Ronald W. Hoham

EVIDENCE A PHOTOPROTECTIVE FOR SECONDARY CAROTENOIDS OF SNOW ALGAE1

Snow algae occupy a unique habitat in high altitude and polar environments. These algae are often subject to extremes in nutrient availability, acidity, solar irradiance, desiccation, and ambient temperature. This report documents the accumulation of secondary carotenoids by snow algae in response to the availability of nitrogenous nutrients. Unusually large accumulations of astaxanthin esters in extra‐chloroplastic lipid globules produce the characteristics red pigmentation typical of some snow algae (e.g. Chlamydomonas nivalis (Bauer) Wille). Consequently these compounds greatly reduce the amount of light available for absorption by the light‐harvesting pigment‐protein complexes, thus potentially limiting photoinhibition and photodamage caused by intense solar radiation. The esterification of astaxnthin with fatty acids represents a possible mechanism by which this chromophore can be concentrated within cytoplasmic globules to maximize its photoprotective efficiency.

A COMBINED 18S rDNA AND rbcL PHYLOGENETIC ANALYSIS OF CHLOROMONAS AND CHLAMYDOMONAS (CHLOROPHYCEAE, VOLVOCALES) EMPHASIZING SNOW AND OTHER COLD‐TEMPERATURE HABITATS1

An extensive phylogenetic analysis of the biflagellate genera, Chlamydomonas Ehrenberg and Chloromonas Gobi emend. Wille, was undertaken using 18S rDNA and rbcL gene sequence analysis. Emphasis was placed on 21 cold‐tolerant taxa of which 10 are from snow. These taxa occurred in four distinct clades each in the 18S rDNA and rbcL phylogenies, and when taken together suggest at least five distinct origins in cold habitats. Most of these taxa occur in a single clade (A), and all snow species occurred in this clade. In the rbcL and combined rbcL–18S rDNA analyses, the snow taxa fell into three groups. Two groups occurred in subclade 1: Chlamydomonas augustae Skuja CU, Chlamydomonas augustae UTEX, and Chlamydomonas sp.‐A and Chloromonas clathrata Korshikov, Chloromonas rosae Ettl CU, and Chloromonas rosae v. psychrophila var. nov. The third snow group, subclade 2, included three species with unique cell divisions, Chloromonas brevispina (Fritsch) Hoham, Roemer et Mullet, Chloromonas pichinchae (Lagerheim) Wille, and Chloromonas sp.‐D, and the basal Chloromonas nivalis (Chodat) Hoham et Mullet with normal cell divisions. This suggests that the snow habitat has been colonized at least twice and possibly three times in the history of these biflagellates. In the 18S rDNA tree, one cold‐tolerant Chloromonas species fell outside clade A: Chloromonas subdivisa (Pascher et Jahoda) Gerloff et Ettl. In the rbcL tree, three cold‐tolerant Chloromonas species fell outside clade A: Chloromonas subdivisa, Chloromonas sp.‐ANT1, and Chloromonas sp.‐ANT3. These results support previous findings that pyrenoids have been gained and lost several times within this complex.

Two new species of green snow algae from Upstate New York, Chloromonas chenangoensis sp. nov. and Chloromonas tughillensis sp. nov. (Volvocales, Chlorophyceae) and the effects of light on their life cycle development

