Abstract
Mucorales comprises fungi commonly isolated as saprobes from soil, dung, stored grains and plants. Although these fungi have been studied in several countries, there are relatively a few reports of them in semi-arid areas. Therefore, the aims of the present study were to assess and compare the Mucorales communities in dung from different species and breeds of herbivores in the semi-arid of Pernambuco, based on the frequency of occurrence and species richness of these fungi. Samples of dung collected in the cities of Arcoverde, Serra Talhada and Sertânia were incubated in moist chambers in triplicate. Altogether, 24 taxa of Mucorales distributed in the genera Absidia, Circinella, Cunninghamella, Lichtheimia, Mucor, Pilobolus, Rhizopus and Syncephalastrum were identified. The highest species richness was found in sheep excrement. Mucor circinelloides f. griseo-cyanus was the most common taxon, followed by M. ramosissimus. The similarity of the composition of Mucorales species was greatest between the excrements of Guzerá and Sindi breeds (bovine). All mucoralean species isolated are being cited for the first time from animal dung found in Caatinga and a new species of Mucor was recorded. An identification key for species of Mucorales from dung in the semi-arid region of Brazil is provided.
Keywords:
Caatinga; Dung; Herbivore; Mucoromycotina; Taxonomy
Introduction
Mucoromycotina Benny, one of the four subphyla proposed to accommodate certain species of traditional Zygomycota C. Moreau (phylum no longer accepted in the new classification due to its polyphyletic nature),11 Hibbett DS, Binder M, Bischoff JF, et al. A higher-level phylogenetic classification of the Fungi. Mycol Res. 2007;3:509-547.
2 Hoffmann K, Voigt K, Kirk PM. Mortierellomycotina subphyl. nov., based on multi-gene genealogies. Mycotaxon. 2011;115(1):353-363.-33 Humber RA. Entomophthoromycota: a new phylum and reclassification for entomophthoroid fungi. Mycotaxon. 2012;120:477-492. covers most saprobes fungi characterized by the production of thick-walled spores of sexual origin, known as zygospores. The order Mucorales Fr., the largest in number of species within the Mucoromycotina, includes fungi forming coenocytic hyphae with septa either at the base of reproductive structures or irregularly distributed in older cultures. Species of this order produce asexual structures such as sporangiophores, sporangia, sporangiospores, sporangiola, merosporangia and merospores.44 Santiago ALCM de A, dos Santos PJP, Maia LC. Mucorales from the semiarid of Pernambuco, Brazil. Braz J Microbiol. 2013;44(1):299-305. These fungi are commonly isolated from soil, stored grains, plants, and animal excrement, especially that of herbivores and rodents.55 Alexopoulos CJ, Mims CW, Blackwell M. Introductory Mycology. 4th ed. New York: John Wiley & Sons; 1996.
Coprophilous fungi are those that live in or are associated with fecal material, including soil contaminated with feces. These microorganisms are essential for the maintenance of ecosystems and are directly involved in the decomposition of fecal waste, participating in carbon, nitrogen and energy cycles.66 Krug JC, Benny GL, Keller HW. Coprophilous fungi. In: Mueller GM, Bills GF, Foster MS, eds. Biodiversity of Fungi. Hardbound: Elsevier Academic Press; 2004:468–499.,77 Richardson MJ. Records of coprophilous fungi from the Lesser Antilles and Puerto Rico. Caribb J Sci. 2008;44(2):206-214. According to Dix and Webster,88 Dix NJ, Webster J. Fungal Ecology. London: Chapman & Hall; 1995. fungi may occur in excrement as obligate and optional coprophilous. The obligate coprophilous have spores that require the action of gastric enzymes to break their dormancy, while secondary or facultative coprophilous spores do not need to go through the digestive tract of animals to germinate.99 Richardson MJ. Coprophilous fungi. Field Mycol. 2003;4(2):41-43. Among the Mucorales, only Pilobolus species are considered obligate coprophilous, although other taxa from this order are common in dung.66 Krug JC, Benny GL, Keller HW. Coprophilous fungi. In: Mueller GM, Bills GF, Foster MS, eds. Biodiversity of Fungi. Hardbound: Elsevier Academic Press; 2004:468–499.
The Brazilian semiarid region occupies an area of approximately 969,589.4 km2, comprising eight northeastern states: Alagoas, Bahia, Ceará, Paraíba, Pernambuco, Piauí, Rio Grande do Norte, Sergipe and part of the state of Minas Gerais.1010 Drumond MA, Kiill LHP, Nascimento CES. Inventário e sociabilidade de espécies arbóreas e arbustivas da Caatinga na Região de Petrolina, PE. Bras Florest. 2002;74:37-43. Semiarid regions are formed, mostly by typical vegetation of Caatinga, an exclusively Brazilian domain that consists of heterogeneous phytophysiognomic systems characteristically xerophilic, with vegetation ranging from tree to shrub.1111 Gusmão LFP, Barbosa FR, Barbosa FF. Fungos Conidias. In: Gusmão LFP, Maia LC, eds. Diversidade e caracterização dos fungos no semi-árido. Vol 1. Recife: Associação Plantas do Nordeste; 2006:27–47. The diversity of fungi in Semi-arid ecosystems is admittedly higher than previously thought.44 Santiago ALCM de A, dos Santos PJP, Maia LC. Mucorales from the semiarid of Pernambuco, Brazil. Braz J Microbiol. 2013;44(1):299-305.,1111 Gusmão LFP, Barbosa FR, Barbosa FF. Fungos Conidias. In: Gusmão LFP, Maia LC, eds. Diversidade e caracterização dos fungos no semi-árido. Vol 1. Recife: Associação Plantas do Nordeste; 2006:27–47. However, it is estimated that 41.1% of Caatinga regions have not yet been inventoried.1111 Gusmão LFP, Barbosa FR, Barbosa FF. Fungos Conidias. In: Gusmão LFP, Maia LC, eds. Diversidade e caracterização dos fungos no semi-árido. Vol 1. Recife: Associação Plantas do Nordeste; 2006:27–47. Consequently, data related to the fungal community and the ecological relationship between microorganisms and substrates in these ecosystems are still scarce.
