Hidden levels of phylodiversity in Antarctic green algae: further evidence for the existence of glacial refugia

1. Introduction

Recent work on terrestrial metazoans in Antarctica has questioned the long-held ‘recolonization hypothesis’ that expanded ice-cover during successive Neogene and Late Pleistocene glacial maxima has resulted in almost complete extinction of biota, followed by extensive colonization after glacial retreat (e.g. Stevens et al. 2006; Convey & Stevens 2007; Convey et al. 2008; McGaughran et al. 2008; Pugh & Convey 2008). Instead, the data support the ‘glacial refugia hypothesis’, with various groups of organisms, including mites, springtails and copepods, showing a high degree of endemism and regionalization, suggesting that they were able to survive in isolated ice-free refugia (see Convey & Stevens 2007; Convey et al. 2008 for an overview). To date, the focus has largely been on terrestrial organisms for which coastal oases and nunataks, the Transantarctic mountains and specific regions such as the McMurdo Dry Valleys—some of which are known to have been ice-free since at least the Mid-to-Late Miocene (up to approx. 14 Ma; Boyer 1979; Prentice et al. 1993)—probably acted as important refuges.

Evidence for the widespread persistence of refugia in which aquatic organisms could survive glaciation (e.g. coastal low-latitude regions) is, however, still elusive (Hodgson et al. 2001, 2005; Gibson & Bayly 2007). Life in Antarctic aquatic ecosystems is largely microbial and confined to benthic mats consisting of cyanobacteria, and to a lesser extent diatoms and green algae (Sabbe et al. 2004; de los Rios et al. 2004). Micro-organisms form an interesting test case in the light of the refugium hypothesis in that they are expected, within the limits of their environmental tolerance, to show ubiquitous distribution patterns as a result of their virtually unlimited dispersal capacity (but see ongoing discussion on microbial biogeography in, for example, Martiny et al. 2006). Molecular studies of cyanobacteria, using 16S rRNA gene sequencing, suggest that most isolates have a long association with the Antarctic environment (Taton et al. 2003, 2006). Likewise, morphological studies on diatoms suggest that in some areas at least 40 per cent of the species are Antarctic endemics (Schmidt et al. 1990; Sabbe et al. 2004; Spaulding et al. in press). These high levels of endemism may point to the importance of relatively low dispersal rates and long-term survival in suitable habitats confined to isolated glacial refugia. In contrast to diatoms and cyanobacteria, the green algal component of microbial mats has remained virtually unstudied. The available data are largely restricted to morphological taxonomic inventories on the continent, such as Victoria Land (Cavacini 2001; Adams et al. 2006), the Antarctic Peninsula (Mataloni & Pose 2001) and maritime Antarctica (Fermani et al. 2007; Zidarova 2007). Broady (1996) suggested that most Antarctic terrestrial green algae are cosmopolitan taxa. Morphological delimitation of green algal species and higher taxa is, however, contentious, especially in groups that are morphologically depauperate and exhibit convergent evolution towards reduced morphology (e.g. coccoid forms). Several cases of incorrect taxon delineation and cryptic species diversity are being disclosed by application of molecular phylogeny. A well-known example is that of the coccoid alga Chlorella, which has been shown to be polyphyletic, with the different taxa placed in separate green algal classes (Huss et al. 1999). DNA sequence data are therefore well placed to provide more insight into the green algal diversity on the Antarctic continent. The currently available molecular data are, however, fragmentary, consisting of a number of isolated taxonomic and ecophysiological studies on individual taxa (e.g. Pyramimonas australis: Moro et al. 2002; Scenedesmus sp.: Lesser et al. 2002; Pocock et al. 2004; lichen symbionts: Romeike et al. 2002; eukaryotic clone libraries: Christner et al. 2003; de la Torre et al. 2003; Fell et al. 2006).

The current study was designed to assess whether Antarctic freshwater green algae are mainly cosmopolitan species as a result of their fast colonization rates (Broady 1996), supporting the recolonization hypothesis, or are endemic to Antarctica or particular Antarctic regions, supporting the ‘glacial refugia hypothesis’ (cf. Convey et al. 2008). To this end, we employed molecular phylogenetic techniques, including molecular clock analysis of nuclear encoded 18S rRNA gene sequences of original Antarctic microchlorophyte isolates, along with an extensive set of other green algal sequences.

