Marine and Freshwater Cyanophages in a Laurentian Great Lake: Evidence from Infectivity Assays and Molecular Analyses of g20 Genes

While it is well established that viruses play an important role in the structure of marine microbial food webs, few studies have directly addressed their role in large lake systems. As part of an ongoing study of the microbial ecology of Lake Erie, we have examined the distribution and diversity of viruses in this system. One surprising result has been the pervasive distribution of cyanophages that infect the marine cyanobacterial isolate Synechococcus sp. strain WH7803. Viruses that lytically infect this cyanobacterium were identified throughout the western basin of Lake Erie, as well as in locations within the central and eastern basins. Analyses of the gene encoding the g20 viral capsid assembly protein (a conservative phylogenetic marker for the cyanophage) indicate that these viruses, as well as amplicons from natural populations and the ballast of commercial ships, are related to marine cyanophages but in some cases form a unique clade, leaving questions concerning the native hosts of these viruses. The results suggest that cyanophages may be as important in freshwater systems as they are known to be in marine systems.     

The effects of predation and unionid burrowing on bivalve communities in a Laurentian Great Lake coastal wetland

Unionid (Mollusca: Unionidae) densities have declined dramatically throughout the Laurentian Great Lakes after the introduction of dreissenid mussels (Mollusca: Dreissenidae). Recent surveys in some Great Lake coastal wetlands have found abundant unionid populations, but the factors that reduce zebra mussels on unionids in these habitats are not well understood. In 2001–2002, we tested effects of predation and unionid burrowing on corbiculids, sphaeriids and dreissenids in a Great Lake coastal wetland in western Lake Erie. In one experiment, we reduced access by molluscivores using exclosures with two mesh sizes (1.3 cm × 1.3 cm; 5 cm × 10 cm) and sampled bivalves after 15 months. Small mesh exclosures had higher numbers of dreissenids, Corbicula fluminea and sphaeriids (54.9, 3.8, 22.6 individuals/m², respectively) than large mesh exclosures (0.0, 1.13, 0.13 individuals/m², respectively) or open controls (0.3, 1.0, 0.1 individuals/m², respectively). Numbers of dreissenids on C. fluminea were higher in small mesh exclosures (3.8 dreissenids/Corbicula) than in large mesh exclosures (0.1 dreissenids/Corbicula) or cageless controls (0␣dreissenids/Corbicula). In a second experiment, we held two species of live unionids (Leptodea fragilis, Quadrula quadrula) and immobile Pyganodon grandis shells in exclosures (2.5 cm × 2.5 cm mesh) with either 5 cm, 10 cm, or 20 cm deep sediments and sampled bivalves after 2 months. There were fewer dreissenids on L. fragilis than P. grandis shells, but there was no difference in the number of dreissenids on Q. quadrula and P. grandis shells. Numbers of attached dreissenids were higher inside (189–494 dreissenids/unionid) than outside (8–11 dreissenids/unionid) exclosures, and densities of sphaeriid and C. fluminea clams were also higher inside (21.8, 4.7 individuals/m², respectively) than outside (0.4, 0.9 individuals/m², respectively) exclosures. Numbers of attached dreissenids were higher on unionids that could burrow below the sediments (20 cm depth) than unionids in shallow sediments (5 cm depth) for unexplained reasons. Our data suggest that molluscivores can play a pivotal role in limiting numbers of bivalves including dreissenids in coastal wetlands.     

Regulation of phytoplankton dynamics in a Laurentian Great Lakes estuary

The composition and dynamics of phytoplankton populations were examined in Old Woman Creek estuary, Lake Erie (USA). The centric bacillariophytes,Cyclotella atomus Hust.,Cyclotella meneghiniana Kütz., andAulacoseira alpigena (Grun.) Krammer, and the cryptophytes,Cryptomonas erosa Ehren. andRhodomonas minuta var. nannoplanctonica Skuja, dominated the phytoplankton most of the year. Chlorophytes, euglenophytes, and cyanophytes were observed less frequently. Estuarine and Lake Erie phytoplankton were considered distinct populations; lake taxa were largely confined to the estuary mouth and present only in low biomass. Maxima and minima of estuarine phytoplankton coincided with meteorological and hydrological forcing in the form of rainfall and subsequent storm-water inflows, respectively. Distinct population dynamics between the upper and lower estuary following storm events were attributed to the presence/absence of refugia serving as a source for repopulation by opportunistic taxa, fluctuating light conditions in the water column resulting from influx of particulate matter and resuspension of bottom sediments, and nutrient inputs associated with surface runoff and sub-surface interflow. Additionally, agricultural herbicides introduced by storm-water inflows potentially may affect and/or control the growth and physiological responses of individual taxa.     