R.W. Hoham, J.D. Berman, H.S. Rogers, J.H. Felio, J.B. Ryba and P.R. Miller. 2006. Two new species of green snow algae from Upstate New York, Chloromonas chenangoensis sp. nov. and Chloromonas tughillensis sp. nov. (Volvocales, Chlorophyceae) and the effects of light on their life cycle development. Phycologia 45: 319–330. DOI: 10.2216/04-103.1 Two new species of Volvocalean green snow algae are described from Upstate New York, Chloromonas chenangoensis and Chloromonas tughillensis. According to rbcL sequence analysis, these species belong to a subclade of five snow species of Chloromonas that includes Cr. brevispina, Cr. nivalis and Cr. pichinchae. These species have zoosporangia/gametangia (cell packs) that are propelled by flagella. The life cycles of Cr. chenangoensis and Cr. tughillensis include a change in cell shape from oblong to spherical before the loss of the cell wall. Using 8-h laboratory experiments, a peak in the change of cell shape occurred at 4 h for Cr. chenangoensis and between 2 and 4 h for Cr. tughillensis after the onset of light, and this was followed by a decline in change of cell shape for both species. Spherical cells peaked between 6 and 8 h for Cr. chenangoensis and at 8 h for Cr. tughillensis. Maximum total matings occurred at 4 h for Cr. chenangoensis and 6 h for Cr. tughillensis, which was followed by a peak in quadriflagellate zygotes at 8 h for both species. Mature zygotes (zygospores) were observed only in Cr. tughillensis.

THE OPTIMUM PH OF FOUR STRAINS OF ACIDOPHILIC SNOW ALGAE IN THE GENUS CHLOROMONAS (CHLOROPHYTA) AND POSSIBLE EFFECTS OF ACID PRECIPITATION1

Four axenic strains of snow algae were examined for optimum pH under laboratory conditions using M‐1 growth medium. Growth was measured using cell counts, cell measurements and absorbance readings at 440 nm. Strains C204 and C479A of Chloromonas sp. from the Adirondack Mountains, New York, grew optimally at pH 4.0 to 5.0. Strains C381F and C381G, Chloromonas polyptera (Fritsch) Hoh., Mull. & Roem. from the White Mountains, Arizona, grew optimally at pH 4.5 to 5.0. Growth was significantly higher at pH 4.0 in the northeastern species (Chloromonas sp.), but no significant difference was observed in final growth at pH 4.5, 5.0 and 5.5 between species. It is postulated that the more acidic precipitation in the northeastern United States may be selecting for strains of snow algae with greater tolerance to acidity than in strains from the southwestern United States or that the different pH optima reported are simply species differences. New York strain C204 was also grown in heavily buffered AM medium where it had an optimum pH of 5.0, but cells became irregularly shaped and tended to clump at pH 6.0 to 7.0. Growth of C204 in AM medium was significantly lower than in M‐1 medium for snow algae. These findings justify the use of M‐1 medium for this type of experimentation.

The effects of irradiance levels and spectral composition on mating strategies in the snow alga, Chloromonas sp.-D, from the Tughill Plateau, New York State

Studies have related changes in snow albedo to snow crystal structure and to the presence of surface debris (i.e. pine needles), but there has been less attention given to the existence of algae in snow. An increase in the number of snow algae could also decrease albedo and increase snowmelt rates. The primary purpose of this paper is to document how solar irradiance serves to control the developing stages of algae in snow. Snow algae do not appear near the surface until there is meltwater in the snowpack. Low levels of solar irradiance penetrate through the snowpack and germinate snow algal resting stages that lie underneath, and snow algal growth is enhanced by available gases and nutrients. Flagellate cells swim through the snowpack in the meltwater around the snow crystals, and cells are positioned according to irradiance and spectral differences. In this study, Chloromonas sp.-D strains 582C and 582D. isolated from the upper 20 cm of snowpacks in the Tughill Plateau, Whetstone Gulf State Park, NY, were used to investigate mating strategies under different irradiance levels and spectral compositions in the laboratory, and the irradiance levels used in the experiments correlated with those recorded from the upper 20 cm of snow. Using similar irradiance levels, blue light regimes produced more matings than green and red light regimes. There were no trends in mating when comparing green and red light regimes. When red light regimes of higher photon irradiance (85 μmol m -2 s -1 ) were compared with those of blue light regimes of lower irradiance (30 μmol m -2 s -1 ). more mating occurred under red light. A photon irradiance of 95 μmol m -2 s -1 [photosynthetically active radiation (PAR) of 400-700 nm] produced the most mating under both wide-spectrum (WS) and cool-white (CW) regimes, but more mating occurred under CW in all irradiances tested. Mating pairs of three types were observed: oblong-oblong (o-o), oblong-sphere (o-s) and sphere-sphere (s-s). Cell packs that produced mating types and O-O mating pairs diminished through the eight-hour time duration of the experiments. However, o-s and s-s mating pairs peaked at six to seven hours after the experiments began, while quadriflagellate zygotes produced from the mating pairs continued to increase throughout the eight hours.

