Icelandic Spring Research Articles

Draft genome of Thermoanaerobacter thermohydrosulfuricus strain AK152, a novel thermophilic and anaerobic bacterium isolated from a hot spring in Iceland.

We present the draft genome of the bacterium Thermoanaerobacter thermohydrosulfuricus strain AK152, a thermophilic, endospore-spore-forming, anaerobe isolated from a hot spring in Grensdalur, in Southwestern Iceland. This assembled genome will lay the foundation for identifying the carboxylic and amino acid fermentation pathways, suggesting biotechnological applications for this strain.

Biotechnological Prospects of Thermoanerobacter AK15: End-Product Formation from Carbohydrates, Amino Acids, and Lignocellulosic and Macroalgae Hydrolysates.

The conversion of lignocellulosic and algal biomass by thermophilic bacteria has been an area of active investigation. Thermoanaerobacter species have proven to be particularly capable in the production of bioethanol and biohydrogen from lignocellulosic biomass, although detailed studies of their abilities to utilize the full gamut of carbohydrate, amino acids, and proteins encountered in biomass hydrolysates are seldom comprehensively examined. Here, we re-evaluate the ability of Thermoanaerobacter strain AK15, a highly ethanologenic strain previously isolated from a hot spring in Iceland. Similar to other Thermoanaerobacter species, the strain degraded a wide range of mono- and di-saccharides and produced a maximum of 1.57 mol ethanol per mol of glucose degraded at high liquid-gas phase ratios. The ability of strain AK15 to utilize amino acids in the presence of thiosulfate is limited to the branched-chain amino acids as well as serine and threonine. Similar to other Thermoanaerobacter species, strain AK15 produces a mixture of branched-chain fatty acids and alcohols, making the strain of interest as a potential source of longer-chain alcohols. Finally, the strain was also shown to use butyrate as an electron sink during glucose degradation resulting in the reduced product butanol, in addition to end-products produced from glucose. Thus, strain AK15 is a promising candidate for ethanol and higher-order alcohols from a range of lignocellulosic and algal biomass.

Crystal structure of DNA polymerase I from Thermus phage G20c.

This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SβαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SβαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3'-5' exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SβαR motif, was first determined to 2.19 Å resolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Å resolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.

Invertebrate communities in springs across a gradient in thermal regimes.

In many respects, freshwater springs can be considered as unique ecosystems on the fringe of aquatic habitats. This integrates their uniqueness in terms of stability of environmental metrics. The main objective of our study was to evaluate how environmental variables may shape invertebrate diversity and community composition in different freshwater spring types and habitats within. In order to do so, we sampled invertebrates from 49 springs in Iceland, where we included both limnocrene and rheocrene springs. At each site, samples were taken from the benthic substrate of the spring (“surface”) and the upwelling groundwater at the spring source (“source”). To collect invertebrates from the spring sources we used a modified method of “electrobugging” and Surber sampler for collecting invertebrates from the surface. In total, 54 invertebrate taxa were identified, mostly Chironomidae (Diptera). Chironomid larvae also dominated in terms of abundance (67%), followed by Ostracoda (12%) and Copepoda (9%). The species composition in the surface samples differed considerably between rheocrene and limnocrene springs and was characterised by several indicator species. Alpha diversity was greater at the surface of springs than at the source, but the beta diversity was higher at the source. Diversity, as summarized by taxa richness and Shannon diversity, was negatively correlated with temperature at the surface. At the source, on the other hand, Shannon diversity increased with temperature. The community assembly in springs appears to be greatly affected by water temperature, with the source community of hot springs being more niche-assembled (i.e., affected by mechanisms of tolerance and adaptation) than the source community of cold springs, which is more dispersal-assembled (i.e., by mechanisms of drift and colonization).