Assortative mating in poison-dart frogs based on an ecologically important trait

The origin of new species can be influenced by both deterministic and stochastic factors. Mate choice and natural selection may be important deterministic causes of speciation (as opposed to the essentially stochastic factors of geographic isolation and genetic drift). Theoretical models predict that speciation is more likely when mate choice depends on an ecologically important trait that is subject to divergent natural selection, although many authors have considered such mating/ecology pleiotropy, or "magic-traits" to be unlikely. However, phenotypic signals are important in both mate choice and ecological processes such as avoiding predation. In chemically defended species, it may be that the phenotypic characteristics influencing mate choice are the same signals being used to transmit a warning to potential predators, although few studies have demonstrated this in wild populations. We tested for assortative mating between two color morphs of the Strawberry Poison-Dart Frog, Dendrobates pumilio, a group with striking geographic variation in aposematic color patterns. We found that females significantly prefer individuals of their own morph under two different light treatments, indicating strong assortative mating based on multiple coloration cues that are also important ecological signals. This study provides a rare example of one phenotypic trait affecting both ecological viability and nonrandom mating, indicating that mating/ecology pleiotropy is plausible in wild populations, particularly for organisms that are aposematically colored and visually orienting.     

Marine and freshwater cyanophages in a Laurentian Great Lake: evidence from infectivity assays and molecular analyses of g20 genes

While it is well established that viruses play an important role in the structure of marine microbial food webs, few studies have directly addressed their role in large lake systems. As part of an ongoing study of the microbial ecology of Lake Erie, we have examined the distribution and diversity of viruses in this system. One surprising result has been the pervasive distribution of cyanophages that infect the marine cyanobacterial isolate Synechococcus sp. strain WH7803. Viruses that lytically infect this cyanobacterium were identified throughout the western basin of Lake Erie, as well as in locations within the central and eastern basins. Analyses of the gene encoding the g20 viral capsid assembly protein (a conservative phylogenetic marker for the cyanophage) indicate that these viruses, as well as amplicons from natural populations and the ballast of commercial ships, are related to marine cyanophages but in some cases form a unique clade, leaving questions concerning the native hosts of these viruses. The results suggest that cyanophages may be as important in freshwater systems as they are known to be in marine systems.     

Allozymic variation in tubificid oligochaetes from the Laurentian Great Lakes

Aquatic oligochaetes are an important component of the benthos in many freshwater habitats. Yet, virtually nothing is known about the population genetics of this group. Electrophoretic surveys of allozymic variation of selected members of the Family Tubificidae were conducted at six locations in the Laurentian Great Lakes of North America. Branchiura sowerbyi was the dominant member of the oligochaete fauna at two sites, and was found to be monomorphic at all enzyme loci that were examined. In contrast, members of the genus Limnodrilus, which included L. cervix, L. claparedianus, L. hoffmeisteri, L. maumeensis, and L. udekemianus showed considerable allozymic variation at several enzyme loci. L. udekemianus exhibited tri- and tetra-allelic heterozygous electromorph banding patterns at the monomeric Pgm locus, along with unbalanced heterozygous patterns at both Pgm and the dimeric Pgi locus.Genetic distance analyses suggest that L. cervix, L. claparedianus, and L. maumeensis are closely-related (genetic identifies ranged from 0.92–0.85), and may represent subspecies rather than distinct species. Breeding studies need to confirm this assertion. This survey represents the first attempt to characterize allozymic variation of aquatic oligochaetes in North America. Additional work should focus on elucidating taxonomic ambiguities within this group via both morphological and biochemical genetic studies.     

