abstract: Human activity has increased loading of reactive nitrogen (N) in the environment, with important and often deleterious impacts on biodiversity, climate, and human health. Since the fate of N in the ecosystem is mainly controlled by microorganisms, understanding the factors that shape microbial communities becomes relevant and urgent. In arid land soils, these microbial communities and factors are not well understood. I aimed to study the role of N cycling microbes, such as the ammonia-oxidizing bacteria (AOB), the recently discovered ammonia-oxidizing archaea (AOA), and various fungal groups, in soils of arid lands. I also tested if niche differentiation among microbial populations is a driver of differential biogeochemical outcomes. I found that N cycling microbial communities in arid lands are structured by environmental factors to a stronger degree than what is generally observed in mesic systems. For example, in biological soil crusts, temperature selected for AOA in warmer deserts and for AOB in colder deserts. Land-use change also affects niche differentiation, with fungi being the major agents of N2O production in natural arid lands, whereas emissions could be attributed to bacteria in mesic urban lawns. By contrast, NO3- production in the native desert and managed soils was mainly controlled by autotrophic microbes (i.e., AOB and AOA) rather than by heterotrophic fungi. I could also determine that AOA surprisingly responded positively to inorganic N availability in both short (one month) and long-term (seven years) experimental manipulations in an arid land soil, while environmental N enrichment in other ecosystem types is known to favor AOB over AOA. This work improves our predictions of ecosystem response to anthropogenic N increase and shows that paradigms derived from mesic systems are not always applicable to arid lands. My dissertation also highlights the unique ecology of ammonia oxidizers and draws attention to the importance of N cycling in desert soils. / Dissertation/Thesis / Ph.D. Biology 2013

Anthropogenic activities perturb the global carbon and nitrogen cycle with large implications for the earth’s climate. Land use activities deliver excess carbon and nitrogen to aquatic ecosystems. In the boreal biome, this is mainly due to forestry and atmospheric deposition. Yet, impacts of these anthropogenically mediated inputs of carbon and nitrogen on the processing and emissions of greenhouse gases from recipient streams and lakes are largely unknown. Understanding the ecosystem-scale response of aquatic greenhouse gas cycling to land use activities is critical to better predict anthropogenic effects on the global climate system and design more efficient climate change mitigation measures. This thesis assesses the effects of forest clearcutting and nitrate enrichment on greenhouse gas emissions from boreal inland waters. It also advances methods to quantify sources and sinks of these emissions. Short-term clearcut and nitrate enrichment effects were assessed using two whole-ecosystem experiments, carried out over four years in nine headwater catchments in boreal Sweden. In these experiments, I measured or modeled air-water fluxes of carbon dioxide (CO2), methane (CH4) and nitrous oxide (N2O), combining concentration, ebullition and gas-transfer velocity measurements in groundwater, streams and lakes. By using Swedish national monitoring data, I also assessed broad-scale effects of forest clearcutting by relating CO2 concentrations in 439 forest lakes to the areal proportion of catchment forest clearcuts. To improve quantifications of CO2 sources and sinks in lakes, I analyzed time series of oxygen concentrations and water temperature in five lakes on conditions under which whole-lake metabolism estimates can be inferred from oxygen dynamics given the perturbing influence of atmospheric exchange, mixing and internal waves. The experiments revealed that aquatic greenhouse gas emissions did not respond to nitrate addition or forest clearcutting. Importantly, riparian zones likely buffered clearcut-induced increases in groundwater CO2 and CH4 concentrations. Experimental results were confirmed by monitoring data showing no relationship between CO2 patterns across Swedish lakes and clearcut gradients. Yet, conclusions on internal vs. external CO2 controls largely depended on whether spatially or temporally resolved data was used. Partitioning CO2 sources and sinks in lakes using time series of oxygen was greatly challenged by physical transport and mixing processes. Conclusively, ongoing land use activities in the boreal zone are unlikely to have major effect on headwater greenhouse gas emissions. Yet, system- and scale specific effects cannot be excluded. To reveal these effects, there is a large need of improved methods and design of monitoring programs that account for the large spatial and temporal variability in greenhouse gas dynamics and its controls by abiotic and biotic factors.

