William J. Henley

Phylogenetic analysis of the ‘Nannochloris-like’ algae and diagnoses of Picochlorum oklahomensis gen. et sp. nov. (Trebouxiophyceae, Chlorophyta)

Abstract A broadly halotolerant new isolate of a small asexual coccoid chlorophyte and six new, related freshwater isolates provided the impetus for a phylogenetic analysis of the so-called ‘Nannochloris-like’ algae within the Trebouxiophyceae. Previous taxonomic disagreements concerning this group had not been rigorously tested with molecular phylogenetic analyses. We show with 1 8S ribosomal DNA (rDNA) sequence phylogeny that 19 of 22 isolates previously assigned to either Nannochloris or Nanochlorum fall within a diverse sister clade to a clade including the four ‘true’ Chlorella species sensu lato. In addition, Marvania geminata, Gloeotila contorta, Chlorella sp. Yanaqocha RA1, Koliella spiculiformis, ‘Chlorella minutissima’ C-1. 1.9, and new Koliella, Gloeotila and Marvania isolates were included in the Nannochloris-like clade. Distinct freshwater and marine or saline lineages comprise at least three major subclades, generally corresponding to cell division pattern. Seven of 14 marine or saline isolates are known (and the others presumed) to divide by autosporulation. Eight freshwater isolates divide by binary fission, including two Koliella, two Gloeotila, N. bacillaris, Chlorella sp. Yanaqocha RA1, and two new unassigned isolates. Four freshwater isolates divide by budding or autosporulation (three Marvania, including CCAP 251/1b, previously assigned to N. coccoides). The autosporic taxa N. eucaryotum UTEX 2502 (marine) and C. minutissima C-1.1.9 (freshwater), which have nearly identical 18S rDNA sequences, are deeper-branching than the freshwater and marine or saline lineages. We propose including the 13 marine or saline, autosporic taxa (excluding N. eucaryotum UTEX 2502) in the new genus Picochlorum until distinctive morphological or biochemical characters are identified that would indicate multiple genera corresponding to subclades. Such characters exist in the freshwater lineages, supporting retention of Koliella, Gloeotila, Marvania and Nannochloris as distinct genera, although each is currently represented by few isolates. Nannochloris at this time may be restricted to N. bacillaris and Chlorella sp. Yanaqocha RA1. We also describe halotolerant P. oklahomensis Hironaka sp. nov. Based on 18S rDNA sequence and lack of chlorophyll b, Nannochloris sp. UTEX 2379 should be reassigned to the Eustigmatophyceae.

RESPONSE TO SALINITY AND HEAT STRESS IN TWO HALOTOLERANT CHLOROPHYTE ALGAE1

Two new isolates of halotolerant chlorophyte algae from the Salt Plains National Wildlife Refuge in Oklahoma, USA, tentatively identified as Dunaliella sp. Teodoresco and Nannochloris sp. Naumann, were characterized with respect to interaction between growth salinity and short‐term heat tolerance. Cells were cultured at 23–25° C over a wide range of salinity. In both species, salinity alone had little effect on maximum photochemical yield (measured by pulse modulated fluorescence) and integrity of the light harvesting system (77 K fluorescence emission spectra). In contrast, Nannochloris exhibited decreasing growth rate (μ), light‐saturated photosynthetic capacity (Pcellmax), respiration (Rd), light‐harvesting efficiency (αcell), and chl content with increasing salinity. Cultures were heated for 2 h near their upper temperature limits (41.5° C for Dunaliella and 45° C for Nannochloris grown at 50 psu). Dunaliella was progressively more heat‐tolerant with increasing salinity. Photochemical yield of cells at 100 and 50 psu was inhibited by about 15% and 40%, respectively, and largely recovered within 30 min after return to 23° C. Thermal inhibition of photochemical yield in Nannochloris was about 45% at both 50 and 100 psu, but recovery was slower at 100 psu. At 20 psu, both species were almost 90% inhibited by high temperature and required more than a day to recover. In both species, 2 h of heating increased the PSI:PSII fluorescence emission ratio (714:690 nm) at all salinities. This ratio largely recovered within 24 h in Dunaliella at 50 and 100 psu and partially recovered in Nannochloris at 100 psu, but cells of both species heated at 20 psu were chlorotic the next day.

