BJM 158 1–8 ARTICLE IN PRESS b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y x x x (2 0 1 6) xxx–xxx ht tp://www.bjmicrobiol.com.br/ 1 Environmental Microbiology 2 Coprophilous Mucorales (ex Zygomycota) from 3 three areas in the semi-arid of Pernambuco, Brazil a a b 4 Q1 Carlos Alberto Fragoso de Souza , Diogo Xavier Lima , Luciana M.S. Gurgel , a,∗ 5 André Luiz Cabral Monteiro de Azevedo Santiago a 6 Universidade Federal de Pernambuco, Departamento de Micologia, Recife, PE, Brazil b 7 Instituto Agronômico de Pernambuco (IPA), Recife, PE, Brazil 8 9 a r t i c l e i n f o a b s t r a c t 10 11 Article history: Mucorales comprises fungi commonly isolated as saprobes from soil, dung, stored grains 12 Received 31 October 2015 and plants. Although these fungi have been studied in several countries, there are rela- 13 Accepted 20 June 2016 tively a few reports of them in semi-arid areas. Therefore, the aims of the present study 14 Available online xxx 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 Associate Editor: André Rodrigues and species richness of these fungi. Samples of dung collected in the cities of Arcoverde, 15 Serra Talhada and Sertânia were incubated in moist chambers in triplicate. Altogether, 24 16 Keywords: taxa of Mucorales distributed in the genera Absidia, Circinella, Cunninghamella, Lichtheimia, 17 Caatinga Mucor, Pilobolus, Rhizopus and Syncephalastrum were identified. The highest species richness 18 Dung was found in sheep excrement. Mucor circinelloides f. griseo-cyanus was the most common 19 Herbivore taxon, followed by M. ramosissimus. The similarity of the composition of Mucorales species 20 Mucoromycotina was greatest between the excrements of Guzerá and Sindi breeds (bovine). All mucoralean 21 Taxonomy 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. © 2016 Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de Microbiologia. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). fungi characterized by the production of thick-walled spores 26 Introduction of sexual origin, known as zygospores. The order Mucorales Fr., 27 the largest in number of species within the Mucoromycotina, 28 22 Mucoromycotina Benny, one of the four subphyla proposed includes fungi forming coenocytic hyphae with septa either at 29 23 to accommodate certain species of traditional Zygomycota the base of reproductive structures or irregularly distributed 30 24 C. Moreau (phylum no longer accepted in the new classifica- in older cultures. Species of this order produce asexual struc- 31 1–3 25 tion due to its polyphyletic nature), covers most saprobes tures such as sporangiophores, sporangia, sporangiospores, ∗ Corresponding author. E-mail: [email protected] (A.L. Santiago). http://dx.doi.org/10.1016/j.bjm.2016.09.008 1517-8382/© 2016 Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de Microbiologia. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Souza CA, et al. Coprophilous Mucorales (ex Zygomycota) from three areas in the semi-arid of Pernambuco, BJM 158 1–8 Brazil. Braz J Microbiol. (2016), http://dx.doi.org/10.1016/j.bjm.2016.09.008 BJM 158 1–8 ARTICLE IN PRESS 2 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y x x x (2 0 1 6) xxx–xxx 4 32 sporangiola, merosporangia and merospores. These fungi Table 1 – Food supplied to herbivores from Arcoverde, 33 are commonly isolated from soil, stored grains, plants, ver- Sertânia and Serra Talhada, PE. 34 tebrates, invertebrates and animal excrement, especially that Herbivore Food 5 35 of herbivores and rodents. (breed) 36 Coprophilous fungi are those that live in or are associated Cane silage Corn silage Mineral salt Graze 37 with fecal material, including soil contaminated with feces. Anglo Nubiano − + − − 38 These microorganisms are essential for the maintenance of Moxotó − + − − 39 ecosystems and are directly involved in the decomposition Santa Inês − + − − − − − 40 of fecal waste, participating in carbon, nitrogen and energy Morada Nova + − 6,7 8 Holandês + + − 41 cycles. According to Dix and Webster, fungi may occur − Sindi + + + 42 in excrement as obligate and optional coprophilous. The − Girolando + + − 43 obligate coprophilous have spores that require the action Guzerá − − − + 44 of gastric enzymes to break their dormancy, while sec- 45 ondary or facultative coprophilous spores do not need to go 9 Dung samples 86 46 through the digestive tract of animals to germinate. Among 47 the