Concerning Mucorales in Brazil, 74 taxa, belonging to 20 genera, have been reported. Of these, 42 were isolated from herbivore dung, which is a highly favorable substrate for the growth of these fungi.1212 Santiago ALCM de A. Mucorales. In: Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro.; 2015. Available from http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB120276.
http://floradobrasil.jbrj.gov.br/jabot/f...
Although a number of authors have documented the occurrence of coprophilous Mucorales in Brazil,1313 Trufem SFB, Viritato A. Mucorales do Estado de São Paulo. Mucoraceae coprófilas. Rikia. 1985;12:113-123.
14 Alves MH, Trufem SFB, Milanez AI. Táxons de Mucor Fresen. (Zygomycota) em fezes de herbívoros, Recife, PE, Brasil. Rev Bras Bot. 2002;25(2):147-160.-1515 Santiago ALCM de A, Trufem SFB, Malosso E, dos Santos PJP, Cavalcanti MA de Q. Zygomycetes from herbivore dung in the ecological reserve of Dois Irmãos, Northeast Brazil. Braz J Microbiol. 2011;42:89-95. there are no reports addressing the diversity and ecology of these fungi on dung in semi-arid areas. Therefore, the aims of the present study were to assess and compare the Mucorales communities in dung from different species and breeds of herbivores in the semi-arid region of Pernambuco, based on the frequency of occurrence and species richness of these fungi.
Materials and methods
Study areas
Samples of herbivore dung were collected at the Instituto Agronômico de Pernambuco (IPA) in Sertânia-PE (8°03′38″ S, 37°13′32″ W) [animals: caprine (Capra hircus L., breeds Anglo-Nubiano and Moxotó), and ovine (Ovis aries L., breeds Santa Inês and Morada Nova], and Arcoverde (8°25′00″ S, 37°04′00″ W) [animal: bovine (Bos taurus L., breeds Holandês, Girolando and Sindi]. Bos taurus L. dung samples (Breed Guzerá Leiteiro) were collected at the IPA in the city of Serra Talhada (7°95′67″ S, 38°29′71″ W).
Dung samples
The samples were collected monthly, from September 2013 to April 2014, using sterilized spatulas. They were placed in cellophane autoclaved paper bags and kept in polystyrene boxes with ice until they arrived in the laboratory. All samples were collected in the morning, usually after the animals’ first meal of the day.
Food supplied to herbivores
The composition of food supplied to herbivores from Arcoverde, Sertânia and Serra Talhada is provided in Table 1.
Isolation, purification and identification
Dung samples from each animal were incubated in moist chambers at 28 ± 2 °C for 15 days under alternating light and dark periods, during which time mycelial growth was observed. Fragments of the grown colonies were transferred to malt extract agar (MEA) medium (Merck - EMB),1616 Benny GL. The methods used by Dr. R.K. Benjamin, and other Mycologists to isolate Zygomycetes. Aliso. 2008;26:37-61. supplemented with chloramphenicol (100 mg L−1). After growth, the fungi were transferred to test tubes containing the same culture medium without antibiotic.
The specimens were identified by observing their macroscopic (color, appearance and diameter of colony) and microscopic (microstructures) characteristics as described by Hesseltine and Ellis,1717 Hesseltine CW, Ellis JJ. The genus Absidia: Gongronella and cylindrical-spored species of Absidia. Mycologia. 1964;56:568-601. Schipper,1818 Schipper MAA. On certain species of Mucor with a key to all accepted species. Stud Mycol. 1978;17:1-69. Hesseltine and Fennel,1919 Hesseltine CW, Fennel DI. The genus Circinella. Mycologia. 1995;47:193-211. Zheng and Chen,2020 Zheng R-y, Chen G-q. A monograph of Cunninghamella. Mycotaxon. 2001;80:1-75. Hoffmann et al.,2121 Hoffmann K, Discher S, Voigt K. Revision of the genus Absidia (Mucorales, Zygomycetes) based on physiological, phylogenetic, and morphological characters; thermotolerant Absidia spp. form a coherent group, Mycocladiaceae fam. nov. Mycol Res. 2007;111:1169-1183. Zheng et al.2222 Zheng R-y, Chen G-q, Huang H, Liu X-y. A monograph a Rhizopus. Sydowia. 2007;59(2):273-372. and Santiago et al.2323 Santiago ALCM de A, Hoffmann K, Lima DX, et al. A new species of Lichtheimia (Mucoromycotina, Mucorales) isolated from Brazilian soil. Mycol Progr. 2014;13:343-352.