Boreal forests are often strongly nitrogen (N) limited. However, human activities are leading to increased N inputs into these ecosystems, through atmospheric N deposition and forest fertilization. N input into boreal forests can promote net primary productivity, increase herbivore and pathogen damage, and shift plant species composition and community structure. Genetic diversity has been suggested as a key mechanism to promote a plant species’ stability within communities in response to environmental change. Within any plant population, specific traits (e.g. growth and defense traits) can vary substantially among individuals, and a greater variation in traits may increase chances for the persistence of at least some individuals of a population, when environmental conditions change. One aspect of plant chemistry that can greatly vary among different genotypes (GTs) are condensed tannin (CTs). These secondary metabolites have been suggested to affect plant performance in many ways, e.g. through influencing plant growth, the interactions of plants with herbivores and pathogens, and through affecting litter decomposition, and hence the return of nutrients to plants. To investigate how genotypic variation in foliar CT production may mediate the effects that anthropogenic N enrichment can have on plant performance and litter decomposition, I performed a series of experiments. For these experiments, aspen (Populus tremula) GTs with contrasting abilities to produce foliar CTs (i.e. low- vs. high-tannin producers) were grown under 3 N conditions, representing ambient N (+0 kg ha-1), upper level atmospheric N deposition (+15 kg ha-1), and forest fertilization rates (+150 kg ha-1). This general experimental set-up was once established in a field-like environment, from which natural enemies were excluded, and once in a field, in which enemies were present. In my first two studies, I investigated tissue chemistry and plant performance in both environments. I observed that foliar CT levels decreased in response to N in the enemy‑free environment (study I), but increased with added N when enemies were present (study II). These opposing responses to N may be explained by differences in soil N availability in the two environments, or by induction of CTs after enemy attack. Enemy damage generally increased in response to N, and was higher in low-tannin than in high-tannin plants across all N levels. Plant growth of high‑tannin plants was restricted under ambient and low N conditions, probably due to a trade-off between growth and defense. This growth constraint for high‑tannin plants was weakened, when high amounts of N were added (study I and II), and when enemy levels were sufficiently high, so that benefits gained through defense could outweigh the costs of defense production (study II). Despite those general responses of low- and high‑tannin producers to added N, I also observed a number of individual responses of GTs to N addition, which in some case were not connected to the intrinsic ability of the GTs to produce foliar CTs. In study III, gene expression levels in young leaves and phenolic pools of the plants that were grown in the enemy‑free environment were studied. This study revealed that gene control over the regulation of the phenylpropanoid pathway (PPP) was distributed across the entire pathway. Moreover, PPP gene expression was higher in high-tannin GTs than in low‑tannin GTs, particularly under ambient N. At the low N level, gene expressions declined for both low- and high-tannin producers, whereas at the high N level expression at the beginning and the end of the PPP was upregulated and difference between tannin groups disappeared. Furthermore, this study showed that phenolic pools were frequently uncorrelated, and that phenolic pools were only to some extent related to tannin production and gene expression. In study IV, I investigated the decomposability of litter from the field plants. I found that N enrichment generally decreased mass loss, but there was substantial genetic variation in decomposition rates, and GTs were differentially responsive to added N. Study IV further showed that CTs only had a weak effect on decomposition, and other traits, such as specific leaf area and the lignin:N ratio, could better explain genotypic difference in mass loss. Furthermore, N addition caused a shift in which traits most strongly influenced decomposition rates. Collectively, the result of these studies highlight the importance of genetic diversity to promote the stability of species in environments that experience anthropogenic