Field photosynthetic activity of lichens in the Windmill Islands oasis, Wilkes Land, continental Antarctica

In order to ascertain whether the major species of continental antarctic macrolichens are photosynthetically active during summer conditions, the chlorophyll fluorescence of three lichen species [Umbilicarin decussata (Vill. Zahlbr., Pseudephebe minuscula (Ny-l. ex Arnold) Brodo and Hawksw. and Usnea sphacetala R. Br.] was monitored in the vicinity of Casey Station. Wilkes Land, continental Antarctica using a PAM-2000 modulated fluorescence system. Lichens were studied when in equilibrium with the atmosphere as well as when moistened by snow showers. Photochemical quantum yield was estimated as ΔF/F′_m and related to thallus water content as well as microclimatic conditions. Lichens were photosynthetically active only when moistened by snow fall or by run-off from snow melt. The levels of photosynthetic activity in the field for all species were influenced by microenvironmental conditions and patterns in response were site and species specific. Highest levels of photosynthetic efficiency occurred when thalli were at intermediate water contents. Photosynthetic activity was reduced by cold as well as warm, bright conditions. Highest thallus water contents occurred during the middle of the day after substantial “falls of snow. P. minuscula maintained highest thallus water contents at all sites and appears to have a high water compensation point which is related to its observed distribution patterns. Umbilicaria decussata studied in the laboratory did not become photosynthetically active even when exposed to 95% relative humidity (RH) for 51 h and. when dehydrating after artificial wetting, showed an optimum thallus water content for photosynthesis of ca 90% dry weight and a thallus water compensation point of about 35% dry weighl. In the field U. decussata did not become pholosynthetically active except when moistened by snow. Usnea sphacelata exposed to the atmosphere had low thallus water contents (ca 30%) which was not related to RH. The results indicate that the lichens are photosynthetically inactive for most of the summer period and are totally reliant on snow as a water supply. This i.s important when modelling carbon gain and growth rates of continental antarctic lichens.     

Symbiosis as a successful strategy in continental Antarctica: performance and protection of Trebouxia photosystem II in relation to lichen pigmentation

Lichens as symbiotic associations consisting of a fungus (the mycobiont) and a photosynthetic partner (the photobiont) dominate the terrestrial vegetation of continental Antarctica. The photobiont provides carbon nutrition for the fungus. Therefore, performance and protection of photosystem II is a key factor of lichen survival. Potentials and limitations of photobiont physiology require intense investigation to extend the knowledge on adaptation mechanisms in the lichen symbiosis and to clarify to which extent photobionts benefit from symbiosis. Isolated photobionts and entire lichen thalli have been examined. The contribution of the photobiont concerning adaptation mechanisms to the light regime and temperature conditions was examined by chlorophyll a fluorescence and pigment analysis focusing on the foliose lichen Umbilicaria decussata from North Victoria Land, continental Antarctica. No photoinhibition has been observed in the entire lichen thallus. In the isolated photobionts, photoinhibition was clearly temperature dependent. For the first time, melanin in U. decussata thalli has been proved. Though the isolated photobiont is capable of excess light protection, the results clearly show that photoprotection is significantly increased in the symbiotic state. The closely related photobiont of Pleopsidium chlorophanum, a lichen lacking melanin, showed a higher potential of carotenoid-based excess light tolerance. This fact discriminates the two photobionts of the same Trebouxia clade. Based on the results, it can be concluded that the successful adaptation of lichens to continental Antarctic conditions is in part based on the physiological potential of the photobionts. The findings provide information on the success of symbiotic life in extreme environments.     

Lecanoric acid,a secondary lichen substance with antioxidant properties from <Emphasis Type="Italic">Umbilicaria antarctica</Emphasis> in maritime Antarctica (King George Island)

Eight lichen species, Cetraria aculeata, Cladonia furcata, Pseudephebe pubescens, Sphaerophorus globosus, Stereocaulon alpinum, Umbilicaria antarctica, Usnea antarctica and Usnea aurantiacoatra, were collected from King George Island, maritime Antarctica, for the evaluation of antioxidant activities. Anti-linoleic acid peroxidation activity, free radical scavenging activity, reducing power and superoxide anion scavenging activity were assessed of methanol and acetone extract of the lichens in vitro. Extract of Umbilicaria antarctica, Cladonia furcata, Sphaerophorus globosus and Usnea antarctica were found to have strong in vitro antioxidant properties. In general, acetone extract exhibited stronger activities than methanol extract. The activity-guided bioautographic TLC and HPLC analysis demonstrated that lecanoric acid was the main antioxidant compound in the acetone extract of Umbilicaria antarctica, the most potent antioxidant lichen species among the test species. The results suggested that several Antarctic lichens and their substances can be used as novel bioresources of natural antioxidants.     