2. Material and methods

3. Results

We characterized 61 isolates of green algae from 13 maritime and 30 continental Antarctic samples (table 1). All strains were isolated as non-flagellated unicells (coccal morphology). Sequencing of the 18S rRNA gene yielded 14 distinct sequences. Ten of these were detected in one Antarctic region only, two were detected in two regions and two were detected in three regions (table 2). No sequences were found in more than three of the five regions sampled. Twelve additional GenBank sequences of freshwater and terrestrial green algae from different Antarctic regions were incorporated in our dataset, along with a broad representation of green algae from other regions. The alignment of 191 sequences was 1854 sites in total, including 950 phylogenetically informative characters. ML and BI phylogenetic analyses yielded similar tree topologies, which are congruent with published 18S phylogenies of green algae (Lewis & Lewis 2005; Pröschold & Leliaert 2007). The ML phylogram, with indication of ML bootstrap values and BI posterior probabilities, is shown in fig. S1 in the electronic supplementary material. The chronogram, obtained by rate-smoothing the ML tree using penalized likelihood and calibrated by a conservative and more liberal node age of the Chlorophyta–Streptophyta split (700 and 1500 Ma, respectively), is shown in figure 2.

Phylogenetic analysis shows that the freshwater Antarctic isolates are mainly distributed among the chlorophytan classes Chlorophyceae and Trebouxiophyceae, and only a single Antarctic strain (Trichosarcina mucosa, a filamentous green alga) belongs to the Ulvophyceae (figure 2). Apart from the Chlorella vulgaris NJ-7 strain, identical to an isolate from a eutrophic pond in The Netherlands, none of the Antarctic sequences was identical to non-Antarctic sequences currently available in GenBank. Uncorrected p-distances with the most closely related non-Antarctic sequences ranged from 0.002 to 0.034 (table S2, electronic supplementary material). Of the 26 distinct Antarctic sequences, 15 are Trebouxiophyceae. Within this clade, six strains (VI11, VI2, VPL1-3, NJ-18, NJ-7 and Ant-1) belong to the Chlorellaceae (Huss et al. 1999) and are intermixed with temperate members of the clade. Likewise, the remaining trebouxiophycean Antarctic isolates are scattered among temperate freshwater green algae. One strain, VPL5-6, was most closely related to a Paradoxia isolate from the Siberian permafrost, although its exact phylogenetic position could not be determined with satisfactory statistical support. The 18S sequence of isolate SC2-2 from the Schirmacher Oasis was extremely divergent, resulting in a deep branch with uncertain phylogenetic affinity within the trebouxiophycean lineage. Fourteen unique sequences are distributed in the Chlorophyceae clade. Within the Chlorococcales clade, two isolates from Lake Fryxell (VII3 and VI12) are most closely related to strain II4 from Ace Lake, which was found to be sister to a snow alga from Spitzbergen. Strain VPL9-6 is also resolved within the Chlorococcales but its phylogenetic affinity within this clade is uncertain. Four distinct sequences fall within the Scenedesmus/Desmodesmus clade along with temperate and Arctic members. The remaining Antarctic chlorophycean strains are spread mainly among temperate freshwater representatives.

While the phylogram (fig. S1, electronic supplementary material) reflects the different levels of sequence divergence found between the Antarctic sequences and their closest non-Antarctic relatives, the time-calibrated phylogeny (chronogram; figure 2) provides absolute age estimates for evolutionary events. Estimated ages of the Antarctic lineages (determined as the node separating the Antarctic sequence from its non-Antarctic sister) range from 2.7–9.9 Ma for a number of chlamydomonad isolates (Chlorophyceae) to over 17–84 Ma for the majority of the sequences and 330–708 Ma for the trebouxiophycean isolate SC2-2.

4. Discussion

From five well-distributed regions in both maritime and continental Antarctica, 14 distinct microchlorophyte sequences were recovered. Although there is no absolute threshold of 18S sequence divergence for defining green algal taxa, most Antarctic sequences are divergent enough to be considered distinct species, genera or even higher-order taxa (Lewis & Flechtner 2004; Lewis & Lewis 2005). Our results thus indicate a wide phylogenetic diversity of apparently endemic Antarctic lineages at different taxonomic levels, which has several important implications.

