Phylogenetic divergence in a local population of the ectomycorrhizal fungus Cenococcum geophilum

Cenococcum geophilum is a widely distributed mycorrhizal species associated with diverse gymnosperm and angiosperm hosts. In previous studies, a significant amount of genetic and genotypic diversity has been detected in this species, despite the fact that C. geophilum is not thought to reproduce by meiotic or mitotic spores. We conducted a phylogenetic analysis of 103 C. geophilum isolates from a California oak woodland and seven non-California isolates using a glyceraldehyde 3-phosphate dehydrogenase gene. In addition, a subset of isolates was analyzed using sequences from ITS-rDNA, a Group I intron located in the 3' end of the SSU-rDNA and a portion of the mitochondrial SSU-rDNA. Phylogenetically distinct lineages, or cryptic species, of C. geophilum were detected at the scale of a single soil sample within our field site. As much genetic diversity was found within a soil sample as was found for isolates collected across the USA. Our results help explain the large amount of physiological, phenotypic, and genetic differences reported among isolates of C. geophilum from similar as well as diverse geographic regions. The ecological role that these sympatric cryptic species play remains to be determined.     

Genetic diversity and differential in vitro responses to Ni in <Emphasis Type="Italic">Cenococcum geophilum</Emphasis> isolates from serpentine soils in Portugal

Amplified fragment length polymorphism (AFLP) analysis was used to investigate the genetic diversity in isolates of the ectomycorrhizal fungus Cenococcum geophilum from serpentine and non-serpentine soils in Portugal. A high degree of genetic diversity was found among C. geophilum isolates; AFLP fingerprints showed that all the isolates were genetically distinct. We also assessed the in vitro Ni sensitivity in three serpentine isolates and one non-serpentine isolate. Only the non-serpentine isolate was significantly affected by the addition of Ni to the growth medium. At 30 microg g(-1) Ni, radial growth rate and biomass accumulation decreased to 73.3 and 71.6% of control, respectively, a highly significant inhibitory effect. Nickel at this concentration had no significant inhibitory effect on serpentine isolates, and so the fitness of serpentine isolates, as evaluated by radial growth rate and biomass yield, is likely unaffected by Ni in the field. In all isolates, the Ni concentration in the mycelia increased with increasing Ni concentration in the growth medium, but two profiles of Ni accumulation were identified. One serpentine isolate showed a linear trend of Ni accumulation. At the highest Ni exposure, the concentration of Ni in the mycelium of this isolate was in the hyperaccumulation range for Ni as defined for higher plants. In the remaining isolates, Ni accumulation was less pronounced and seems to approach a plateau at 30 microg g(-1) Ni. Because two profiles of Ni accumulation emerged among our Ni-insensitive serpentine isolates, this result suggests that different Ni detoxification pathways may be operating. The non-serpentine isolate whose growth was significantly affected by Ni was separated from the other isolates in the genetic analysis, suggesting a genetic basis for the Ni-sensitivity trait. This hypothesis is further supported by the fact that all isolates were maintained on medium without added Ni to avoid carry-over effects. However, because AFLP analysis failed to distinguish between serpentine and non-serpentine isolates, we cannot conclude that Ni insensitivity among our serpentine isolates is due to evolutionary adaptation. Screening a larger number of isolates, from different geographical origins and environments, should clarify the relationships between genetic diversity, morphology, and physiology in this important species.     

Molecular evolution and quantitative variation for chemosensory behaviour in the nematode genus Caenorhabditis

Caenorhabditis elegans is a model organism in biology, yet despite the tremendous information generated from genetic, genomic and functional analyses, C. elegans has rarely been used to address questions in ecological genetics. Here, we analyse genetic variation for chemosensory behaviour, an ecologically important trait that is also genetically well characterized, at both the phenotypic and molecular levels within three species of the genus Caenorhabditis. We show that the G-protein ODR-3 plays an important role in chemosensory avoidance behaviour and identify orthologues of odr-3 in C. briggsae and C. remanei. Both quantitative genetic analysis of chemosensory behaviour and molecular population genetic analysis of odr-3 show that there is little genetic variation among a worldwide collection of isolates of the primarily selfing C. elegans, whereas there is substantially more variation within a single population of the outcrossing C. remanei. Although there are a large number of substitutions at silent sites within odr-3 among the three species, molecular evolution at the protein level is extremely conserved, suggesting that odr-3 plays an important role in cell signalling during chemosensation and/or neuronal cilia development in C. remanei and in C. briggsae as it does in C. elegans. Our results suggest that C. remanei may be a more suitable subject for ecological and evolutionary genetic studies than C. elegans.     