Photobiont selectivity for lichens and evidence for a possible glacial refugium in the Ross Sea Region, Antarctica

Lichens are a symbiosis consisting of heterotrophic, fungal (mycobiont) and photosynthetic algal or cyanobacterial (photobiont) components. We examined photobiont sequences from lichens in the Ross Sea Region of Antarctica using the internal transcribed spacer region of ribosomal DNA and tested the hypothesis that lichens from this extreme environment would demonstrate low selectivity in their choice of photobionts. Sequence data from three targeted lichen species (Buellia frigida, Umbilicaria aprina and Umbilicaria decussata) showed that all three were associated with a common algal haplotype (an unnamed Trebouxia species) which was present in all taxa and at all sites, suggesting lower selectivity. However, there was also association with unique, local photobionts as well as evidence for species-specific selection. For example, the cosmopolitan U. decussata was associated with two photobiont species, Trebouxia jamesii and an unnamed species. The most commonly collected lichen (B. frigida) had its highest photobiont haplotype diversity in the Dry Valley region, which may have served as a refugium during glacial periods. We conclude that even in these extreme environments, photobiont selectivity still has an influence on the successful colonisation of lichens. However, the level of selectivity is variable among species and may be related to the ability of some (e.g. B. frigida) to colonise a wider range of habitats.     

Sensitivity of Photosystem 2 of Antarctic Lichens to High Irradiance Stress: Fluorometric Study of Fruticose (Usnea antarctica) and Foliose (Umbilicaria decussata) Species

Two lichen species collected in maritime Antarctica (King George Island) were exposed under laboratory conditions to excess irradiance to evaluate the response of photosystem 2 (PS2). The response was measured on fully hydrated lichen thalli at 5 °C by means of a modulated fluorometer using chlorophyll (Chl) fluorescence induction curve supplemented with analysis of quenching mechanisms. Chl fluorescence parameters [i.e. ratio of variable to maximum Chl fluorescence (F_V/F_M), quantum yield of PS2 photochemical reactions (₂), quenching coefficients] were evaluated before and several times after exposition to high irradiance in order to characterise the extent of photoinhibition, fast and slow phase of recovery. Strong irradiance (2 000 mol m^–2 s^–1) caused high degree of photoinhibition, particularly higher in fruticose (Usnea antarctica) than in foliose (Umbilicaria decussata) lichen species. Fast phase of recovery from photoinhibition, corresponding to regulatory mechanisms of PS2, was more apparent in U. decussata and ₂ than in U. antarctica and F_V/F_M and ₂ within 40 min after photoinhibitory treatment. It was followed by a slow phase lasting several hours, corresponding to repair and re-synthesis processes. After photoinhibitory treatment, recovery of non-photochemical quenching (NPQ) was faster and more pronounced in U. decussata than in U. antarctica. Significant differences were found between the two species in the rate of recovery in fast-(q_E) and slow-recovering (q_T+I) component of NPQ.     

New Insights into the Biological Activity of Lichens: Bioavailable Secondary Metabolites of Umbilicaria decussata as Potential Anticoagulants

This study reports the in vitro anticoagulation activity of acetonic extract (AE) of 42 lichen species and the identification of potential bioavailable anticoagulant compounds from Umbilicaria decussata as a competent anticoagulant lichen species. Lichens’ AEs were evaluated for their anticoagulant activity by monitoring activated partial thromboplastin time (APTT) and prothrombin time (PT) assays. A strong, positive correlation was observed between total phenolics concentration (TPC) of species and blood coagulation parameters. U. decussata was the only species with the longest clotting time in both APTT and PT assays. The research was moved forward by performing in vivo assays using rats. The results corroborated the dose-dependent impact of U. decussata’s AE on rats’ clotting time. Major secondary metabolites of U. decussata and their plasma-related bioavailability were also investigated using LC-ESI-MS/MS. Atranol, orsellinic acid, D-mannitol, lecanoric acid, and evernic acid were detected as possible bioavailable anticoagulants of U. decussata. Our findings suggest that U. decussata might be a potential anticoagulant lichen species that can be used for the prevention or treatment of coagulation-related issues such as cardiovascular diseases (CVDs).     