Late-summer phytoplankton in western Lake Erie (Laurentian Great Lakes): bloom distributions,toxicity, and environmental influences

Phytoplankton abundance and composition and the cyanotoxin, microcystin, were examined relative to environmental parameters in western Lake Erie during late-summer (2003–2005). Spatially explicit distributions of phytoplankton occurred on an annual basis, with the greatest chlorophyll (Chl) a concentrations occurring in waters impacted by Maumee River inflows and in Sandusky Bay. Chlorophytes, bacillariophytes, and cyanobacteria contributed the majority of phylogenetic-group Chl a basin-wide in 2003, 2004, and 2005, respectively. Water clarity, pH, and specific conductance delineated patterns of group Chl a, signifying that water mass movements and mixing were primary determinants of phytoplankton accumulations and distributions. Water temperature, irradiance, and phosphorus availability delineated patterns of cyanobacterial biovolumes, suggesting that biotic processes (most likely, resource-based competition) controlled cyanobacterial abundance and composition. Intracellular microcystin concentrations corresponded to Microcystis abundance and environmental parameters indicative of conditions coincident with biomass accumulations. It appears that environmental parameters regulate microcystin indirectly, via control of cyanobacterial abundance and distribution.     

The development of ecosystem objectives for the Laurentian Great Lakes

Historically management of human use of ecosystems has been based around engineering and chemical approaches and through the construction of treatment facilities, effluent controls and setting chemical concentrations, both at end of pipe and in the aquatic environment. However, the general continued degradation of many ecosystems shows these approaches alone are insufficient. In the Laurentian Great Lakes the Great Lakes Water Quality Agreement was first signed in 1972 and ratified in 1978 and in 1987 tacitly acknowledged the problems with a chemical only approach by requiring the development of ecosystem objectives in the 1978 agreement. Furthermore, the agreement specifically identified numerical ecosystem objectives in the 1987 agreement. The evolution of ecosystem objectives in the Great Lakes has expanded from the strictly numerical objectives such as production of lake trout and abundance of the amphipod Pontoporeia hoyi. More recent developments in ecosystem objectives have been the inclusion of indicators for wildlife, habitat, human health and stewardship.Prepared as a discussion paper presented to the United Nations Economic Commission for Europe's seminar on an Ecosystems Approach to Water Management (May 27–31, 1991).     

A replicated climate change field experiment reveals rapid evolutionary response in an ecologically important soil invertebrate

Whether species can respond evolutionarily to current climate change is crucial for the persistence of many species. Yet, very few studies have examined genetic responses to climate change in manipulated experiments carried out in natural field conditions. We examined the evolutionary response to climate change in a common annelid worm using a controlled replicated experiment where climatic conditions were manipulated in a natural setting. Analyzing the transcribed genome of 15 local populations, we found that about 12% of the genetic polymorphisms exhibit differences in allele frequencies associated to changes in soil temperature and soil moisture. This shows an evolutionary response to realistic climate change happening over short‐time scale, and calls for incorporating evolution into models predicting future response of species to climate change. It also shows that designed climate change experiments coupled with genome sequencing offer great potential to test for the occurrence (or lack) of an evolutionary response.     

Pathway position constrains the evolution of an ecologically important pathway in aspens (Populus tremula L.)

Many ecological interactions of aspens and their relatives (Populus spp.) are affected by products of the phenylpropanoid pathway synthesizing condensed tannins (CTs), whose production involves trade‐offs with other ecologically important compounds and with growth. Genes of this pathway are candidates for investigating the role of selection on ecologically important, polygenic traits. We analysed sequences from 25 genes representing 10 steps of the CT synthesis pathway, which produces CTs used in defence and lignins used for growth, in 12 individuals of European aspen (Populus tremula). We compared these to homologs from P. trichocarpa, to a control set of 77 P. tremula genes, to genome‐wide resequencing data and to RNA‐seq expression levels, in order to identify signatures of selection distinct from those of demography. In Populus, pathway position exerts a strong influence on the evolution of these genes. Nonsynonymous diversity, divergence and allele frequency shifts (Tajima's D) were much lower than for synonymous measures. Expression levels were higher, and the direction of selection more negative, for upstream genes than for those downstream. Selective constraints act with increasing intensity on upstream genes, despite the presence of multiple paralogs in most gene families. Pleiotropy, expression level, flux control and codon bias appear to interact in determining levels and patterns of variation in genes of this pathway, whose products mediate a wide array of ecological interactions for this widely distributed species.     

