**6. Yeast species identification**

Macroscopic and microscopic identification of yeasts is frequently inaccurate due to the high similarity that yeast may show at a glance, either in colony or in a microscopic field. The best way to identify and differentiate yeast species as well as strains is by molecular techniques that are being used since 2000. A variety of techniques have been developed and this fact has greatly boosted the number of new species of yeasts identified. Molecular analyses of the variable D1/D2 regions of the 26S rDNA, 18S, 5.8S and mitochondrial small subunit rDNAs gene, as well as ITS sequencing and RFLP-ITS are very useful ways to identify yeast species and invaluable tools for phylogenetic studies [21, 22].

The D1/D2 domain of the LSU rRNA gene was PCR amplified directly from whole yeast cell suspensions [23], and using the primers NL1 and NL4 [24]. Initial amplification reactions were carried out in Ecuador (The Catholic University Yeast Collection-CLQCA) and Brazil (Collec‐ tion of Microorganisms and Cells of UFMG). Amplified fragments were checked by agarose gel electrophoresis. The PCR purified products, were further sequenced with the external amplification primers NL1 and NL4. Finally, a sequence similarity search was conducted using NCBI Blast tool. Yeasts with more than 99% identity were considered members of the same species.

#### **6.1. An overview of the yeast species in four Islands of the Galápagos Archipelago**

As a result of the collections carried out in Santa Cruz, Isabela, Floreana, and San Cristóbal Islands 881 isolates were recovered from a wide variety of substrates. Currently we can report 614 yeast isolates already identified, while 267 isolates are in process of identification.

The number of yeast isolates collected per island is shown in Table 1.


**Table 1.** Number of yeast isolates collected in four islands of the Galápagos Archipelago during an expedition carried out in October 2009

The difference in the abundance of yeast isolates collected in each island is due to different conditions of time and logistics of the expedition and does not have any relationship with the abundance or diversity of yeasts in each island.

The present chapter reports the isolates and biodiversity recovered in a number of substrates including: flowers, rotten wood, excrement, insects, fruits, exudates, leafs, one sugar cane mill (Santa Cruz Island) and others. Figure 1 shows the shares of yeast isolates by substrate we have preserved in the Catholic University Yeasts Collection (CLQCA).

#### **6.2. Yeast species in Ecuador Mainland and Galápagos Islands**

yeasts biodiversity within the still natural environments of four inhabited islands. This chapter is the first report of such expedition that explored Santa Cruz, Isabela, Floreana and San Cristóbal Islands, where a number of substrates were sampled by using different culture media

Yeast sampling in substrates like flowers, fruits, excrement or fugus was carried out using sterile cotton wool swabs, to inoculate in liquid and solid YM media. In the case of sampling the insect's gut content a technique of catching the living insect in plastic bags for further inoculation by the living insect walking on the surface of Petri dishes containing YM agar medium. Eventually the insects were liberated alive. Additionally a number of substrates were collected in plastic sterile tubes for further culturing in selective culture broths such as YNB-CMC, YNB-D-xylose; YNB-xylan; YNB-L-arabinose, and YNB-raffinose the CLQCA labora‐ tory in Quito, Ecuador. The selective culture media were used especially in the search of yeast strains that exhibit some biotechnological potential use in xylose fermentation as well as

Macroscopic and microscopic identification of yeasts is frequently inaccurate due to the high similarity that yeast may show at a glance, either in colony or in a microscopic field. The best way to identify and differentiate yeast species as well as strains is by molecular techniques that are being used since 2000. A variety of techniques have been developed and this fact has greatly boosted the number of new species of yeasts identified. Molecular analyses of the variable D1/D2 regions of the 26S rDNA, 18S, 5.8S and mitochondrial small subunit rDNAs gene, as well as ITS sequencing and RFLP-ITS are very useful ways to identify yeast species

The D1/D2 domain of the LSU rRNA gene was PCR amplified directly from whole yeast cell suspensions [23], and using the primers NL1 and NL4 [24]. Initial amplification reactions were carried out in Ecuador (The Catholic University Yeast Collection-CLQCA) and Brazil (Collec‐ tion of Microorganisms and Cells of UFMG). Amplified fragments were checked by agarose gel electrophoresis. The PCR purified products, were further sequenced with the external amplification primers NL1 and NL4. Finally, a sequence similarity search was conducted using NCBI Blast tool. Yeasts with more than 99% identity were considered members of the same

**6.1. An overview of the yeast species in four Islands of the Galápagos Archipelago**

The number of yeast isolates collected per island is shown in Table 1.

As a result of the collections carried out in Santa Cruz, Isabela, Floreana, and San Cristóbal Islands 881 isolates were recovered from a wide variety of substrates. Currently we can report 614 yeast isolates already identified, while 267 isolates are in process of identification.

and techniques.

species.

cellulose degradation/fermentation.

200 Biodiversity - The Dynamic Balance of the Planet

**6. Yeast species identification**

and invaluable tools for phylogenetic studies [21, 22].

The Catholic University Yeasts Collection in Quito, in its database presents 118 yeast species belonging to the Ecuador mainland and the Galápagos Islands as shown in Table 4. By establishing a comparison between these two regions, there is not a big difference in number of species registered in the mainland and the islands (82 and 78 respectively). It is important to remark that the yeasts registers in this work were taken only from natural environments and substrates (no clinical or industrial samples are taken into account).

In Mainland Ecuador, about 50% of the characterized isolates preserved in the CLQCA (c.a. 250 yeast isolates) belong to four species: *Candida tropicalis, Meyerozyma guilliermondii, Kodamaea ohmeri,* and *Pichia kudriavzevii.* In contrast, the more represented isolates from the Galápagos Islands in the CLQCA (c.a. 615 yeast isolates) correspond to the species: *Candida tropicalis, Hanseniaspora* sp*., Pichia norvegensis, Candida parazyma, Kodamaea transpacifica, Hanseniaspora uvarum, Barnettozyma californica, Candida intermedia,* and *Galactomyces geotrichum*.

*Candida tropicalis* in both cases is the most abundant yeast species registered in CLQCA: in mainland it is about 21% of the total identified isolates, while in the Galápagos Islands it represents about 18%. *C. tropicalis* is a cosmopolite yeast species that is ubiquitous in a wide range of substrates: from beetles to fermented beverages, but predominantly it is found in rotten vegetal matter, flowers and excrements.

Between the three regions of mainland there are also registers of 10 species that have been collected from different substrates. These species are quite adaptable to a wide range of climatic conditions and substrates. Table 2 shows the species that are ubiquitous in Amazonia, Andes and Pacific Coast.

Matching the coincidences of yeast species between any individual mainland region with the Galápagos Islands yeast isolates it is noticeable an increase in the number of coincident yeast species if compared to the matching between the three regions within the mainland (Table 3).


**N° SPECIES FOUND IN MAINLAND AND THE ISLANDS**

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*Candida pseudointermedia*

 *Candida quercitrusa Candida saopaulonensis Candida sinolaborantium*

 *Candida theae Candida tropicalis Cryptococcus humícola Cryptococcus laurentii Debaryomyces hansenii Debaryomyces nepalensis Galactomyces geotrichum*

 *Hanseniaspora sp. Kazachstania exigua Kodamaea ohmeri*

 *Pichia fermentans Pichia kluyveri Pichia kudriavzevii Pichia manshurica Pichia occidentalis Pichia terricola*

*Meyerozyma guilliermondii*

*Rhodosporidium paludigenum*

 *Trichosporon coremiiforme Wickerhamomyces anomalus Yamadazyma mexicana*

**Table 3.** Yeast species represented both in mainland and Galápagos Islands

 *Rhodotorula glutinis Rhodotorula mucilaginosa Saccharomyces cerevisiae Torulaspora delbrueckii Trichosporon asahii*

**Table 2.** Ubiquitous yeast species within Ecuadorian mainland

This increase represents almost four times the amount of the yeasts species shown in Table 2 (i.e. 38 yeast species) according to updated CLQCA registers.