Distributional and ecophysiological study on the Antarctic lichens species pair <Emphasis Type="Italic">Usnea antarctica</Emphasis>/<Emphasis Type="Italic">Usnea aurantiaco</Emphasis>-<Emphasis Type="Italic">atra</Emphasis>

The factors that control lichen distribution in Antarctica are still not well understood, and in this investigation we focused on the distribution, local and continental, and gas exchange of a species pair, closely related lichens with differing reproductive strategies, Usnea aurantiaco-atra (fertile) and Usnea antarctica (sterile, sorediate). The local distributions of these species were recorded along an altitudinal gradient of nearly 300 m at South Bay, Livingston Island, and microclimate was also recorded over 1 year. The photosynthetic responses to temperature, light and thallus water content were determined under controlled conditions in the laboratory. The species were almost identical in their photosynthetic profiles. Locally, on Livingston Island, U. antarctica was confined to low altitude sites which were warmer and drier, whilst U. aurantiaco-atra was present at all altitudes. This contrasts with its distribution across Antarctica where U. antarctica grows 9° latitude further south than U. aurantiaco-atra. Temperature appears not to be the main controller of distribution in these species, but dryness of habitat, which will influence length of activity periods, may be important.     

子囊菌亚门石耳科属级分类及拉诺属地位的研究

关于子囊菌亚门石耳科的属级分类问题地衣学家持有不同的观点。这些观点使属级分类出现了单属系统,二属系统,三属系统以及四属系统与五属系统。现在新二属系统,即石耳属一疱脐衣属系统已被越来越多的地衣学家和子囊菌学家所接受。但是,因为由Dodge (1968)描述的单种属拉诺属(Llanoa)的模式种与早已为人熟知的网脊石耳很相似,而近年来,这一单种属又被一些作者所接受、因此、我们不得不对该属模式种所依据的原始材料进行必要的复查。复查结果表明,拉诺属的模式种卖为网脊石耳的异名。因此,拉诺属也就成为石耳属的异名。此外,基于石耳科中三十五种地衣和五十二项特征的聚类分析结果也支持新的二属系统。     

The influence of physical factors and ornithogenic products on plant and arthropod abundance at an Inland Nunatak group in Antarctica

Summary Robertskollen is a group of geologically similar nunataks in western Dronning Maud Land, Antarctica, with breeding colonies of snow petrels Pagodroma nivea at some of the nunataks. Transects determining macro-plant structure and cover were conducted along an a priori determined bird-influence gradient to assess the relative importance of ornithogenic products and physical factors on plant assemblages. Fine-scale (intra-transect) dispersion patterns of plants were determined primarily by physical factors affecting water availability (i.e. the amount of drift-snow available and the effects of shelter and shade on evaporation rates). Coarse-scale (inter-transect) analysis of plant dispersion patterns, however, showed significant responses along the bird-influence gradient, with consistent responses between nunataks. Plant cover was directly related to bird-influence, but was depressed within bird colonies, apparently due to excessive nutrient enrichment. Mites (Acari) were the only arthropods recorded at Robertskollen and total mite abundance was related to plant cover, although specific responses varied. Thallus diameters of the abundant lichen Umbilicaria decussata decreased with decreasing bird influence, suggesting that nutrients limit plant growth away from bird colonies. Soil concentrations of major plant nutrients, N, P and K, were significantly greater close to bird colonies. We conclude that nutrients in ornithogenic products have direct causal effects on the abundance and dispersion of the biota of inland Antarctic nunataks, and that the effects of nutrient enrichment are detectable at different spatial scales from the effects of physical factors.     

