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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The genetic diversity of all available culture strains of the Tribonemataceae (Stramenopiles, Xanthophyceae) from Antarctica was assessed using the chloroplast-encoded psbA /rbcL spacer region sequences, a highly variable molecular marker, to test for endemism when compared with their closest temperate relatives. There was no species endemic for Antarctica, and no phylogenetic clade corresponded to a limited geographical region. However, species of the Tribonemataceae may have Antarctic populations that are distinct from those of other regions because the Antarctic strain spacer sequences were not identical to sequences from temperate regions. Spacer sequences from five new Antarctic isolates were identical to one or more previously available Antarctic strains, indicating that the Tribonemataceae diversity in Antarctic may be rather limited. Direct comparisons of the spacer sequences and phylogenetic analyses of the more conserved rbcL gene revealed that current morphospecies were inadequate to describe the actual biodiversity of the group. For example, the genus Xanthonema, as currently circumscribed, was paraphyletic. Fortunately, the presence of distinctive sequence regions within the psbA/rbcL spacer, together with differences in the rbcL phylogeny, provided significant autoapomorphic criteria to re-define the Tribonemataceae species.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Microalgae dominate the terrestrial habitats of Antarctica in terms of biomass and species diversity (Elster and Benson, 2004; Broady, 2005). These algae play a key role in cycling soil nutrients in the isolated Antarctic ecosystem, and they provide a source of organic matter through their photosynthesis (Tibbles and Harris, 1996; Tscherko et al., 2003). Long periods of snow cover, low temperatures and low water availability are major controlling factors for Antarctic microbial life (Tscherko et al., 2003), but not all terrestrial habitats are hostile. These terrestrial algae may be continually dispersed to Antarctica from other continents or they may be endemic to Antarctica. The goal of our study was to investigate the diversity and endemicity of yellow-green microalgae belonging to the family Tribonemataceae (Xanthophyceae, Heterokontophyta).

The composition and diversity of Antarctic microalgal communities of terrestrial and freshwater localities have been studied frequently (e.g. Seaburg et al., 1979; Broady, 1986; Pankow et al., 1991; Broady and Smith, 1994; Mataloni et al., 2000). Surveys frequently list the occurrence of the yellow-green alga Xanthonema (homonym Heterothrix Pascher; Silva, 1979), which forms unbranched filaments lacking obvious H-shaped cell wall segments (Pascher, 1932; Ettl, 1978; Ettl and Gärtner, 1995). Antarctic Xanthonema have been isolated into culture from water-flushed soils near glaciers, stone surfaces (hypolithic) and moss, where they are epiphytes (Broady et al., 1997; Broady, 2005). Xanthonema usually occurs in small amounts and it grows intermixed with green algae and cyanobacteria; often it is detectable only using a culturing approach. Most Antarctic culture strains belonging to the Tribonemataceae have been assigned to the genus Xanthonema and they have been assigned to six species using a traditional morphology-based approach. A few strains could not be assigned to a species due to phenotypic plasticity (Broady et al., 1997). Apart from Xanthonema, other Xanthophyceae isolated from terrestrial locations in Antarctica include the genera Tribonema, Bumilleriopsis and Heterococcus (all Tribonemataceae), the filamentous Heterotrichella (Heteropediaceae) and a few non-filamentous genera (Andreoli et al., 1999; Mataloni et al., 2000; Broady, 2005).

Several studies have used SSU rRNA gene sequences to investigate the diversity of cyanobacteria in Antarctica (see Komárek et al., 2008 for review), and a few molecular environmental studies, using SSU rRNA, have investigated the eukaryotic algal diversity (Lawley et al., 2004; Fell et al., 2006). The latter detected xanthophycean algae only at the class or generic levels. So far, there has been only one study for inferring phylogenetic relationships of Antarctic Xanthophyceae, and it was based on the chloroplast-encoded rbcL and psaA genes and culture strains (Maistro et al., 2007). The nuclear-encoded SSU rRNA gene, widely used for prokaryotes and some eukaryotes, has been too conservative for xanthophycean species level phylogenies (Bailey and Andersen, 1998; Negrisolo et al., 2004). Because of its limited use, only six 18S rDNA sequences for Tribonemataceae are in GenBank, whereas 26 rbcL sequences available (Maistro et al., 2007). The rbcL data showed that Xanthonema strains were distributed on two or three deeply diverging clades (Maistro et al., 2007; Rybalka et al., 2007), and preliminary results for Antarctic strains showed that they were intermixed with strains from other geographical regions. This finding makes the Tribonemataceae an appropriate group to investigate endemicity among different culture strains. We conducted a more thorough study of Antarctic strains using a highly variable molecular marker for Xanthophyceae, the chloroplast-encoded psbA/rbcL spacer region (Andersen and Bailey, 2002; Rybalka et al., 2007). We tested for endemism or cosmopolitanism by comparing the Antarctic strains to those from temperate regions on other continents (e.g., Lawley et al., 2004). We also compared morphological and molecular data for these taxa, in an effort to provide accurate identifications and more stable scientific names.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The psbA/rbcL spacer region sequences could not be aligned across all taxa because they are so variable (see below), and therefore rbcL gene sequences were used in phylogenetic analyses to establish their evolutionary relationships. The phylogenetic analyses showed that the Antarctic filamentous Xanthophyceae were distributed in three deeply diverging clades, which we name Xanthonema 1, Xanthonema 2 and Tribonema 1 (Fig. 1). These algae were only distantly related to Bumilleria sicula and Bumilleriopsis filiformis, which showed that the clades were not misidentified taxa belonging to other genera in the Tribonemataceae (Fig. 1). Within the Xanthonema clade 1, the rbcL sequences for Antarctic strains B4-1 and B8-5 were virtually identical, i.e. they differed in only one position. The two strains differed by only 1–2 bp from strain CCAP 808/3 (isolated from a snow field in Alaska) and from GenBank AJ874710 (from identical Antarctic strains, Ohtani 889 and Ling 906; Maistro et al., 2007). Strains A19 and Broady773 from Antarctica and three non-Antarctic strains (CCAP 836/2, SAG 836-1 and UTEX 353) had identical rbcL sequences. Other closest relatives to the Antarctic strains were non-Antarctic strains (UTEX 155, CCAP 808/2, CCALA 516) and Antarctic strain PAB 421 (Fig. 1). Interestingly, UTEX 155 was a duplicate strain of SAG 836-1, i.e. both represented the same isolate that had been kept at two different culture collections. However, the SAG 836-1 rbcL sequence differed by 15 and 14 positions from an existing GenBank sequence of strain UTEX 155 (AF084612) and our sequence (EF455920). Finally, Antarctic strains Broady 395 and Broady 601 and the temperate strain CCALA 517 had nearly identical sequences (8 and 9 rbcL bp separated the Antarctic strains from the temperate strain).