Limited influence of a wind power project submarine cable on a Laurentian Great Lakes fish community

Previous research has identified the generation of electromagnetic fields (EMFs) emanating from renewable energy project transmission cables to be a potential stressor to aquatic communities. In this study, we investigated whether the presence of a high voltage submarine transmission cable affected the spatial pattern and composition of nearshore and offshore fishes at a Laurentian Great Lakes site. The transmission cable investigated in this study runs 7.8 km along the lakebed of Lake Ontario, carrying electricity from the Wolfe Island wind power project to the city of Kingston, Ontario. In autumn of 2011, both nearshore electrofishing and deeper‐water fisheries acoustic surveys were conducted along transects at varying distances to the cable. For both habitat types, no detectable effects of the cable on the fish community were found. Local habitat variables, including substrate or depth, were more important in explaining variation in fish density than proximity to the cable. Common species encountered during the surveys were round goby (Neogobius melanostomus) in the nearshore and alewife (Alosa pseudoharengus) in the deeper channel. American eel (Anguilla rostrata), thought to be an electromagnetically sensitive species, was also encountered during the surveys including in close proximity to the cable. More robust impact assessments require sampling fishes before a cable installation, over greater time frames (additional seasons or years), and habitats that support more diverse native assemblages.     

Recolonization and possible recovery of Burrowing Mayflies (Ephemeroptera: Ephemeridae: Hexagenia spp.) in Lake Erie of the Laurentian Great Lakes

Burrowing mayflies of the genus Hexagenia spp. were widely distributed (ca. 80% of sites) and abundant (ca. 160 nymphs/m²) in the western basin of Lake Erie of the Laurentian Great Lakes in 1929–1930, prior to a period of anoxia in the mid 1950s. Nymphs were absent or rare in the basin between 1961 and 1973–1975. In 1979–1991, nymphs were infrequently found (13–46% of sites) in low abundance (3–40 nymphs/m²) near shore (<7.5 km from shore), but were absent or rare offshore (0–7% of sites at 0–1 nymphs/m²). Increased abundance occurred offshore between 1991 (0% of sites) and 1993 (52% of sites at 7/m²). Annual sampling, beginning in 1995, indicates that nymphs increased in both nearshore and offshore waters. By 1997, nymphs were found throughout the lake (88% of sites) at a mean density 40-fold greater (392/m²) than that observed in 1993 (11/m²). In 1998, the distribution of nymphs remained the same as 1997 (88% of sites) but density declined 3-fold (392 to 134/m²). These data indicate that mayflies have recolonized sediments of western Lake Erie and that their abundance may be similar to levels observed before their disappearance in the mid 1950s. However, prior to the mid 1950s, densities were greater in offshore than nearshore waters, but between 1979 and 1998 greater densities occurred near shore than offshore. In addition, there were two areas in the 1990s where low densities consistently occurred. Therefore, recovery of nymphs in western Lake Erie may not have been complete in 1998. At present we do not know the cause for the sudden recolonization of nymphs in large portions of western Lake Erie. Undoubtedly, pollution-abatement programs contributed to improved conditions that would have ultimately led to mayfly recovery in the future. However, the explosive growth of the exotic zebra mussel, Dreissena polymorpha, undoubtedly diverted plankton foods to bottom substrates which could have increased the speed at which Hexagenia spp. nymphs recolonized sediments in western Lake Erie in the 1990s.     

Characterising functionally important and ecologically meaningful genetic diversity using a candidate gene approach

Over the past two decades the fields of molecular ecology and population genetics have been dominated by the use of putatively neutral DNA markers, primarily to resolve spatio-temporal patterns of genetic variation to inform our understanding of population structure, gene flow and pedigree. Recent emphasis in comparative functional genomics, however, has fuelled a resurgence of interest in functionally important genetic variation that underpins phenotypic traits of adaptive or ecological significance. It may prove a major challenge to transfer genomics information from classical model species to examine functional diversity in non-model species in natural populations, but already multiple gene-targeted candidate loci with major effect on phenotype and fitness have been identified. Here we briefly describe some of the research strategies used for isolating and characterising functional genetic diversity at candidate gene-targeted loci, and illustrate the efficacy of some of these approaches using our own studies on red grouse (Lagopus lagopus scoticus). We then review how candidate gene markers have been used to: (1) quantify genetic diversity among populations to identify those depauperate in genetic diversity and requiring specific management action; (2) identify the strength and mode of selection operating on individuals within natural populations; and (3) understand direct mechanistic links between allelic variation at single genes and variance in individual fitness.     