TWO SNOW SPECIES OF THE QUADRIFLAGELLATE GREEN ALGA CHLAINOMONAS (CHLOROPHYTA, VOLVOCALES): ULTRASTRUCTURE AND PHYLOGENETIC POSITION WITHIN THE CHLOROMONAS CLADE1

The quadriflagellate snow alga Chlainomonas Christen, distributed in New Zealand and North America, has several unusual structural attributes. A process assumed to be cytokinesis involves extrusion of protoplasm from the parent through a narrow canal, C. kolii (J. T. Hardy et Curl) Hoham produces a net‐like outer envelope rather than a cell wall, and the flagellar basal apparatus of Chlainomonas consists of two semi‐independent pairs of basal bodies. Structural connections between basal body pairs appear minimal, but a connecting system different from that observed in other genera exists within each pair. Phylogenetic analysis using rbcL sequences places Chlainomonas in the Chloromonas clade, other known members of which are all biflagellate. Chlainomonas is split into two robust lineages, with New Zealand collections sharing an origin with northern North American collections. Although the quadriflagellate condition is regarded as ancestral in the Chlorophyceae, we speculate—based on ultrastructural and molecular data presented here—that Chlainomonas represents a derived form that has arisen from fusion of two ancestral biflagellate cells. Other explanations (for example, that Chlainomonas represents a diploid form of a biflagellate species) are remotely possible but are presently at odds with extensive observations of field material. Improvements in techniques for experimental manipulation of these sensitive cryophiles will be required to fully characterize their structure and progress our understanding of their biology.

The importance of light and photoperiod in sexual reproduction and geographical distribution in the green snow alga, Chloromonas sp.-D (Chlorophyceae, Volvocales)

The effects of premating light regimes on sexual reproduction and the production of spherical cells in Chloromonas sp.-D, a unicellular green snow alga, were studied using cross-mating strains 582C and 582D isolated from snowpacks associated with mixed hardwood-softwood forests in Whetstone Gulf State Park, Tughill Plateau, NY. Two preacclimation regimes were used, Vita-Lite as controls (530-700 nm peak) and blue light as experimentals (430-460 nm peak) prior to the mating experiments. In blue light, an increase in the number of matings and spherical cells (spheres) produced in the life cycle was observed as the photoperiod increased. This implies that longer photoperiods of blue light are more optimal for sexual reproduction in Chloromonas sp.-D than shorter ones. Under Vita-Lite, there was a significant increase in the number of matings and spheres with the extended 20 : 4 photoperiod compared with the shorter 14 : 10 photoperiod. Under blue light, significantly more matings and spheres occurred than under Vita-Lite using the same irradiance level of 95 μmol photons m -2 s -1 (photosynthetically active radiation [PAR] of 400-700 nm) for the 14: 10 and 20: 4 photoperiods. The results of these experiments suggest that Chloromonas sp.-D. known only from the Tughill Plateau, NY, is not reproducing optimally at this site where it grows and reproduces under an approximate 14: 10 photoperiod in early April. However, in the upper 10 cm of snow in the Tughill Plateau, a blue light irradiance level of 95 μmol photons m 2 s -1 occurs, which is optimal for this species. When these conditions are combined with a 14: 10 photoperiod, the Tughill Plateau appears to be sub-optimal for mating and production of spherical cells. Since Chloromonas sp.-D does not appear to have a dependence on a dark cycle, this would allow it to expand its geographical distribution. It may reproduce more optimally under blue light (95 μmol photons m -2 s -1 ) with an extended photoperiod (>20:4 h, light:dark) in high latitude field sites such as Lake Bienville, Quebec, in eastern North America where other species of Chloromonas are known in snow associated with coniferous forests.

