The freshwater alga Mucidosphaerium sphagnale (Chlorellaceae: Chlorophyta) in New Zealand: the first record outside Europe

ABSTRACT

Mucidosphaerium sphagnale, a new record for New Zealand, is reported from Awarua Wetland. The genus Mucidosphaerium has recently been separated from the well-known Dictyosphaerium, a colonial green alga with cells connected by thin, branching stalks, to encompass those forms with near-spherical adult cells. The previous name for this taxon, Dictyosphaerium sphagnale, is not recorded from New Zealand; however, Mucidosphaerium pulchellum (syn. Dictyosphaerium pulchellum), which has been reported here many times and is very difficult to distinguish without DNA sequences, may represent previously misidentified instances of M. sphagnale. The M. sphagnale is reported here from a Sphagnum bog, as is typical of its European range, whereas M. pulchellum in New Zealand appears to have been reported exclusively from lakes and rivers. The organism reported here is therefore quite likely a genuine new occurrence. Co-introduction to New Zealand with foreign Sphagnum is a possibility.

KEYWORDS:

• Algae
• Dictyosphaerium
• Mucidosphaerium sphagnale
• New Zealand
• Sphagnum
• wetland

Introduction

The genus Dictyosphaerium Nӓgeli comprises colonial green algae in which the cells are united by thin, branching stalks originating at the centre of an approximately spherical, mucilaginous colony (Komárek & Perman 1978). Dictyosphaerium has been widely reported in New Zealand, mostly from lakes. Two species are relatively common, according to Cassie (1984): Dictyosphaerium ehrenbergianum Nӓgeli (12 sites) and Dictyosphaerium pulchellum H.C.Wood (20 sites); other species reported from New Zealand are Dictyosphaerium planktonicum Tiffany & Ahlstrom (one site) and Dictyosphaerium primarium Skuja (two sites). The most recent checklist by Broady et al. (2012) included no additional records but noted the transfer of D. planktonicum to Lobocystis planctonica (Tiffany & Ahlstrom) Fott, and D. primarium to Dictyosphaerium subsolitarium Van Goor.

Dictyosphaerium has undergone a prolonged series of taxonomic revisions. Those incorporating DNA sequence data (which are perhaps more reliable) have led to the establishment of the genus Hindakia C.Bock, Prӧschold & Krienitz for forms belonging to the Chlorella clade (Bock et al. 2010; Krienitz et al. 2010), and more recently the splitting of the remaining forms in the Parachlorella clade into those with ellipsoidal mature cells (Dictyosphaerium) from those with near-spherical mature cells: the genus Mucidosphaerium C. Bock, Prӧschold & Krienitz (Bock et al. 2011). One of the species previously reported from New Zealand, D. pulchellum, has been transferred to this genus.

Here the occurrence of a further species, Mucidosphaerium sphagnale (Hindák) C. Bock, Prӧschold & Krienitz (syn. Dictyosphaerium sphagnale Hindák), previously known only from Europe, is reported from New Zealand for the first time.

Materials and methods

Taxon sampling, culturing and observation

Water was squeezed from Sphagnum collected from a pool in the Awarua Wetland complex, 46.553364°S, 168.637025°E, into a sterile container on 7 November 2011. The sample was kept on ice until transferred to the laboratory after 3 days in the field, when it was used to inoculate sterile MLA medium (Bolch & Blackburn 1996) containing 0.5% agarose. Cells were grown at room temperature under natural light and repeatedly subcultured using standard aseptic techniques to obtain a unialgal strain. Specimens were examined using a Leica DMLB compound microscope with Nomarksi differential interference contrast and a Canon DS126271 digital camera.

Electron microscopy

Cells were prepared for the transmission electron microscope by fixing in 3% glutaraldehyde, 0.1 m cacodylate buffer, for 3 h. After washing for 1 h in three changes of 0.1 m cacodylate buffer, specimens were post-fixed in 1% osmium tetroxide, 0.1 m cacodylate buffer, for 2 h. The specimens were dehydrated in ethanol and embedded in Spurr’s resin. Sections were cut, stained in uranyl acetate and lead citrate, and examined with a JEOL 1200 EX microscope.