土生空团菌的遗传多样性分析

为了研究土生空团菌的遗传多样性,对来自中国、美国、瑞士和法国等菌株的rDNA ITS区进行序列分析,利用Popgene32和phylip软件进行数据计算和聚类分析。序列分析表明土生空团菌rDNA ITS区的序列长度为422–447bp,遗传距离在0.000–0.051之间。居群结构和聚类分析结果表明:(1)土生空团菌有一定的遗传多样性,且遗传差异主要来自于居群内;(2)基因流Nm>1,遗传漂变不是导致土生空团菌居群遗传分化的主要因素;(3)土生空团菌的遗传分化受到地理环境的影响,而与宿主来源没有明显相关性。     

Genetic structure of Cenococcum geophilum populations in primary successional volcanic deserts on Mount Fuji as revealed by microsatellite markers

Polymorphic simple sequence repeat (SSR) markers were used to investigate the genetic structure in a Cenococcum geophilum population associated with Salix reinii in an early successional volcanic desert at Gotenba, on the south-eastern slope of Mount Fuji in Japan, and in three other populations associated with the same host at more developed sites on the mountain, one at Fujinomiya and two at Subashiri. The genotype richness of C. geophilum tended to be higher in more developed vegetation patches as well as in more developed sites, suggesting that genotype richness increased with advanced succession because new genotypes might have been introduced into these sites over time. High genotypic similarity was observed between the Gotenba and Fujinomiya populations but not between the Gotenba and Subashiri populations, suggesting that C. geophilum genotypes in Gotenba were introduced from the direction of Fujinomiya. Genotypes in the Gotenba population were clearly distinguishable into two groups. The absence of any intermediate genotype suggests the absence of frequent recombination in this C. geophilum population associated with early successional vegetation.     

A review of the molecular evidence for ballast water introduction of the toxic dinoflagellates Gymnodinium catenatum and the Alexandrium “tamarensis complex” to Australasia

The potential of ballast water to act as a major introduction vector for toxic dinoflagellates and other phytoplankton is beyond doubt; however, evidence that links the suspected introduced species with a source population is less convincing, especially without supporting historical and biochemical data, or consideration of palaeobiogeographical scenarios that may explain current species distributions. This paper presents new molecular data based on LSU-rDNA and rDNA-ITS sequences that demonstrate an unequivocal and recent link between Temperate Asian and Australasian populations of the toxic dinoflagellates Gymnodinium catenatum and toxic strains of the Alexandrium “tamarensis complex”. We integrate our data with supporting evidence from historical distribution records, sediment dating studies, toxin profiles, mating studies and previous molecular studies. We contrast the observed patterns of genetic and biochemical variation with those expected from various palaeobiogeographical scenarios explaining the evolution and natural dispersal of both species. While definitive proof is impossible, the total evidence indicates that these toxic dinoflagellates were introduced to Australasia during the past 100 years, most probably via ballast water from bulk-cargo shipping from Japan and/or south-east Asia.     

Genetic diversity of Vibrio cholerae in Chesapeake Bay determined by amplified fragment length polymorphism fingerprinting

Vibrio cholerae is indigenous to the aquatic environment, and serotype non-O1 strains are readily isolated from coastal waters. However, in comparison with intensive studies of the O1 group, relatively little effort has been made to analyze the population structure and molecular evolution of non-O1 V. cholerae. In this study, high-resolution genomic DNA fingerprinting, amplified fragment length polymorphism (AFLP), was used to characterize the temporal and spatial genetic diversity of 67 V. cholerae strains isolated from Chesapeake Bay during April through July 1998, at four different sampling sites. Isolation of V. cholerae during the winter months (January through March) was unsuccessful, as observed in earlier studies (J. H. L. Kaper, R. R. Colwell, and S. W. Joseph, Appl. Environ. Microbiol. 37:91-103, 1979). AFLP fingerprints subjected to similarity analysis yielded a grouping of isolates into three large clusters, reflecting time of the year when the strains were isolated. April and May isolates were closely related, while July isolates were genetically diverse and did not cluster with the isolates obtained earlier in the year. The results suggest that the population structure of V. cholerae undergoes a shift in genotype that is linked to changes in environmental conditions. From January to July, the water temperature increased from 3 degrees C to 27.5 degrees C, bacterial direct counts increased nearly an order of magnitude, and the chlorophyll a concentration tripled (or even quadrupled at some sites). No correlation was observed between genetic similarity among isolates and geographical source of isolation, since isolates found at a single sampling site were genetically diverse and genetically identical isolates were found at several of the sampling sites. Thus, V. cholerae populations may be transported by surface currents throughout the entire Bay, or, more likely, similar environmental conditions may be selected for a specific genotype. The dynamic nature of the population structure of this bacterial species in Chesapeake Bay provides new insight into the ecology and molecular evolution of V. cholerae in the natural environment.     