change. / Boreala skogar är ofta mycket kväve (N) begränsade. Men mänskliga aktiviteter leder till ökad N tillförsel i dessa ekosystem, både genom depostition av N från atmosfären och skogsgödsling. N-tillförsel i boreala skogar kan främja netto primärproduktionen men även leda till ökade skador från naturliga fiender (herbivorer och patogener) samt skiftningar i växtartsammansättning. Genetisk mångfald har föreslagits som en viktig mekanism för att främja en växtarts stabilitet inom samhällen som upplever miljöförändringar. Inom varje växtpopulation kan specifika egenskaper (t.ex. tillväxt och försvar) varierar kraftigt mellan individer och en större variation i egenskaper kan öka chanserna för att åtminstone några individer från en population överlever ifall miljöförhållandena förändras. En aspekt av växtkemi som i hög grad kan variera mellan olika genotyper (GT) är bladens kondenserade tanniner (KT). Dessa sekundära metaboliter har föreslagits påverka växtens prestationsförmåga på många sätt, t.ex. genom att påverka tillväxt, interaktioner mellan växter och herbivorer eller patogener och genom att påverka förna nedbrytning, och följaktligen återbördandet av näringsämnen till kretsloppet. För att undersöka hur genotypiska variation i KT produktion kan påverka de effekter som antopogent N kan ha på växtens prestationsförmåga och förna nedbrytning, utförde jag en serie experiment. Jag studerade olika asp (Populus tremula) GT med olika förmåga att producera KT (låg- och hög-tannin producenter). Växterna odlades i tre olika N förhållanden, som representerade ambient N nivå (+0 kg ha-1), atmosfärisk N deposition = låg nivå (+15 kg ha-1), och skogsgödsling = hög nivå (150 kg ha‑1). Dessa GT etablerades i en fält-liknande miljö där naturliga fiender uteslutits och i ett fält där naturliga fiender var närvarande. I mina första två studierna undersökte jag vävnadskemi och växternas prestationsförmåga i de båda miljöerna. Jag observerade att KT nivåerna sjönk till följd av N‑tillsats i den fiende-fria miljön (studie I), men ökade med N-tillsats ifall fiender var närvarande (studie II). Dessa motsatta reaktioner på N-tillsats kan förklaras av skillnader i N-tillgång mellan de två odlingsplatserna eller genom ökad KT produktion som respons på angrepp. Skador orsakade av herbivorer och patogener ökade generellt till följd av N‑tillsats och var högre i låg-tannin än i hög‑tannin producerande GT oavsett N‑förhållande. Tillväxten hos växter från hög‑tannin GT begränsades i ambient- och låg N-tillsats förhållanden, troligen på grund av att avvägning mellan tillväxt och försvar förskjutits emot försvar. Den begränsade tillväxten i hög-tannin växter minskade om stora mängder N tillsattes (studie I och II) och om antalet fiender var tillräckligt högt så att nyttan av försvaret kunde uppväga kostnaderna för försvarsproduktionen (studie II). Trots dessa generella respons hos låg- och hög-tannin GT till följd av N‑tillsats observerade jag även ett antal individuella respons hos GT som i vissa fall var orelaterade till växters förmåga att producera KT. I studie III undersöktes genuttrycksnivåer och fenolinnehåll i blad från växter som odladats i en miljö där naturliga fiender exkluderats. Denna studie visade att fenylpropanoidsyntesvägen (FPV) regleras genom kontroll av många av de undersökta FPV-generna. Dessutom var FPV genuttryck högre i hög-tannin GT än i låg-tannin GT, särskilt vid ambient N. Vid låg N-tillsats minskade genuttrycket av FPV-gener i både låg- och hög-tannin producenter, medan hög N-tillgång ledde till att gener i början och slutet av FPV uppreglerades och till att skillnaderna mellan tannin grupperna försvann. Dessutom visade studien att de separata fenol-poolerna ofta var okorrelerade med varandra och att fenol-poolerna bara till viss del var korrelerade med KT produktion och FPV-genutryck. I studie IV undersökte jag nedbrytningshastigheten för förnan från fältodlade aspar. Jag upptäckte att N-tillsats generellt minskade viktförlusten men att det fanns en betydande genetisk variation mellan GT och att dessa även var olika mottagliga för tillsatt N. Studie IV visade vidare att KT endast hade en svag effekt på nedbrytning och att andra egenskaper såsom specifik bladyta och lignin:N ratio kunde bättre förklara den genotypiska skillnaden i viktförlust. Dessutom orsakade N‑tillsats en förskjutning av vilka egenskaper som mest påverkade förnans nedbrytningshastighet. Sammanfattningsvis visar mina studier på vikten av genetisk mångfald för att främja växtartens stabilitet i miljöer som upplever antropogena förändringar.