THE FAR SHOALS OF NEPTUNISM: WILLIAM H. KEATING AND THE ST. PETER SANDSTONE IN THE AMERICAN MIDWEST

ABSTRACT William H. Keating (1799–1840) served as mineralogist on Major Stephen H. Long’s 1823 expedition to the source of the St. Peter’s (Minnesota) River, concluding, on the basis of grain shape, that the St. Peter Sandstone, at what was later to be its type section, Fort Snelling, in the state of Minnesota, was a chemical precipitate from seawater. This appears to be an echo of the Neptunist teachings of Abraham Gottlob Werner (1749–1817), as interpreted in the American Midwest. While endorsed by prominent geologist James Hall and others, and given some plausibility by the analogy of siliceous sinter depositing from hot springs in Iceland, the ‘purity’ criterion used by supporters of the theory was found fallacious with further advances in sedimentology such as those by Charles L. Dake and George A. Thiel. The word ‘purity,’ which was industrial-commercial parlance, merged into the concept of ‘maturity’ in the ternary sandstone classifications of the 1940s. Because the St. Peter Sandstone appears so featureless, actors projected their latent biases onto the blank walls of the sandstone outcrops.

Virulence of Icelandic Pyrenophora teres f. teres populations and resistance of Icelandic spring barley lines

The causal agent of the barley net blotch disease, Pyrenophora teres, is known for its high level of diversity due to sexual reproduction. Different pathotypes, defined by a virulence combination, even within the same fields are frequently found and virulence between locations can vary considerably. Evaluation of virulence patterns of a pathogen population is essential for breeding resistant cultivars suitable for specific locations. To identify virulence patterns in Icelandic Pyrenophora teres f. teres (Ptt) isolates, twenty single spore isolates of Ptt were collected from seven locations in Iceland and analysed with AFLP markers. Principle Coordinate Analysis (PCoA) revealed Icelandic Ptt isolates clustering away from reference isolates from Austria, Finland, Sweden, Switzerland, UK, and USA. Hierarchical clustering grouped the Icelandic isolates into three distinct groups. Furthermore, the virulence of these twenty isolates was tested on 16 barley differential lines and revealed high variation in their virulence. Twenty-one barley cultivars commonly used in Iceland showed high susceptibility towards inoculation with Icelandic Ptt isolates.

The Winter-Type Allele of HvCEN Is Associated With Earliness Without Severe Yield Penalty in Icelandic Spring Barley (Hordeum vulgare L.).

Icelandic barley genotypes have shown extreme earliness both in flowering and maturity compared to other north European genotypes, whereas earliness is a key trait in adapting barley to northern latitudes. Four genes were partially re-sequenced, which are Ppd-H1, HvCEN, HvELF3, and HvFT1, to better understand the mechanisms underlying this observed earliness. These genes are all known to play a part in the photoperiod response. The objective of this study is to correlate allelic diversity with flowering time and yield data from Icelandic field trials. The resequencing identified two to three alleles at each locus which resulted in 12 haplotype combinations. One haplotype combination containing the winter-type allele of Ppd-H1 correlated with extreme earliness, however, with a severe yield penalty. A winter-type allele in HvCEN in four genotypes correlated with earliness combined with high yield. Our results open the possibility of marker-assisted pyramiding as a rapid way to develop varieties with a shortened time from sowing to flowering under the extreme Icelandic growing conditions and possibly in other arctic or sub-arctic regions.

Specific adaptation for early maturity and height stability in Icelandic spring barley

AbstractCereal production in important growing regions is negatively influenced by climate change. This can be countered by expanding cereal production northwards in Scandinavia and Iceland, where today, barley (Hordeum vulgare L.) is primarily used as feed, as it rarely reaches malting quality. This study explores genetic factors underlying the ability of barley to mature fully in low temperature and long photoperiod. A panel of 84 spring barley lines were grown in controlled environments with different day lengths and temperatures, partially mimicking the target environment. The panel was screened for accumulated heat sum to heading, maturity, and height, all traits of importance for adaptation to the northern periphery. Subgroups with different stability and heat sum requirements were found, and day‐length‐neutral lines were identified. Height was temperature controlled, with lower temperature resulting in taller plants. The results were coupled to a genome‐wide association study (GWAS). Despite the small panel size, the Mat‐a locus was identified to have the strongest association with heat sum to heading; Ppd‐H1, Mat‐a, FT1, and DHAR2 with heat sum to maturity; and GA20ox1 with height. Early maturing lines with height stability have successfully been developed in Iceland, and this study confirms their performance in controlled environments for the first time. It provides insight to the mechanisms behind early maturity that will increase our ability to further adapt barley and other cereals to the northern climate. This will facilitate breeding work toward combining early maturity and height stability with traits such as quality, further enabling the northward expansion of grain production.

