From Alaska to Antarctica: Species boundaries and genetic diversity of Prasiola (Trebouxiophyceae), a foliose chlorophyte associated with the bipolar lichen-forming fungus Mastodia tessellata

Highlights

• A molecular survey was conducted of lichenised Prasiola specimens from Alaska, Tierra del Fuego and Antarctica.

• Genetic data were generated for two nuclear loci (nrITS, RPL10A) and a chloroplast locus (tufA).

• A discovery-validation approach was used for Prasiola species delimitation.

• We identified two Prasiola species forming lichens with Mastodia tessellata.

• Lichenized Prasiola species showed contrasting distribution ranges.

Abstract

Symbiotic associations between green algae (Chlorophyta) and fungi give rise to morphologically and eco-physiologically distinct entities, or so-called, lichens. In one of the most peculiar of these associations, the partners are species of the macroscopic genus Prasiola (Trebouxiophyceae) and the ascomycete Mastodia tessellata (Verrucariaceae). This is the only known case of a lichen symbiosis involving a foliose green alga. Despite intense research targeted at understanding the biology of this particular association, little is known about the genetic variability of its symbionts. This study focuses on the photobiont partner of this lichen and was designed to explore and compare its genetic diversity along a latitudinal axis from Alaska to Antarctica. Molecular sequence data were generated for three loci: two nuclear markers (nrITS, RPL10A) and one plastid-encoded marker (tufA). The usefulness of the Prasiola nrITS and RPL10A data was examined at the species and intraspecific levels. We used the population assignment tests implemented in BAPS and STRUCTURE and two algorithmic species delimitation procedures (ABGD, GMYC) to generate species boundary discovery hypotheses, which were subsequently tested using Bayes factors. Population genetic differentiation and structure were also assessed through fixation indices, polymorphism statistics and haplotype networks. Based on the results of the species validation method, we propose that at least two species of Prasiola associate with the lichen-forming fungus Mastodia tessellata. Of these, P. borealis is broadly distributed in Alaska, Tierra del Fuego and the Antarctic Peninsula, whereas the second, undescribed, species is restricted to the Antarctic Peninsula. We detected significant phylogeographic substructure in P. borealis, including greater haplotype diversity in the Tierra del Fuego populations. Our findings provide new data that will be useful to unravel the cryptic diversity and phylogeographic patterns of the green alga partners of lichens.

Introduction

With ca. 4500 described species, the green algae are an extraordinarily diverse group of eukaryotic macro- and microorganisms that date back to 700–1500 Mya (Guiry, 2012, Herron et al., 2009). Difficulties in the systematics of these organisms are two-fold. First, morphological homoplasy and stasis along with phenotypic plasticity are common at different phylogenetic depths (e.g. Fraser et al., 2009, Škaloud and Rindi, 2013, Verbruggen, 2014). And second, the multiple species concepts that phycologists have applied to accommodate the singularities of their respective groups of interest often prevent establishing broadly accepted species delimitation criteria across the whole lineage (Leliaert et al., 2014).

The systematics of green algae associated with lichen-forming fungi is no exception. While fungal species delimitation based on traditional morpho-anatomical and chemical characters is relatively straightforward, the circumscription of symbiont algae is particularly challenging. For over a decade, studies have been unravelling the diversity of lichen-associated photobionts within a phylogenetic framework. The consequence of this research effort has been the revision of traditional species concepts and even the description of new taxa (Kroken and Taylor, 2000, Nelsen et al., 2011, Škaloud and Peksa, 2010, Vančurová et al., 2015). So far, the use of numerous coalescent-based methods to document and describe species based on DNA sequences (see Fujita et al., 2012) has been limited, most studies having focused on selectivity and specificity (Leavitt et al., 2015, Sadowska-Dés et al., 2014). However, in many groups of chlorophytes involved in lichen symbioses, further evidence based on multi-locus data is required.