The following research has focused on the utility of Ulva as an indicator of seawater nitrogen loading. Evaluation was made in three ways: 1) Observation of large-scale geographic variation in nitrogen status in natural populations around New Zealand in summer and winter, 2) Laboratory-based experimental assessment of the biochemical responses of N-indices in Ulva to nitrogen enrichment, and 3) Culturing standardized test-Ulva under low nutrient conditions which could be deployed into a variety of field situations. Seawater inorganic nutrient (nitrate, nitrite, ammonium and phosphate) concentrations and nitrogen (N)-indices (free amino acids, chlorophyll and total tissue nitrogen) in natural Ulva populations from 32 sites around New Zealand were compared. Sites were divided into 6 environmental categories: sheltered rural, exposed rural, rock pools, sheltered urban, exposed urban, and nitrogen-enriched urban sites. Seawater nutrient concentrations were highly variable between all sites in summer and winter. However, in the summer enriched urban sites had the highest mean total inorganic nitrogen concentrations and Ulva with the highest mean levels of all N-indices compared with any other environmental category. In the winter, Ulva contained more nitrogen (reflected in all N-indices) compared with Ulva in the summer, particularly in populations growing in colder southern seawater on more exposed coasts. The increase in Ulva N-status was not explained by increased seawater inorganic nitrogen concentrations. With univariate and multivariate statistical approaches it was shown that there was a significant effect of seawater temperature and site exposure on N-status in Ulva. Compared with other N-indices, stable nitrogen isotopes (δ15N) from Ulva growing in enriched urban sites had the widest range (4.77 ± 0.04 ‰ to 15.16 ± 0.03 ‰) of values compared with all other categories in both summer and winter. Conversely, Ulva from exposed rural sites had the lowest range of δ15N values compared with any other category (6.7 ± 0.1 to 8.8 ± 0.1 ‰) and showed no seasonal change in mean values (7.8 ‰ and 7.6 ‰ for summer and winter, respectively). In addition, δ15N values in Ulva were the only N-index that showed a significant difference between urban and rural categories. To test the relationship between inorganic nitrogen concentration in seawater and the responses of biochemical nitrogen indices in Ulva pertusa, several experiments were conducted in an outdoor, flow-through culture apparatus, in summer and winter. In this apparatus effects of ammonium concentration, nitrogen source (nitrate and ammonium), light and seawater motion were investigated. Of the same three N-indices examined in natural Ulva populations (free amino acids, chlorophyll and total tissue nitrogen), increases in free amino acids, particularly asparagine, provided the strongest indicator of increases in nitrogen availability. In addition, while tissue nitrogen and chlorophyll also increased with seawater nitrogen concentration, it was apparent that these indices were also strongly influenced by light, and probably season. Rates of ammonium assimilation provided no overall measure of the availability of nitrogen in seawater and were clearly affected by season. Similarly, growth rates in Ulva only showed a response to nitrogen addition in summer months. Stable isotopes of nitrogen (δ15N) in Ulva provided a clear distinction between natural and synthetic nitrogen sources, but more importantly, showed only minor fractionation (ranging from 1.3 ‰ to -1.9 ‰) of 15N supplied from synthetic nitrate and ammonium under both light-saturating and light-limiting conditions. To further develop Ulva as a standardized test-organism it was cultured in low-nutrient (non-polluted) seawater to deplete internal storage pools of nitrogen. Each month the resulting test-Ulva was then placed in surface-moored growth enclosures at a range of coastal sites around Auckland and then monitored for one year. In winter there were increases in seawater inorganic nitrogen concentrations and concomitant increases in free amino acid content. However, tissue nitrogen and chlorophyll content in test-Ulva showed