Despite the fact that it is really difficult to address an accurate center of origin of the yeast species occurring in the natural zones of the Galápagos Islands (the islands are now visited by thousands of tourists along the year), we must assume that the Ecuadorian mainland must be the center of origin of most of the yeast species occurring in the Galápagos based upon the Porter's report [18] where about 30% of the vegetal species in the Galápagos Islands have had a Neotropical origin. From our data, about 31% of yeast species have been found both in Ecuador Mainland and the Galápagos Islands. These figures reveal the disharmony of taxa between mainland and oceanic islands [16].

Table 3 shows the species that matched at least once between any single regions of Ecuador's mainland with yeast species found in anyone of the four islands explored.



This increase represents almost four times the amount of the yeasts species shown in Table 2

**N° YEASTS SPECIES** *Candida intermedia Candida tropicalis*

 *Hanseniaspora* sp*. Kodamaea ohmeri Pichia kluyveri Pichia kudriavzevii Pichia manshurica*

*Galactomyces geotrichum*

 *Saccharomyces cerevisiae Torulaspora delbrueckii*

Despite the fact that it is really difficult to address an accurate center of origin of the yeast species occurring in the natural zones of the Galápagos Islands (the islands are now visited by thousands of tourists along the year), we must assume that the Ecuadorian mainland must be the center of origin of most of the yeast species occurring in the Galápagos based upon the Porter's report [18] where about 30% of the vegetal species in the Galápagos Islands have had a Neotropical origin. From our data, about 31% of yeast species have been found both in Ecuador Mainland and the Galápagos Islands. These figures reveal the disharmony of taxa

Table 3 shows the species that matched at least once between any single regions of Ecuador's

**N° SPECIES FOUND IN MAINLAND AND THE ISLANDS**

mainland with yeast species found in anyone of the four islands explored.

 *Aureobasidium pullulans Barnettozyma californica*

 *Candida carvajalis Candida humilis Candida intermedia Candida oleophila Candida orthopsilosis Candida parapsilosis*

(i.e. 38 yeast species) according to updated CLQCA registers.

**Table 2.** Ubiquitous yeast species within Ecuadorian mainland

Biodiversity - The Dynamic Balance of the Planet

between mainland and oceanic islands [16].

On the other hand, mainland Ecuador is one of the richest biodiversity zones in the world [21], due to the varied environments as consequence of the topography that provokes the occurrence of uncountable micro-ecosystems as a consequence of a number of biotic and abiotic factors such as the altitude variation (from 0 to more than 6000 m.a.s.l), geographic location, marine currents, as well as the natural history involving the Neotropic which provides biogeographic unique features to this region of the planet [21].

**N° YEAST SPECIES ECUADOR MAINLAND GALAPAGOS ISLANDS**

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 *Candida orthopsilosis Candida parapsilosis Candida parazyma Candida pomicola*

 *Candida pseudointermedia Candida pseudolambica Candida quercitrusa Candida rugosa*

*Candida saopaulonensis*

*Candida sinolaborantium*

 *Candida sonorensis Candida sorbosivorans Candida sorboxylosa Candida stellimalicola Candida tallmaniae Candida theae Candida tropicalis*

*Candida trypodendroni*

*Cryptococcus rajastharensis*

*Cryptococcus saitoi*

 *Candida xylopsoci Candida zeylanoides Clavispora lusitaniae Clavispora opuntiae Cryptococcus albidus Cryptococcus flavescens Cryptococcus flavus Cryptococcus humicola Cryptococcus laurentii*

*Candida silvae*

The total yeast biodiversity currently registered both in Ecuadorian mainland and the Galápagos Islands (i.e. 118 yeast species identified) is shown in Table 4. The shaded cases represent the register of occurrence of the species either in the mainland or in the Galápagos Islands.



On the other hand, mainland Ecuador is one of the richest biodiversity zones in the world [21], due to the varied environments as consequence of the topography that provokes the occurrence of uncountable micro-ecosystems as a consequence of a number of biotic and abiotic factors such as the altitude variation (from 0 to more than 6000 m.a.s.l), geographic location, marine currents, as well as the natural history involving the Neotropic which provides biogeographic

The total yeast biodiversity currently registered both in Ecuadorian mainland and the Galápagos Islands (i.e. 118 yeast species identified) is shown in Table 4. The shaded cases represent the register of occurrence of the species either in the mainland or in the Galápagos

**N° YEAST SPECIES ECUADOR MAINLAND GALAPAGOS ISLANDS**

unique features to this region of the planet [21].

Biodiversity - The Dynamic Balance of the Planet

Islands.

 *Aureobasidium pullulans Barnettozyma californica*

 *Candida albicans Candida apicola Candida asparagi Candida boidinii Candida boleticola Candida bombi Candida carpophila Candida carvajalis Candida cylindracea Candida dendronema Candida ecuadorensis Candida gigantensis Candida glabrata Candida humilis Candida intermedia Candida leandrae Candida naeodendra Candida natalensis Candida oleophila*


**N° YEAST SPECIES ECUADOR MAINLAND GALAPAGOS ISLANDS**

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 *Pichia fermentans Pichia kluyveri Pichia kudriavzevii Pichia manshurica Pichia nakasei Pichia norvegensis Pichia occidentalis Pichia terricola*

 *Rhodosporidium babjevae Rhodosporidium paludigenum*

 *Rhodotorula mucilaginosa Rhodotorula slooffiae*

 *Saccharomyces cerevisiae Saccharomycodes ludwigii Saccharomycopsis fodiens Saccharomycopsis vini Saturnispora quitensis Scheffersomyces stipitis Sporidiobolus ruineniae Sporopachydermia* sp*. Torulaspora delbrueckii Trichosporon asahii*

 *Trichosporon coremiiforme Trichosporon dermatis Trichosporon insectorum Trichosporon jirovecii Trichosporon laibachii*

 *Rhodotorula glutinis Rhodotorula minuta*

*Rhodotorula* sp*.*


**N° YEAST SPECIES ECUADOR MAINLAND GALAPAGOS ISLANDS**

 *Debaryomyces hansenii Debaryomyces nepalensis*

Biodiversity - The Dynamic Balance of the Planet

 *Filobasidium uniguttulatum Galactomyces geotrichum*

*Hanseniaspora guilliermondii*

 *Dekkera anomala Dekkera bruxellensis*

*Geotrichum silvicola*

 *Hanseniaspora meyeri Hanseniaspora opuntiae*

 *Hanseniaspora* sp*. Hanseniaspora uvarum Hanseniaspora valbyensis*

 *Kazachstania exigua Kazachstania unispora Kluyveromyces lactis*

*Kluyveromyces marxianus*

 *Kodamaea transpacifica Kurtzmaniella zeylanoides Kwoniella mangrovensis*

 *Metschnikowia kipukae Metschnikowia koreensis Metschnikowia reukaufii Meyerozyma guilliermondii*

*Pichia fabianii*

*Kodamaea ohmeri*

 *Lindnera* sp*. Lindnera fabianii Lindnera jadinii Lindnera saturnus*


Ecuadorian mainland *C. humilis* was isolated from mango fruit, chicha de jora (corn fermented beverage), spider network, rotten wood and some flowers from the Asteraceae family. As for *C. intermedia* and *C. parapsilosis* are considered widespread yeasts that can be found in clinical samples, caterpillar frass, beer contaminants and other substrates. In the case of Ecuadorian isolates of these both species, the origin is predominantly found in vegetal substrates and in

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209

In the case of *C. saopaulonensis* we can find that in the mainland Ecuador this species was isolated from a Heliconiaceae as well as the type strain collected in Brazil [26]; in contrast, this yeast species was found in nine different flowers from Asteraceae family in Santa Cruz Island, which is an interesting case of adaptation to new ecological niches in oceanic islands [16].

