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This Paper Must Be Cited As Document downloaded from: http://hdl.handle.net/10251/121824 This paper must be cited as: Molins, A.; Moya, P.; García-Breijo, F.; Reig-Armiñana, J.; Barreno, E. (2018). A multi-tool approach to assess microalgal diversity in lichens. isolation, Sanger sequencing, HTS and ultrastructural correlations. The Lichenologist. 50(1):123-138. https://doi.org/10.1017/S0024282917000664 The final publication is available at http://doi.org/10.1017/S0024282917000664 Copyright Cambridge University Press Additional Information The Lichenologist A multi -tool approach to assess microalgal diversity in lichens: isolation, Sanger sequencing, HTS and ultrastructural correlations Journal: The Lichenologist ManuscriptManuscript ID LICH-Mar-17-SP-0768.R1 For Review Manuscript Type: Standard Paper Date Submitted by the Author: n/a Complete List of Authors: Molins , Arántzazu; Botanica Moya, Patricia; Botanica García-Breijo, Francisco; Universitat Politecnica de Valencia Reig-Armiñana, Jose Barreno, Eva Keywords: coexistence, propagation, 454-pyrosequencing, Trebouxia Cambridge University Press Page 1 of 40 The Lichenologist 1 ORIGINAL ARTICLES 2 3 Title: A multi-tool approach to assess microalgal diversity in lichens: isolation, Sanger 4 sequencing, HTS and ultrastructural correlations. 5 6 Authors:Manuscript Arántzazu Molins 1, ┼┼┼, Patricia Moya For 1, ┼┼┼, Francisco Review J. García-Breijo 2, José Reig- 7 Armiñana 1 and Eva Barreno 1. 8 9 1 Dpto. Botánica, ICBIBE & Jardí Botànic, Fac. CC. Biológicas, Universitat de València, C/ 10 Dr. Moliner 50. 46100-Burjassot, Valencia, Spain. 11 12 2 Dpto. Ecosistemas Agroforestales, Universidad Politécnica de Valencia, Camino de Vera s/n, 13 46022-Valencia, Spain. 14 15 Running Head: Algal diversity by a multi-tool approach 16 17 For correspondence: A. Molins, Dpto. Botánica, ICBIBE, Fac. CC. Biológicas, Universitat de 18 València, C/ Dr. Moliner 50. 46100-Burjassot, Valencia, Spain 19 Phone number: +34 963544376; Fax number +34 963544082; email: [email protected] . 20 21 ┼ These two authors contributed equally to this work. 22 23 1 Cambridge University Press The Lichenologist Page 2 of 40 24 25 ABSTRACT 26 Lichen thalli represent the most relevant examples of fungal–algal interactions. Studies 27 that describe phycobiont diversity within entire thalli are based mainly on Sanger sequencing. 28 In some Manuscriptlichen species, this technique could undereForstimate Review the intrathalline coexistence of 29 multiple microalgae. In this study, different multi-tool approaches were applied to two lichen 30 taxa, Circinaria hispida and Flavoparmelia soredians , to detect algal coexistence. Here, we 31 combined Sanger sequencing, a specific polymerase chain reaction (PCR) primer, 454- 32 pyrosequencing, phycobiont isolation and ultrastructural characterisation. Furthermore, we 33 compared pyrenoid ultrastructural features of lichenised phycobionts with microalgae isolated 34 in culture. An improved methodology was used to isolate and propagate phycobionts which, 35 in combination with fast genetic identification, resulted in a considerable reduction in time 36 and cost to complete the process. This isolation method, coupled with a specific PCR primer, 37 allowed for the detection of coexisting algae in C. hispida (four Trebouxia lineages). 454- 38 pyrosequencing detected only a fraction of such diversity, while Sanger sequencing identified 39 only the primary phycobiont. Ultrastructural features of the isolated algae were observed by 40 transmission electron microscopy; the maintenance of the pyrenoid characteristics suggested 41 the existence of different Trebouxia lineages. In F. soredians , a single Trebouxia lineage was 42 identified using all these approaches. 43 In cases of lichens with algal coexistence, a combination of different molecular and 44 ultrastructural approaches may be required to reveal the underlying algal diversity within a 45 single thallus. The approaches proposed in this study provide information about the 46 relationship between molecular and ultrastructural data, and they represent an improvement in 2 Cambridge University Press Page 3 of 40 The Lichenologist 47 the delimitation of taxonomical features which is needed to recognise intrathalline Trebouxia 48 diversity. 49 50 KEY WORDS 51 coexistence;Manuscript propagation; 454-pyrosequencing; For Trebouxia. Review 52 53 INTRODUCTION 54 Lichens are classical symbiotic associations (holobiont) that result in a complex 55 organism with a specific biological organisation (Chapman & Margulis 1998). Such 56 associations involve at least two very different organisms, a heterotrophic fungus (mycobiont) 57 and a photosynthetic (photobiont). Photobionts can be cyanobacteria (cyanobionts) or green 58 microalgae (phycobionts). The presence of microalgal communities, lichen-associated fungi, 59 bacteria and yeast highlight the intrathalline complexity of lichen thalli (Arnold et al . 