image

Figure 1. Phylogenetic analysis of Antarctic strains of Tribonemataceae using a data set of complete rbcL sequences (1467 bp long; 295/236 variable/parsimony informative sites). Sequences in bold are the five new Antarctic isolates. Strains isolated from Antarctica are denoted by an asterisk. Sequences except those where accession numbers are given are reported for the first time in this study. Capital letters next to groups of sequences indicate psbA/rbcL spacer sequence similarity groups (see Table 1). Strains that have simply been added to the figure because their sequences were identical with those that were used in the phylogenetic analyses are marked by ‘+’. The phylogeny shows a Bayesian consensus tree. Thick lines indicate those internal branches that were resolved by maximum likelihood, maximum parsimony, minimum evolution distance and Bayesian analyses. High support for internal branches that connect almost identical sequences is indicated by a filled circle. Numbers at branches are bootstrap values for minimum evolution distance (> 70%; upper left), posterior probabilities from Bayesian analyses (> 0.95; upper right) and weighted maximum parsimony (below). Three unicellular Xanthophyceae served as an outgroup.

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In Xanthonema clade 2, the Antarctic strains A16-5 and Turner 907 shared almost identical rbcL sequences (1 bp difference). They were related to two temperate freshwater strains, CCALA 518 and SAG 60.94 (Fig. 1). The Antarctic strain Broady 759 was more distantly related (Fig. 1). Its rbcL sequence was identical with that from the authentic strain of Xanthonema sessile (ASIB V98; AJ874329), and Broady and colleagues (1997) found that this Antarctic strain was identical to temperate strain ASIB V98 at the morphological level. Therefore, we assigned strain Broady 759 to X. sessile.

The Xanthonema clade 3 was formed by strain Broady 735, isolated from soil attached to vegetables imported from New Zealand into Antarctica (Broady et al., 1997), and two temperate Ukrainian strains (SAG 2181 and SAG 2182). The three strains varied by one position. Total pairwise rbcL sequence differences among the three Xanthonema clades were 63–112 bp, which was approximately the same magnitude as between representatives of three different genera. That is, there were 69–103 bp differences among Xanthonema debile CCALA517, B. filiformis SAG 809-2 and Tribonema sp. SAG 21.94.

The Tribonema clade 1 contained Antarctic strains A21 and Ohtani 887 (identical sequences) as well as Antarctic strain SAG 2165 (4 bp difference). They were most closely related to two temperate freshwater strains (SAG 21.94, SAG 23.94), which were assigned to two distinct species of Tribonema (Table 1). There were no more than four and eight positions different between the rbcL sequences of these Antarctic and temperate strains.

Table 1.  Strains of Tribonemataceae grouped according to similarities of their psbA/rbcL spacers and clades as inferred from the rbcL phylogeny shown in Fig. 1.
rbcL cladepsbA/rbcL spacer similarity group/ new species designationStrain and previous species designationLocationLength of psbA/rbcL spacer
  • a.

    Epitype strain.

  • b.

    Duplicate strains.

  • c.

    Only a 5′ partial psbA/rbcL spacer sequences was obtained.

  • New isolates are in bold. (A) Strains isolated from Antarctica.