Water Distribution in Foliose Lichen Species: Interactions between Method of Hydration, Lichen Substances and Thallus Anatomy

Three lichens (Neofuscelia pokornyi, N. pulla and Xanthoriaparietina) from a semi-arid habitat were examined using low-temperaturescanning electron microscopy to evaluate the effects of hydrationmethod, lichen substances and thallus anatomy on the water distributionof hydrated thalli. In theNeofuscelia species, extracellularwater within the thallus was observed in association with cracksin its otherwise impervious upper cortex, while X. parietinashowed abundant extracellular water between medullary hyphae.Spraying the thalli followed by maintenance for 14–20h in a water-saturated atmosphere led to the disappearance ofthe external water film in X. parietina but not in the Neofusceliaspecies. Surface water was abundant in specimens of all speciesimmediately after spraying for 15 min. No extracellular waterwas observed inside the thallus 14–20 h after spraying,but after rinsing with acetone its presence was detected inall three species. Hydric strategy correlated with cortex hygroscopicity:X. parietina, an aero-hygrophytic species, had a more hygroscopicupper cortex than theNeofuscelia species, which are substrate-hygrophytic.The hygroscopicity of the upper cortex was linked with the amountof extracellular water in the thalline interior. Differencesbetween X. parietina and Neofuscelia in the polarity and distributionof their lichen substances agreed with species differences inthe presence and distribution of free water both as a film overthe surface and inside the thallus. Lichen substances appearto play a role in the maintenance of air-filled intrathallinespaces in species whose anatomy, habitat, or both, favour water-loggedconditions. Copyright 2000 Annals of Botany Company Lichen, water relations, semi-arid, lichen substances, LTSEM, thallus anatomy, extracellular water, Neofuscelia pokornyi(Körb.) Essl., Neofuscelia pulla(Ach.) Essl., Xanthoria parietina(L.) Th. Fr     

Vegetation and ecology of ice-free areas of Northern Victoria Land,Antarctica

Summary At Birthday Ridge, a small ice free area in northern Victoria Land (70°48S, 167°00E), cryptogamic vegetation is mostly confined to gaps between granitc rocks. The sheltering effect on lichens and mosses was analyzed by continuous measurements of the microclimate at various levels between the rocks. Although warming by solar radiation was favourable for the existence of cryptogams, rocks strongly insolated were mostly devoid of lichens and mosses. Lichens in the soaked active state were heated to above air temperature but did not reach more than 10°C. The presence of lichens was dependent on the moisture conditions of the habitat. It was observed that snow, the only source of moisture, accumulated in summer only in deeper levels between rocks, and that the snow rapidly melted on contact with the lichens. After a snow shower,Usnea sulphurea gained 67% andUmbilicaria decussata 94% of their maximum water capacity. During one quarter of the time period of 7 days the lichens were soaked and therefore capable of carrying out photosynthesis. The lichens in soaked state had always less than optimum temperatures for net photosynthesis. The rock gaps at Birthday Ridge form oases, the only localities where moisture is provided, and temperature is high enough to enable growth of lichens and mosses.Bryum is also able to exist in the upper 3 cm of soil.     

Ecological and physiological investigations in continental Antarctic cryptogams

Summary Microclimate and CO₂ exchange of the lichen Usnea sphacelata were measured during summen on a hill near Casey Station, Bailey Peninsula, Wilkes Land, Antarctica. Within a period of 52 days (November 10 until December 31, 1985), 8 diurnal courses of net photosynthesis were measured in naturally snow-covered lichen thalli, and 9 diurnal courses in thalli experimentally sprayed with melt water. Photosynthetic performance of a light-form of Usnea sphacelata was compared with that of a shade-form. Net photosynthesis was reversibly depressed in snow-covered lichen thalli of both forms when irradiance was higher than 600 mol m^–2 s^–1 photosynthetic active radiation (PAR), the depression persisting several hours after a period of strong light. These responses suggest photoinhibition. Models of photosynthesis were established for the light-form by non-linear regressions with field data from water-sprayed thalli (Model W) and field data measured in snow-covered lichens (SNO I, SNO II). Model SNO I is based on median values of photosynthetic rates and SNO II on maximum values for each light/temperature combination. Photosynthetic rates were calculated using model W; the results showed values approximately three times higher than measured in the field with naturally moistened thalli. Photosynthetic rates according to model SNO II fitted the data of naturally moistened lichens measured during the day, before strong light (> 600 mol m^–2s^–1 PAR) caused reversible decrease of net photosynthesis. Model SNO I fitted the data measured during and after a phase of strong irradiance. Model SNO I demonstrated that light stress was highest at temperatures below 2 °C. This study has shown that long-term calculation of the photosynthetic productivity must take into account decreases in net photosynthesis rate caused by strong light, as well as effects of water content and temperature. For the investigated period of the austral summer, a carbon production of 3.44 gm^–2 was estimated for U. sphacelata.     