Molecular analysis
Culture grown in test tubes containing malt extract were incubated at 28 °C for 6 days to obtain fungal biomass. The material was transferred to 2 mL microtubes with screw caps. Subsequently, 0.5 g acid-washed glass beads of two different diameters (150-212 µm and 425-600 µm, 1:1; Sigma, USA) were added to each tube. The material was crushed by stirring at high speed in a FastPrep homogenizer. The genomic DNA extraction procedure was conducted as described by Góes-Neto et al.2424 Góes-Neto A, Loguercio-Leite C, Guerrero RT. DNA extraction from frozen field collected and dehydrated herbarium fungal basidiomata: performance of SDS and CTAB-base methods. Biotemas. 2005;18:19-32. The mycelium was washed with chloroform:isoamyl alcohol (24:1) and then homogenized in 2% cetyltrimethylammonium bromide buffer. The DNA was precipitated in isopropanol, washed with 70% ethanol, and resuspended in 50 µL ultrapure water.
The primer pairs LR1/LSU2 were used for the amplification of the large subunit (LSU) of nuclear ribosomal DNA (rDNA).2323 Santiago ALCM de A, Hoffmann K, Lima DX, et al. A new species of Lichtheimia (Mucoromycotina, Mucorales) isolated from Brazilian soil. Mycol Progr. 2014;13:343-352.,2525 van Tuinen D, Zhao B, Gianinazzi-Pearson V. PCR in studiesof AM fungi: from primers to application. In: Varma AK, ed.Mycorrhizal Manual. Berlin: Springer; 1998:387–399. The polymerase chain reactions were carried out as described by Oliveira et al.2626 Oliveira RJV, Lima TEF, Cunha IB, et al. Corniculariella brasiliensis, a new species of coelomycetes in the rhizosphere of Caesalpinia echinata (Fabaceae, Caesalpinioideae) in Brazil. Phytotaxa. 2014;178(3):197-204. The newly obtained sequence was deposited in the National Center for Biotechnology Information GenBank database (accession number KX133009).
Frequency of occurrence (FO)
FO was calculated using the following equation: FO = Ji/k, where FO is the frequency of occurrence of the species i, Ji is the number of samples in which the species i has occurred and k is the total number of soil samples.2727 Brower JE, Zar JH, Von Ende CN. Field and Laboratory Methods for General Ecology. Dubuque: McGraw Hill; 1990.
Statistical analysis
Differences in the associations of Mucorales occurring among different herbivores dung were determined using Similarity Analysis (ANOSIM Primer v.6), in which the matrix of Bray-Curtis similarity was plotted as described by Clarke and Gorley.2828 Clarke KR, Gorley RN. Primer v.6 User Manual/Tutorial. Plymouth: Primer-E Ltd; 2006. Differences between the number of species of Mucorales associated with different breeds of animals and the different months of the year were assessed using the Chi-Square (χ 2) test (adjustment of compliance with expected equality proportions), according to the following formula: χ 2 = Σ[(o − e)2/i], where o is the observed frequency for each class and e is the expected frequency for that class.2929 Melo RFR, Bezerra JL, Cavalcanti MA de Q. Diversity of coprophilous ascomycetes from captive wild animals in Dois Irmãos State Park, Brazil. Nova Hedwig. 2012;94(2):153-162. The significance level was set at 0.05 in the analysis.
Results
Twenty-four taxa within the genera Absidia, Circinella, Cunninghamella, Lichtheimia, Mucor, Pilobolus, Rhizopus and Syncephalastrum were identified in bovine, caprine and ovine dung found in the semi-arid region of Pernambuco. The highest number of taxa was observed in samples from Morada Nova (14 taxa) and Santa Inês (12) (ovine), followed by Holandês (10) and Girolando (8) (bovine). The lowest species richness was observed in samples from Guzerá (bovine) (3) (Table 2). Mucor exhibited the highest number of species (9), followed by Pilobolus (5) (Table 2).
Richness and frequency of occurrence (FO) of Mucorales in herbivore dung from Arcoverde, Sertânia and Serra Talhada, PE.
Mucor circinelloides f. griseo-cyanus occurred in high prevalence (FO = 59.35%) in dung from the bovines, caprine and ovine examined, followed by M. ramosissimus (FO = 32.8%) and M. circinelloides f. circinelloides (FO = 31.25%). Cunninghamella echinulata var. echinulata, Mucor sp., P. minutus and R. arrhizus var. arrhizus were the least common (FO = 1.56%) (Table 2).
The highest number of taxa was observed in dung samples from Morada Nova (14) and Santa Inês sheep breeds (12) (Table 2). However, according to the χ 2 test, there were no significant differences in the species richness of Mucorales in dung from the different breeds of animals (p = 0.0926), but differences in the number of species isolated across sampling per months of sampling were significant (p = 0.0458).
Most Mucorales occurred in the excrement of bovine, caprine and ovine, with the exception of Absidia cylindrospora var. cylindrospora, C. echinulata var. echinulata, Mucor sp. and P. minutus, which were only found in bovine dung, while Lichtheimia brasiliensis, M. variosporus, R. arrhizus var. arrhizus and R. stolonifer were only observed in sheep feces.