Xanthonema 1A/X. sp. AB4-1Xanthonema or Bumilleria (= SAG 2183)King George Island (A)439
B8-5Xanthonema sp. (= SAG 2184)King George Island (A)440
Ling 906 Xanthonema debile (= SAG 2190)King George Island (A)440
Ohtani 889 X. debile (= SAG 2191)King George Island (A)440
CCAP 808/3 Bumilleria sp.USA, cold-tolerant439
B/X. hormidioidesA19Xanthonema sp. (= SAG 2179)King George Island (A)431
Broady 773 Xanthonema pascheri (= SAG 2188)Ross Island (A)429
CCAP 836/2 Heterothrix hormidioides (= SAG 2288a,b)Europe, temperate422
SAG 836-1 X. debileEurope, temperate422
UTEX 353bH. hormidioidesEurope, temperaten.a.c
C/X. bristolianumCCALA 516 Xanthonema bristolianum (= SAG 2285a)Europe, cold-tolerant449
D/X. debileUTEX 155 Heterothrix debilis (= SAG 2289a)Europe, temperate430
E/X. exileBroady 395 X. debile (= SAG 2185)Vestfold Hills (A)451
Broady 601 X. debile (= SAG 2186)Ross Island (A)451
CCALA 517 Xanthonema exile (= SAG 2286a)Europe, temperate385
Xanthonema 3 (new genus)F/‘X.’ sp. FBroady 735 Xanthonema cf. bristolianum (= SAG 2187)New Zealand, cold-tolerant451
SAG 2182 Xanthonema sp.Europe, temperate451
SAG 2181 Xanthonema cf. exileEurope, temperate451
Xanthonema 2 (new genus)G/Heterothrix mucicolaA16-5Xanthonema or Tribonema (= SAG 2192)King George Island (A)476
Turner 907 Xanthonema hormidioides (= SAG 2194)Southern Victoria Land (A)476
CCALA 518 H. mucicolaEurope, temperate475
SAG 60.94 Xanthonema cf. bristolianumEurope, temperate492
H/‘X.’sessileBroady 759 Xanthonema sessile (= SAG 2193a)Ross Island (A)418
Tribonema 1I/T. sp. IA21Xanthonema tribonematoides (= SAG 2172)King George Island (A)534
Ohtani 887 X. tribonematoidesKing George Island (A)n.a.c
SAG 2165 Tribonema sp.Adelaide Island (A)536
SAG 21.94 Tribonema ulotrichoidesEurope, temperate536
SAG 23.94 Tribonema virideEurope, temperate536

psbA/rbcL spacer

The psbA/rbcL spacer sequences were determined to investigate the close relationships of strains within a single species or among closely related species. The spacer contained large regions of hypervariable nucleotides that were unalignable in entirety over the four major clades. However, a short region of about 30 positions at the 5′-end (pos. 93–122 of reference sequence EF455930) and a second region of about 212 bp at the 3′-end (pos. 319–531 of reference sequence EF455930) could be aligned (Fig. 2). The Xanthonema clade 1 psbA/rbcL spacer sequences were also aligned about 45 bp further downstream (pos. 169 of EF455930) into the hypervariable region, and five groups (A–E) were revealed (Table 1; Fig. 2). Group A strains (from Antarctica and an Alaskan snow field) were almost identical except for a single position and a single 1 bp indel. Group A did not correspond to any morphological species, and therefore it was designated Xanthonema sp. A.

image

Figure 2. Alignment of psbA/rbcL spacer sequences of studied strains of Tribonemataceae grouped according to sequence similarity (groups A–I, see Table 1). Parts of the more conserved 5′- and 3′-ends are shown as well as a large part of the hypervariable region in between. Numbers refer to sequence positions of strain B4-1 (Accession No. EF455930).

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Five strains comprised group B (Table 1; Fig. 2). Two strains were from Antarctica (A19 and Broady 773) and their sequences were almost identical, i.e. they differed in a single sequence position (Fig. 2) and by two indels of 1 bp each. Both Antarctic strains were distinct from the three temperate strains (which had identical spacer regions) by the presence/absence of a short indel (7–9 bp) at the 5′-end of the psbA/rbcL spacer as well as at two other sequence positions. Two of the three temperate strains with identical spacer sequences (CCAP 836/2, UTEX 353) were duplicate authentic strains for Xanthonema hormidioides. The third strain (SAG 836-1) was an authentic strain for X. debile. The authentic strains were (putatively) established by Vischer when he described both Xhormidioides and X. debile (Vischer, 1936; 1945). However, a duplicate authentic strain of X. debile, UTEX 155, was distinctly different (group D), indicating that SAG 836-1 became mislabelled during its history. Therefore, all five group B strains were named X. hormidioides.

Group E (Antarctic strains Broady 395 and 601), whose rbcL sequences were identical, also had identical psbA/rbcL spacer sequences (Fig. 2). They differed from their closest relative (CCALA 517) by 15 sequence positions and an insertion of 65 bp. The CCALA 517 strain was identified as X. exile; it was isolated from a temperate locality, as was the culture used by Klebs (1896) when he described that species, and therefore we used that name for group E. Xanthonema exile was the type species for the genus Xanthonema, and therefore species in clade 1 must be named Xanthonema.

Group C and D sequences from strains CCALA 516 and UTEX 155 were rather distinct as well as strain CCAP 808/2, i.e. they could not be meaningfully aligned (Table 1; Fig. 2). Group C (CCALA 516) was identified as Xanthonema bristolianum and we used that name. Group D contained the authentic strain (UTEX 155) for X. debile and thus the group was assigned this name. Strain CCAP 808/2, which was identified as Bumilleria exilis, was not closely related to strain CCALA 517 (Fig. 2). Because we designated CCALA 517 as the epitype for X. exile (see below), CCAP 808/2 had to be assigned a different species that we designated Xanthonema sp. B. Note that strain PAB 421, used in the rbcL phylogenetic analyses, was not included because the strain became extinct before the psbA/rbcL spacer sequence could be determined.