Phylogenetic revision of the Strophomenida,a diverse and ecologically important Palaeozoic brachiopod order

The order Strophomenida was an ecologically abundant and taxonomically diverse group of Palaeozoic brachiopods that originated in the earliest Ordovician and went extinct in the Carboniferous. During their long geological range, the Strophomenida survived two of the ‘Big Five’ mass extinction events, the Late Ordovician and the Late Devonian, suggesting that they are potentially informative taxa for studying the evolutionary effects of these two distinct mass extinctions, each with drastically different forcing mechanisms. However, while there have been previous phylogenetic studies on smaller groups within the Strophomenida, the phylogenetic relationships of the whole group are still largely unknown. The group has been divided into two major superfamilies, the Strophomenoidea (strophomenoids) and the Plectambonitoidea (plectambonitoids). Despite being treated as separate clades, the plectambonitoids may form a paraphyletic grade into the strophomenoids. We present a detailed higher‐level parsimony‐based phylogenetic analysis of the Strophomenida, consisting of 69 characters and 62 exemplar species sampled from the majority of the taxonomically defined families/subfamilies. Several species of basal chonetids (strophochonetids) were also included in this analysis, as they may be closely related to the Strophomenida and share several characters with both the plectambonitoids and strophomenoids. The phylogenetic analysis suggests the plectambonitoids, as originally defined, are paraphyletic to the monophyletic strophomenoids. The basal chonetids are reconstructed as a monophyletic group that is sister to the strophomenoids, suggesting that their proper placement might be within the Strophomenida. The topology also suggests that at least 17 of the taxonomically defined strophomenoid and plectambonitoid families are likely to be monophyletic. The Plectambonitidae and the Taffiidae as defined are paraphyletic, and the Grorudiidae and Leptostrophiidae are polyphyletic. Furthermore, subfamilies Leptodontellinae, Dicoelostrophiinae, Palaeostrophomeninae and Aegiromeninae are raised to the level of family. When analysed within this phylogenetic context, the Late Ordovician mass extinction event had little effect on the large‐scale evolution of the group.     

Forest transition in an ecologically important region: Patterns and causes for landscape dynamics in the Niger Delta

The influence of human related actives such as oil and gas exploration, intensified logging of trees and over exploitation of forest resources for food, have negatively impacted the once flourishing and ecologically diverse forest system of the Niger Delta region in Nigeria. Relevant information on the transitional changes of forested landscapes in the delta is poor compared to other tropical forests such as the Brazilian and Columbian Amazonian forest where numerous research studies have been conducted. Consequently, this study aimed at investigating the spatial extent and rates of forest transition in the Niger Delta region taking into consideration the patterns, causes and implications of the landscape dynamics. The study determined the spatial extent and rates of forest transition in the study area using remotely sensed data from 1986 and 2007. The results indicated that the spatial extent of deforestation, unchanged forest cover and afforestation were 1.38, 2.39, and 1.15 million hectares, respectively, while the annual deforestation and afforestation rates were 0.95 and 0.75% which are high compared to other areas in the humid tropics. The annual rate of change in forest cover was determined as ?0.13% indicating an overall reduction in the spatial extent of forest cover for the entire delta. Changes in the spatial structure of forests were investigated using landscape metrics and the results showed there was a substantial increase in forest fragmentation. The variations in population dynamics and poverty indicators between different states of the Niger Delta were unable to explain the observed patterns of forest change. Instead, the authors observed that the main determinants of forest dynamics were the variations in state forest management policies and the influence of the oil and gas industry on the economies of the states. High rates of afforestation were found in states that have limited oil resources and were more economically dependent on forest products, while states with high deforestation rates were found in the main oil-producing parts of the study site. Using the present trend of forest transition dynamics, a 20-year forward simulation was generated using the Markov algorithm. The results concerning forest transition in the study area point to the urgent need for appropriate environmental policy development and implementation for the Niger Delta region.     

Bacterial symbionts as mediators of ecologically important traits of insect hosts

1. Bacterial symbionts play a prominent role in insect nutritional ecology by aiding in digestion of food or providing nutrients that are limited or lacking in the diet. Thereby, endosymbionts open niches to their insect host that would otherwise be unavailable. 2. Currently, several other ecologically relevant traits mediated by endosymbionts are being investigated, including enhanced parasite resistance, enhanced heat tolerance, and influences on insect–plant interactions such as manipulation of plant physiology to the benefit of the insect. 3. Traits mediated by endosymbionts are often identified by correlative studies where traits are found to be altered in the presence of a particular symbiont. Recent developments in genomic tools offer the opportunity for studying the impact of bacteria–insect symbioses under natural conditions in a population and community ecology context. In vivo experiments specifically testing putative functions of endosymbionts in parallel to population‐level studies on the prevalence of endosymbionts allow disentangling host versus symbiont contribution to phenotypic variability observed in individuals. Effects of symbionts on host phenotype are often large and relevant to host fitness, e.g. by significantly enhancing survival or fecundity in a context‐dependent manner. 4. Predominantly vertically transmitted endosymbionts contribute to the heritable genetic variation present in a host species. Phenotypic variation on which selection can act may be due to differences either among host genomes, symbiont genomes, or genotype × genotype interactions. Therefore the holobiont, i.e. the host including all symbionts, should be regarded as the unit of selection as the association between host and symbionts may affect the fitness of the holobiont depending on the environment.     