A New Record of Oxytricha granulifera granulifera Foissner and Adam, 1983 (Protozoa, Ciliophora, Oxytrichidae) From a Hot Spring in Iceland, With Notes on Its Abnormal Form During Cultivation

We isolated a population of Oxytricha granulifera granuliferaFoissner and Adam (1983) from a hot spring in Iceland. The pure culture of this isolate was established at room temperature in the laboratory. This allowed for a detailed investigation, informed by integrated approaches, of the isolate’s morphology and morphogenesis, as well as molecular phylogeny. Results showed that the morphological and morphogenetic characteristics of the Iceland population are consistent with those of other populations. During the 3-year long period of laboratory cultivation, some abnormal individuals appeared repeatedly in the culture system. Interestingly, the morphological characteristics of these abnormal cells were rather stable, and were as follows: 1) body slender and elliptical-shaped; 2) remarkably shortened adoral zone and significantly reduced number of adoral membranelles; and 3) loss of undulating membranes. Resting cysts, binary fission and conjugate reproduction were not found in abnormal specimens. Although the morphology of abnormal individuals changed significantly, the sequences of the SSU rDNA of the normal and abnormal morphotypes were the same. Phylogenetic analyses showed that the two morphotypes clustered in a clade with other populations of O. granulifera granulifera.

Temperature and precipitation at migratory grounds influence demographic trends of an Arctic-breeding bird.

Anthropogenic climate disruption, including temperature and precipitation regime shifts, has been linked to animal population declines since the mid-20th century. However, some species, such as Arctic-breeding geese, have thrived during this period. An increased understanding of how climate disruption might link to demographic rates in thriving species is an important perspective in quantifying the impact of anthropogenic climate disruption on the global state of nature. The Greenland barnacle goose (Branta leucopsis) population has increased tenfold in abundance since the mid-20th century. A concurrent weather regime shift towards warmer, wetter conditions occurred throughout its range in Greenland (breeding), Ireland and Scotland (wintering) and Iceland (spring and autumn staging). The aim of this study was to determine the relationship between weather and demographic rates of Greenland barnacle geese to discern the role of climate shifts in the population trend. We quantified the relationship between temperature and precipitation and Greenland barnacle goose survival and productivity over a 50year period from 1968 to 2018. We detected significant positive relationships between warmer, wetter conditions on the Icelandic spring staging grounds and survival. We also detected contrasting relationships between warmer, wetter conditions during autumn staging and survival and productivity, with warm, dry conditions being the most favourable for productivity. Survival increased in the latter part of the study period, supporting the possibility that spring weather regime shifts contributed to the increasing population trend. This may be related to improved forage resources, as warming air temperatures have been shown to improve survival rates in several other Arctic and northern terrestrial herbivorous species through indirect bottom-up effects on forage availability.

<strong>Trichoptera diversity in Icelandic springs</strong>

The aim of this study was to characterize the Trichoptera fauna of freshwater springs in Iceland and to relate distribution of caddis larvae to environmental properties of the springs. Out of a total of 48 springs sampled, Trichoptera larvae were found in only eleven. Larval densities were low, as was species diversity. Only three of the 12 species known to occur in Iceland were found: Apatania zonella, Limnephilus griseus, and Limnephilus affinis. The occurrence of A. zonella in springs in North-Iceland may suggest that springs might play a role as refugia for this species that is otherwise excluded from the area by larvae of the predatory caddis species Potamophylax cingulatus, which seems to be absent from spring habitats. Caddis larval abundance was higher in rheocrene springs and in springs with sandy substrate, and decreased with increasing water temperature. Presence or absence of Trichoptera larvae, on the other hand, was not associated with any of the environmental variables measured.