Genetic diversity in Tetrachaetum elegans, a mitosporic aquatic fungus

Tetrachaetum elegans Ingold is a saprobic aquatic hyphomycete for which no sexual stage has yet been described. It occurs most commonly during the initial decay of tree leaves in temperate freshwater habitats and typically sporulates under water. Dispersal of the aquatic fungus takes place primarily in the water column and has a large passive component. Differences in substrate composition (e.g. quality of leaf litter) may also play a role in the distribution of different species or genotypes. The population genetic structure of T. elegans was studied using amplified fragment length polymorphism (AFLP) multilocus fingerprints. The populations were isolated from the leaf litter of three different tree genera, sampled in nine streams distributed throughout a mixed deciduous forest. Molecular markers were developed for 97 monosporic isolates using four selective primer pairs. A total of 247 fragments were scored, of which only 32 were polymorphic. Significant stream differentiation was detected for the isolates considered in this study. Analysis of molecular variance revealed that 20% of the genetic variation observed was the result of differences between streams. No correlation between genetic and geographical distances was found but a few multilocus genotypes were observed in different locations. Altogether these results suggest that environmental barriers play a role in the population structure of this aquatic fungus. No clear-cut effect of leaf litter composition on genetic variation could be demonstrated. Finally, tests of linkage disequilibrium between the 32 polymorphic AFLP loci as well as simulations did not provide a final answer regarding clonality in T. elegans. Indeed, it was possible to reject linkage equilibrium at different sampling levels and show that full linkage was unlikely.     

黄淮海地区玉蜀黍黑粉菌群体遗传结构分析

利用17个多态性ISSR引物对2017年在我国黄淮海地区采集的41株玉蜀黍黑粉菌Ustilago maydis样本进行了种群遗传结构分析。基于UPGMA法和贝叶斯模型对玉蜀黍黑粉菌群体遗传结构分析显示,采集的样本可以分为两大群,且种群的划分表现出了与来源地理纬度的一致性。因此按照各样本所属地理纬度将样本分为北纬33°-34°、北纬35°-36°、北纬37°-38° 3大种群。结果表明,分离自北纬33°-34°地区的玉蜀黍黑粉菌遗传多样性最丰富,Nei’s基因多样性（H）为0.2216,Shannon指数（I）为0.3300。分子方差分析（AMOVA）显示,变异中来自种群内部的变异为86%,种群间变异14%（φpt=0.141）。     

Evidence of host-associated populations of Cryptosporidium parvum in Italy

Recent studies have revealed extensive genetic variation among isolates of Cryptosporidium parvum, an Apicomplexan parasite that causes gastroenteritis in both humans and animals worldwide. The parasite's population structure is influenced by the intensity of transmission, the host-parasite interaction, and husbandry practices. As a result, C. parvum populations can be panmictic, clonal, or even epidemic on both a local scale and a larger geographical scale. To extend the study of C. parvum populations to an unexplored region, 173 isolates of C. parvum collected in Italy from humans and livestock (calf, sheep, and goat) over a 10-year period were genotyped using a multilocus scheme based on 7 mini- and microsatellite loci. In agreement with other studies, extensive polymorphism was observed, with 102 distinct multilocus genotypes (MLGs) identified among 173 isolates. The presence of linkage disequilibrium, the confinement of MLGs to individual farms, and the relationship of many MLGs inferred using network analysis (eBURST) suggest a predominantly clonal population structure, but there is also evidence that part of the diversity can be explained by genetic exchange. MLGs from goats were found to differ from bovine and sheep MLGs, supporting the existence of C. parvum subpopulations. Finally, MLGs from isolates collected between 1997 and 1999 were also identified as a distinct subgroup in principal-component analysis and eBURST analysis, suggesting a continuous introduction of novel genotypes in the parasite population.     