*C. sinolaborantium* [27] is a yeast species that in the Ecuadorian mainland as well as in the Galápagos Islands was found chiefly in vegetal substrates except for one strain that was collected in *Drosophila* sp. Nevertheless the type strain was isolated from the gut of handsome fungus beetle in Panamá, and two other strains were isolated from a cerambycid larva. The original study was carried out in insect's guts. The results obtained by [27] suggest that this yeast species is part of the microbial community that can be found in the intestine of insects

Another remarkable yeast species found in this survey both in mainland and the Galápagos Islands is *C. tropicalis.* This ubiquitous species is by far the most abundant in isolates number that has been collected since 2006 in our surveys in Ecuador; *C. tropicalis* is practically distrib‐ uted in every kind of substrate, which is a sign of the high adaptability of this species. As part of a study on traditional fermented beverages we were able to find this yeast species even in unexpected substrates such as cassava fermented beverages which are produced in Yasuní National Park, located in the deep Ecuadorian Amazonia. This millenary beverage is still being produced in the same traditional way by autochthonous tribes of Waorani people [28]. In the literature *C. tropicalis* is reported to be collected from clinical samples. This species belongs to the *Lodderomyces* clade, where *C. albicans, C. theae*[29] and other potential pathogens have been accommodated. Nevertheless its presence by itself does not mean a hazard of infection in human, at least to those who possess a strong immune response due to permanent exposition

A very few understood yeast species in terms of its ecology is *Debaryomyces nepalensis*, since it was isolated from a number of different substrates like soil, fermenting tobacco, spoiled sake and others [25]. The isolates from the Galápagos were found in rotten wood, but also in flowers, orchids, and leafs among others. This yeast species was also collected in a range of altitudes

*Galactomyces geotrichum*[31] is another yeast species which is widespread along the four natural regions of Ecuador. Its distribution has been registered from 75 to 3500 m.a.s.l. This fact demonstrates its high adaptability to a number of ecosystems and growth temperatures. It is a vigorous yeast species characterized by its fast growth in laboratory conditions. The strength of this yeast is a feature that has been useful in the prevention of microbial infections in greenhouse crops [25] because of its highly competitive way of occupying micro substrates

(beetles) whose excretions are dropped on flowers and other vegetal matter.

archaeological fermentation pots from ancient cultures.

to *Candida* sp. cell wall antigens [30].

in Ecuadorian territory: from 150 to 1820 m.a.s.l.

**Table 4.** Species occurrence in Mainland Ecuador and/or the Galápagos Islands

It is noticeable that in terms of biodiversity registered in Ecuador's mainland and the Galápa‐ gos Islands, the figures are very similar. Nevertheless, it should be understood that the total sample that is herein analyzed, represents only a fraction of the total yeasts species which may be found in this biodiverse region of the planet. At this point of the research the registers of yeast species in Ecuador are still in constant updating as well as the registers in the rest of the world. We believe that less than 5% of the world's yeast biodiversity has been described [21, 25]. A big effort and a long time of research are needed to try fulfilling the CLQCA database. Despite the data analyzed in this first overview of the biodiversity of yeasts in the four regions of Ecuador are still in process, we can certainly draw some features of the yeast biota of the Galápagos and its closest mainland territory.

#### **6.3. A brief ecological approach to the most remarkable yeast species in Galápagos and Mainland**

The yeast species that are part of the communities found both in the Galápagos Islands and the mainland are: *Aureobasidium pullulans, Barnettozyma californica, C. carvajalis, C. humilis, C. intermedia, C. oleophila, C. orthopsilosis, C. parapsilosis, C. pseudointermedia, C. saopaulensis, C. sinolaborantium, C. tropicalis, Debaryomyces nepalensis, Galactomyces geotrichum, Hanseniaspora* sp., *Pichia terricola, Rhodotorula glutinis*, and *Rh. mucilaginosa*.

Strains recovered of *B. californica* in other studies includes substrates such as soil, ox dung, insect frass, pond water, sewage water, river water, and stream water [25]. In the Ecuadorian collection this species was found in flowers, beetles captured in flowers of *Ipomoea alba*, giant turtle's dung, sugar cane bagasse, fungus, and rotten wood. Insects may have played a role in the dispersion of this species from mainland into the Galápagos.

*Candida humilis* in the Galápagos Islands has been found in rotten wood and flowers, but other registers of this species such as the strains CBS 6312 and CBS 6099 reported in The Yeasts a Taxonomic Studies [25] which were found in frass and gut of auger beetles. In this case we have maybe another evidence of insects as vectors of this yeast species and the occupation of the habitats in the Islands may not be so different than in mainland for this yeast species. In Ecuadorian mainland *C. humilis* was isolated from mango fruit, chicha de jora (corn fermented beverage), spider network, rotten wood and some flowers from the Asteraceae family. As for *C. intermedia* and *C. parapsilosis* are considered widespread yeasts that can be found in clinical samples, caterpillar frass, beer contaminants and other substrates. In the case of Ecuadorian isolates of these both species, the origin is predominantly found in vegetal substrates and in archaeological fermentation pots from ancient cultures.

**N° YEAST SPECIES ECUADOR MAINLAND GALAPAGOS ISLANDS**

It is noticeable that in terms of biodiversity registered in Ecuador's mainland and the Galápa‐ gos Islands, the figures are very similar. Nevertheless, it should be understood that the total sample that is herein analyzed, represents only a fraction of the total yeasts species which may be found in this biodiverse region of the planet. At this point of the research the registers of yeast species in Ecuador are still in constant updating as well as the registers in the rest of the world. We believe that less than 5% of the world's yeast biodiversity has been described [21, 25]. A big effort and a long time of research are needed to try fulfilling the CLQCA database. Despite the data analyzed in this first overview of the biodiversity of yeasts in the four regions of Ecuador are still in process, we can certainly draw some features of the yeast biota of the

**6.3. A brief ecological approach to the most remarkable yeast species in Galápagos and**

The yeast species that are part of the communities found both in the Galápagos Islands and the mainland are: *Aureobasidium pullulans, Barnettozyma californica, C. carvajalis, C. humilis, C. intermedia, C. oleophila, C. orthopsilosis, C. parapsilosis, C. pseudointermedia, C. saopaulensis, C. sinolaborantium, C. tropicalis, Debaryomyces nepalensis, Galactomyces geotrichum, Hanseniaspora*

Strains recovered of *B. californica* in other studies includes substrates such as soil, ox dung, insect frass, pond water, sewage water, river water, and stream water [25]. In the Ecuadorian collection this species was found in flowers, beetles captured in flowers of *Ipomoea alba*, giant turtle's dung, sugar cane bagasse, fungus, and rotten wood. Insects may have played a role in

*Candida humilis* in the Galápagos Islands has been found in rotten wood and flowers, but other registers of this species such as the strains CBS 6312 and CBS 6099 reported in The Yeasts a Taxonomic Studies [25] which were found in frass and gut of auger beetles. In this case we have maybe another evidence of insects as vectors of this yeast species and the occupation of the habitats in the Islands may not be so different than in mainland for this yeast species. In

 *Trichosporon multisporon Wickerhamiella occidentalis Wickerhamomyces anomalus Wickerhamomyces onychis Yamadazyma mexicana*

208 Biodiversity - The Dynamic Balance of the Planet

**117** *Yarrowia lipolytica*

**Mainland**

**118** *Zygotorulaspora florentina*

**Table 4.** Species occurrence in Mainland Ecuador and/or the Galápagos Islands

Galápagos and its closest mainland territory.

sp., *Pichia terricola, Rhodotorula glutinis*, and *Rh. mucilaginosa*.

the dispersion of this species from mainland into the Galápagos.

In the case of *C. saopaulonensis* we can find that in the mainland Ecuador this species was isolated from a Heliconiaceae as well as the type strain collected in Brazil [26]; in contrast, this yeast species was found in nine different flowers from Asteraceae family in Santa Cruz Island, which is an interesting case of adaptation to new ecological niches in oceanic islands [16].