Growth and photosynthesis of marine Synechococcus (Cyanophyceae) under iron stress

Prokaryotic picoplankton such as Synechococcus are relatively abundant in putatively Fe‐limited high‐nutrient, low‐chlorophyll (HNLC) regions of the oceans. The physiology of Synechococcus under Fe stress has been studied less than eukaryotic algae. Recent evidence suggests that although biomass and growth rates of Synechococcus are not typically Fe limited in situ, cells may still exhibit symptoms of Fe stress. We grew Synechococcus A2169 and WH7803 in laboratory batch cultures in the artificial medium Aquil and enriched natural seawater, at a series of Fe concentrations and Fe:macronutrient ratios, and with either nitrate or ammonium as the sole nitrogen source. Cell yields, and in some experiments exponential specific growth rate (μ), were more readily Fe limited in the Atlantic isolate WH7803 than in the equatorial Pacific isolate A2169. In both strains, final cell yields spanned about an order of magnitude and decreased continuously with Fe concentration from 900 to 3.6 nM (150 μM N, 10 μM P), whereas μ decreased much less and only at Fe concentrations below 90 nM. Synechococcus yield was controlled by both absolute Fe concentration and Fe:macronutrient ratio, but μ was determined primarily by absolute Fe concentration. Contrary to theoretical predictions, neither yield nor μ was higher in Fe‐limited cells grown in ammonium compared to nitrate. Under severe Fe stress, cellular chlorophyll (Chl) content and light‐saturated gross photosynthetic capacity (Pcellm) decreased proportionately, and dark respiration (Rcelld) increased, such that net Pcellm was extremely low but gross PChlm was unchanged. This is the first report of an absolute increase in Rcelld under Fe stress in phytoplankton.

ALGAL COMMUNITY DYNAMICS AND HALOTOLERANCE IN A TERRESTRIAL, HYPERSALINE ENVIRONMENT1

We studied the algal community of the Great Salt Plains (GSP), an expansive (65 km2) salt flat situated in north‐central Oklahoma, USA that has been designated as the Salt Plains Microbial Observatory (SPMO) by the National Science Foundation. The GSP offered a unique opportunity to study a terrestrial, hypersaline algal community that experiences wide‐ranging environmental conditions. We were able to show that ammonium‐N, rather than salinity, was the most important predictor of total algal biomass. However, salinity was found to be a significant controlling variable in diatom distribution at the GSP, where diatom abundance was negatively correlated with porewater salinity concentrations. Overall, chlorophytes (likely dominated by Dunaliella spp.) were the most abundant algal group at the consistently hypersaline (>300 ppt) south site. Diatom and cyanobacterial biomass were on average highest at the central site, which experienced greater fluctuations in salinity. While taxonomic diversity was limited to three algal groups (chlorophytes, diatoms, and cyanobacteria), the salinity preferences and halotolerance ranges of isolated strains were quite variable. Although porewater salinities at the GSP are commonly near saturation (>300 ppt), the large majority of isolates had halotolerance ranges below 150 ppt. This suggests that algae at the GSP rarely achieve maximum growth rates, and could only do so when intermittent rain events reduce salinity to optimal levels. Because the vast majority of our strains were isolated from salt‐saturated soil samples, maintaining viability (rather than growth efficiency) appears to be the most successful adaptation to the extreme conditions at the GSP.