Snow Algae: The Effects of Chemical and Physical Factors on Their Life Cycles and Populations

Algae, fungi and bacteria are microorganisms commonly found in snow and ice habitats (Kol 1968, Horner 1985, Hoham and Duval 1999). These microbes may encounter conditions of extreme temperature, acidity, irradiation levels, minimal nutrients and desiccation when liquid water is no longer available (Hoham 1992). When liquid water is present, it allows for their growth and reproduction (Pollock 1970), and special adaptations and mechanisms for surviving in cold temperatures occur (Bidigare et al. 1993, Hoham and Duval 1999). Microorganisms play a fundamental role in the biogeochemistry of snow and ice (Jones 1991) and are involved in the primary production, respiration, nutrient cycling, decomposition, metal accumulation and food webs associated with these habitats (Fjerdingstad et al. 1978, Hoham 1980, Jones 1991, Hoham et al. 1993, Hoham and Duval 1999).

Optimum Growth Temperatures of Three Species of Green Chloromonas Snow Algae from Upstate New York and the White Mountains, Arizona

ABSTRACT The optimum temperatures of three species of snow algae were studied using four strains of Chloromonas (Cr.) rosae v. psychrophila, six strains of Cr. tughillensis, and one strain of Cr. chenangoensis. These axenic strains were from Upstate New York except for two of Cr. rosae v. psychrophila from the White Mountains, Arizona. Temperatures tested were from 2.5 to 20°C. The high elevation subalpine Cr. rosae v. psychrophila from New York and Arizona grew from 4 to 20°C and had the greatest cell counts at 4 to 15°C. In contrast, the subalpine to temperate low elevation strains of Cr. tughillensis grew from 2.5 to 10°C and optimally at 2.5 or 5°C, and Cr. chenangoensis grew from 2.5 to 7.5°C and optimally at 2.5 and 5°C. Chloromonas tughillensis and Cr. chenangoensis belong to a genetic subclade with low temperature optima, whereas Cr. rosae v. psychrophila belongs to a subclade with broad temperature optima. In acclimation experiments, there were no significant differences in cell counts when acclimating two Adirondack, New York, strains of Cr. rosae v. psychrophila for two weeks prior to experiments vs. using non-acclimated strains that were moved from 4°C directly to 4, 10, 15, or 20°C. For Cr. tughillensis, four of six strains had significantly higher cell counts when grown at 2.5°C after acclimation at 7.5°C for five months. These are the first reports of temperature optima of snow algae from eastern North America.

The Optimum pH of the Green Snow Algae, Chloromonas Tughillensis and Chloromonas Chenangoensis, from Upstate New York

ABSTRACT The optimum pH of two species of snow algae from Upstate New York were assessed by studying three axenic strains of Chloromonas tughillensis in a pH range of 3.0–7.0 and three non-axenic strains of Cr. chenangoensis in a pH range of 3.0–8.0. Growth was examined at 0.5 pH intervals. Cell counts at the termination of the experiments differed among strains and among pH intervals in individual strains for both species, and strains of Cr. tughillensis responded differently to changes in pH (p < 0.001) while strains of Cr. chenangoensis did not (p  =  0.193). Cell counts and absorbance data for Cr. tughillensis indicated an optimum pH of 4.9–6.1 using regression analysis. Strains of Cr. chenangoensis exhibited higher but insignificantly different counts between pH 7.0 and 8.0 with maxima at pH 7.5, but pH optima were not determined. When the range was expanded to include pH 8.5–9.0, an optimal pH of 7.0–8.0 was determined for strain CU 722B, and this is the first snow alga reported to have an optimum alkaline pH. The highest absorbance values, however, occurred between pH 3.0–4.5 and pH 7.0–8.0. The pH values recorded in the field were 5.0–5.3 for Cr. tughillensis and 6.7–7.6 for Cr. chenangoensis.