*C. sinolaborantium* [27] is a yeast species that in the Ecuadorian mainland as well as in the Galápagos Islands was found chiefly in vegetal substrates except for one strain that was collected in *Drosophila* sp. Nevertheless the type strain was isolated from the gut of handsome fungus beetle in Panamá, and two other strains were isolated from a cerambycid larva. The original study was carried out in insect's guts. The results obtained by [27] suggest that this yeast species is part of the microbial community that can be found in the intestine of insects (beetles) whose excretions are dropped on flowers and other vegetal matter.

Another remarkable yeast species found in this survey both in mainland and the Galápagos Islands is *C. tropicalis.* This ubiquitous species is by far the most abundant in isolates number that has been collected since 2006 in our surveys in Ecuador; *C. tropicalis* is practically distrib‐ uted in every kind of substrate, which is a sign of the high adaptability of this species. As part of a study on traditional fermented beverages we were able to find this yeast species even in unexpected substrates such as cassava fermented beverages which are produced in Yasuní National Park, located in the deep Ecuadorian Amazonia. This millenary beverage is still being produced in the same traditional way by autochthonous tribes of Waorani people [28]. In the literature *C. tropicalis* is reported to be collected from clinical samples. This species belongs to the *Lodderomyces* clade, where *C. albicans, C. theae*[29] and other potential pathogens have been accommodated. Nevertheless its presence by itself does not mean a hazard of infection in human, at least to those who possess a strong immune response due to permanent exposition to *Candida* sp. cell wall antigens [30].

A very few understood yeast species in terms of its ecology is *Debaryomyces nepalensis*, since it was isolated from a number of different substrates like soil, fermenting tobacco, spoiled sake and others [25]. The isolates from the Galápagos were found in rotten wood, but also in flowers, orchids, and leafs among others. This yeast species was also collected in a range of altitudes in Ecuadorian territory: from 150 to 1820 m.a.s.l.

*Galactomyces geotrichum*[31] is another yeast species which is widespread along the four natural regions of Ecuador. Its distribution has been registered from 75 to 3500 m.a.s.l. This fact demonstrates its high adaptability to a number of ecosystems and growth temperatures. It is a vigorous yeast species characterized by its fast growth in laboratory conditions. The strength of this yeast is a feature that has been useful in the prevention of microbial infections in greenhouse crops [25] because of its highly competitive way of occupying micro substrates and ecological niches. As for the substrates where this yeast species was found in this survey, these includes rotten wood, flowers, turtle's feces, insects, vegetal residues, fruits, etc. Re‐ markably, by 1970 just a few strains of this species were collected from soil samples in Puerto Rico [25].

Both yeast species are ubiquitous being found in a wide range of latitudes, including in Antarctica glaciers (data not published). Some registers of *R. glutinis* are in atmosphere, trees, leaves, grapes, soil, spoiled leather, sea water, water supply of a brewery, sputum of pneu‐ monia patient, exudates, limph nodes, feces, pasteurized beer and a number of other substrates [41]. These two yeast species are not fermentative, which is a trait of basidiomycetous yeast

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From Ecuador we were able to collect 46 strains belonging to these two species. Astonishingly, 18 out of 46 were isolated from ancient-dormant yeast communities found in fermentation vessels and other utensils used by ancient cultures from the Andes used in their daily life. The studies of Microbial Archaeology [42] yielded other yeast species which will be taken later on

Other substrates where *R. glutinis* and *R. mucilaginosa* were isolated from are fermented beverages, sugar cane juice, insects (Orthoptera, Coleoptera, Hemiptera), as well as in flowers,

*C. carvajalis* [43] was the first yeast species described from Ecuador. This yeast was found in the course of a yeast biodiversity survey in the Amazonia. The substrate sampled was rotten wood and fallen leaf debris, collected around crude oil wells, close to Dayuma town. One Isolate of this species was collected in Santa Cruz Island from *Psidium guajava* mucilage. The closest relatives of *C. carvajalis* are *C. asparagi*, *C. fructus*, and *C. musae*. This group of yeasts belongs to the *Clavispora* clade. In Mainland Ecuador it has not collected any *C. asparagi* [44] isolate, but in the Galápagos Islands we have one register from Santa Cruz Island where this

**6.4. Ecology of the yeasts species in the Galápagos Islands and Mainland Ecuador**

The yeast isolates that were identified, characterized and preserved in the CLQCA since 2006 represent an invaluable platform for studies in ecology of yeasts form Ecuador. Our data permit a better understanding of the situation of the yeast species and communities in natural environments of Ecuador, which is a contribution for the knowledge of the yeast ecology and biology. In this collaborative work other laboratories and centers have been involved, namely the Collection of Microorganisms and Cells at the Universidade Federal de Minas Gerais in Belo Horizonte, Brazil; the National Collection of Yeast Cultures in Norwich, England; the Department of Biology at the University of Western Ontario, In Ontario, Canada, among the

In this part of the chapter we will analyze not only the yeast species occurrence in Ecuador, but also the relationships we have found respect to the substrates where the yeasts were collected; the ecological roles of the species as reported by other authors and contrasting information we got in our own work. Finally, we will present some ecological similitudes and differences in the roles and behavior of yeasts depending on their biogeographic zones where

species.

in this chapter.

species was collected from a nitidulid beetle.

more active collaborators in this survey.

and moss.

they occur.

The genus *Hanseniaspora* was also found in flower samples from the Galápagos and Mainland Ecuador. This genus is characterized by its bipolar budding, presenting apiculate and ovoid cells that can even be long ovoidal or elongate [32]. This genus is composed by a number of species: *H. clermontiae, H. guilliermondii, H. lachancei, H. meyeri, H. occidentalis, H. opuntiae, H osmophila, H. pseudoguilliermondii, H. uvarum, H. valbyensis, H. vinae, H. singularis and H. thailandica.* In Ecuador mainland it was found *H. meyeri*, *H. guilliermondii*, *H. valvyensis, H. opuntiae,* and *H. uvarum*. Nevertheless, we couldn't yet identify the Galápagos Islands isolates to the species level.

In terms of the substrates where the yeast species have been found both in mainland and the islands there is a wide range of sources: insects, flowers, leaves, feces, sugar cane mills, fungus, fruit, moss, and a number of samples taken from endemic plants in the Galápagos Islands, including *Miconia robinsoniana*, *Scalesia* sp., *Opuntia* sp. *Castela galapageia*, etc. This genus is widely represented in the CLQCA, where about 100 isolates were collected from all the regions in mainland and Galápagos. The isolates from Galápagos represent about 60% of the total isolates of this genus in the CLQCA.

In the case of *Pichia terricola* [33] has been scarcely represented in the mainland with only one isolate in the Andean province of Pichincha. The range of altitude in which this species is distributed in the Ecuadorian territory, according to the registers of the CLQCA, is from 100 to 1120 m.a.s.l., and it's found in a variety of habitats, from dry forests in the Galápagos Islands to cloudy forests in the mainland. The description of this species was based on soil samples from South Africa, but it was also found in cherry juice, from pressed grapes, spoiled figs, and wine [25]. This is an ethanol assimilating yeast, since it can use this compound as an alternative carbon source. This yeast can also use other carbon compounds such as glycerol and glucose, as well as succinate.

This yeast species was collected in flowers, rotten wood, fruit, insects, and exude of trees. In Galápagos the microhabitats for this species are quite different to those of the original description in 1957, where *P. terricola* was named based on the soil samples where it was found in South Africa by [33]. The appellative given to this species illustrates a frequent issue found in taxonomic accommodation; the species names derived from the substrates where the isolates were found, may not provide an accurate idea of its ecological niche. Frequently, yeasts isolates may represent allochthonous members of microbial communities [34].

*Rhodotorula glutinis* [35] and *Rhodotorula mucilaginosa* [36] are both basidiomycetous yeast species characterized by the production of carotenoid pigments which protect them from the UV irradiation that provokes damage in DNA [37-39]; especially in zones where the solar rays are particularly severe, like in the equatorial latitude, the need for a protective strategy can make the difference for yeast cells that are exposed to aggressive light stress [40].