Xanthonema clade 2 was deeply divergent from Xanthonema clade 1, and the clade represented a new genus distinct from Xanthonema. Clade 2 had two groups, G and H (Table 1; Fig. 2). Within group G, the Antarctic strains A16-5 and Turner 907 had identical spacer sequences. They differed from the temperate strain of that group, CCALA 518, by 18 sequence positions, an indel of one nucleotide and another indel of two nucleotides. The other temperate strain of group G, SAG 60.94, appeared to be even more distant from the two Antarctic strains based upon the spacer region, i.e. with differences in 15 sequence positions (Fig. 2) and five indels of 1–17 bp long. However, the hypervariable regions of all group G strains were still easily aligned (Fig. 2). Group G contained strain CCALA 518 that represented Heterothrix mucicola; therefore, we temporarily assigned the group G strains to this species until a new genus can be described (see taxonomic discussion below). In group H, Antarctic strain Broady 759 (assigned X. sessile; see above) was more distantly related because its psbA/rbcL spacer was shorter and the spacer could not be aligned with those from group G (Fig. 2).

Xanthonema clade 3 (group F) sequences aligned well (Fig. 2) but the clade was deeply divergent, and strains appeared to belonging to a second new genus (Fig. 1). Broady 735, from New Zealand soil material, had no more than three and eight sequence differences when compared with the two Ukrainian temperate strains (SAG 2181 and SAG 2182; Fig. 2). The strains in group F did not correspond to any described morphological species, and therefore we temporarily named the strains ‘Xanthonema’ sp. F until a new genus and species is described.

Tribonema clade 1 had a total of 16 variable spacer sequence positions. Isolate A21 was identical with strain Ohtani 887; however, only a 5′ partial sequence, 356 bp, could be obtained for the latter strain (Fig. 2). Antarctic strain SAG 2165 differed from strain A21 by nine sequence positions and two indels of 1 bp each. Both had only 9/12 and 6/5 sequence differences with their next closest temperate relatives, strains SAG 21.94 and SAG 23.94. Strains in group I could not be assigned a single species name because group I contained two Tribonema species and therefore we temporarily named the group Tribonema sp. I until an unambiguous species name can be found.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Endemism

The Tribonemataceae (Stramenopiles, Xanthophyceae) diversity, based upon all available Antarctic culture strains plus five new isolates, was assessed using the conserved rbcL gene and the highly variable non-coding psbA/rbcL spacer. The spacer region allows a detailed comparison of the Antarctic strains with those from temperate regions at or even below the level of species and therefore the spacer can be used to test for endemism. The term endemism refers to a group of organisms that is confined to a single geographical region, or with respect to Antarctic microorganisms, endemics have genotypes found only in Antarctica (Vincent, 2000). However, endemism applied to microorganisms is a controversial topic (Lawley et al., 2004). It has long been suspected that microbial dispersal is not restricted by geographic boundaries (Baas-Becking, 1934; Staley and Gosink, 1999; Finlay, 2002). Currently, there is limited evidence for microbial endemism in Antarctic terrestrial environments (e.g. Roser et al., 1993; Broady, 1996; 2005), but most previous studies have been based solely on traditional morphological and/or culture approaches. In a pioneering study of eukaryotic diversity in Antarctic soils, Lawley and colleagues (2004) used the rather conserved 18S rRNA gene sequences, and they found a high degree of similarity for Antarctic isolates with non-Antarctic organisms. However, they found it impossible to confirm that their taxa were conspecific with isolates from other geographic locations because 18S rRNA is so highly conserved. In our study, the Antarctic strains were distributed among six psbA/rbcL spacer sequence similarity groups, but each group also included strains from other geographic regions. The Xanthonema group A strains shared almost identical psbA/rbcL spacers despite they were from quite distant locations, but of similar ecology, i.e. from Alaska (strain CCAP 808/3) and from Antarctica (Table 1). Group A strains may represent a new species of Xanthonema that is cold-adapted because it is known only from cold regions. Also within X. hormidioides (group B) and group F, there were pairs of strains from distant locations within Antarctica (and thus of similar ecology; strains A19/Broady 773 and strains A16-5/Turner 907) that shared identical hyper variable spacer regions with each other. The high similarity in the psbA/rbcL spacer regions clearly indicates conspecificity, i.e. the strains from Antarctica cannot be distinguished from temperate strains at the species level. We conclude that there is no Tribonemataceae species endemism in Antarctica and that the high genetic similarity reflects a similar ecology rather than a geographical closeness. Furthermore, no clades in the rbcL phylogeny were restricted to a geographical region, indicating that the Tribonemataceae species are generally widespread where suitable habitats exist.

