Bullera) vrieseae (Landell et al. 2015); Candida aechmeae and Candida vrieseae (Landell et al. 2010); Candida bromeliacearum and Candida ubatubensis (Ruivo et al. 2005); Candida heliconiae, C. picinguabensis, and C. saopaulonensis (Ruivo et al. 2006); Hagleromyces aurorensis (Sousa et al. 2014); Hannaella pagnoccae (Landell et al. 2014); Kaz. bromeliacearum (Araujo et al. 2012); Kaz. rupicola (Safar et al. 2013); Kockovaella libkindii (Gomes et al. 2016); and Occultifur brasiliensis (Gomes et al. 2015).

Fig. 2.3
figure 3

Bromeliads. A, rupestrian bromeliads; B, bromeliads and other epiphytes on a tree; C, bromeliad growing on soil showing various phytotelmata surrounding central tank and leaf litter in the center; D, unshaded bromeliad with photoautotrophic growth in its tank

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Table 2.5 Yeasts found in more than five samples of phytotelmata
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2.7 Concluding Remarks

Yeasts in aquatic ecotones are rich in species diversity and with high population levels compared with open waters. More than 270 yeast species and many other unidentified yeasts, often representing new taxa, were reported from aquatic ecotones covered in this chapter. Cultivation of yeasts from these waters is complicated by large populations of filamentous fungi and competition between different yeasts while growing on culture media. Using various enrichment cultures and solid media with and without different antifungal antibiotics to inhibit parts of the fungal populations, including some yeasts, should improve isolation of yeasts from aquatic ecotones. No medium, even if all yeasts can grow on it in pure culture, will allow cultivation of all yeasts in the mixed populations of an environmental sample. The methods of cultivation, isolation, and enumeration of yeasts have been reviewed by Boundy-Mills (2006) and Kurtzman et al. (2011b). A single nutrient-rich medium favors fast-growing species that can later inhibit development of others on the isolation medium. Various media with inhibitors and nutrients favoring different species should be used, and the frequency of presence in various samples, rather than counts, used to indicate the relative importance of different species. Indicator dyes, such as bromocresol green at pH 4, added to media can inhibit some species and assist in selection of colonies for further study. Culture-independent methods with DNA analysis can detect the species present and their relative population (Xu 2006; Abarenkov et al. 2010), although this methodology does not yield cultures for further studies or applications. Culture-independent methods have detected yeasts in the presence of large populations of filamentous fungi, but yeasts known to be abundant by cultivation methods are often not detected as prevalent by these methods. The ITS region has been proposed as the universal bar code for next-generation sequencing methodology applied in fungal diversity studies and has shown detection of yeasts (Arfi et al. 2012a, b; Schoch et al. 2011). The detection of yeast taxa could be improved when sequences of large subunit rDNA (D1/D2 region) are used, probably due to the large database available on this region for yeasts (Bokulich et al. 2014). A polyphasic approach of cultivation and culture-independent methods should provide better information on yeast communities in aquatic ecotones. The exceptional species richness found, especially for mangroves and phytotelmata, yielded many new species descriptions. This should encourage further studies of yeast ecology and bioprospecting for yeasts in aquatic ecotone habitats.