Both yeast species are ubiquitous being found in a wide range of latitudes, including in Antarctica glaciers (data not published). Some registers of *R. glutinis* are in atmosphere, trees, leaves, grapes, soil, spoiled leather, sea water, water supply of a brewery, sputum of pneu‐ monia patient, exudates, limph nodes, feces, pasteurized beer and a number of other substrates [41]. These two yeast species are not fermentative, which is a trait of basidiomycetous yeast species.

and ecological niches. As for the substrates where this yeast species was found in this survey, these includes rotten wood, flowers, turtle's feces, insects, vegetal residues, fruits, etc. Re‐ markably, by 1970 just a few strains of this species were collected from soil samples in Puerto

The genus *Hanseniaspora* was also found in flower samples from the Galápagos and Mainland Ecuador. This genus is characterized by its bipolar budding, presenting apiculate and ovoid cells that can even be long ovoidal or elongate [32]. This genus is composed by a number of species: *H. clermontiae, H. guilliermondii, H. lachancei, H. meyeri, H. occidentalis, H. opuntiae, H osmophila, H. pseudoguilliermondii, H. uvarum, H. valbyensis, H. vinae, H. singularis and H. thailandica.* In Ecuador mainland it was found *H. meyeri*, *H. guilliermondii*, *H. valvyensis, H. opuntiae,* and *H. uvarum*. Nevertheless, we couldn't yet identify the Galápagos Islands isolates

In terms of the substrates where the yeast species have been found both in mainland and the islands there is a wide range of sources: insects, flowers, leaves, feces, sugar cane mills, fungus, fruit, moss, and a number of samples taken from endemic plants in the Galápagos Islands, including *Miconia robinsoniana*, *Scalesia* sp., *Opuntia* sp. *Castela galapageia*, etc. This genus is widely represented in the CLQCA, where about 100 isolates were collected from all the regions in mainland and Galápagos. The isolates from Galápagos represent about 60% of the total

In the case of *Pichia terricola* [33] has been scarcely represented in the mainland with only one isolate in the Andean province of Pichincha. The range of altitude in which this species is distributed in the Ecuadorian territory, according to the registers of the CLQCA, is from 100 to 1120 m.a.s.l., and it's found in a variety of habitats, from dry forests in the Galápagos Islands to cloudy forests in the mainland. The description of this species was based on soil samples from South Africa, but it was also found in cherry juice, from pressed grapes, spoiled figs, and wine [25]. This is an ethanol assimilating yeast, since it can use this compound as an alternative carbon source. This yeast can also use other carbon compounds such as glycerol and glucose,

This yeast species was collected in flowers, rotten wood, fruit, insects, and exude of trees. In Galápagos the microhabitats for this species are quite different to those of the original description in 1957, where *P. terricola* was named based on the soil samples where it was found in South Africa by [33]. The appellative given to this species illustrates a frequent issue found in taxonomic accommodation; the species names derived from the substrates where the isolates were found, may not provide an accurate idea of its ecological niche. Frequently, yeasts isolates

*Rhodotorula glutinis* [35] and *Rhodotorula mucilaginosa* [36] are both basidiomycetous yeast species characterized by the production of carotenoid pigments which protect them from the UV irradiation that provokes damage in DNA [37-39]; especially in zones where the solar rays are particularly severe, like in the equatorial latitude, the need for a protective strategy can

make the difference for yeast cells that are exposed to aggressive light stress [40].

may represent allochthonous members of microbial communities [34].

Rico [25].

210 Biodiversity - The Dynamic Balance of the Planet

to the species level.

as well as succinate.

isolates of this genus in the CLQCA.

From Ecuador we were able to collect 46 strains belonging to these two species. Astonishingly, 18 out of 46 were isolated from ancient-dormant yeast communities found in fermentation vessels and other utensils used by ancient cultures from the Andes used in their daily life. The studies of Microbial Archaeology [42] yielded other yeast species which will be taken later on in this chapter.

Other substrates where *R. glutinis* and *R. mucilaginosa* were isolated from are fermented beverages, sugar cane juice, insects (Orthoptera, Coleoptera, Hemiptera), as well as in flowers, and moss.

*C. carvajalis* [43] was the first yeast species described from Ecuador. This yeast was found in the course of a yeast biodiversity survey in the Amazonia. The substrate sampled was rotten wood and fallen leaf debris, collected around crude oil wells, close to Dayuma town. One Isolate of this species was collected in Santa Cruz Island from *Psidium guajava* mucilage. The closest relatives of *C. carvajalis* are *C. asparagi*, *C. fructus*, and *C. musae*. This group of yeasts belongs to the *Clavispora* clade. In Mainland Ecuador it has not collected any *C. asparagi* [44] isolate, but in the Galápagos Islands we have one register from Santa Cruz Island where this species was collected from a nitidulid beetle.

#### **6.4. Ecology of the yeasts species in the Galápagos Islands and Mainland Ecuador**

The yeast isolates that were identified, characterized and preserved in the CLQCA since 2006 represent an invaluable platform for studies in ecology of yeasts form Ecuador. Our data permit a better understanding of the situation of the yeast species and communities in natural environments of Ecuador, which is a contribution for the knowledge of the yeast ecology and biology. In this collaborative work other laboratories and centers have been involved, namely the Collection of Microorganisms and Cells at the Universidade Federal de Minas Gerais in Belo Horizonte, Brazil; the National Collection of Yeast Cultures in Norwich, England; the Department of Biology at the University of Western Ontario, In Ontario, Canada, among the more active collaborators in this survey.

In this part of the chapter we will analyze not only the yeast species occurrence in Ecuador, but also the relationships we have found respect to the substrates where the yeasts were collected; the ecological roles of the species as reported by other authors and contrasting information we got in our own work. Finally, we will present some ecological similitudes and differences in the roles and behavior of yeasts depending on their biogeographic zones where they occur.

#### **6.5. Yeast species by substrate**

We intended to compare the communities of yeasts that are present in the different substrates we have sampled in mainland and the islands. The most common substrates represented in this survey are: flowers, rotten wood, beetles, excrement, rotten vegetal matter, fruits, insects (*Drosophila* sp. and Nitidulid beetles), exudates, leaves, fungus, and human related substrates such as artisanal sugar cane mills. Some substrates like bodies of water, moss, wood, etc. are not analyzed due to the fact that in mainland we do not have the correspondent substrates to compare with.

Another issue is the number of isolates that were collected by substrate. Given the fact that the number of collections in the field made by each substrate varies substantially from one substrate to the other, a calculation was employed to obtain the average yeast isolates obtained

An Overview of the Yeast Biodiversity in the Galápagos Islands and Other Ecuadorian Regions

Where *<sup>A</sup>*¯ represents the average number of isolates per substrate, *<sup>N</sup>* represents the number of isolates with morphological differences gotten from each substrate; and, *S* corresponds to the number of samples taken from each substrate. In that way it was possible to determine the number of potentially different species based upon the macroscopic morphology. This approach provides an idea about the composition of the communities in terms of the com‐ plexity, based on the morphological differences from its members in a variety of substrates.