Considering that L. ramosa is difficult to be separated from L. corymbifera (Cohn) Vuill. and L. ornata (A.K. Sarbhoy) Alastr.-Izq. & Walther based exclusively on morphological characters, the LSU rDNA region of our L. ramosa was sequenced. The Blastn analysis showed that our sequence (KX133009) was 99% similar to L. ramosa (CBS 582.65 - NG042518.1), confirming the identity of our specimen.
The species composition was most similar between Guzerá and Sindi (bovine) (75%), followed by Girolando (bovine) and Morada Nova (ovine) (63.63%) and Holandês (bovine) and Anglo-Nubiano (caprine) (60%) (Fig. 1).
Dendrogram of Bray-Curtis similarity for species composition of Mucorales from the herbivore dung of different animals. Species composition was more similar between Guzerá and Sindi dung, followed by Girolando and Morada Nova dung. Excrements of Anglo-Nubiano and Holandês were less similar.
Identification key for species of Mucorales from dung in the semi-arid region of Brazil
Discussion
Altogether, 24 taxa of Mucorales were identified in the dung of herbivores from Arcoverde, Sertânia and Serra Talhada, PE, Brazil. All species identified in the present study are being cited for the first time in the dung of herbivores in Caatinga areas. However, most of the species identified herein have been reported by other authors in excrement from other domains in Brazil, indicating that they are not endemic to the Caatinga.1414 Alves MH, Trufem SFB, Milanez AI. Táxons de Mucor Fresen. (Zygomycota) em fezes de herbívoros, Recife, PE, Brasil. Rev Bras Bot. 2002;25(2):147-160.,1515 Santiago ALCM de A, Trufem SFB, Malosso E, dos Santos PJP, Cavalcanti MA de Q. Zygomycetes from herbivore dung in the ecological reserve of Dois Irmãos, Northeast Brazil. Braz J Microbiol. 2011;42:89-95.,3030 Trufem SFB. Mucorales do Estado de São Paulo. 4. Espécies coprófilas. Rickia. 1984;11:65-75.
With the exception of M. indicus and Mucor sp., all taxa of this genus isolated in the present study as well as Circinella muscae, P. crystallinus, P. kleinii, P. longipes, R. arrhizus var. arrhizus and S. racemosum were reported by Alves et al.1414 Alves MH, Trufem SFB, Milanez AI. Táxons de Mucor Fresen. (Zygomycota) em fezes de herbívoros, Recife, PE, Brasil. Rev Bras Bot. 2002;25(2):147-160. and by Santiago et al.1515 Santiago ALCM de A, Trufem SFB, Malosso E, dos Santos PJP, Cavalcanti MA de Q. Zygomycetes from herbivore dung in the ecological reserve of Dois Irmãos, Northeast Brazil. Braz J Microbiol. 2011;42:89-95. in the dung of herbivores in the Atlantic Forest in Recife, PE. Lichtheimia ramosa (as A. ramosa), C. muscae, M. hiemalis, P. crystallinus, P. kleinii and P. longipes were described by Trufem3030 Trufem SFB. Mucorales do Estado de São Paulo. 4. Espécies coprófilas. Rickia. 1984;11:65-75. and Trufem and Viriato1313 Trufem SFB, Viritato A. Mucorales do Estado de São Paulo. Mucoraceae coprófilas. Rikia. 1985;12:113-123. in the Atlantic Forest in São Paulo state. Fifteen species reported by the above mentioned authors were observed in the present study, although feces from different herbivores were analyzed in different studies, indicating that the majority of Mucorales do not exhibit specificity for a particular type of animal dung.3131 Richardson MJ. Diversity and occurrence of coprophilous fungi. Mycol Res. 2001;105(4):387-402.
32 Nyberg Å, Persson IL. Habitat differences of coprophilous fungi on moose dung. Mycol Res. 2002;106(11):1360-1366.-3333 Delgado-Ávila AE, Urdaneta-García LM, Piñeiro-Chávez AJ. Coprophilous fungi of Zulia state, Venezuela. Divisions: Myxomycota, Zygomycota and Basidiomycota. Rev Cient Fac Cien Vet Univ Zulia. 2005;15(1):5763.
A recent phylogenetic study on Mucorales that included critical species of this order3434 Walther G, Pawłowska J, Alastruey-Izquierdo A, et al. DNA barcoding in Mucorales: an inventory of biodiversity. Persoonia. 2013;30:11-47. strongly evidenced that M. ramosissimus is a synonym of M. circinelloides f. circinelloides or f. janssenii. In fact, both species are morphologically very similar to each other. However, considering that our isolates exhibited morphological characteristics, such as small colonies up to 2 mm high, sporangiophores with a frequent swelling below the sporangia and columellae applanate, that were very similar to those described by Schipper3535 Schipper MAA. On Mucor circinelloides, Mucor racemosus and related species. Stud Mycol. 1976;12:1-40. for M. ramosissimus, and considering that M. ramosissimus is still a valid species, we prefer to maintain M. ramosissimus in our manuscript.