similar increases (possibly saturating) across all sites suggesting that seasonal increases in these N-indices were also due to other seasonal factors (e.g., surface irradiance and / or seawater temperature). On the other hand, the total free amino acid pool showed strong differences between a low-nitrogen reference site and the other study sites all year round. It was probable that test-Ulva was integrating differences in tidally-averaged nitrogen loading that were not reliably detected in instantaneous seawater samples. In addition to N-indices in test-Ulva, levels of tissue heavy metals and stable isotopes of nitrogen showed strong differences with higher values of both typically found in urban environments compared with values found in non-polluted reference sites. It is concluded that several abiotic and biotic factors affect nitrogen status in Ulva, but the average nitrogen concentration in seawater, and the physical factors of temperature, light and water motion, appear to be the overarching determinants. It is further suggested that in combination with Ulva tissue δ15N values, tissue nitrogen and the free amino acid pool, as quantitative biochemical measures of nitrogen availability, are likely to provide useful information on both the amount and composition of nitrogen entering coastal environments. / Foundation for Research, Science and Technology. Auckland Regional Council.

The following research has focused on the utility of Ulva as an indicator of seawater nitrogen loading. Evaluation was made in three ways: 1) Observation of large-scale geographic variation in nitrogen status in natural populations around New Zealand in summer and winter, 2) Laboratory-based experimental assessment of the biochemical responses of N-indices in Ulva to nitrogen enrichment, and 3) Culturing standardized test-Ulva under low nutrient conditions which could be deployed into a variety of field situations. Seawater inorganic nutrient (nitrate, nitrite, ammonium and phosphate) concentrations and nitrogen (N)-indices (free amino acids, chlorophyll and total tissue nitrogen) in natural Ulva populations from 32 sites around New Zealand were compared. Sites were divided into 6 environmental categories: sheltered rural, exposed rural, rock pools, sheltered urban, exposed urban, and nitrogen-enriched urban sites. Seawater nutrient concentrations were highly variable between all sites in summer and winter. However, in the summer enriched urban sites had the highest mean total inorganic nitrogen concentrations and Ulva with the highest mean levels of all N-indices compared with any other environmental category. In the winter, Ulva contained more nitrogen (reflected in all N-indices) compared with Ulva in the summer, particularly in populations growing in colder southern seawater on more exposed coasts. The increase in Ulva N-status was not explained by increased seawater inorganic nitrogen concentrations. With univariate and multivariate statistical approaches it was shown that there was a significant effect of seawater temperature and site exposure on N-status in Ulva. Compared with other N-indices, stable nitrogen isotopes (δ15N) from Ulva growing in enriched urban sites had the widest range (4.77 ± 0.04 ‰ to 15.16 ± 0.03 ‰) of values compared with all other categories in both summer and winter. Conversely, Ulva from exposed rural sites had the lowest range of δ15N values compared with any other category (6.7 ± 0.1 to 8.8 ± 0.1 ‰) and showed no seasonal change in mean values (7.8 ‰ and 7.6 ‰ for summer and winter, respectively). In addition, δ15N values in Ulva were the only N-index that showed a significant difference between urban and rural categories. To test the relationship between inorganic nitrogen concentration in seawater and the responses of biochemical nitrogen indices in Ulva pertusa, several experiments were conducted in an outdoor, flow-through culture apparatus, in summer and winter. In this apparatus effects of ammonium concentration, nitrogen source (nitrate and ammonium), light and seawater motion were investigated. Of the same three N-indices examined in natural Ulva populations (free amino acids, chlorophyll and total tissue nitrogen), increases