From a general point of view, we can say that the potential number of different species isolated is predominant in those substrates where the conditions favor the yeast reproduction and activity. Thus, in the sugar cane mill we could recover an average of 5 yeast isolates per sampling (regardless its biodiversity), while in flowers we could recover an average of 1.9 yeast isolates per sample. The abundance of yeasts also seems to be higher in insect's guts as well as in rotten wood: from the guts of beetles staying within *Datura* sp. and *Ipomoea* sp. flowers we could recover 3.1 and 4 isolates in average, which is slightly lower than the number of isolates from the sugar cane mill. In rotten wood the number of isolates was relatively high since we could recover an average of 3.5 yeast colonies. Table 5 resumes the average of isolates recovered by substrate in the Galápagos Islands. Nevertheless, these data does not provide

A further analysis of these figures reveals that the occurrence of yeast isolates in the samples has not a direct relationship with the biodiversity that can be found in each substrate, consid‐ ering that macroscopic differences of yeast colonies does not necessarily represent different yeast species, but strains of a same species. A clear example of that is the biodiversity of yeasts found in the sugar cane mill (5 possible different species) which reports the highest average of isolates per sample (5 isolates), compared to flowers which present a rather low average of isolates per sample (1.9 isolates) but the highest biodiversity (14.2 species per sample). In Figure 2 the correlation between the number of samples, the number of isolates per sample, and the

The insects were the substrates that yielded the highest biodiversity of yeasts in the Galápagos Islands (38 species), followed by rotten wood (35 species) samples and flowers (28 species). The substrates like trees exudates, rotten plant matter (leaves and fruits), fruits, fungus, leaves, and excrement, yielded from 7 to 11 different yeast species, while the sugar mill and fermented sugar cane juice sampled presented 5 isolates. Nonetheless, the highest average number of

species per sample was registered in flowers, followed by insects and rotten wood.

enough information about the biodiversity of yeasts living in the substrates.

number of species by sample is drawn.

*<sup>S</sup>* (1)

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213

*<sup>A</sup>*¯ <sup>=</sup> *<sup>N</sup>*

by substrate. This calculation was done using the equation (1):

The data were homogenized in order to get comparable ecological niches in Galápagos and Ecuador mainland. Consequently, a comparison of the yeast communities found in analogous substrates sampled both in the islands and the mainland is made in this section. matter, fruits, insects (*Drosophila* sp. and Nitidulid beetles), exudates, leaves, fungus, and human related substrates such as artisanal sugar cane mills. Some substrates like bodies of water, moss, wood, etc. are not analyzed due to the fact that in mainland we do not have the correspondent substrates to compare with.

As a consequence of this grouping of substrates, the number of species having correspondence between mainland and the islands decreased from 38 to 22, since the variety of substrates in mainland is much larger than those sampled in the Galapagos, furthermore, it was not possible to match all the samples for a comparison. The data were homogenized in order to get comparable ecological niches in Galápagos and Ecuador mainland. Consequently, a comparison of the yeast communities found in analogous substrates sampled both in the islands and the mainland is made in this section. As a consequence of this grouping of substrates, the number of species having

Nevertheless, an independent analysis of the species found only in mainland and the Galápa‐ gos was also performed in order to get some biodiversity plots of the yeast communities. Based on that, we were able to analyze the situation of yeast biodiversity in Ecuador, taking into account the general characteristics of oceanic islands. correspondence between mainland and the islands decreased from 38 to 22, since the variety of substrates in mainland is much larger than those sampled in the Galapagos, furthermore, it was not possible to match all the samples for a comparison. Nevertheless, an independent analysis of the species found only in mainland and the Galápagos was also performed in order to get some biodiversity plots of the yeast

The substrates from which more samples were taken are flowers, insects and rotten wood. This fact responds to the specific objectives of the expeditionaries who centered their interest in the surveys of these substrates. Nonetheless, other substrates were also sampled with different intensity. The share of the samples taken in the expedition is shown in Figure 1. communities. Based on that, we were able to analyze the situation of yeast biodiversity in Ecuador, taking into account the general characteristics of oceanic islands. The substrates from which more samples were taken are flowers, insects and rotten wood. This fact responds to the specific objectives of the expeditionaries who centered their interest in the surveys of these substrates. Nonetheless, other substrates were also sampled with

different intensity. The share of the samples taken in the expedition is shown in Figure 1.

Figure 1. Substrates sampled in the basis of the number of samples taken in the Galápagos Islands.

**Figure 1.** Substrates sampled in the basis of the number of samples taken in the Galápagos Islands.

Another issue is the number of isolates that were collected by substrate. Given the fact that the number of collections in the field made by each substrate varies substantially from one substrate to the other, a calculation was employed to obtain the average yeast isolates obtained by substrate. This calculation was done using the equation (1):

**6.5. Yeast species by substrate**

212 Biodiversity - The Dynamic Balance of the Planet

to match all the samples for a comparison.

account the general characteristics of oceanic islands.

13%

8%

6%

6%

the correspondent substrates to compare with.

compare with.

We intended to compare the communities of yeasts that are present in the different substrates we have sampled in mainland and the islands. The most common substrates represented in this survey are: flowers, rotten wood, beetles, excrement, rotten vegetal matter, fruits, insects (*Drosophila* sp. and Nitidulid beetles), exudates, leaves, fungus, and human related substrates such as artisanal sugar cane mills. Some substrates like bodies of water, moss, wood, etc. are not analyzed due to the fact that in mainland we do not have the correspondent substrates to

The data were homogenized in order to get comparable ecological niches in Galápagos and Ecuador mainland. Consequently, a comparison of the yeast communities found in analogous

matter, fruits, insects (*Drosophila* sp. and Nitidulid beetles), exudates, leaves, fungus, and human related substrates such as artisanal sugar cane mills. Some substrates like bodies of water, moss, wood, etc. are not analyzed due to the fact that in mainland we do not have

As a consequence of this grouping of substrates, the number of species having correspondence between mainland and the islands decreased from 38 to 22, since the variety of substrates in mainland is much larger than those sampled in the Galapagos, furthermore, it was not possible

The data were homogenized in order to get comparable ecological niches in Galápagos and Ecuador mainland. Consequently, a comparison of the yeast communities found in analogous substrates sampled both in the islands and the mainland is made in this section.

Nevertheless, an independent analysis of the species found only in mainland and the Galápa‐ gos was also performed in order to get some biodiversity plots of the yeast communities. Based on that, we were able to analyze the situation of yeast biodiversity in Ecuador, taking into

As a consequence of this grouping of substrates, the number of species having correspondence between mainland and the islands decreased from 38 to 22, since the variety of substrates in mainland is much larger than those sampled in the Galapagos, furthermore,

The substrates from which more samples were taken are flowers, insects and rotten wood. This fact responds to the specific objectives of the expeditionaries who centered their interest in the surveys of these substrates. Nonetheless, other substrates were also sampled with different

The substrates from which more samples were taken are flowers, insects and rotten wood. This fact responds to the specific objectives of the expeditionaries who centered their interest in the surveys of these substrates. Nonetheless, other substrates were also sampled with different intensity. The share of the samples taken in the expedition is shown in Figure 1.

**Share of substrates sampled**

41%

Flower Insecta Rotten wood Rotten vegetal matter

Excrement Fruit Exude Fungus Leaf

Sugar cane mill

Figure 1. Substrates sampled in the basis of the number of samples taken in the Galápagos Islands.

 Nevertheless, an independent analysis of the species found only in mainland and the Galápagos was also performed in order to get some biodiversity plots of the yeast communities. Based on that, we were able to analyze the situation of yeast biodiversity in

intensity. The share of the samples taken in the expedition is shown in Figure 1.

16%

**Figure 1.** Substrates sampled in the basis of the number of samples taken in the Galápagos Islands.

4% 3% 2% 1%

Ecuador, taking into account the general characteristics of oceanic islands.

it was not possible to match all the samples for a comparison.

substrates sampled both in the islands and the mainland is made in this section.

$$
\overline{A} = \frac{N}{S} \tag{1}
$$

Where *<sup>A</sup>*¯ represents the average number of isolates per substrate, *<sup>N</sup>* represents the number of isolates with morphological differences gotten from each substrate; and, *S* corresponds to the number of samples taken from each substrate. In that way it was possible to determine the number of potentially different species based upon the macroscopic morphology. This approach provides an idea about the composition of the communities in terms of the com‐ plexity, based on the morphological differences from its members in a variety of substrates.