Ultrastructural and molecular characterization of a bacterial symbiosis in the ecologically important scale insect family Coelostomidiidae

Scale insects are important ecologically and as agricultural pests. The majority of scale insect taxa feed exclusively on plant phloem sap, which is carbon rich but deficient in essential amino acids. This suggests that, as seen in the related aphids and psyllids, scale insect nutrition might also depend upon bacterial symbionts, yet very little is known about scale insect-bacteria symbioses. We report here the first identification and molecular characterization of symbiotic bacteria associated with the New Zealand giant scale Coelostomidia wairoensis, using fluorescence in situ hybridization (FISH), transmission electron microscopy (TEM) and 16S rRNA gene-based analysis. Dissection and FISH confirmed the location of the bacteria in large, paired, multilobate organs in the abdominal region of the insect. TEM indicated that the dominant pleomorphic bacteria were confined to bacteriocytes in the sheath-enclosed bacteriome. Phylogenetic analysis revealed the presence of three distinct bacterial types, the bacteriome-associated B-symbiont (Bacteroidetes), an Erwinia-related symbiont (Gammaproteobacteria) and Wolbachia sp. (Alphaproteobacteria). This study extends the current knowledge of scale insect symbionts and is the first microbiological investigation of the ecologically important coelostomidiid scales.     

Microbial communities of the Laurentian Great Lakes reflect connectivity and local biogeochemistry

The Laurentian Great Lakes are a vast, interconnected freshwater system spanning strong physicochemical gradients, thus constituting a powerful natural laboratory for addressing fundamental questions about microbial ecology and evolution. We present a comparative analysis of pelagic microbial communities across all five Laurentian Great Lakes, focusing on Bacterial and Archaeal picoplankton characterized via 16S rRNA amplicon sequencing. We collected samples throughout the water column from the major basins of each lake in spring and summer over 2 years. Two oligotypes, classified as LD12 (Alphaproteobacteria) and acI-B1 (Actinobacteria), were among the most abundant in every sample. At the same time, microbial communities showed distinct patterns with depth during summer stratification. Deep hypolimnion samples were frequently dominated by a Chloroflexi oligotype that reached up to 19% relative abundance. Stratified surface communities differed between the colder, less productive upper lakes (Superior, Michigan, Huron) and warmer, more productive lower lakes (Erie, Ontario), in part due to an Actinobacteria oligotype (acI-C2) that averaged 7.7% of sequences in the lower lakes but <0.2% in the upper lakes. Together, our findings suggest that both hydrologic connectivity and local selective pressures shape microbial communities in the Great Lakes and establish a framework for future investigations.     

Dynamics of organohalogen production by the ecologically important fungus Hypholoma fasciculare

The ecologically important white rot basidiomycete Hypholoma fasciculare was previously shown to produce large amounts of adsorbable organic halogens (AOX). The purposes of this study were to identify the time period of AOX production in relation to the primary and secondary metabolic phases of the growth cycle of the fungus, to determine the maximal specific AOX production rates and final AOX yields on the different substrates and to account for the measured AOX in identifiable compounds. The AOX production was observed to take place during the transition between the primary and secondary metabolic phases of the growth cycle of the fungus. The maximum AOX production rates ranged from 0.63 to 3.23 mg AOX per gram of dry mycelium per day and the final AOX yields ranged from 0.88 and 1.50 percent of dry weight of mycelium on five different substrates including natural woody substrates. The AOX produced by the fungus was stable in all five substrates, even after prolonged incubation periods. However, the composition of the AOX changed drastically. Initially most of the AOX was accounted for by the compound 3,5-dichloro-p-anisyl alcohol; however, after prolonged incubation this compound was largely converted into 3,5-dichloro-p-anisic acid in N-rich medium and into unidentified organohalogens in N-limited medium.