HYPERSALINE SOIL SUPPORTS A DIVERSE COMMUNITY OF DUNALIELLA (CHLOROPHYCEAE)1

Numerous isolates of the green halophile Dunaliella were studied as part of a survey of microbial diversity at the Great Salt Plains (GSP) in Oklahoma, USA. The GSP is a large (∼65 km2) salt flat with extreme temporal and spatial fluctuations in salinity and temperature. Although the flagellate halophile Dunaliella is common worldwide, nearly all cultured isolates are from saline habitats that are primarily aquatic rather than primarily terrestrial. The diverse GSP Dunaliella strains exhibit three morphotypes: a predominantly motile form, a motile form with a prominent palmelloid phase (nonmotile, mucilage rich), and a palmelloid form with a weakly motile phase. All had broad salinity optima well below typical in situ salinities at the GSP, and two of the palmelloid isolates grew as well in freshwater as in highly saline media. Molecular phylogenetic and evolutionary analyses revealed that Dunaliella from the GSP (and two similar habitats in the Great Basin, USA) are allied with D. viridis Teodor. but possess phylogenetic diversity in excess of existing global isolates from aquatic habitats. In addition, isolates from primarily terrestrial habitats exhibit statistically higher rates of nucleotide substitution than the phylogenetically homogeneous set of primarily aquatic Dunaliella taxa. We hypothesize that dynamically extreme saline soil habitats may select for different and more diverse Dunaliella lineages than more stable saline aquatic habitats. We also propose Dunaliella as a tractable microbial model for in situ testing of evolutionary and phylogeographic hypotheses.

The effect of ampicillin plus streptomycin on growth and photosynthesis of two halotolerant chlorophyte algae

Streptomycin and ampicillin are antibiotics commonly used to eliminate prokaryotes from the cultures of eukaryotic algae. We studied the effects of 25 mg l−1 streptomycin plus 50 mg l−1 ampicillin on the growth and photosynthesis of two broadly halotolerant algae, Picochlorum oklahomensis and Dunaliella sp. (Chlorophyceae). We measured growth rate, oxygen evolution, chlorophyll fluorescence kinetics, and pigment content in low (150 μmol photons m−2 s−1) and high (600 μmol photons m−2 s−1) light grown batch cultures. Our results show only a minor effect of the antibiotics on P. oklahomensis, and none on Dunaliella sp., so this combination of antibiotics is suitable for maintenance of stock cultures for physiological experiments. We also show that these antibiotics can be used in turbidostat cultures of P. oklahomensis, which otherwise tend to succumb to bacteria.

In situ studies of algal biomass in relation to physicochemical characteristics of the Salt Plains National Wildlife Refuge, Oklahoma, USA

This is the first in a series of experiments designed to characterize the Salt Plains National Wildlife Refuge (SPNWR) ecosystem in northwestern Oklahoma and to catalogue its microbial inhabitants. The SPNWR is the remnant of an ancient ocean, encompassing ~65 km2 of variably hypersaline flat land, fed by tributaries of the Arkansas River. Relative algal biomass (i.e., chlorophyll concentrations attributed to Chlorophyll-a-containing oxygenic phototrophs) and physical and chemical parameters were monitored at three permanent stations for a one-year period (July 2000 to July 2001) using a nested block design. Salient features of the flats include annual air temperatures that ranged from -10 to 40°C, and similar to other arid/semi-arid environments, 15–20-degree daily swings were common. Shade is absent from the flats system; intense irradiance and high temperatures (air and sediment surface) resulted in low water availability across the SPNWR, with levels of only ca. 15 % at the sediment surface. Moreover, moderate daily winds were constant (ca. 8–12 km h-1), sometimes achieving maximum speeds of up to 137 km h-1. Typical of freshwater systems, orthophosphate (PO43-) concentrations were low, ranging from 0.04 to <1 μM; dissolved inorganic nitrogen levels were high, but spatially variable, ranging from ca. 250–600 μM (NO3- + NO2-) and 4–166 μM (NH4+). Phototroph abundance was likely tied to nutrient availability, with high-nutrient sites exhibiting high Chl-a levels (ca. 1.46 mg m-2). Despite these harsh conditions, the phototrophic microbial community was unexpectedly diverse. Preliminary attempts to isolate and identify oxygenic phototrophs from SPNWR water and soil samples yielded 47 species from 20 taxa and 3 divisions. Our data indicate that highly variable, extreme environments might support phototrophic microbial communities characterized by higher species diversity than previously assumed.