From a general point of view, we can say that the potential number of different species isolated is predominant in those substrates where the conditions favor the yeast reproduction and activity. Thus, in the sugar cane mill we could recover an average of 5 yeast isolates per sampling (regardless its biodiversity), while in flowers we could recover an average of 1.9 yeast isolates per sample. The abundance of yeasts also seems to be higher in insect's guts as well as in rotten wood: from the guts of beetles staying within *Datura* sp. and *Ipomoea* sp. flowers we could recover 3.1 and 4 isolates in average, which is slightly lower than the number of isolates from the sugar cane mill. In rotten wood the number of isolates was relatively high since we could recover an average of 3.5 yeast colonies. Table 5 resumes the average of isolates recovered by substrate in the Galápagos Islands. Nevertheless, these data does not provide enough information about the biodiversity of yeasts living in the substrates.

A further analysis of these figures reveals that the occurrence of yeast isolates in the samples has not a direct relationship with the biodiversity that can be found in each substrate, consid‐ ering that macroscopic differences of yeast colonies does not necessarily represent different yeast species, but strains of a same species. A clear example of that is the biodiversity of yeasts found in the sugar cane mill (5 possible different species) which reports the highest average of isolates per sample (5 isolates), compared to flowers which present a rather low average of isolates per sample (1.9 isolates) but the highest biodiversity (14.2 species per sample). In Figure 2 the correlation between the number of samples, the number of isolates per sample, and the number of species by sample is drawn.

The insects were the substrates that yielded the highest biodiversity of yeasts in the Galápagos Islands (38 species), followed by rotten wood (35 species) samples and flowers (28 species). The substrates like trees exudates, rotten plant matter (leaves and fruits), fruits, fungus, leaves, and excrement, yielded from 7 to 11 different yeast species, while the sugar mill and fermented sugar cane juice sampled presented 5 isolates. Nonetheless, the highest average number of species per sample was registered in flowers, followed by insects and rotten wood.

**SUBSTRATE**

Flower 1.9 Rotten wood 3.5 Beetle in *Datura* sp./*Ipomoea* sp. 3.1 Turtle´s excrement 2.6 Rotten leaf/fruit 2.3 Fruit 2.1 Drosophila sp. in *Datura* sp./*Ipomoea* sp./*Psidium guajava* 4 Exude 2.2 Leaf 3 Fungus 2.1 Sugar cane mill 5

**Table 5.** Abundance of yeast isolates recovered from different substrates in the Galápagos Islands

biodiversity is much higher and the communities are richer.

any other isolate of this yeast species.

**Galápagos**

3a, 3b, and 3c.

**6.6. Analyzing the yeast communities in Ecuador Mainland and The Archipelago of**

Disharmony and relictualism are both characteristics of oceanic islands [16]. The yeast diversity in the Galápagos Islands seems to be highly influenced by Ecuador mainland: the yeast species that are represented in the Galápagos and mainland accounts for 31% of the total species represented in mainland. Coincidently, this is the figure that corresponds to the iterative plant species in the mainland and the islands [18]. Another feature is the competence for ecological niches in the Galápagos which is not as severe as in mainland, where the

With the data we got, it is not possible to analyze relictualism in the islands, since there is not any fossil register of ancient yeasts in the mainland which now occurs in the islands. Never‐ theless, we do have one example of what we could consider a relictual species that has collected in Galápagos from the surface of a leaf. The same yeast species was resuscitated from ancient chicha (corn beer) fermentation vessels during a Microbial Archaeology survey in Quito in 2008 [21, 42]. The substrate is catalogued as an archaeological piece from 680 a.D. and the yeast species is *Candida theae* [29]. After intensive sampling in Ecuador mainland, we did not found

To analyze the state of the yeast communities in the islands and in mainland, we performed a series of comparisons in order to find the composition of the communities in the correspondent substrates in mainland, islands and a combination of both. The results are shown in Figures

**AVERAGE OF ISOLATES BY NUMBER OF SUBSTRATE SAMPLED**

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215

An Overview of the Yeast Biodiversity in the Galápagos Islands and Other Ecuadorian Regions

Galápagos Islands (38 species), followed by rotten wood (35 species) samples and flowers (28 species). The substrates like trees exudates, rotten plant matter (leaves and fruits), fruits, fungus, leaves, and excrement, yielded from 7 to 11 different yeast species, while the sugar mill and fermented sugar cane juice sampled presented 5 isolates. Nonetheless, the **Figure 2.** Correlation between number of samples from substrates, number of isolates per sample, and biodiversity of yeasts in substrates

The insects were the substrates that yielded the highest biodiversity of yeasts in the

**Figure 2**. Correlation between number of samples from substrates, number of isolates per

sample, and biodiversity of yeasts in substrates

highest average number of species per sample was registered in flowers, followed by insects and rotten wood. On the other hand, the fermentation vessels and utensils where sugar cane juice is extracted and then transformed into fermented liquor containing high ethanol degree, constitutes a restrictive environment for yeast, where only a few species such as *Saccharomyces cerevisiae* can be found, owing to its high tolerance to ethanol provided by the fatty acids contained in the cells [45]. The rather poor biodiversity of this kind of yeast community is inversely proportional with the population abundance of the community. This inversely proportional relationship confirms that the highest biodiversity of yeasts can be found in those substrates where yeast communities play a fundamental ecological role. Most yeast species isolated from flowers are supposedly nectar-inhabiting yeasts. Dense yeast communities often occur in the floral nectar of animal-pollinated plants, where they can behave as parasites of plant-pollinator mutualisms [46-50]. **Analyzing the yeast communities in Ecuador Mainland and The Archipelago of Galápagos**  On the other hand, the fermentation vessels and utensils where sugar cane juice is extracted and then transformed into fermented liquor containing high ethanol degree, constitutes a restrictive environment for yeast, where only a few species such as *Saccharomyces cerevi‐ siae* can be found, owing to its high tolerance to ethanol provided by the fatty acids contained in the cells [45]. The rather poor biodiversity of this kind of yeast community is inversely proportional with the population abundance of the community. This inversely proportional relationship confirms that the highest biodiversity of yeasts can be found in those substrates where yeast communities play a fundamental ecological role. Most yeast species isolated from flowers are supposedly nectar-inhabiting yeasts. Dense yeast commun‐ ities often occur in the floral nectar of animal-pollinated plants, where they can behave as parasites of plant-pollinator mutualisms [46-50].


**Table 5.** Abundance of yeast isolates recovered from different substrates in the Galápagos Islands

**Figure 2**. Correlation between number of samples from substrates, number of isolates per

<sup>45</sup> **BIODIVERSITY AND ABUNDANCE OF YEASTS BY** 

**SUBSTRATE FROM GALÁPAGOS** 

Average number of isolates

Average number of Species

per sample

by Sample

Number of species

The insects were the substrates that yielded the highest biodiversity of yeasts in the Galápagos Islands (38 species), followed by rotten wood (35 species) samples and flowers (28 species). The substrates like trees exudates, rotten plant matter (leaves and fruits), fruits, fungus, leaves, and excrement, yielded from 7 to 11 different yeast species, while the sugar mill and fermented sugar cane juice sampled presented 5 isolates. Nonetheless, the highest average number of species per sample was registered in flowers, followed by

**Figure 2.** Correlation between number of samples from substrates, number of isolates per sample, and biodiversity of

On the other hand, the fermentation vessels and utensils where sugar cane juice is extracted and then transformed into fermented liquor containing high ethanol degree, constitutes a restrictive environment for yeast, where only a few species such as *Saccharomyces cerevisiae* can be found, owing to its high tolerance to ethanol provided by the fatty acids contained in the cells [45]. The rather poor biodiversity of this kind of yeast community is inversely proportional with the population abundance of the community. This inversely proportional relationship confirms that the highest biodiversity of yeasts can be found in those substrates where yeast communities play a fundamental ecological role. Most yeast species isolated from flowers are supposedly nectar-inhabiting yeasts. Dense yeast communities often occur in the floral nectar of animal-pollinated plants, where they can