A re-examination of the phylogenetic relationship between the causal agents of carrot black rot, Alternaria radicina and A. carotiincultae

The phylogenetic relationship between Alternaria radicina and A. carotiincultae was reexamined based on morphology, sequence analysis of rDNA (ITS and mitochondrial small subunit [mtSSU]), protein coding genes (actin [ACT], beta-tubulin, chitin synthase [CHS], translation elongation factor [EF-1a], Alternaria allergen a1 [Alt a1], and glyceraldehyde-3-phosphate dehydrogenase [gpd]), and RAPD and ISSR analysis of total genomic DNA. Although some morphological characters overlapped to a degree, with A. radicina isolates expressing moderate variation and A. carotiincultae isolates being highly uniform, A. carotiincultae could be differentiated from A. radicina based on significantly greater growth rate on potato dextrose agar (PDA) or acidified PDA (APDA) and average number of transverse septa per conidium. Sequence of rDNA and two protein coding genes, ACT and CHS, were invariant between species. However polymorphism with the EF-1a, beta-tubulin, and Alt a1 gene strictly separated the population of A. radicina and A. carotiincultae as distinct lineages, as did RAPD and ISSR analysis. The polymorphic gpd gene did not strictly separate the two species. However isolates of A. radicina encompassed several haplotypes, one of which was the exclusive haplotype possessed by A. carotiincultae isolates, suggesting evidence of incomplete lineage sorting. The results suggest that A. carotiincultae is closely related to A. radicina but is a recently divergent and distinct lineage, which supports its status as a separate species.     

Population genetic structure of Carchesium polypinum (Ciliophora: Peritrichia) in four Chinese lakes inferred from ISSR fingerprinting: high diversity but low differentiation

Although the peritrichous ciliate Carchesium polypinum is common in freshwater, its population genetic structure is largely unknown. We used inter-simple sequence repeat (ISSR) fingerprinting to analyze the genetic structure of 48 different isolates of the species from four lakes in Wuhan, central China. Using eight polymorphic primers, 81 discernible DNA fragments were detected, among which 76 (93.83%) were polymorphic, indicating high genetic diversity at the isolate level. Further, Nei's gene diversity (h) and Shannon's Information index (I) between the different isolates both revealed a remarkable genetic diversity, higher than previously indicated by their morphology. At the same time, substantial gene flow was found. So the main factors responsible for the high level of diversity within populations are probably due to conjugation (sexual reproduction) and wide distribution of swarmers. Analysis of molecular variance (AMOVA) showed that there was low genetic differentiation among the four populations probably due to common ancestry and flooding events. The cluster analysis and principal component analysis (PCA) suggested that genotypes isolated from the same lake displayed a higher genetic similarity than those from different lakes. Both analyses separated C. polypinum isolates into subgroups according to the geographical locations. However, there is only a weak positive correlation between the genetic distance and geographical distance, suggesting a minor effect of geographical distance on the distribution of genetic diversity between populations of C. polypinum at the local level. In conclusion, our studies clearly demonstrated that a single morphospecies may harbor high levels of genetic diversity, and that the degree of resolution offered by morphology as a marker for measuring distribution patterns of genetically distinct entities is too low.     

Isolation of new species of the genus Actinomadura on selective media with streptomycin and bruneomycin]

In the screening programme using selective media with streptomycin (25, 50 and 100 gamma/ml) and bruneomycin (0.5, 1 and 2 gamma/ml) 3 unusual actinomycetous cultures forming short, rarely long chains of straight, hook-like or spiral spores with 1 or rarely 2 turns were isolated. The study of the cell wall composition of the isolates showed that the walls contained meso-diaminopimelic acid and a sugar madurose. On the basis of the morphological structure and the cell wall composition the isolates were classified as belonging to Actinomadura. Comparison of the cultures with the Actinomadura species described in the literature showed that the isolates were new species of the genera, i. e. Am. coerulea sp. nov., Am. cremea sp. nov. and Am. salmonea sp. nov.     