in free amino acids, particularly asparagine, provided the strongest indicator of increases in nitrogen availability. In addition, while tissue nitrogen and chlorophyll also increased with seawater nitrogen concentration, it was apparent that these indices were also strongly influenced by light, and probably season. Rates of ammonium assimilation provided no overall measure of the availability of nitrogen in seawater and were clearly affected by season. Similarly, growth rates in Ulva only showed a response to nitrogen addition in summer months. Stable isotopes of nitrogen (δ15N) in Ulva provided a clear distinction between natural and synthetic nitrogen sources, but more importantly, showed only minor fractionation (ranging from 1.3 ‰ to -1.9 ‰) of 15N supplied from synthetic nitrate and ammonium under both light-saturating and light-limiting conditions. To further develop Ulva as a standardized test-organism it was cultured in low-nutrient (non-polluted) seawater to deplete internal storage pools of nitrogen. Each month the resulting test-Ulva was then placed in surface-moored growth enclosures at a range of coastal sites around Auckland and then monitored for one year. In winter there were increases in seawater inorganic nitrogen concentrations and concomitant increases in free amino acid content. However, tissue nitrogen and chlorophyll content in test-Ulva showed similar increases (possibly saturating) across all sites suggesting that seasonal increases in these N-indices were also due to other seasonal factors (e.g., surface irradiance and / or seawater temperature). On the other hand, the total free amino acid pool showed strong differences between a low-nitrogen reference site and the other study sites all year round. It was probable that test-Ulva was integrating differences in tidally-averaged nitrogen loading that were not reliably detected in instantaneous seawater samples. In addition to N-indices in test-Ulva, levels of tissue heavy metals and stable isotopes of nitrogen showed strong differences with higher values of both typically found in urban environments compared with values found in non-polluted reference sites. It is concluded that several abiotic and biotic factors affect nitrogen status in Ulva, but the average nitrogen concentration in seawater, and the physical factors of temperature, light and water motion, appear to be the overarching determinants. It is further suggested that in combination with Ulva tissue δ15N values, tissue nitrogen and the free amino acid pool, as quantitative biochemical measures of nitrogen availability, are likely to provide useful information on both the amount and composition of nitrogen entering coastal environments. / Foundation for Research, Science and Technology. Auckland Regional Council.

The following research has focused on the utility of Ulva as an indicator of seawater nitrogen loading. Evaluation was made in three ways: 1) Observation of large-scale geographic variation in nitrogen status in natural populations around New Zealand in summer and winter, 2) Laboratory-based experimental assessment of the biochemical responses of N-indices in Ulva to nitrogen enrichment, and 3) Culturing standardized test-Ulva under low nutrient conditions which could be deployed into a variety of field situations. Seawater inorganic nutrient (nitrate, nitrite, ammonium and phosphate) concentrations and nitrogen (N)-indices (free amino acids, chlorophyll and total tissue nitrogen) in natural Ulva populations from 32 sites around New Zealand were compared. Sites were divided into 6 environmental categories: sheltered rural, exposed rural, rock pools, sheltered urban, exposed urban, and nitrogen-enriched urban sites. Seawater nutrient concentrations were highly variable between all sites in summer and winter. However, in the summer enriched urban sites had the highest mean total inorganic nitrogen concentrations and Ulva with the highest mean levels of all N-indices compared with any other environmental category. In the winter, Ulva contained more nitrogen (reflected in all N-indices) compared with Ulva in the summer, particularly in populations growing in colder southern seawater