Some of the species isolated in the present study have also been reported in animal dung from other countries. Masunga3636 Masunga GS, Andresen Ø, Taylor JE, Dhillion SS. Elephant dung decomposition and coprophilous fungi in two habitats of semi-arid Botswana. Mycol Res. 2006;110:1214-1226. isolated C. elegans and P. crystallinus from the dung of elephants in Africa, while Abdullah3737 Abdullah SK. Coprophilous mycoflora on different dung types in southern desert of Iraq. Sydowia. 1982;35:1-5. reported P. kleinii in donkey, sheep and camel dung collected in Iraq. Studies have shown that this group of fungi has a wide distribution and can adapt to different environmental conditions.3131 Richardson MJ. Diversity and occurrence of coprophilous fungi. Mycol Res. 2001;105(4):387-402.,3838 Ebersohn C, Eicker A. Determination of the coprophilous fungal fruit body successional phases and the delimitation of species association classes on dung substrates of African game animals. Bot Bull Acad Sin. 1997;38:183-190.,3939 Caretta G, Piontelli E, Savino E, Bilgheroni A. Some coprophilous fungi from Kenya. Mycopathologia. 1998;142:25-134.
Lichtheimia brasiliensis and M. indicus have been commonly isolated from soils, although they have not as yet been found in dung.2323 Santiago ALCM de A, Hoffmann K, Lima DX, et al. A new species of Lichtheimia (Mucoromycotina, Mucorales) isolated from Brazilian soil. Mycol Progr. 2014;13:343-352.,4040 Szwedek-Trzaska A, Glowacka A. Seeking ways to eradicate potentially pathogenic fungi isolated from soil. Pol J Environ Stud. 2011;20(5):1313-1318.,4141 Lima DX, Santiago ALCM de A, Souza-Motta CM. Diversity of Mucorales in natural and degraded semi-arid soils. Rev Bras Bot. 2015;38:1-7. However, these species are now reported for the first time in the dung of herbivores, thereby expanding our knowledge about the diversity of Mucorales. Mucor sp. exhibit morphological and genetic characteristics that differ from other taxa of the genus and will be described and published as a new species in a subsequent paper.
Among the isolated genera, Mucor was the most representative in number of taxa, with nine species and four forms, followed by Pilobolus, with five species. According to Krug et al.,66 Krug JC, Benny GL, Keller HW. Coprophilous fungi. In: Mueller GM, Bills GF, Foster MS, eds. Biodiversity of Fungi. Hardbound: Elsevier Academic Press; 2004:468–499.Mucor is characterized by facultative coprophilous species, while Pilobolus is characterized by obligatory coprophilous species. Since the species of the former genus are very common in soil samples,4242 Domsch KH, Gams W, Anderson TH. Compendium of Soil Fungi. San Francisco: IHW-Verlag; 2007. it is possible that the presence of these taxa in the inventoried excrement was due to the transition of propagules from the soil to the dung, as mentioned by Santiago et al.1515 Santiago ALCM de A, Trufem SFB, Malosso E, dos Santos PJP, Cavalcanti MA de Q. Zygomycetes from herbivore dung in the ecological reserve of Dois Irmãos, Northeast Brazil. Braz J Microbiol. 2011;42:89-95. All species of Mucorales reported here are being cited for the first time as coprophilous in the Caatinga. In Brazil, the occurrence of Mucor species and/or Piloloblus in herbivore dung was reported by Batista and Pontual,4343 Batista AC, Pontual D. Alguns fungos coprófilos de Pernambuco. Bol Secr Agric Ind Comér. 1948;15:27-44. Trufem,3030 Trufem SFB. Mucorales do Estado de São Paulo. 4. Espécies coprófilas. Rickia. 1984;11:65-75. Trufem and Viriato,1313 Trufem SFB, Viritato A. Mucorales do Estado de São Paulo. Mucoraceae coprófilas. Rikia. 1985;12:113-123. Richardson,3131 Richardson MJ. Diversity and occurrence of coprophilous fungi. Mycol Res. 2001;105(4):387-402. Alves et al.1414 Alves MH, Trufem SFB, Milanez AI. Táxons de Mucor Fresen. (Zygomycota) em fezes de herbívoros, Recife, PE, Brasil. Rev Bras Bot. 2002;25(2):147-160. and Santiago et al.1515 Santiago ALCM de A, Trufem SFB, Malosso E, dos Santos PJP, Cavalcanti MA de Q. Zygomycetes from herbivore dung in the ecological reserve of Dois Irmãos, Northeast Brazil. Braz J Microbiol. 2011;42:89-95.. The greatest representation of Mucor and Pilobolus in relation to the number of taxa in the dung analyzed was confirmed by Santiago et al.1515 Santiago ALCM de A, Trufem SFB, Malosso E, dos Santos PJP, Cavalcanti MA de Q. Zygomycetes from herbivore dung in the ecological reserve of Dois Irmãos, Northeast Brazil. Braz J Microbiol. 2011;42:89-95.