DNA extraction, PCR amplification and sequencing

A Petri plate containing an approximately 3-week-old culture of strain LCR-Awa6/2 was verified microscopically to be unialgal. Algae were scraped from the plate into an Eppendorf tube, manually disrupted using a sterile plastic pestle in lysis buffer from a Maxwell extraction cartridge, and the resulting mixture was transferred back to the cartridge and subjected to extraction in an automated Maxwell^®16 instrument (Promega Corporation). The resulting samples were processed using Zymo Clean and Concentrate columns, according to the manufacturer’s instructions (Zymo Research). The resulting genomic DNA was used as a template in polymerase chain reactions (PCR) incorporating the primers and conditions of Mikhailyuk et al. (2008) for the nuclear ITS1-5.8S-ITS2 region. Sequencing of these products was carried out by Landcare Research New Zealand Ltd. Electropherograms were checked using Sequencher 4.8 (Gene Codes Corporation). The sequence of strain LCR-Awa6/2 is available at https://www.ncbi.nlm.nih.gov/, accession number KX812540.

Phylogenetic analysis

The sequence was aligned to the internal transcribed spacer (ITS) alignment of Bock et al. (2011; submission 10727 at https://treebase.org) using Profile Alignment Mode in ClustalX 1.8 (Thompson et al. 1997) and checked by eye. The resulting data set contained 843 base positions, 371 of which were variable, including 295 parsimony-informative sites.

The aligned data set was used in two phylogenetic analyses. 1. Bayesian inference in MrBayes v3.2 (Ronquist & Huelsenbeck 2003), using the GTR + G substitution model selected using MEGA6 (Tamura et al. 2013): two parallel runs were completed, each of 2.5 million cycles, with default burn in, and parameter estimates were compared with their associated variances to assess the effectiveness of modelling. 2. Maximum Parsimony heuristic bootstrap analysis in PAUP*4.0b10 (Swofford 2002) was carried out using the following settings: branches collapsed if maximum length = 0, DELTRAN character state optimisation, Maxtrees set at 10,000, tree bisection and reconnection branch-swapping algorithm, 10 random sequence additions, and assignment of character states not observed in terminal taxa allowed at internal nodes. Non-parametric bootstrap values for nodes were calculated on the basis of 1000 replicates.

Results

Illustrative material is provided in Figure 1, and the results of molecular analyses are shown in Figure 2.

Figure 1. Mucidosphaerium sphagnale strain LCR-Awa6/2, viewed by light and electron microscopy. A–G, Light microscopy. A, Mature colony with large, near-spherical cells. B, Colony with a single autosporangium (arrow), showing the elongated shape of autospores. C, Colony enlargement: several of the mature cells have formed autosporangia; on release the autospores remain attached. D–G, Formation of a new colony: on initial release, the four autospores remain attached at their narrower ends; lengthening of the stalks follows, and usually rotation of the cell such that the pyrenoid (arrows) moves from a lateral to a basal position. H, Electron microscopy of the pyrenoid, which is surrounded by a bilenticular starch sheath and bisected by a single thylakoid (arrow), as observed in other members of the Parachlorella clade. Scale in G = 10 µm. Scale in H = 0.5 µm. Use scale in G for A–G.

Figure 2. Bayesian phylogeny of the ITS1-5.8S-ITS2 region based on the secondary structure alignment of Bock et al. (2011) with Mucidosphaerium sphagnale LCR-Awa6/2 added (shown in bold). Strain numbers follow the names, with GenBank Accession numbers in brackets. The species of Mucidosphaerium established by Bock et al. are clearly resolved. Support values above branches are Bayesian Posterior Probabilities / Maximum Parsimony bootstrap percentages. Scale bar represents 0.1 substitutions per site.

Mucidosphaerium sphagnale (Hindák) C. Bock, Pröschold & Krienitz

(Figure 1)

Reference: Bock et al. (2011), p. 642, fig. 6G.