Effect of thiobencarb on growth and photosynthesis of the soil alga Protosiphon botryoides (Chlorophyta)

The effects of the herbicide thiobencarb (Saturn) were tested on the growth and physiology of the chlorophyte Protosiphon botryoides isolated from an Egyptian paddy. Assays were conducted using 16-day batch cultures. Chlorophyll and dry weight biomass yields were significantly reduced at 2–3 mg L-1 thiobencarb, and dark respiration increased and protein decreased significantly at 3 mg L-1. Reductions in exponential specific growth rate (μ) were generally small, but in some cases significant. Thiobencarb also slightly, but significantly, reduced the 77 K fluorescence parameter Fv/Fm, an indicator of maximum photosynthetic efficiency. No consistent dose-dependent changes occurred in chlorophyll per unit dry weight, total carbohydrate or gross photosynthetic capacity. Whereas half of the added thiobencarb was recovered from control (uninoculated) medium, it was largely absent from cells and culture medium after sixteen days, indicating biodegradation by the alga or associated bacteria. P. botryoides recovered fully within sixteen days following subculture in thiobencarb-free medium. Independently varying phosphate and nitrate nine-fold had no clear effect on the sensitivity of P. botryoides to thiobencarb.

Diatom Genus Diversity and Assemblage Structure in Relation to Salinity at the Salt Plains National Wildlife Refuge, Alfalfa County, Oklahoma

Abstract Despite the common geographic occurrence of inland (athalassic) saline habitats, their biota has not been extensively studied. Diatoms have been estimated to contribute as much as 25% to the earths primary production (Werner, 1977). However, in hypersaline systems the proportion of in situ carbon fixation by diatoms is likely to be higher. We used substrate samples taken from the Salt Plains National Wildlife Refuge, an athalassic hypersaline ecosystem, to investigate the relationship between diatom genus diversity, composition and salinity. These samples range in salinity from 14 to 306 ppt and contain 21 genera. Six genera (Cymbella, Mastogloia, Psammodictyon, Amphora, Navicula and Nitzschia) comprise 97% of the diatoms counted in all samples. Diatom genus diversity shows an inverse relationship with salinity, while genus richness shows no clear relationship with salinity. Hence, loss in diversity is the result of dominance by fewer taxa at higher salinities. The relative abundance of the genus Navicula is positively correlated with salinity, with it dominating the highest salinity sites. We used a canonical correspondence analysis (CCA) to explore the relationship between salinity and relative abundance of diatom genera. The primary variables influencing diatom genus composition at the Salt Plains were found to be the variability of salinity within a site and the overall magnitude of salinity.

Phylogenetic analysis of Dunaliella (Chlorophyta) emphasizing new benthic and supralittoral isolates from Great Salt Lake

Dunaliella, a commercially important chlorophyte, is globally distributed in saline habitats. Morphological species have not been definitively reconciled with phylogenetic analyses. Considerable genetic diversity continues to be discovered in new isolates, especially from soil and benthic habitats. Twenty‐nine new isolates from Great Salt Lake, Utah, many from benthic or supralittoral habitats, were phylogenetically analyzed using ITS1+5.8S+ITS2 in comparison to a broad sampling of available sequences. A few new isolates align in one branch of a bifurcated monophyletic Dunaliella salina clade and several cluster within monophyletic D. viridis. Several others align with relatively few unnamed strains from other locations, comprising a diverse clade that may represent two or more new species. The overall Dunaliella clade is relatively robust, but the nearest outgroups are ambiguously placed with extremely long branches. About half of the isolates, all from benthic or supralittoral habitats, have been persistently sarcinoid in liquid media since isolation. This trait is spread across the Dunaliella phylogeny. The morphology of two sarcinoid strains was documented with light microscopy, revealing an extensive glycocalyx. Clumping behavior of unicellular and sarcinoid strains was unaffected by presence or absence of Mg2+ or Ca2+, addition of lectin‐inhibiting monosaccharides, or water‐soluble factors from morphologically opposite strains. Results from this investigation have significantly expanded our current understanding of Dunaliella diversity, but it seems likely that much remains to be discovered with additional sampling.