Despite the close similarity of Antarctic and temperate strains, we did not find a single case where an Antarctic strain was identical in its psbA/rbcL spacer region to that of an isolate from a temperate location. The fewest differences between Antarctic and temperate strains were within X. hormidioides (group B) and Tribonema sp. 1 (group I). Xanthonema exile (group E) and ‘Xanthonema’ sp. F (group F) also had Antarctic and temperate strains with psbA/rbcL spacer regions distinct from each other. The differences between the Antarctic and temperate strains were greater than those within either the Antarctic or the temperate strains. These findings imply that the Antarctic and temperate strains represent two different populations of a single species. It further implies that the Antarctic strains for a given species share a common evolutionary history. That is, there was just one colonization event in Antarctica for each group of strains with nearly identical spacer regions or, if multiple colonization events occurred, then the invasions were too recent to produce significant divergence (Whitaker et al., 2003; Prosser et al., 2007). It is not possible to calculate divergence times because there are no reliable fossil records for the Tribonemataceae or the Xanthophyceae. It would be intriguing to investigate ancient soil samples for the presence of Tribonemataceae DNA because the old soils may have been isolated for long time. Also, it would be interesting to test for phenotypic differences between the Antarctic and temperate populations, e.g. with respect to cold tolerance.

Tribonemataceae diversity in Antarctica

Only six different psbA/rbcL spacer groups were recovered for the Antarctic Tribonemataceae. Although the number of culture strains currently available (15) is rather limited, it is remarkable that the psbA/rbcL spacer sequences of the five newly established Antarctic isolates were identical (or nearly so) to corresponding sequences of previously available strains. Furthermore, only two of the six known groups were not recovered by the new isolates (groups E and H). Remarkably, the diversity represented by the new isolates was found in a relatively small but ecologically diverse area, the Admiralty Bay region on King George Island. The material was collected from valleys with soils of different moisture content, i.e. slopes or tops of moraines at various distances from a glacier and the seacoast. Guano and bird excrements further influenced at least two sites (Tscherko et al., 2003). We conclude that Tribonemataceae diversity is rather limited in Antarctica, but once a species initially colonizes the continent, the species spreads by adapting to various habitats. Certainly, a more extensive field sampling is needed to provide more conclusive data and to confirm this hypothesis. Further studies should also include a culture-independent approach to recover Tribonemataceae taxa that are difficult or impossible to culture. The psbA/rbcL spacer PCR amplification approach used here appears to be selective for Xanthophyceae (Andersen and Bailey, 2002) and would facilitate an environmental molecular study.

Limits of current morphospecies

Species of the Tribonemataceae have been defined by morphological data alone even though the family is morphology-poor (Ettl, 1978; Ettl and Gärtner, 1995; Lokhorst, 2003; Zuccarello and Lokhorst, 2005). We conclude that the morphological features traditionally used to define taxa are inadequate to describe the actual biodiversity in this group. This is clearly exemplified with the five newly established Antarctic isolates. Strains A19 and A16-5 were distantly related in the rbcL phylogeny (Fig. 1), but they had only minor morphological differences (Fig. 3A and B). Conversely, strains B4-1 and B8-5 shared almost identical hypervariable spacer sequences. However, strain B4-1 exhibited an internal thick cap-like structure between two adjacent cells (Fig. 3C), a morphological character that is used to define Bumilleria (Ettl, 1978). Both strains fragment readily into short filaments with two to four cells (Fig. 2C and D), which is typical for Bumilleria, but strains B4-1 and B8-5 were rather distinct from the clade representing true Bumilleria and Bumilleriopsis (Fig. 1). The presence of H-shaped wall fragments (Fig. 3A and E) has been the classical diagnostic feature for Tribonema (Ettl, 1978). However, two strains with obvious such H-pieces, A19 and A21, are not closely related to each other and only one (A21; Fig. 3E and F) is related to true representatives of Tribonema (Fig. 1). We conclude that the morphological features are more likely to be homoplasious than the nucleotides of the spacer region.

image

Figure 3. Morphological features of five new strains of Tribonemataceae isolated from soils of King George Island, Antarctica. Terminal H-pieces at fragmented filaments obvious in strain A19 (A). Short and thin filaments with no terminal H-pieces visible at fragment ends in strain A16-5 (B). Internal cap-like thick structure (arrows) present at readily fragmented filaments of strain B4-1 (C). Strain B8-5 also exhibits fragmentation into short filaments of two to four cells, but lacks the cap-like structure (D). Strain A21 exhibits prominent H-pieces at ends of filament fragments (E) and has large cells that are somehow rounded and thickened in their middle (F). Scale 10 μm (A–F).

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Molecular phylogenetic species concept

The Tribonemataceae are rarely (if ever) sexual and therefore the biological species concept is not applicable (e.g. Mayr and Ashlock, 1991). Johansen and Casamatta (2005) proposed the ‘monophyletic species concept’ (autoapomorphic species concept sensuMishler and Theriot, 2000a) for asexual taxa because they consider it the most theoretically sound and well suited. The monophyletic species concept recognizes species as the smallest monophyletic groups worthy of taxonomic recognition. Monophyly within a species is recognized by the presence of autapomorphies (i.e. unique derived characters present only in those species; Mishler and Theriot, 2000a,b; Johansen and Casamatta, 2005). Johansen and Casamatta (2005) propose, in cases where morphological data are insufficient, that a monophyletic species can be identified by a maximum likelihood analysis using molecular sequence data. They proposed that in cyanobacteria distinctive 16S-23S rRNA ITS secondary structures and genetic distances in 16S rRNA gene sequences were molecular evidences for genetic separation of species. We propose that the psbA/rbcL spacer, in combination with the rbcL gene analysed with maximum likelihood, represent autoapomorphic criteria for distinguishing taxa within the Tribonemataceae. Within one of our species, the psbA/rbcL spacer sequences are readily aligned over their entire lengths, and differences are limited to single base pairs and short indels. For each species, the rbcL sequences have few (< 10 bp) or no differences, and the strains of each species form a monophyletic clade (Fig. 1). Between two tribonematacean species, the entire psbA/rbcL spacer sequences cannot be aligned (Fig. 2). We also note that for species within the same genus, the spacer sequences can be aligned further downstream at the 5′-end than is possible between genera. We recognize genera when the species form a deeply diverging clade in the rbcL phylogeny. For example, groups A–E form the Xanthonema clade 1, which corresponds to the genus Xanthonema sensu strictu (see below).