Basionym: Dictyosphaerium sphagnale Hindák

Specimens examined

Cultured strain LCR-Awa6/2, isolated from material collected at Awarua Wetland, 46.553364°S, 168.637025°E, pond water containing Sphagnum sp., 7 November 2011.

Colonies approximately spherical, 7–30 µm diameter, containing cells united by thin dichotomously or tetrachotomously branching stalks originating from the colony centre; unicells occasionally observed. Cells ellipsoidal to pyriform, fusiform or semilunate when young, broadly ovoid to spherical when mature, 2.1–4.9 µm wide, 3.6–5.5 µm long, length : width approximately 1.7 at autospore release, reducing to approximately 1.1 at maturity. Chloroplast parietal, cup-shaped, with a single pyrenoid surrounded by two hemispherical starch grains. Pyrenoid matrix bisected by a single thylakoid. Pyrenoid variably positioned (although often lateral) in young cells, which are joined at their narrow ends by very short mucilaginous stalks that lengthen with age, at which time the pyrenoid is usually located at the side of the cell to which the stalk is connected. Colonies 4–16 celled, up to approximately 30 µm in diameter.

DNA data

The ITS sequence of this strain was closest to two strains of M. sphagnale (CB 2008/19 and CB 2008/44, p-distance = 0.005 in each case; p-distance to CB 2008/15 = 0.008). Both methods of analysis placed the strain in a clade comprising M. sphagnale with high support (Figure 2). The phylogeny was largely congruent with that of Bock et al. (2011), despite the use of shorter sequences (ITS region only).

Distribution

A new record for New Zealand. This appears to be the first record of the species outside Europe.

Discussion

The recently established genus Mucidosphaerium is readily distinguished from Dictyosphaerium morphotypes by its near-spherical mature cells (Bock et al. 2011). Spherical-celled species in New Zealand have previously been identified as D. pulchellum H.C.Wood (now Mucidosphaerium pulchellum (H.C.Wood) C. Bock, Pröschold & Krienitz) and D. primarium Skuja (since subsumed into D. subsolitarium Van Goor; Cassie Cooper 2001). Cells of the latter are spherical (suggesting that this species will prove to belong in Mucidosphaerium should molecular data become available), but considerably smaller at maturity than those found in the present material; Lineham (1983) noted a maximum diameter of 3 µm in specimens from Te Waihora/Lake Ellesmere. New Zealand records of D. primarium/subsolitarium can be excluded by size as possible previous observations of M. sphagnale.

Records of M. pulchellum are more difficult to distinguish and could represent misidentified records of M. sphagnale. According to the original description of Wood (1873), the cell diameter of M. pulchellum is 0.00025″, or approximately 6 µm. This is outside the size range of the specimens reported here; however, later authors state size ranges such as 5–8 µm (Komárek & Perman 1978), 6–8 µm (Bock et al. 2011), or 5 µm for cells in colonies (Lineham 1983) for this species; the latter measurement is the upper size limit observed in the Awarua strain. As far as can be determined, only two records of M. pulchellum from New Zealand contain descriptions and/or illustrations. Cells in the image by Kloos (1976) correspond to approximately 5–9 µm at the stated scale, and those of Lineham (1983) are 5–7 µm. There seems to be only slight overlap between these ranges and those of the Awarua material, which is in fair agreement with the description of Bock et al. (2011) for M. sphagnale (adult cells 4–6(–7.5) µm in diameter; confirmed with online images of strain CCAP 222/13 at www.ccap.ac.uk). It seems safest to conclude that morphology does not reliably discriminate these two species, a view shared by Bock et al. (2011), who stated that the size overlap between all species of Mucidosphaerium means that DNA sequence data are necessary to separate them; DNA data unequivocally place the New Zealand strain in M. sphagnale (Figure 2). The use of morphology at the level of light microscopy does not, therefore, rule out previous records of M. pulchellum being misidentified collections of M. sphagnale.