Mucorales, only Pilobolus species are considered obligate The samples were collected monthly, from September 2013 to 87 48 coprophilous, although other taxa from this order are common 6 April 2014, using sterilized spatulas. They were placed in cel- 88 49 in dung. lophane autoclaved paper bags and kept in polystyrene boxes 89 50 The Brazilian semiarid region occupies an area of approx- 2 with ice until they arrived in the laboratory. All samples were 90 51 imately 969,589.4 km , comprising eight northeastern states: collected in the morning, usually after the animals’ first meal 91 52 Alagoas, Bahia, Ceará, Paraíba, Pernambuco, Piauí, Rio Grande 10 of the day. 92 53 do Norte, Sergipe and part of the state of Minas Gerais. 54 Semiarid regions are formed, mostly, by typical vegetation 93 55 of Caatinga, an exclusively Brazilian domain that consists Food supplied to herbivores 56 of heterogeneous phytophysiognomic systems characteris- 94 57 tically xerophilic, with vegetation ranging from tree to The composition of food supplied to herbivores from 11 95 58 shrub. The diversity of fungi in semiarid ecosystems is Arcoverde, Sertânia and Serra Talhada is provided in Table 1. 4,11 59 admittedly higher than previously thought. However, it 96 60 is estimated that 41.1% of Caatinga regions have not yet Isolation, purification and identification 11 61 been inventoried. Consequently, data related to the fun- 97 62 gal community and the ecological relationship between Dung samples from each animal were incubated in moist ◦ ± 98 63 microorganisms and substrates in these ecosystems are still chambers at 28 2 C for 15 days under alternating light 64 scarce. and dark periods, during which time mycelial growth was 99 100 65 Concerning Mucoromycotina in Brazil, 74 taxa, belonging observed. Fragments of the grown colonies were transferred 16 101 66 to 20 genera, have been reported. Of these, 42 were isolated to malt extract agar (MEA) medium (Merck – EMB), supple- −1 102 67 from herbivore dung, which is a highly favorable substrate for mented with chloramphenicol (100 mg L ). After growth, the 12 103 68 the growth of these fungi. Although a number of authors fungi were transferred to test tubes containing the same cul- 104 69 have documented the occurrence of coprophilous Mucorales ture medium without antibiotic. 13–15 105 70 in Brazil, there are no reports addressing the diversity and The specimens were identified by observing their macro- 106 71 ecology of these fungi on dung in semi-arid areas. Therefore, scopic (color, appearance and diameter of colony) and 107 72 the aims of the present study were to assess and compare the microscopic (microstructures) characteristics as described by 17 18 19 108 73 Mucorales communities in dung from different species and Hesseltine and Ellis, Schipper, Hesseltine and Fennel, 20 21 22 109 74 breeds of herbivores in the semi-arid region of Pernambuco, Zheng and Chen, Hoffman et al., Zheng et al. and Santi- 23 110 75 based on the frequency of occurrence and species richness of ago et al. 76 these fungi. Molecular analysis 111 Materials and methods Culture grown in test tubes containing malt extract were 112 ◦ incubated at 28 C for 6 days to obtain fungal biomass. The 113 77 Study areas material was transferred to 2 mL microtubes with screw caps. 114 Subsequently, 0.5 g acid-washed glass beads of two differ- 115 ␮ ␮ 78 Samples of herbivore dung were collected at the Instituto ent diameters (150–212 m and 425–600 m, 1:1; Sigma, USA) 116 ◦ 79 Agronômico de Pernambuco (IPA) in Sertânia-PE (8 03 38 S, were added to each tube. The material was crushed by stir- 117 ◦ 80 37 13 32 W) [animals: caprine (Capra hircus L., breeds Anglo ring at high speed in a FastPrep homogenizer. The genomic 118 81 Nubiano and Moxotó), and ovine (Ovis aries L., breeds DNA extraction procedure was conducted as described by 119 ◦ 24 82 Santa Inês and Morada Nova], and Arcoverde (8 25 00 S, Góes-Neto et al. The mycelium was washed with chlo- 120 ◦ 83 37 04 00 W) [animal: bovine (Bos taurus L., breeds Holandês, roform:isoamyl alcohol (24:1) and then homogenized in 2% 121 84 Girolando and Sindi]. Bos taurus L. dung samples (Breed Guzerá cetyltrimethylammonium bromide buffer. The DNA was pre- 122 85 Leiteiro) were collected at the IPA in the city of Serra Talhada cipitated in isopropanol, washed with 70% ethanol, and 123 ◦ ◦ ␮ (7 95 67 S, 38 29 71 W). resuspended in 50 L ultrapure water.