on more exposed coasts. The increase in Ulva N-status was not explained by increased seawater inorganic nitrogen concentrations. With univariate and multivariate statistical approaches it was shown that there was a significant effect of seawater temperature and site exposure on N-status in Ulva. Compared with other N-indices, stable nitrogen isotopes (δ15N) from Ulva growing in enriched urban sites had the widest range (4.77 ± 0.04 ‰ to 15.16 ± 0.03 ‰) of values compared with all other categories in both summer and winter. Conversely, Ulva from exposed rural sites had the lowest range of δ15N values compared with any other category (6.7 ± 0.1 to 8.8 ± 0.1 ‰) and showed no seasonal change in mean values (7.8 ‰ and 7.6 ‰ for summer and winter, respectively). In addition, δ15N values in Ulva were the only N-index that showed a significant difference between urban and rural categories. To test the relationship between inorganic nitrogen concentration in seawater and the responses of biochemical nitrogen indices in Ulva pertusa, several experiments were conducted in an outdoor, flow-through culture apparatus, in summer and winter. In this apparatus effects of ammonium concentration, nitrogen source (nitrate and ammonium), light and seawater motion were investigated. Of the same three N-indices examined in natural Ulva populations (free amino acids, chlorophyll and total tissue nitrogen), increases in free amino acids, particularly asparagine, provided the strongest indicator of increases in nitrogen availability. In addition, while tissue nitrogen and chlorophyll also increased with seawater nitrogen concentration, it was apparent that these indices were also strongly influenced by light, and probably season. Rates of ammonium assimilation provided no overall measure of the availability of nitrogen in seawater and were clearly affected by season. Similarly, growth rates in Ulva only showed a response to nitrogen addition in summer months. Stable isotopes of nitrogen (δ15N) in Ulva provided a clear distinction between natural and synthetic nitrogen sources, but more importantly, showed only minor fractionation (ranging from 1.3 ‰ to -1.9 ‰) of 15N supplied from synthetic nitrate and ammonium under both light-saturating and light-limiting conditions. To further develop Ulva as a standardized test-organism it was cultured in low-nutrient (non-polluted) seawater to deplete internal storage pools of nitrogen. Each month the resulting test-Ulva was then placed in surface-moored growth enclosures at a range of coastal sites around Auckland and then monitored for one year. In winter there were increases in seawater inorganic nitrogen concentrations and concomitant increases in free amino acid content. However, tissue nitrogen and chlorophyll content in test-Ulva showed similar increases (possibly saturating) across all sites suggesting that seasonal increases in these N-indices were also due to other seasonal factors (e.g., surface irradiance and / or seawater temperature). On the other hand, the total free amino acid pool showed strong differences between a low-nitrogen reference site and the other study sites all year round. It was probable that test-Ulva was integrating differences in tidally-averaged nitrogen loading that were not reliably detected in instantaneous seawater samples. In addition to N-indices in test-Ulva, levels of tissue heavy metals and stable isotopes of nitrogen showed strong differences with higher values of both typically found in urban environments compared with values found in non-polluted reference sites. It is concluded that several abiotic and biotic factors affect nitrogen status in Ulva, but the average nitrogen concentration in seawater, and the physical factors of temperature, light and water motion, appear to be the overarching determinants. It is further suggested that in combination with Ulva tissue δ15N values, tissue nitrogen and the free amino acid pool, as quantitative biochemical measures of nitrogen availability, are likely to provide useful information on both the amount and composition of nitrogen entering coastal environments. / Foundation for Research, Science and Technology. Auckland Regional Council.