Contrasting patterns of lichen photobiont phylodiversity have been observed at high phylogenetic levels. For instance, the lichen-forming fungus family Parmeliaceae, with more than 2500 species showing a wide variety of ecological and geographic ranges, is strictly associated with green micro-algae of the genus Trebouxia Puymaly (e.g. Fernández-Mendoza et al., 2011, Leavitt et al., 2015). Likewise, nearly all Peltigerales associate with Nostoc Vaucher ex Bornet & Flahault cyanobacterium either as a primary or secondary photobiont (Rikkinen, 2013), while orders including many tropical species such as Arthoniales and Ostropales have symbiotic relationships preferentially with the Trentepohliales (Nelsen et al., 2011). In contrast, members of the widespread family Verrucariaceae associate with at least seven photobiont genera from three different phyla (reviewed in Thüs et al., 2011). The relationship between the lichen-forming fungus Mastodia tessellata (Hook. f. & Harv.) Hook. f. & Harv. and a macroscopic green alga of the genus Prasiola (C. Agardh) Meneghini (Prasiolales, Trebouxiophyceae) has long drawn the attention of biologists as the only known lichen with a foliose photobiont (Kohlmeyer et al., 2004, Pérez-Ortega et al., 2010). Descriptions of this symbiosis have ranged from a mycophycobiosis or putative ‘fungal infestation’ (Parader and Ahmadjian, 2000, Reed, 1902, Rindi et al., 2007) to a primitive or borderline lichen (Kohlmeyer et al., 2004, Kováčik and Pereira, 2001, Lud et al., 2001). Based on electron microscopy observations, Pérez-Ortega et al. (2010) claimed that the fungal partner of this lichen provokes the altered arrangement of algal cells, and that the cells of both bionts undergo intimate interactions albeit with negligible impacts on macromorphology. These authors also highlighted the complexity of this symbiotic relationship, which rather than being strictly attributed to parasitism, mutualism or saprophytism, may be described as a dynamic equilibrium in which the photobiont under certain conditions is able to ‘escape’ from the mycobiont.

Despite the long-standing debate about the nature and ecological implications of this uncommon association, little is known about the genetic variability of these symbionts. In early work, it was acknowledged that two species of Prasiola associated with Ascomycetes. In the Northern Hemisphere, Reed (1902) designated as Prasiola borealis Reed, green algae colonized by a fungus he described as Guignardia alaskana Reed. Lichenised Prasiola specimens from the Antarctic and sub-Antarctic regions were initially ascribed to Prasiola crispa ssp. antarctica (Kützing) Knebel (Kohlmeyer et al., 2004, Kováčik and Pereira, 2001, Lud et al., 2001). However, more recent studies have shown that both P. delicata Setchell & N.L. Gardner and P. borealis associate with Mastodia tessellata in the Northern and both Hemispheres respectively (Moniz et al., 2012a, Moniz et al., 2014, Pérez-Ortega et al., 2010). Further, Moniz et al. (2012b) used molecular data to resurrect Prasiola antarctica Kützing (=P. crispa ssp. antarctica) to accommodate a distinct Antarctic lineage, yet the specimens of this species examined so far show no distinct signs of fungal presence meaning that the species of Antarctic Prasiola associated with M. tessellata remain unknown.

In this study, we compiled a comprehensive molecular dataset extracted from specimens of Prasiola collected in Alaska, Tierra del Fuego and the Antarctic Peninsula. Three molecular markers were selected for the alga, including the plastid-encoded elongation factor Tu (tufA) gene, the nuclear ribosomal Internal Transcribed Spacer (nrITS), and the nuclear RPL10A gene. The latter encodes the protein RPL10, required to join together 40S and 60S subunits into a functional 80S ribosome (Eisinger et al., 1997). The markers tufA and nrITS have been often used in barcode and phylogenetic studies of several groups of green algae (e.g. Leliaert et al., 2009, Moya et al., 2015, Rindi et al., 2011, Sadowska-Dés et al., 2014, Saunders and Kucera, 2010). In contrast, the RPL10A gene has been seldom used for evolutionary analyses in Chlorophyta (del Campo et al., 2013). Moreover, there are no literature data available on nrITS and RPL10A markers for Prasiola.

The aims of our study were: (1) to test the use of nrITS and RPL10A markers for evolutionary analyses at intermediate and low taxonomic levels in Prasiola, (2) to explore the population structure of the Mastodia tessellata photobiont, (3) to propose and validate species boundaries for lichenized Prasiola using a multi-locus approach, and (4) to shed light on the genetic structure and differentiation within each delimited species across its distribution range.