On the other hand, the fermentation vessels and utensils where sugar cane juice is extracted and then transformed into fermented liquor containing high ethanol degree, constitutes a restrictive environment for yeast, where only a few species such as *Saccharomyces cerevi‐ siae* can be found, owing to its high tolerance to ethanol provided by the fatty acids contained in the cells [45]. The rather poor biodiversity of this kind of yeast community is inversely proportional with the population abundance of the community. This inversely proportional relationship confirms that the highest biodiversity of yeasts can be found in those substrates where yeast communities play a fundamental ecological role. Most yeast species isolated from flowers are supposedly nectar-inhabiting yeasts. Dense yeast commun‐ ities often occur in the floral nectar of animal-pollinated plants, where they can behave as

**Analyzing the yeast communities in Ecuador Mainland and The Archipelago of** 

sample, and biodiversity of yeasts in substrates

behave as parasites of plant-pollinator mutualisms [46-50].

parasites of plant-pollinator mutualisms [46-50].

insects and rotten wood.

yeasts in substrates

214 Biodiversity - The Dynamic Balance of the Planet

**Galápagos** 

#### **6.6. Analyzing the yeast communities in Ecuador Mainland and The Archipelago of Galápagos**

Disharmony and relictualism are both characteristics of oceanic islands [16]. The yeast diversity in the Galápagos Islands seems to be highly influenced by Ecuador mainland: the yeast species that are represented in the Galápagos and mainland accounts for 31% of the total species represented in mainland. Coincidently, this is the figure that corresponds to the iterative plant species in the mainland and the islands [18]. Another feature is the competence for ecological niches in the Galápagos which is not as severe as in mainland, where the biodiversity is much higher and the communities are richer.

With the data we got, it is not possible to analyze relictualism in the islands, since there is not any fossil register of ancient yeasts in the mainland which now occurs in the islands. Never‐ theless, we do have one example of what we could consider a relictual species that has collected in Galápagos from the surface of a leaf. The same yeast species was resuscitated from ancient chicha (corn beer) fermentation vessels during a Microbial Archaeology survey in Quito in 2008 [21, 42]. The substrate is catalogued as an archaeological piece from 680 a.D. and the yeast species is *Candida theae* [29]. After intensive sampling in Ecuador mainland, we did not found any other isolate of this yeast species.

To analyze the state of the yeast communities in the islands and in mainland, we performed a series of comparisons in order to find the composition of the communities in the correspondent substrates in mainland, islands and a combination of both. The results are shown in Figures 3a, 3b, and 3c.


**SPECIES SUBSTRATE SPECIES SUBSTRATE**

An Overview of the Yeast Biodiversity in the Galápagos Islands and Other Ecuadorian Regions

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217

*Hanseniaspora uvarum Saccharomycopsis fodiens Pichia norvegensis Saccharomycopsis vini Candida sonorensis Trichosporon insectorum Cryptococcus flavescens Sporopachydermia* **sp***. Aureobasidium pullulans Candida theae* 

*Trichosporon laibachii Cryptococcus rajastharensis* 

*Candida leandrae* Flower *Candida rugosa* Rotten Wood *Candida zeylanoides* Insecta *Cryptococcus flavus* Rotten vegetal

*Kluyveromyces lactis* Fruit *Kurtzmaniella zeylanoides* Exude *Kwoniella mangrovensis* Leaf

*Metschnikowia kipukae* Sugar cane mill

(b)

*Candida parazyma Geotrichum silvicola Kodamaea transpacifica Wickerhamiella occidentalis Clavispora opuntiae Cryptococcus laurentii Candida trypodendroni Yamadazyma mexicana Candida natalensis Candida cylindracea Candida sorbosivorans Rhodosporidium paludigenum* 

*Candida albicans Candida dendronema Candida naeodendra Candida silvae Kazachstania unispora Lindnera* **sp***. Lindnera saturnus Scheffersomyces stipitis Trichosporon multisporon Candida asparagi* 

An Overview of the Yeast Biodiversity in the Galápagos Islands and Other Ecuadorian Regions http://dx.doi.org/10.5772/58303 217

**SPECIES SUBSTRATE**

*Torulaspora delbrueckii* Flower *Trichosporon asahii* Rotten Wood *Candida carvajalis* Insecta *Cryptococcus humicola* Excrement *Debaryomyces hansenii* Rotten vegetal

*Hanseniaspora meyeri* Fruit *Rhodotorula glutinis* Exudate *Candida orthopsilosis* Leaf *Candida saopaulonensis* Fungus

(a)

Sugar cane mill

*Candida tropicalis Hanseniaspora* **sp***. Candida intermedia Candida parapsilosis Galactomyces geotrichum Saccharomyces cerevisiae Kodamaea ohmeri* 

216 Biodiversity - The Dynamic Balance of the Planet

*Meyerozyma guilliermondii Wickerhamomyces anomalus Barnettozyma californica Candida quercitrusa Pichia kudriavzevii Kazachstania exigua Pichia kluyveri* 

*Candida sinolaborantium* 

*Candida pseudointermedia Debaryomyces nepalensis Pichia manshurica Trichosporon coremiiforme Pichia fermentans Rhodotorula mucilaginosa* 

*Pichia terricola* 

*Candida humilis Pichia occidentalis* 

(b)

**7. Ecological parameters and models developed**

composition [53].

to *Drosophila* flies [34].

those which are represented in both ecosystems.

yeasts.

The degree of specialization of a yeast species is given by its metabolic abilities and tolerance to environmental factors such as UV radiation [40], inhibition substances and even microbial predators [34], etc. Leaves are exposed to fluctuations of temperature and relative humidity values, which may have an impact on the yeast communities. Large fluxes of UV radiation are also one of the most prominent features of the leaves, fruits and other substrates in the environment to which microorganisms have presumably had to adapt [21, 51]. Many plants contain a number of compounds whose adaptive significance may be a defense against invertebrates and microorganisms; for instance, all parts of *Datura* sp. contain toxic belladonna alkaloids, the concentration of which is highest in the petioles of the flowers [52]. These compounds also act, in some cases, as selective agents which shape the yeast community

An Overview of the Yeast Biodiversity in the Galápagos Islands and Other Ecuadorian Regions

http://dx.doi.org/10.5772/58303

219

The yeasts which are highly adaptable to different ecological niches—like *Candida tropicalis* are the ones that display a wide range of responses and developed defense strategies [45]. This particularly abundant and cosmopolitan yeast species is the most frequently isolated in mainland and in the islands according to CLQCA database. This yeast species has been found in flowers, rotten wood, insects, excrement, rotten vegetal matter, and fungus (Figure 3a). *Candida tropicalis* has been reported to grow even at pH so high as 10 [41], which is a remarkable

On the other hand there are yeasts that show a narrower repertoire of metabolism and even metabolic deficiencies such as *Saccharomycopsis fodiens*, a predacious yeast species that is deficient in sulfate uptake and require supplementation of organic sulfur sources [54]. This yeast species is highly specialized in predation of other yeasts and fungi. Nevertheless it appears to be quite rare in the environment and very few is known about its natural history and ecology. Only three isolates from Costa Rica, Australia and the Galápagos islands were found so far. A further expedition in Taiwan registered other isolates of this species associated

**7.1. How the ecological niches are occupied by yeasts in mainland and the islands?**

It is certainly ventured to establish in an accurate way the ecological relationships of yeasts in the islands and the mainland with general data and relatively few samples. For this report we have addressed this issue by means of a global comparison of yeast species number that has been found in different substrates both in mainland and the islands. Despite the kind of substrate, we marked the repetitions of registers in substrates from each one of the species. This analysis gave us a general scheme of the degree of adaptation that is currently facing the

In Figure 4 it is seen the general state of the yeasts based on the number of substrates they occur. This analysis was performed on those species found in mainland and the islands and

feature in terms of tolerance facing unusually hard environmental conditions.

**Figure 3.** (a) Shared species between Galápagos Islands and Mainland Ecuador and its occurrence in different sub‐ strates; (b) Species found in Galápagos and the substrates these yeast colonize; (c) Species found in Ecuador Mainland and the substrates these yeasts colonize
