Molecular and morphological diversity of Trebouxia microalgae in sphaerothallioid Circinaria spp. lichens¹

Abstract

Three vagrant (Circinaria hispida, Circinaria gyrosa, and Circinaria sp. 'paramerae') and one crustose (semi-vagrant, Circinaria sp. 'oromediterranea') lichens growing in very continental areas in the Iberian Peninsula were selected to study the phycobiont diversity. Mycobiont identification was checked using nrITS DNA barcoding: Circinaria sp. 'oromediterranea' and Circinaria sp. 'paramerae' formed a new clade. Phycobiont diversity was analyzed in 50 thalli of Circinaria spp. using nrITS DNA and LSU rDNA, with microalgae coexistence being found in all the species analyzed by Sanger sequencing. The survey of phycobiont diversity showed up to four different Trebouxia spp. as the primary phycobiont in 20 thalli of C. hispida, in comparison with the remaining Circinaria spp., where only one Trebouxia was the primary microalga. In lichen species showing coexistence, some complementary approaches are needed (454 pyrosequencing and/or ultrastructural analyses). Five specimens were selected for high-throughput screening (HTS) analyses: 22 Trebouxia OTUs were detected, 10 of them not previously known. TEM analyses showed three different cell morphotypes (Trebouxia sp. OTU A12, OTU S51, and T. cretacea) whose ultrastructure is described here in detail for the first time. HTS revealed a different microalgae pool in each species studied, and we cannot assume a specific pattern between these pools and the ecological and/or morphological characteristics. The mechanisms involved in the selection of the primary phycobiont and the other microalgae by the mycobiont are unknown, and require complex experimental designs. The systematics of the genus Circinaria is not yet well resolved, and more analyses are needed to establish a precise delimitation of the species.

Symbiotic microalgal diversity within lichenicolous lichens and crustose hosts on Iberian Peninsula gypsum biocrusts

“…Even though these results should be interpreted with caution due to the small number of specimens analysed, our study reveals that including metabarcoding analyses could be key to understanding the phycobiont relationship in these systems. Photobiont switching seems to be a ubiquitous phenomenon in lichens and appears to play a vital role in lichen adaptation to environmental conditions 46 , 47 , 60 – 64 . Association with a wide range of symbionts may help lichens survive under harsh environmental conditions 65 – 67 , such as those characterizing gypsum biocrusts in a low-dry ombroclimate.…”

“…Several authors reported that sequences showing double peaks, or polymorphic sequences, in electrophoretograms were removed from their analyses 41 , 44 , 53 ; perhaps those removed sequences constituted specimens showing algal coexistence in these lichen species, which were not taken into account. The ability of Sanger sequencing to address a wide variety of inquiries in lichen ecology and evolution is not in question, but it is important to point out that phycobiont coexistence could vary depending on the lichen species and could be the general rule in some lichen species 37 , 38 , 46 – 48 , 74 . We hypothesized that coexistence could be related to, or favoured by, specific thallus architectures, such as the crustose type, and this factor should be considered when studies about patterns of fungal-algal associations are carried out.…”

“…The coexistence of multiple Trebouxia species within a single lichen thallus was studied in depth in the epiphytic species Ramalina farinacea from the Mediterranean region 34 – 36 and in other lichens with diverse geographic origins and growth forms 37 – 45 . However, it was not analysed in depth in crustose lichen species 46 – 48 , and in the specific case of gypsum crustose parasitic lichens, little information is available related to algal diversity.…”

Symbiosis between river and dry lands: phycobiont dynamics on river gravel bars

“…Illumina metabarcoding as well as the Sanger sequencing uncovered URa28 as a predominant phycobiont of Stereocaulon alpinum OTU2. In most cases, the phycobiont determined by the Sanger sequencing corresponds with a predominant phycobiont according to the high-throughput sequencing (Molins et al 2018; Paul et al 2018).…”

“…Even though the two mycobiont species-level lineages OTU2 and OTU35 differ in predominant phycobiont pool, they share the pool of other intrathalline algae, unlike two Circinaria spp. collected at the same location shared the predominant phycobiont, but showed a completely different pool of other intrathalline algae (Molins et al 2018). The Stereocaulon OTUs differ significantly in the frequency of intrathalline algae (Fig.…”