Diversity of the lichenized fungi in King George Island, Antarctica, revealed by phylogenetic analysis of partial large subunit rDNA sequences

Lichens are predominant and important components of flora in the terrestrial ecosystem of Antarctica. However, relatively few researches on the phylogenetic position of Antarctic lichen-forming fungi have been accomplished. In this study, partial sequences of nuclear large subunit rDNAs from 50 Antarctic specimens were obtained and the phylogeny was reconstructed. Antarctic lichen species were distributed among 4 orders, including the monophyletic order Agyrales, paraphyletic orders Pertusariales and Teloschistales, and polyphyletic order Lecanorales. Species diversity was highest in the order Lecanorales, followed by Teloschistales and Pertusariales. Based on the phylogeny and sequence similarity analyses, it is proposed that the taxonomy of Stereocaulon alpinum, Physcia caesia, Usnea aurantiacoatra, and Cladonia species should be revised by careful examination of their phenotypic and molecular characteristics. Six species known to be endemic to Antarctica, Catillaria corymbosa, Himantormia lugubris, Leptogium puberulum, Pertusaria pertusa, Rhizoplaca aspidophora, and Umbilicaria antarctica, formed unique lineages, implying independent origins in the Antarctic area.     

Trade‐off relationship between productivity and thallus toughness in Laminariales (Phaeophyceae)

Trade‐off relationships are considered key to understanding the mechanisms supporting the coexistence of multiple species within kelp beds. Thus, information on trade‐offs is expected to contribute to conservation of kelp bed diversity. To test the existence of a trade‐off between productivity and thallus toughness, thallus traits and relationships between the traits were examined for seven species of Laminariales including 24 populations. For each population, photosynthetic capacity per unit biomass (as A _mass) and nitrogen (i.e., photosynthetic nitrogen‐use efficiency, PNUE), nitrogen content (as N _mass), thallus mass per unit thallus area (as TMA) and force required to penetrate the thallus (as F _p, a common index of leaf toughness in land plants by punch test) were determined. A _mass increased with increasing N _mass. Blades with high N _mass showed high A _mass. These blades may invest a large proportion of nitrogen to the photosynthetic parts, and consequently exhibit high metabolic rates. Moreover, blades with high N _mass tended to be associated with low TMA, and N _mass decreased with increasing TMA. A significant negative correlation was observed between TMA and A _mass because of the linkage of high A _mass with high N _mass and high N _mass associated with low TMA, while a significant positive correlation was observed between TMA and F _p. The two correlations indicate the existence of a trade‐off between productivity and thallus toughness in Laminariales. PNUE showed a significant negative correlation with TMA, which also showed a significant positive correlation with F _p as the index of thallus toughness, and therefore a trade‐off relationship between productivity and thallus toughness.     

Evidence of high annual growth rate for lichens in the maritime Antarctic

On Livingston Island (South Shetland Islands, Antarctica) young moraines, now roughly 45 years old, were investigated in 1991 and 2002. More than 500 thalli of the 6 most abundant species, Acarospora macrocyclos, Bellemerea sp. Buellia latemarginata, Caloplaca sublobulata, Rhizocarpon geographicum and Usnea antarctica, previously measured in 1991, were measured again in 2002, which allowed an accurate measure of thallus growth rates. From our results, we believe it important to emphasize that: (1) lichen colonization probably took place at the end of the 1950s, soon after the last glacier retreat. (2) No relation between thallus growth rate and boulder size could be established. (3) Higher annual growth rates than those previously estimated were observed for R. geographicum and Bellemerea sp. and the former had one of the highest growth rates ever reported for this species. We speculate whether this high growth rate may be related to the rapid retreat of the Livingston Island ice cap and glaciers during the last recent years, and which have been attributed to climate warming on the Antarctic Peninsula region.     