The following research has focused on the utility of Ulva as an indicator of seawater nitrogen loading. Evaluation was made in three ways: 1) Observation of large-scale geographic variation in nitrogen status in natural populations around New Zealand in summer and winter, 2) Laboratory-based experimental assessment of the biochemical responses of N-indices in Ulva to nitrogen enrichment, and 3) Culturing standardized test-Ulva under low nutrient conditions which could be deployed into a variety of field situations. Seawater inorganic nutrient (nitrate, nitrite, ammonium and phosphate) concentrations and nitrogen (N)-indices (free amino acids, chlorophyll and total tissue nitrogen) in natural Ulva populations from 32 sites around New Zealand were compared. Sites were divided into 6 environmental categories: sheltered rural, exposed rural, rock pools, sheltered urban, exposed urban, and nitrogen-enriched urban sites. Seawater nutrient concentrations were highly variable between all sites in summer and winter. However, in the summer enriched urban sites had the highest mean total inorganic nitrogen concentrations and Ulva with the highest mean levels of all N-indices compared with any other environmental category. In the winter, Ulva contained more nitrogen (reflected in all N-indices) compared with Ulva in the summer, particularly in populations growing in colder southern seawater on more exposed coasts. The increase in Ulva N-status was not explained by increased seawater inorganic nitrogen concentrations. With univariate and multivariate statistical approaches it was shown that there was a significant effect of seawater temperature and site exposure on N-status in Ulva. Compared with other N-indices, stable nitrogen isotopes (δ15N) from Ulva growing in enriched urban sites had the widest range (4.77 ± 0.04 ‰ to 15.16 ± 0.03 ‰) of values compared with all other categories in both summer and winter. Conversely, Ulva from exposed rural sites had the lowest range of δ15N values compared with any other category (6.7 ± 0.1 to 8.8 ± 0.1 ‰) and showed no seasonal change in mean values (7.8 ‰ and 7.6 ‰ for summer and winter, respectively). In addition, δ15N values in Ulva were the only N-index that showed a significant difference between urban and rural categories. To test the relationship between inorganic nitrogen concentration in seawater and the responses of biochemical nitrogen indices in Ulva pertusa, several experiments were conducted in an outdoor, flow-through culture apparatus, in summer and winter. In this apparatus effects of ammonium concentration, nitrogen source (nitrate and ammonium), light and seawater motion were investigated. Of the same three N-indices examined in natural Ulva populations (free amino acids, chlorophyll and total tissue nitrogen), increases in free amino acids, particularly asparagine, provided the strongest indicator of increases in nitrogen availability. In addition, while tissue nitrogen and chlorophyll also increased with seawater nitrogen concentration, it was apparent that these indices were also strongly influenced by light, and probably season. Rates of ammonium assimilation provided no overall measure of the availability of nitrogen in seawater and were clearly affected by season. Similarly, growth rates in Ulva only showed a response to nitrogen addition in summer months. Stable isotopes of nitrogen (δ15N) in Ulva provided a clear distinction between natural and synthetic nitrogen sources, but more importantly, showed only minor fractionation (ranging from 1.3 ‰ to -1.9 ‰) of 15N supplied from synthetic nitrate and ammonium under both light-saturating and light-limiting conditions. To further develop Ulva as a standardized test-organism it was cultured in low-nutrient (non-polluted) seawater to deplete internal storage pools of nitrogen. Each month the resulting test-Ulva was then placed in surface-moored growth enclosures at a range of coastal sites around Auckland and then monitored for one year. In winter there were increases in seawater inorganic nitrogen concentrations and concomitant increases in free amino acid content. However, tissue nitrogen and chlorophyll content in test-Ulva showed similar increases (possibly saturating) across all sites suggesting that seasonal increases in these N-indices were also due to other seasonal factors (e.g., surface irradiance and / or seawater temperature). On the other hand, the total free amino acid pool showed strong differences between a low-nitrogen reference site and the other study sites all year round. It was probable that test-Ulva was integrating differences in tidally-averaged nitrogen loading that were not reliably detected in instantaneous seawater samples. In addition to N-indices in test-Ulva, levels of tissue heavy metals and stable isotopes of nitrogen showed strong differences with higher values of both typically found in urban environments compared with values found in non-polluted reference sites. It is concluded that several abiotic and biotic factors affect nitrogen status in Ulva, but the average nitrogen concentration in seawater, and the physical factors of temperature, light and water motion, appear to be the overarching determinants. It is further suggested that in combination with Ulva tissue δ15N values, tissue nitrogen and the free amino acid pool, as quantitative biochemical measures of nitrogen availability, are likely to provide useful information on both the amount and composition of nitrogen entering coastal environments. / Foundation for Research, Science and Technology. Auckland Regional Council.