2009; 60 U´Ren et al . 2010, 2012; Park et al . 2015; Aschenbrenner et al . 2016; Muggia et al. 2016a; 61 Spribille et al . 2016; Moya et al . 2017). 62 Lichen literature has reported the occurrence of different phycobiont lineages inside 63 the thalli in several lichen species (Blaha et al . 2006; Ohmura et al . 2006; Piercey-Normore 64 2006; Muggia et al . 2010, 2014; Schmull et al . 2011; Molins et al . 2013; Leavitt et al . 2015). 65 The lichen Ramalina farinacea has proven to be a suitable model to study the multiplicity of 66 microalgae in lichen thalli due to the recurrent coexistence of Trebouxia sp. TR9 and T. 67 jamesii in long-distance populations (del Campo et al. 2010; Casano et al. 2011). Moreover, a 68 recent study using a deep sequencing technique revealed a much higher microalgal diversity 3 Cambridge University Press The Lichenologist Page 4 of 40 69 associated with a single lichen thallus of Ramalina farinacea (22 operational taxonomic units 70 [OTUs]) (Moya et al . 2017). 71 In order to understand the functions of microalgal communities in lichen thalli, 72 phycobiont isolation is a key step in the experimental design. The isolation and culture of 73 symbioticManuscript organisms offers an opportunity to undersFortand theReview mechanisms underlying the 74 selection of partners in symbiotic interactions (Yoshimura et al . 2002). Cultures of lichen 75 microalgae involve time-consuming techniques necessary for the successful propagation of 76 these symbionts (Rafat et al . 2015). However, phycobiont isolation is an essential procedure 77 to study lichen physiology, perform morphologic characterisation and obtain pure material for 78 molecular identification. 79 Studies dealing with phycobiont diversity in lichen thalli are primarily based on 80 Sanger sequencing. However, this technique could underestimate phycobiont diversity in 81 some lichen species (del Campo et al . 2010; Molins et al . 2013). There has been an increase 82 in the use of deep sequencing techniques to analyse the diversity of microalgal communities 83 (Bates et al . 2012; Park et al . 2015) because these techniques allow for the detection of a vast 84 number of genotypes that would otherwise remain underestimated in conventional PCR 85 amplifications (Stewart & Cavanaugh 2009). 86 To understand the hidden intrathalline phycobiont diversity and the role of each 87 phycobiont type in a symbiotic relationship, multi-tool and multidisciplinary approaches are 88 needed to obtain comparative results (Catalá et al. 2015). In particular, we focused on three 89 specific aims: i) to upgrade a methodology to propagate phycobionts that, coupled to fast 90 molecular identification, will significantly reduce the time needed to complete the process; ii) 91 to evaluate the phycobiont diversity using Sanger sequencing, 454-pyrosequencing and 4 Cambridge University Press Page 5 of 40 The Lichenologist 92 isolation; and iii) to compare the ultrastructural characterisation between lichenised 93 phycobionts and microalgae isolated in culture, and to correlate these ultrastructural traits 94 using molecular analyses. To achieve these objectives, we selected Circinaria hispida 95 (Mereschk.) A. Nordin, S. Savi ć and Tibell and Flavoparmelia soredians (Nyl.) Hale, two 96 lichen speciesManuscript with different biotypes that occur For in different Review ecosystems. 97 98 MATERIALS AND METHODS 99 Lichen material 100 The Circinaria hispida specimen was collected from Zaorejas (Guadalajara, Spain) 101 (40°46’02”N, 2°11’40”W, 1105 m ), and the Flavoparmelia soredians specimen was collected 102 on Quercus suber cork from Chóvar (Castellón, Spain) (39°51’13,68”N, 0°19’09.60”W). 103 Samples were dried out for one day and then stored at -20ºC until their processing. 104 105 Experimental design 106 Two lichen thalli (one per species) were examined under a stereo microscope to 107 exclude surface contamination. Samples were rehydrated with Milli-Q sterile water one day 108 before being processed, and they were stored in a growth chamber at 21ºC under a 12 h/12 h 109 light/dark cycle (lighting conditions: 15 µmol/m 2s). Lichen thalli were vortexed three times 110 for 5 min at 2,000 rpm with Milli-Q sterile water. Fragments from different parts of each 111 thallus were randomly excised and homogenised together, and each pool was labelled as 112 sample A, B, C or D. The mycobiont and primary phycobiont were identified by Sanger 113 sequencing from sample A. Isolation protocol and Sanger identification was performed for 5 Cambridge University Press The Lichenologist Page 6 of 40 114 sample B. Sample C was analysed
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