Our results indicated that Morada Nova dung (ovine) was the richest in terms of the number of taxa, followed by the dung of Santa Inês (ovine) and Holandês (bovine). However, according to the χ 2 test, no significant difference (p = 0.0926) was found for the species richness of Mucorales in the dung of the different animals analyzed. According to Ebersohn and Eicker3838 Ebersohn C, Eicker A. Determination of the coprophilous fungal fruit body successional phases and the delimitation of species association classes on dung substrates of African game animals. Bot Bull Acad Sin. 1997;38:183-190. and Santiago et al.,1515 Santiago ALCM de A, Trufem SFB, Malosso E, dos Santos PJP, Cavalcanti MA de Q. Zygomycetes from herbivore dung in the ecological reserve of Dois Irmãos, Northeast Brazil. Braz J Microbiol. 2011;42:89-95. differences in the composition of a community can be correlated with abiotic and biotic factors that influence the mycobiota of the substrate. The fact that dung samples were kept in similar experimental conditions (temperature, light incidence, moisture) and were free of mycophagous insects could explain the similarities found for the species richness of Mucorales in dung. However, statistical analysis revealed significant differences in the number of taxa isolated from dung between the different sampling months (p = 0.0458), indicating that seasonality influences the number of species, but not the composition of the Mucorales. The opposite result was observed by Santiago et al.,1515 Santiago ALCM de A, Trufem SFB, Malosso E, dos Santos PJP, Cavalcanti MA de Q. Zygomycetes from herbivore dung in the ecological reserve of Dois Irmãos, Northeast Brazil. Braz J Microbiol. 2011;42:89-95. who reported that the composition of Mucorales species was affected by seasonal changes of the year, differing from the results observed for the number of taxa. Bell4444 Bell A. Fungal succession on dung of the brush-tailed opossum in New Zealand. N Z J Bot. 1975;13:437-462. conducted a three-year study on the mycota in possum dung (Trichosurus Vulpecula Kerr) in New Zealand and attributed the higher incidence of certain fungal species to winter rains.
Concerning the frequency of occurrence of the isolates, M. circinelloides f. griseo-cyanus was the most common taxon (FO = 59.35%), while C. echinulata var. echinulata, Mucor sp. and R. arrhizus var. arrhizus were the least common taxa (FO = 1.56%). Mucor and Rhizopus include many species, coprophilous or not,66 Krug JC, Benny GL, Keller HW. Coprophilous fungi. In: Mueller GM, Bills GF, Foster MS, eds. Biodiversity of Fungi. Hardbound: Elsevier Academic Press; 2004:468–499. while Cunninghamella species primarily colonize other substrates.4242 Domsch KH, Gams W, Anderson TH. Compendium of Soil Fungi. San Francisco: IHW-Verlag; 2007. Most species identified in the present study exhibited low frequencies of occurrence, not exceeding 32.8%. Similar behavior was observed by Richardson,3131 Richardson MJ. Diversity and occurrence of coprophilous fungi. Mycol Res. 2001;105(4):387-402. Nyberg and Persson3232 Nyberg Å, Persson IL. Habitat differences of coprophilous fungi on moose dung. Mycol Res. 2002;106(11):1360-1366. and Santiago et al.1515 Santiago ALCM de A, Trufem SFB, Malosso E, dos Santos PJP, Cavalcanti MA de Q. Zygomycetes from herbivore dung in the ecological reserve of Dois Irmãos, Northeast Brazil. Braz J Microbiol. 2011;42:89-95. in studies of coprophilous fungi from herbivorous dung. Pilobolus kleinii has been commonly associated with a high frequency in herbivorous animal dung in other domains.3131 Richardson MJ. Diversity and occurrence of coprophilous fungi. Mycol Res. 2001;105(4):387-402.,3232 Nyberg Å, Persson IL. Habitat differences of coprophilous fungi on moose dung. Mycol Res. 2002;106(11):1360-1366. However, this species was uncommon in the samples analyzed in the present study (7.81%). It is possible that this taxon is more sensitive to the water and temperature stress that are characteristic of the semi-arid region.
Considering the different breeds of herbivores, species composition was more similar between Guzerá and Sindi (bovines) (75%), which although not sharing the same environment, were exposed to a similar nutritional status and were the only animals kept on pasture (Table 1). According to Santiago et al.,1515 Santiago ALCM de A, Trufem SFB, Malosso E, dos Santos PJP, Cavalcanti MA de Q. Zygomycetes from herbivore dung in the ecological reserve of Dois Irmãos, Northeast Brazil. Braz J Microbiol. 2011;42:89-95. nutritional differences between the animals can influence the mycota of dung. The fact that these animals spent most of their time grazing in the pasture may have contributed to the high similarity found. Curiously, high similarity was also observed between the Girolando (bovine) and Morada Nova (ovine) species, and between Anglo-Nubiano (caprine) and Holandês (bovine) species, despite the fact that they were grazed in different cities (although in the same domain) and have different diets. Studies have shown that although the food given to the animals is a limiting factor for the appearance of certain taxa, other factors, such as the geographic location, humidity and high temperatures may influence the composition of fungal communities in animal dung.3131 Richardson MJ. Diversity and occurrence of coprophilous fungi. Mycol Res. 2001;105(4):387-402.,3838 Ebersohn C, Eicker A. Determination of the coprophilous fungal fruit body successional phases and the delimitation of species association classes on dung substrates of African game animals. Bot Bull Acad Sin. 1997;38:183-190.,4545 Caretta G, Piontelli E. Coprophilous fungi from confined deers in Pavia (Lombardia, Italy). Bol Micol. 1996;11:41-50.