五种南极地衣的Co、Cr、Pb和Cu元素富集能力的差异

随着人类活动对全球环境的影响,环境重金属污染负荷日益增加,重金属元素向南极地区的迁移和富集已成为该地区面临的严重问题之一。地衣是研究重金属元素大气沉降特征的良好材料,但对南极地衣的重金属元素大气沉降富集能力的比较研究甚少。作者以网袋法实验比较采自西南极乔治王岛菲尔德斯半岛的5种南极优势地衣,即:王橙衣Caloplaca regalis、夹心果衣Himantormia lugubris、孔树花Ramalina terebrata、球粉衣Sphaerophorus globosus和簇花石萝Usnea aurantiacoatra对钴(Co)、铬(Cr)、铅(Pb)和铜(Cu)的富集能力。2个月的网袋法实验结果表明:1)5种地衣对Pb元素均表现出富集能力,其中夹心果衣富集能力最强,簇花石萝和孔树花次之;2)簇花石萝和夹心果衣对Cu元素的富集能力相近,球粉衣对Cu元素的富集能力较低,其他2种未表现出对Cu的富集作用;3)Co和Cr元素的富集仅分别在夹心果衣和簇花石萝中检测出来。因此,在综合检测Co、Cr、Cu和Pb的大气沉降时,可考虑簇花石萝和夹心果衣的组合;在单独检测Co、Pb和Cu的大气沉降时,夹心果衣均为最佳的物种。     

POROSIMETRIC STUDY OF THE LICHEN FAMILY UMBILICARIACEAE: ANATOMICAL INTERPRETATION AND IMPLICATIONS FOR WATER STORAGE CAPACITY OF THE THALLUS

The pore system within the thalli of 13 lichen taxa belonging to the family Umbilicariaceae has been studied by means of mercury intrusion porosimetry. A characteristic bimodal pore size distribution with a central depression around 0.05 μm of equivalent pore radius was obtained in all lichen samples. However, clear differences were found among the pore size distributions of each lichen taxa. The total thallus porosity was undoubtedly related to the anatomy of the medulla. In general, a radial plectenchymatic medulla conferred larger porosity to the thallus than an arachnoidal one. Maximum thallus water content closely depended on the total thallus porosity in the five lichens possessing rhizinomorphs. The species with a similar type of medulla could be grouped together in a multivariate analysis that considered three porosimetric parameters and the maximum thallus water content. Umbilicaria cinereorufescens was the most distinct species, with the lowest values of total porosity and water storage capacity and the largest value of thallus density, apparently due to its scleroplectenchymatic medulla. The pore size distribution existing inside the thallus of the species studied is discussed in relation to the often opposing problems of CO₂ exchange and optimal water relations. Some results pointed to a large influence of the micropores (<0.05 μm) on the water storage capacity of the thallus, while the macropores would have a more important role in gas exchange.     

In vitro Culture of Immature Cotyledons of Soya Bean (Glycine max L. Merr.)

An in vitro procedure promoting the rapid growth and proteinincrease of soya bean cotyledons has been developed. The amountof protein synthesized varied greatly depending on the nitrogen(N) source provided. Glutamine was the most effective N source,while inorganic forms of N were ineffective. Growth and proteinsynthesis were both more rapid in vitro than in vivo. Underthe best conditions, soya bean cotyledons increased 8-fold bothin dry weight and in protein in 6 days. The formation of the7S and 11S storage proteins in vitro was similar to that invivo. Hence, this in vitro culture method is appropriate forstudying legume seed storage protein synthesis under controlledconditions.     

Environment determines nitrogen content and stable isotope composition in the sporophyte of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> (Harvey) Suringar

Nitrogen content and δ¹⁵N (nitrogen stable isotope ratio) were measured in different parts of the sporophyte of Undaria pinnatifida (Harvey) Suringar from two bays in the northeastern Japan. There were clear differences between the thalli collected in winter and in summer: high nitrogen content and low average δ¹⁵N were observed for the thalli collected in winter, while the opposite pattern was found for the thalli from summer. In addition, the pattern of internal δ¹⁵N distribution in the thallus changed with season. It is possible that the cause for these seasonal differences in the algae was the seasonal change in environmental conditions, because in winter water is normally rich in nitrogen, while in summer it is poor. U. pinnatifida sporophyte may be useful as an indicator of nitrogen sources in coastal waters, but consideration must be given to the effect of isotope fractionation on δ¹⁵N of the plant, especially in winter. Potential may exist for the use of different parts of the thallus to indicate nitrogen sources at different periods, but more investigation is necessary to accomplish this.