Xanthonema strains from Antarctica, which were originally identified based upon morphological characters, were distributed in three deeply diverging clades of the rbcL phylogeny (Xanthonema clades 1 and 2, Tribonema 1; Fig. 1). The same patterns were found using direct comparisons of the psbA/rbcL spacer sequences, i.e. the rbcL and spacer molecular data sets were congruent. Using the molecular definition for species and genera, the Antarctic Tribonemataceae comprises six species in three genera (Xanthonema, Tribonema and one genus listed here simply as Xanthonema 2). This contrasts with previous morphological studies where the same taxa were placed in approximately 10 species and three genera, Bumilleria, Tribonema and Xanthonema. As shown in the rbcL phylogeny (Fig. 1), strains representing Bumilleria are distinct from the Xanthonema clade 2, providing molecular evidence that clade 2 represents an undescribed new genus. Therefore, we conclude that Xanthonema, as defined by only morphological characters, is paraphyletic within the Tribonemataceae. Maistro and colleagues (2007) obtained similar results in a study using the rbcL and psaA genes (but no psbA/rbcL spacer region). Thus, Xanthonema clade 1 represents the genus Xanthonema because it includes the type species, X. exile (Fig. 1). Xanthonema clade 2 (i.e. Xanthonema mucicola and X. sessile, see below) and clade 3 represent new genera. Preliminary data showed that the clade 2 may even be identified using new morphological characters. That is, in clade 2, zoospores develop into germlings with a holdfast (attachment disk) and stalk (Broady et al., 1997; N. Rybalka, unpublished), whereas clade 1 and clade 3 develop elongated symmetric germlings from zoospores.

Taxonomic conclusions

We have used the molecular data to establish unambiguous identifications for the study organisms from Antarctica, and consequently we establish epitypes for four species names to better anchor the species concept (see below). The type material for Tribonemataceae species is generally an iconotype (ink drawing, light micrograph), and it is impossible to obtain DNA from iconotypes (Hoef-Emden et al., 2007; Williamson et al., 2007). An authentic strain, i.e. the culture strain used during the description of the species, is a valuable resource, but it lacks acceptability by the International Code of Botanical Nomenclature if it is not cryopreserved (Art. 8.4, McNeill et al., 2006). For many Xanthophycean species, their taxonomy has undergone a paradigm shift from morphology to gene sequences. Ideally, one would examine type material for DNA so that molecular data can be directly tied to the type specimen upon which the name is anchored. When type material is ambiguous, the International Code of Botanical Nomenclature allows that epitypes may be established as a means for clarifying and anchoring names (Art. 9.7, McNeil et al., 2006). Therefore, we will attempt to stabilize some Tribonemataceae names by formally establishing epitypes for four species that occur in Antarctica, i.e. X. exile, X. hormidioides, X. sessile and X. mucicola as well as two other species that are closely related to the former, X. bristolianum, and X. debile, in Table 2.

Table 2.  List of cryopreserved Xanthonema culture strains formally proposed as epitype material to stabilize Tribonemataceae names and their holotypes (iconotypes).
EpitypeHolotype (Iconotype)
  • a.

    Permanently preserved in a metabolically inactive state (cells stored in liquid nitrogen vapours at c.−165°C) (Sammlung von Algenkulturen der Universität Göttingen: Albrecht-von-Haller-Institut, Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany).

  • b.

    Authentic strain.

  • c.

    Type species for the genus.

X. bristolianum (Pascher) Silva, strain SAG 2285a (= CCALA 516)Rabenhorst's Krypt.-Fl. Deutschl., 2. Aufl., 11: 924, fig. 778 (1939)
X. debile (Vischer) Silva strain SAG 2289a,b (= UTEX 155)Ber. Schweiz. Bot. Ges. 45:379, figs 2 and 3 (1936)
X. exile (Klebs) Silvac, strain SAG 2286a (= CCALA 517)Beding. Fortpflanz. Alg. u. Pilz. p. 389, pl. II, figs. 15–20 (1896)
X. hormidioides (Vischer) Silva, strain SAG 2288a,b (= CCAP 836/2)Ergeb. Wiss. Unters. Schweiz. Nationalparkes, N.F.1: 499, plate 1, figs. 6 and 14 (1945)
X. sessile (Vinatzer) Ettl et Gärtner, strain SAG 2193a (= Broady 759)Plant Syst. Evol. 123:214, fig. 1 (1975)