Population genetics of Caenorhabditis elegans: the paradox of low polymorphism in a widespread species

Caenorhabditis elegans has become one of the most widely used model research organisms, yet we have little information on evolutionary processes and recent evolutionary history of this widespread species. We examined patterns of variation at 20 microsatellite loci in a sample of 23 natural isolates of C. elegans from various parts of the world. One-half of the loci were monomorphic among all strains, and overall genetic variation at microsatellite loci was low, relative to most other species. Some population structure was detected, but there was no association between the genetic and geographic distances among different natural isolates. Thus, despite the nearly worldwide occurrence of C. elegans, little evidence was found for local adaptation in strains derived from different parts of the world. The low levels of genetic variation within and among populations suggest that recent colonization and population expansion might have occurred. However, the patterns of variation are not consistent with population expansion. A possible explanation for the observed patterns is the action of background selection to reduce polymorphism, coupled with ongoing gene flow among populations worldwide.     

New species and unexpected diversity of socially parasitic bees in the genus Inquilina Michener (Hymenoptera: Apoidea: Apidae)

Abstract  Allodapine bees present particular problems for taxonomy due to a high level of morphological conservatism in adults, even between genera. However, this tribe of bees also presents a unique opportunity to explore the evolution of social parasitism because of the comparatively large number of origins of socially parasitic species. Morphological differences presented here, along with DNA sequence data and molecular phylogenetic analyses, indicate a much larger number of Australian social parasite species in the genus Inquilina than previously anticipated, and suggest that the final number of socially parasitic species may be considerable. We describe five new species and present sequence data that will help elucidate the delineation of further new species. Inquilina provides a unique opportunity to study the evolution of social parasitism in social insects, but further studies will need to encompass both population genetic and phylogenetic approaches.     

Patterns of genetic variation within and between Gibbon species

Gibbons are small, arboreal, highly endangered apes that are understudied compared with other hominoids. At present, there are four recognized genera and approximately 17 species, all likely to have diverged from each other within the last 5-6 My. Although the gibbon phylogeny has been investigated using various approaches (i.e., vocalization, morphology, mitochondrial DNA, karyotype, etc.), the precise taxonomic relationships are still highly debated. Here, we present the first survey of nuclear sequence variation within and between gibbon species with the goal of estimating basic population genetic parameters. We gathered ~60 kb of sequence data from a panel of 19 gibbons representing nine species and all four genera. We observe high levels of nucleotide diversity within species, indicative of large historical population sizes. In addition, we find low levels of genetic differentiation between species within a genus comparable to what has been estimated for human populations. This is likely due to ongoing or episodic gene flow between species, and we estimate a migration rate between Nomascus leucogenys and N. gabriellae of roughly one migrant every two generations. Together, our findings suggest that gibbons have had a complex demographic history involving hybridization or mixing between diverged populations.     

Nine polymorphic microsatellite loci in the Neotropical electric eel Eigenmannia (Teleostei: Gymnotiformes)

Ten microsatellite loci were isolated from a species of the Neotropical electric eel, Eigenmannia, a genus of freshwater fish characterized by small populations and low vagility. Nine microsatellites were polymorphic, the number of alleles ranging from seven to 27, and values of observed heterozygosity ranging from 0.327 to 0.741. These loci were developed for population genetic studies of Eigenmannia sp. 2, however, cross‐amplification carried out with other species of this genus as well as other Gymnotiformes genera indicate that these molecular markers are also potentially useful for population‐level studies in closely related species.     

Phylogeny of Stemphylium spp. based on ITS and glyceraldehyde-3-phosphate dehydrogenase gene sequences

The phylogenetic relationships among 44 isolates representing 16 species of Stemphylium were inferred from ITS and glyceraldehyde-3-phosphate dehydrogenase (gpd) sequence data. The results generally agree with morphological species concepts. There was strong support for monophyly of the genus Stemphylium. Analysis of the gpd fragment in particular was useful for establishing well-supported relationships among the species and isolates of Stemphylium. Species of Stemphylium that appear to have lost the ability to produce a sexual state are scattered among the species with the ability to reproduce sexually (Pleospora spp.). Species that are pathogenic to alfalfa are resolved into two groups. Stemphylium botryosum and two isolates with morphological characters similar to S. globuliferum had identical sequences at both loci. These two loci in S. vesicarium, S. alfalfae and S. herbarum are nearly identical but differ from S. botryosum. The separation of S. vesicarium, S. herbarum and S. alfalfae into separate species by morphometric evidence was not supported by the molecular data. Morphological and developmental characters such as size and shape of conidia, conidiophores, and ascospores, and size and time of maturation of pseudothecia are useful for diagnosing species. However, other morphological characters such as septum development and small variations in conidial wall ornamentation are not as useful.     