Pyrenoid ultrastructure can sometimes be used to distinguish close relatives (e.g. Friedl 1989), but ultrastructural features of these species do not yet appear to have been widely studied. However, pyrenoid ultrastructure of strain LCR-Awa6/2, in which a single bisection of the pyrenoid by thylakoids occurs, appears the same as that observed in other members of the Parachlorella clade, such as Parachlorella kessleri (Fott & Nováková) Krienitz, EH Hegewald, Hepperle, V Huss, T Rohr & M Wolf (Juárez et al. 2011) and Planktochlorella nurekis Škaloud & Nĕmcová (Škaloud et al. 2014). This character state therefore seems likely to be present throughout the group, and may offer little further taxonomic information.

However, these species may be distinguished by their ecology. Mucidosphaerium pulchellum (as D. pulchellum) has been recorded from at least 19 sites in New Zealand (Cassie 1984). All of these are lakes, apart from records from the Waikato River (Hill 1970; Lam 1977; Waikato Valley Authority 1979). In contrast, M. sphagnale (as D. sphagnale) has historically been recorded from bog habitats (Komárek & Fott 1983; Bock et al. 2011). The record of M. sphagnale reported here, from a wetland, is therefore consistent for a first observation of this species in New Zealand. The species does not appear to be common: in a wide-ranging survey of algal diversity in 21 North Island and 17 South Island wetland habitats, D. ehrenbergianum was the only species of Dictyosphaeriaceae recorded, and that only once in the North Island and four times in the South Island (Kilroy & Sorrel 2013). A re-examination of images collected during the survey of that material (C. Kilroy, NIWA, pers. comm. 2016) indicates that the cells were near-spherical rather than ellipsoidal, and not connected at short axes. However, identifications were undertaken on frozen samples, and the effect of freezing on the ellipsoidal cell morphology of D. ehrenbergianum is presently unknown. Consequently, the identity of the Dictyosphaeriaceae in the survey by Kilroy & Sorrel (2013) would require further investigation to resolve.

The species Mucidosphaerium planctonicum C. Bock, Pröschold & Krienitz (Figure 2) has not been found in New Zealand to date. This name should not be confused with Dictyosphaerium planktonicum, a species that has been recorded in New Zealand from Albany (Chapman et al. 1957) but has since been transferred to the genus Lobocystis (Fott 1975). Examination of the original illustration of Tiffany & Ahlstrohm (1931) shows strongly elongate cells, quite different from M. sphagnale.

The new record of M. sphagnale raises the possibility of a recent introduction of this species to New Zealand. If that is the case, how might it have arrived? The presence of introduced Sphagnum in New Zealand, such as Sphagnum subnitens Russ & Warnst., offers a possible route through co-invasion. Although many microbes are associated with Sphagnum (Hingley 1993; Kreutz & Foissner 2006), associations with particular species of the moss appear to be little studied (Kostka et al. 2016). Images of Sphagnum taken during collection of M. sphagnale from Awarua Wetland represent the widespread indigenous species Sphagnum falcatulum Besch. (A. Fife and D. Glenny, Landcare Research, pers. comm.). Sphagnum subnitens is restricted to Westland (Fife 1996), and the distribution of M. sphagnale in New Zealand beyond Awarua Wetland is unknown, so there is currently no evidence for an association. However, it is certainly possible for algae, such as Didymosphenia geminata (Lyngbye) Schmidt, which is sensitive to salt, drying, freezing and the low pH characteristic of a bird gut (Kilroy et al. 2007), to disperse widely following successful introduction (Kilroy & Unwin 2011). Therefore, lack of demonstrated overlap in distributions between introduced Sphagnum and introduced algae does not necessarily negate the former as an invasion vector for the latter.

Acknowledgements

The author thanks M. Schallenberg (University of Otago) for field assistance, R Cole (Department of Conservation) for helpful advice regarding collecting sites, and R Smissen, D Glenny, A Fife, I Breitwieser, R Prebble and two anonymous reviewers for helpful comments on the manuscript. Associate editor: Dr Roberta D'Archino.

Disclosure statement

No potential conflict of interest was reported by the author.

Additional information

Funding

This research was supported by Core funding for Crown Research Institutes from the Ministry of Business, Innovation and Employment’s Science and Innovation Group.