Finally, we summarize some additional taxomic and culturing information. Xanthonema hormidioides was described by Vischer (1945) (as Heterothrix hormidiodes) and duplicate authentic strains CCAP 836/2 and UTEX 353 were derived from a culture established by Vischer when he described the species. Strain SAG 836-1 shared an identical psbA/rbcL spacer sequence with the duplicate strains, UTEX 353 and CCAP 836/2. However, SAG 836-1 was previously named X. debile, and apparently this strain/name was mixed with another strain/name in the past. Subsequently, a second strain was identified as X. hormidioides (Turner 907), but in our analyses, it is distinctly different and belongs to a separate species. Xanthonema sessile was described by Vinatzer (1975) (as Heterothrix sessilis) and it was transferred to Xanthonema by Ettl and Gärtner (1995). An authentic strain (ASIB V98; Vinatzer, 1975) was established, and that strain was previously used in DNA sequence analyses (Maistro et al., 2007). The strain is now extinct (G. Gärtner, pers. comm.) and it was unavailable for this study. However, strain Broady 759 has an identical rbcL sequence and we conclude that both strains represent the same species (Table 2).

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Field sites, isolation of strains and microscopy

Five new strains were isolated from soil material collected by M.O. during the Antarctic summer (20 February 2002) (Table 1). Soils were not covered by snow, and came from two transects along the seashore, Admiralty Bay region, King George Island, maritime Antarctica (A), and near Ecology Glacier (B). The transects were established for botanical study (Olech, 2002). Transect A was 10–20 m inland from the seashore, whereas transect B was located about 370 m farther inland. The sampling sites for the three strains were along transect A, separated by approximately 20–30 m, i.e. strains A16-5 (62°09.993′S, 58°27.582′W; from sandr), A19 (62°10.138′S, 58°27.816′W; at plain near a lake), and A21 (62°10.175′S, 58°27.893 W; close to a lagoon). Strain A21 was collected closest to the glacier (30 m) and A16-5 was farthest from the glacier. Transect B sampling sites were about 20 m apart and yielded strain B4-1 (62°09.942′S, 58°28.073′W; at a small valley between two moraines and farthest from the glacier) and strain B8-5 [62°10.024′S, 58°28.064′W; at bottom of a moraine and closest (200 m) from the glacier]. The sea level soil surface temperatures can be considerably higher than the overlying air temperatures when the soils are not covered by snow. The soils may not even experience freeze–thaw cycles during several months, and air temperatures may reach 15°C during Antarctic summer. Therefore, the newly isolated strains may be represented by mesophilic rather than strictly psychrophilic organisms. Further details about the study area, climate, soil properties and transects are given in Olech (2002) and Tscherko and colleagues (2003). After collection, the soil material was kept frozen in plastic bags at −20°C to avoid possible contamination during transportation and storage prior to the isolation of organisms into culture. Strain A21 was isolated soon after collection by I.K., the other strains were isolated in 2006 by N.R. (A16-5, B4-1 and B8-5) and A.M. (A19). For isolating the algae, a small amount of soil was thawed at room temperature and then sprinkled onto a Petri dish with 1.5% agar enriched with modified Bold's Basal Medium containing triple nitrate (Bischoff and Bold, 1963). Petri dishes were maintained at 18°C and illuminated with white fluorescent bulbs under a light/dark regime of 14 h:10 h and a photon fluence rate of about 25 μmol photons m−2 s−1. Colonies were further isolated and purified using standard microbiological techniques (Guillard, 2005) and the unialgal culture strains were then maintained on agar slants under the same conditions as used for isolation. The five new strains are publicly available from the SAG culture collection under the strain numbers listed in Table 1. Microscopic observations were accomplished using an Olympus BX60 microscope with Nomarski DIC optics and cultures 3–4 weeks old. Micrographs were taken with a ColorView III camera (Soft Imaging System GmbH, Münster, Germany) and processed with the Cell^D image program (Soft Imaging System GmbH, Münster, Germany).

Other Antarctic and reference strains for molecular analyses

Cultures of additional available Antarctic strains were kindly provided by Drs C. Andreoli and P.A. Broady and these strains were accessioned by the SAG culture collection (Table 1). Tribonema strain SAG 2165 was kindly provided by Dr. A. Lukešova. For comparison with the Antarctic strains, closely related reference strains, isolated elsewhere than in Antarctica, were selected from a large rbcL gene sequence phylogeny (N. Rybalka, unpublished). The reference strains were from temperate regions of Europe (Germany, Switzerland, Ukraine) with three exceptions: strain Broady 735 was isolated from soil adherent to plant material that was imported into Antarctica from New Zealand (Broady et al., 1997), strain CCAP 808/3 was isolated from a snow field in Alaska (USA) and CCALA 516 from snow detritus in the Tatra mountains (Slovakia) (Table 1).