Variability of United States Isolates of Macrophomina phaseolina Based on Simple Sequence Repeats and Cross Genus Transferability to Related Genera Within Botryosphaeriaceae

Twelve simple sequence repeat (SSRs) loci were used to evaluate genetic diversity of 109 isolates of Macrophomina phaseolina collected from different geographical regions and host species throughout the United States (US). Genetic diversity was assessed using Nei’s minimum genetic distance, and the usefulness of each locus was determined by calculating the polymorphism information content (PIC). A total of 98 alleles were detected and of these 31 were unique to individual genotypes. Eight of twelve loci were highly informative with PIC values greater than 0.50. The majority of pairwise comparisons of genetic distance were greater than 0.60 indicating moderate to high genetic diversity. Dendrograms based on the genetic dissimilarities were created for the 109 isolates of which 79 were from soybean. Some clustering by host and geography was noted, but, the dendrograms generally grouped isolates independent of host or geography. Additionally, sequencing of the internal transcribed spacer region (ITS) for 10 isolates revealed that all of these isolates were 99% similar. Three SSR loci from M. phaseolina were cross amplified in other genera in the Botryosphaeriaceae. This was the first study of genotyping and assessing genetic diversity of M. phaseolina isolates collected from a widespread host and geographic range across the US with SSRs. With an additional 34 loci publically available for M. phaseolina, the results indicate that previously developed SSRs from one species can be used in future population, ecological, and genetic studies of M. phaseolina and other genera within the Botryosphaeriaceae.     

Communities of culturable yeasts and yeast-like fungi in oligotrophic hypersaline coastal waters of the Arabian Gulf surrounding Qatar

This report is the first investigation of yeast biodiversity from the oligotrophic hypersaline coastal waters of the Arabian Gulf surrounding Qatar. Yeasts and yeast-like fungi, were cultured from seawater sampled at 13 coastal areas surrounding Qatar over a period of 2 years (December 2013–September 2015). Eight hundred and forty-two isolates belonging to 82 species representing two phyla viz., Ascomycota (23 genera) and Basidiomycota (16 genera) were identified by molecular sequencing. The results indicated that the coastal waters of the Qatari oligotrophic marine environment harbor a diverse pool of yeast species, most of which have been reported from terrestrial, clinical and aquatic sources in various parts of the world. Five species, i.e., Candida albicans, C. parapsilosis, C. tropicalis, Pichia kudriavzevii and Meyerozyma guilliermondii (n?=?252/842; 30% isolates) are known as major opportunistic human pathogens. Fifteen species belonging to nine genera (n?=?498/842; 59%) and 12 species belonging to seven genera (n?=?459/842; 55%) are hydrocarbon degrading yeast and pollution indicator yeast species, respectively. Ascomycetous yeasts were predominant (66.38%; 559/842) as compared to their basidiomycetous counterparts (33.6%; 283/842). The most isolated yeast genera were Candida (28%; 236/842) (e.g., C. aaseri, C. boidinii, C. glabrata, C. intermedia, C. oleophila, C. orthopsilosis, C. palmioleophila, C. parapsilosis, C. pseudointermedia, C. rugopelliculosa, C. sake, C. tropicalis and C. zeylanoides), Rhodotorula (12.7%; 107/842), Naganishia (8.4%; 71/842), Aureobasidium (7.4%; 62/842), Pichia (7.3%; 62/842), and Debaryomyces (6.4%; 54/842). A total of eleven yeast species ( n = 38) isolated in this study are reported for the first time from the marine environment. Chemical testing demonstrated that seven out of the 13 sites had levels of total petroleum hydrocarbons (TPH) ranging from 200 to 900 µg/L, whereas 6 sites showed higher TPH levels (>?1000–21000 µg/L). The results suggest that the yeast community structure and density are impacted by various physico-chemical factors, namely total organic carbon, dissolved organic carbon and sulphur.