First, our results clearly contrast with morphological studies, which have suggested that Antarctic green algal communities are dominated by cosmopolitan species (Broady 1996), and instead support the notion that Antarctica has developed a distinct regional flora. Given that convergent evolution has led to high morphological similarity between unrelated microchlorophyte species, it is not unusual that molecular data contrast with morphological data. For example, Fawley et al. (2004) detected a high number of novel 18S rRNA genotypes during a molecular diversity study in freshwater systems in North America. Similarly, in desert areas of western North America, Lewis & Lewis (2005) discovered that green algae are not merely accidental visitors from aquatic environments, but long-term inhabitants of these extreme environments. Our results thus corroborate the recent studies that have revealed a high degree of endemism in other Antarctic micro-organisms such as cyanobacteria (e.g. Taton et al. 2006) and diatoms (Sabbe et al. 2003), and refute the hypothesis that for micro-organisms everything is everywhere (e.g. Finlay & Clarke 1999).

Second, our results point to several independent but rare colonization events over a long time frame and long-term survival in glacial refugia as evidenced by the different branch lengths of the Antarctic lineages. Owing to the lack of reliable green algal fossils, our phylogenetic tree is only calibrated at a single node, using a minimum and maximum age of the Chlorophyta–Streptophyta split at 700 and 1500 Ma, respectively (Douzery et al. 2004; Hedges et al. 2004; Yoon et al. 2004; Berney & Pawlowski 2006; Cavalier-Smith 2006; Roger & Hug 2006; Zimmer et al. 2007; Herron et al. 2009). Nonetheless, this approach allowed us to obtain rough estimates for the divergence times of the different Antarctic microchlorophyte strains. The majority of the lineages (16 out of 26) have estimated ages between 17 and 84 Ma and, based on available sequence data, probably diverged from their closest relatives around the time of the opening of Drake Passage (30–45 Ma) during the Eocene, which initiated the first transient glaciations on the continent. The lineages with longer branch lengths, including SC2-2 (330–708 Ma), have estimated ages that precede the break-up of Gondwana (65–100 Ma). These findings therefore support the hypothesis of the existence of refugia being present during successive glacial cycles (Convey & Stevens 2007; Convey et al. 2008) and conflict with the recolonization hypothesis, which proposes that fast colonization rates have resulted in the dominance of cosmopolitan species on Antarctica. Green algal refugia may include ice-free terrestrial habitats as well as ice habitats such as cryoconites and supraglacial ponds. Geological data do not rule out the presence of glacial refugia for aquatic microorganisms in at least three out of the five major regions included in our study. The McMurdo Dry Valleys have probably had ice-free areas since at least the Mid-to-Late Miocene (up to approx. 14 Ma; Boyer 1979; Prentice et al. 1993), with large glacial lakes being present since the Last Glacial Maximum (LGM; Wagner et al. 2006). In Prydz Bay, parts of the Larsemann Hills, less than 80 km away from the Rauer Islands and the Vestfold Hills, were ice-free during the LGM (Hodgson et al. 2001). Some lakes in this region were shown to contain relict populations of diatoms and copepods (Hodgson et al. 2005; Cromer et al. 2006), while in other lakes taxa currently present in other Antarctic and sub-Antarctic regions became locally extinct during the LGM (Hodgson et al. 2006). In the Lützow Holm Bay region, ice-free conditions in some coastal areas during the LGM can be inferred from ¹⁴C dates of in situ fossils in raised beach deposits (Miura et al. 1998). However, whether freshwater habitats escaped glaciation in this region remains unclear.

Third, our data shed some light on green algal dispersal within the Antarctic continent. Most phylotypes (10 out of 14) were only retrieved from a single ice-free region. This implies that, on the one hand, dispersal rates within Antarctica could be low and that immigrants are competitively excluded (priority effects), and/or, on the other hand, that our sampling of the Antarctic microchlorophyte flora is currently limited by a small number of sampling sites that may differ in their limnological properties. Only two taxa (II11 and VI11), belonging to the genera Chlorella and Scenedesmus, were detected in three of the five regions, suggesting that these taxa may have more easily dispersed over the Antarctic continent. This is in agreement with Lawley et al. (2004), who found a similar lack of overlap between the eukaryotic biota of ‘patterned ground soils’ using a clone library approach from several widely separated Antarctic locations, with only a few taxonomic units showing apparently wider distributions. However, extended taxon sampling and more variable molecular markers (such as rDNA ITS) will be required to elucidate phylogeographic patterns of green algal taxa within Antarctica.

While the methods used for both cultivation and tree construction may have biased our results, they clearly highlight that (Antarctic green algae show a remarkable divergence from taxa from other regions). We are therefore confident that our main conclusion, namely that Antarctica is characterized by a distinct microchlorophyte flora, probably as a result of ancient patterns of isolation and long evolutionary history on the continent, will hold as new data become available.