DNA extraction, PCR amplification and sequencing

DNA was extracted from fresh cultures after breaking the cells with glass beads in a Minibeadbeater cell homogenizer (Biospec, Bartlesvilles, OK, USA) and then using an Invisorb Spin Plant Mini DNA extraction kit (Invitek, Berlin, Germany). The spacer region that lies upstream of the rbcL gene, i.e. between the psbA and rbcL genes, and full-length sequences of the rbcL gene (1469 bp) with adjacent rbcL/rbcS spacers were amplified using the PCR approach of Andersen and Bailey (2002), which was modified to amplify the target sequence in one piece. It was noted that the target sequence was easier to amplify with DNA extracted from actively growing cultures than from cultures older than 4 weeks. The 5′ primer (psbA5, Andersen and Bailey, 2002), or Xan1F (5′-CCCATTAGATTTAGCAGCT-3′) or Xan2F (5′-TCCCATTAGATTTAGCAGCTG-3′), was anchored in the psbA gene and the 3′ primer (RS3; Andersen and Bailey, 2002) was placed in rbcS (downstream of rbcL), thereby amplifying the full-length rbcL gene and the spacer region between the psbA and rbcL genes. The PCR was performed with an initial ‘hot start’ for 5 min at 95°C, proceeded by 35 cycles at 94°C for 1 min, 51°C for 1 min and 72°C for 2 min 30 s. Positive PCR products were pooled, purified using Invisorb Spin PCRapid Kit (Invitek, Berlin, Germany) or NucleoSpin Extract II (Macherey-Nagel, Düren, Germany) and sequenced using the two PCR primers and a set of primers internal to the PCR product, i.e. those of Andersen and Bailey (2002) and the following newly determined ones: 4FA, the reverse complement of 4RA (Andersen and Bailey, 2002) close to the 5′-end, Xan3R (5′-TCAGGTAAAAACTACGGTCGT-3′), Xan3F (5′-ACGACCGTAGTTTTTACCTGA-3′), X5FG (5′-ATGCGTTGGAGAG-3′, pos. 1177–1190) and 8R (5′-GACCTTGTAATCGGTTACACAG-3′) at middle positions, and X7Fm (5′-CTTCAATTTGGTGGTGGTACAA-3′), 9Fma (5′-GGTGGTGGTACA/TATTGGT-3′), 9Rma (5′-GGTGGTGGTACAATTGGT-3′) and X7Rm (5′-CTTCAATTTGGTGGTGGTACAA-3′) close to 3′-end of the PCR product. The sequences were assembled using the program SeqAssem (Hepperle, 2004). The sequences were aligned using ClustalW in BioEdit v.6.0.7 (Hall, 1999) and then manually refined by eye.

Phylogenetic analyses

Model selection, number of rate categories, proportion of invariable sites and the gamma distribution parameter were determined with modeltest version 3.7 (Posada and Crandall, 1998). Phylogenetic analyses of the rbcL sequence data set was performed using maximum likelihood, maximum parsimony, minimum evolution distance (Rzhetsky and Nei, 1992) and Bayesian analyses, with the program paup* version 4.0b10 (Swofford, 2001) or MrBayes version 3.1.2 (Ronquist and Huelsenbeck, 2003). For minimum evolution distance and maximum likelihood analyses, the GTR + I + G model (Tavaré, 1986) was used with estimations of nucleotide frequencies (A = 0.3033, C = 0.1746, G = 0.2112, T = 0.3109), a rate matrix with six different substitution types, assuming a heterogeneous rate of substitutions with a gamma distribution of variable sites (number of rate categories = 4, shape parameter α = 0.5425) and a proportion of invariable sites (pinvar) of 0.5190. Bootstrap resamplings (1000 replications) were performed on minimum evolution distance and maximum parsimony trees. For the Bayesian analysis, the GTR + I + G model (rate matrix with six different substitution types, number of rate categories = 4, and with the nucleotide frequencies, shape parameter α and pinvar estimated from the data) was used; four Markov chains and 2 000 000 generations sampling every 100 generations were used with the first 25% of the sampled trees discarded, leaving 15 000 trees. Posterior probabilities were then calculated from two independent runs using the 50% majority rule consensus of the kept trees.

Nucleotide sequence accession numbers

The sequence alignment is available from EMBL-Align (http://www.ebi.ac.uk), Accession Number ALIGN_001299. The newly determined sequences have been submitted to the DDBJ/EMBL/GenBank databases under following accession numbers: EF455930, EF455931, EF426794, EF426796, EF426797 (new isolates); EF455932, EF455933, EF455935, EF455936, EF455947, EF455951-EF455953 (Antarctic reference strains); EF455922, EF455925, EF455934, EF455937, EF455938, EF455939, EF455948, EF431851, EF455955-EF455957, EF455962, EF455964, EF455966, EF455970, EF455972, EF455975, EF455977, EF455982 (other reference strains); EF455962, EF455970 and EF455972 (outgroup taxa).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

This study was supported by a research scholarship of the DAAD extended to N.R. We appreciate the support extended to N.R. by Dr. G. Berthold, which was made possible through his generous donation to the SAG Culture Collection at the University of Göttingen. Parts of this work were financed by the German Federal Ministry of Education and Research, BMBF (AlgaTerra Project, Grant 01 LC 0026) within the BIOLOG programme. I.K. and N.R. are thankful to National Antarctic Scientific Center of Ukraine for provision of equipment for isolation of Antarctic strains. R.A.A. acknowledges support from USA NSF Grant 0629564. N.R. and T.F. are thankful to Opayi Mudimu, Elke Zufall-Roth, Jessica Schäckermann, Ilse Kunkel and Marlis Heinemann for provision of assistance in molecular methodologies, purification and maintenance of culture strains. We thank Paul Silva for his advice on taxonomic questions.

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  6. Experimental procedures
  7. Acknowledgements
  8. References
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