The present work reports 24 taxa of Mucorales in herbivore dung from the semi-arid of Brazil and is a pioneer study for coprophilous Mucorales in the Caatinga. Considering the adverse conditions of temperature and humidity, typical of semi-arid regions, and comparing the results described herein with those of other authors, it is clear that the species richness of Mucorales in the inventoried areas is high. Abdullah3737 Abdullah SK. Coprophilous mycoflora on different dung types in southern desert of Iraq. Sydowia. 1982;35:1-5. reported only three species of Mucorales in donkey, sheep and camel dung collected in semi-arid regions of southern Iraq. Masunga et al.3636 Masunga GS, Andresen Ø, Taylor JE, Dhillion SS. Elephant dung decomposition and coprophilous fungi in two habitats of semi-arid Botswana. Mycol Res. 2006;110:1214-1226. reported six species of this group in semi-arid areas of Botswana, Africa. Upon comparison of the results of the present study with previous studies of the diversity of coprophilous Mucorales in Atlantic Forest areas, Santiago et al.1515 Santiago ALCM de A, Trufem SFB, Malosso E, dos Santos PJP, Cavalcanti MA de Q. Zygomycetes from herbivore dung in the ecological reserve of Dois Irmãos, Northeast Brazil. Braz J Microbiol. 2011;42:89-95. reported 39 taxa in this domain in Pernambuco, whereas Trufem and Viriato1313 Trufem SFB, Viritato A. Mucorales do Estado de São Paulo. Mucoraceae coprófilas. Rikia. 1985;12:113-123. and Viriato and Trufem4646 Viriato A, Trufem SFB. Mucorales de São Paulo. Espécies Merosporangiadas. Rickia. 1985;12:147-154. together only obtained 23 taxa in the same domain in São Paulo. Thus, it is relevant to continue surveying the fungal diversity in the Caatinga in order to demystify the erroneous idea that the richness of fungal species in this area is low.
Acknowledgments
The authors of the present study would like to express their gratitude to the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE - APQ 0842-2.12/14), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - 458391/2014-0), the Programa de Pesquisa em Biodiversidade do semiárido (MCT/CNPQ/PPBio - 457498/2012-9), and the two anonymous reviewers for critical reading the manuscript.
References
-
1Hibbett DS, Binder M, Bischoff JF, et al. A higher-level phylogenetic classification of the Fungi. Mycol Res. 2007;3:509-547.
-
2Hoffmann K, Voigt K, Kirk PM. Mortierellomycotina subphyl. nov., based on multi-gene genealogies. Mycotaxon 2011;115(1):353-363.
-
3Humber RA. Entomophthoromycota: a new phylum and reclassification for entomophthoroid fungi. Mycotaxon 2012;120:477-492.
-
4Santiago ALCM de A, dos Santos PJP, Maia LC. Mucorales from the semiarid of Pernambuco, Brazil. Braz J Microbiol 2013;44(1):299-305.
-
5Alexopoulos CJ, Mims CW, Blackwell M. Introductory Mycology 4th ed. New York: John Wiley & Sons; 1996.
-
6Krug JC, Benny GL, Keller HW. Coprophilous fungi. In: Mueller GM, Bills GF, Foster MS, eds. Biodiversity of Fungi Hardbound: Elsevier Academic Press; 2004:468–499.
-
7Richardson MJ. Records of coprophilous fungi from the Lesser Antilles and Puerto Rico. Caribb J Sci 2008;44(2):206-214.
-
8Dix NJ, Webster J. Fungal Ecology London: Chapman & Hall; 1995.
-
9Richardson MJ. Coprophilous fungi. Field Mycol 2003;4(2):41-43.
-
10Drumond MA, Kiill LHP, Nascimento CES. Inventário e sociabilidade de espécies arbóreas e arbustivas da Caatinga na Região de Petrolina, PE. Bras Florest. 2002;74:37-43.
-
11Gusmão LFP, Barbosa FR, Barbosa FF. Fungos Conidias. In: Gusmão LFP, Maia LC, eds. Diversidade e caracterização dos fungos no semi-árido. Vol 1. Recife: Associação Plantas do Nordeste; 2006:27–47.
-
12Santiago ALCM de A. Mucorales. In: Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro; 2015. Available from http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB120276
» http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB120276 -
13Trufem SFB, Viritato A. Mucorales do Estado de São Paulo. Mucoraceae coprófilas. Rikia 1985;12:113-123.
-
14Alves MH, Trufem SFB, Milanez AI. Táxons de Mucor Fresen. (Zygomycota) em fezes de herbívoros, Recife, PE, Brasil. Rev Bras Bot. 2002;25(2):147-160.
-
15Santiago ALCM de A, Trufem SFB, Malosso E, dos Santos PJP, Cavalcanti MA de Q. Zygomycetes from herbivore dung in the ecological reserve of Dois Irmãos, Northeast Brazil. Braz J Microbiol. 2011;42:89-95.
-
16Benny GL. The methods used by Dr. R.K. Benjamin, and other Mycologists to isolate Zygomycetes. Aliso 2008;26:37-61.
-
17Hesseltine CW, Ellis JJ. The genus Absidia: Gongronella and cylindrical-spored species of Absidia. Mycologia 1964;56:568-601.
-
18Schipper MAA. On certain species of Mucor with a key to all accepted species. Stud Mycol 1978;17:1-69.
-
19Hesseltine CW, Fennel DI. The genus Circinella. Mycologia 1995;47:193-211.
-
20Zheng R-y, Chen G-q. A monograph of Cunninghamella. Mycotaxon 2001;80:1-75.
-
21Hoffmann K, Discher S, Voigt K. Revision of the genus Absidia (Mucorales, Zygomycetes) based on physiological, phylogenetic, and morphological characters; thermotolerant Absidia spp. form a coherent group, Mycocladiaceae fam. nov. Mycol Res. 2007;111:1169-1183.
-
22Zheng R-y, Chen G-q, Huang H, Liu X-y. A monograph a Rhizopus. Sydowia 2007;59(2):273-372.
-
23Santiago ALCM de A, Hoffmann K, Lima DX, et al. A new species of Lichtheimia (Mucoromycotina, Mucorales) isolated from Brazilian soil. Mycol Progr 2014;13:343-352.
-
24Góes-Neto A, Loguercio-Leite C, Guerrero RT. DNA extraction from frozen field collected and dehydrated herbarium fungal basidiomata: performance of SDS and CTAB-base methods. Biotemas 2005;18:19-32.
-
25van Tuinen D, Zhao B, Gianinazzi-Pearson V. PCR in studiesof AM fungi: from primers to application. In: Varma AK, ed.Mycorrhizal Manual. Berlin: Springer; 1998:387–399.
-
26Oliveira RJV, Lima TEF, Cunha IB, et al. Corniculariella brasiliensis, a new species of coelomycetes in the rhizosphere of Caesalpinia echinata (Fabaceae, Caesalpinioideae) in Brazil. Phytotaxa 2014;178(3):197-204.
-
27Brower JE, Zar JH, Von Ende CN. Field and Laboratory Methods for General Ecology Dubuque: McGraw Hill; 1990.
-
28Clarke KR, Gorley RN. Primer v.6 User Manual/Tutorial. Plymouth: Primer-E Ltd; 2006.
-
29Melo RFR, Bezerra JL, Cavalcanti MA de Q. Diversity of coprophilous ascomycetes from captive wild animals in Dois Irmãos State Park, Brazil. Nova Hedwig. 2012;94(2):153-162.
-
30Trufem SFB. Mucorales do Estado de São Paulo. 4. Espécies coprófilas. Rickia 1984;11:65-75.
-
31Richardson MJ. Diversity and occurrence of coprophilous fungi. Mycol Res. 2001;105(4):387-402.
-
32Nyberg Å, Persson IL. Habitat differences of coprophilous fungi on moose dung. Mycol Res 2002;106(11):1360-1366.
-
33Delgado-Ávila AE, Urdaneta-García LM, Piñeiro-Chávez AJ. Coprophilous fungi of Zulia state, Venezuela. Divisions: Myxomycota, Zygomycota and Basidiomycota. Rev Cient Fac Cien Vet Univ Zulia 2005;15(1):5763.
-
34Walther G, Pawłowska J, Alastruey-Izquierdo A, et al. DNA barcoding in Mucorales: an inventory of biodiversity. Persoonia 2013;30:11-47.
-
35Schipper MAA. On Mucor circinelloides, Mucor racemosus and related species. Stud Mycol. 1976;12:1-40.
-
36Masunga GS, Andresen Ø, Taylor JE, Dhillion SS. Elephant dung decomposition and coprophilous fungi in two habitats of semi-arid Botswana. Mycol Res. 2006;110:1214-1226.
-
37Abdullah SK. Coprophilous mycoflora on different dung types in southern desert of Iraq. Sydowia 1982;35:1-5.
-
38Ebersohn C, Eicker A. Determination of the coprophilous fungal fruit body successional phases and the delimitation of species association classes on dung substrates of African game animals. Bot Bull Acad Sin. 1997;38:183-190.
-
39Caretta G, Piontelli E, Savino E, Bilgheroni A. Some coprophilous fungi from Kenya. Mycopathologia 1998;142:25-134.
-
40Szwedek-Trzaska A, Glowacka A. Seeking ways to eradicate potentially pathogenic fungi isolated from soil. Pol J Environ Stud. 2011;20(5):1313-1318.
-
41Lima DX, Santiago ALCM de A, Souza-Motta CM. Diversity of Mucorales in natural and degraded semi-arid soils. Rev Bras Bot 2015;38:1-7.
-
42Domsch KH, Gams W, Anderson TH. Compendium of Soil Fungi San Francisco: IHW-Verlag; 2007.
-
43Batista AC, Pontual D. Alguns fungos coprófilos de Pernambuco. Bol Secr Agric Ind Comér 1948;15:27-44.
-
44Bell A. Fungal succession on dung of the brush-tailed opossum in New Zealand. N Z J Bot 1975;13:437-462.
-
45Caretta G, Piontelli E. Coprophilous fungi from confined deers in Pavia (Lombardia, Italy). Bol Micol. 1996;11:41-50.
-
46Viriato A, Trufem SFB. Mucorales de São Paulo. Espécies Merosporangiadas. Rickia 1985;12:147-154.
Publication Dates
-
Publication in this collection
Jan-Mar 2017
History
-
Received
31 Oct 2015 -
Accepted
20 June 2016