Investigating the Clitellata (Annelida) of Icelandic springs with alternative barcodes

DNA barcoding is an invaluable tool to identify clitellates, regardless of life stage or cryptic morphology. However, as COI (the standard barcode for animals) is relatively long (658 bp), sequencing it requires DNA of high quality. When DNA is fragmented due to degradation, alternative barcodes of shorter length present an option to obtain genetic material. We attempted to sequence 187 clitellates sampled from springs in Iceland. However, the material had been stored at room temperature for two years, and DNA of the worms had degraded, and only three COI sequences were produced (i.e., <2% success rate). Using two alternative barcodes of 16S (one ca. 320 bp, the other ca. 70 bp long) we increased the number of sequenced specimens to 51. Comparisons of the 16S sequences showed that even the short 70 bp fragment contained enough genetic variation to separate all clitellate species in the material. Combined with morphological examinations we recognized a total of 23 species, where at least 8 are new records for Iceland, some belonging to genera new for Iceland: Cernosvitoviella and Pristina. All the new taxa are included in an updated species list of Icelandic Clitellata. The material revealed some stygophilic species previously known to inhabit springs, but true stygobionts, which are restricted to groundwater habitats, were not found. Our study shows that short 16S fragments can be obtained from DNA too degraded to be used in traditional COI barcoding, and contain enough genetic variation to separate closely related clitellate species.

Chironomidae fauna of springs in Iceland – assessing the ecological relevance behind Tuxen’s spring classification

In 1937, S. L. Tuxen studied the animal community of hot springs in Iceland, and classified springs according to their relative temperature into cold, tepid, and hot. Eighty years after Tuxen’s study, we revisited some of the hot springs in Skagafjörður, Northern Iceland. Our aim was to compare the invertebrate community of 1937 and today, and to assess the stability of hot spring habitats over the years. To test Tuxen’s spring classification on an ecological basis, we furthermore collected chironomid larvae from 24 springs of a broad range of temperature, with samples taken both at the surface area of the spring and at the groundwater level. The chironomid species composition of hot springs differed from that of cold and tepid springs. Whereas Cricotopus sylvestris, Arctopelopia sp., and Procladius sp. characterised the chironomid community in Icelandic hot springs, cold and tepid springs were dominated by Eukiefferiella minor, Orthocladius frigidus and Diamesa spp. Community composition analyses and the exclusive occurrence of taxa in one of the temperature classes validated the ecological relevance of Tuxen’s spring classification for the chironomid species community. Both environmental parameters and invertebrate community of Icelandic hot springs seem to be the same as 80 years ago. Although springs have the potential to provide stable habitats, they are currently under high anthropogenic pressure, and should be increasingly considered in nature conservation.

Characterization of carotenoids in Rhodothermus marinus.

Rhodothermus marinus, a marine aerobic thermophile, was first isolated from an intertidal hot spring in Iceland. In recent years, the R. marinus strain PRI 493 has been genetically modified, which opens up possibilities for targeted metabolic engineering of the species, such as of the carotenoid biosynthetic pathway. In this study, the carotenoids of the R. marinus type‐strain DSM 4252T, strain DSM 4253, and strain PRI 493 were characterized. Bioreactor cultivations were used for pressurized liquid extraction and analyzed by ultra‐high performance supercritical fluid chromatography with diode array and quadropole time‐of‐flight mass spectrometry detection (UHPSFC‐DAD‐QTOF/MS). Salinixanthin, a carotenoid originally found in Salinibacter ruber and previously detected in strain DSM 4253, was identified in all three R. marinus strains, both in the hydroxylated and nonhydroxylated form. Furthermore, an additional and structurally distinct carotenoid was detected in the three strains. MS/MS fragmentation implied that the mass difference between salinixanthin and the novel carotenoid structure corresponded to the absence of a 4‐keto group on the ß‐ionone ring. The study confirmed the lack of carotenoids for the strain SB‐71 (ΔtrpBΔpurAcrtBI’::trpB) in which genes encoding two enzymes of the proposed pathway are partially deleted. Moreover, antioxidant capacity was detected in extracts of all the examined R. marinus strains and found to be 2–4 times lower for the knock‐out strain SB‐71. A gene cluster with 11 genes in two operons in the R. marinus DSM 4252T genome was identified and analyzed, in which several genes were matched with carotenoid biosynthetic pathway genes in other organisms.

Do biomass indices from Icelandic groundfish surveys reflect changes in the population of female lumpfish (Cyclopterus lumpus)?

The female portion of the population of lumpfish (Cyclopterus lumpus) in Iceland is currently monitored using a biomass index calculated using catch data from the Icelandic spring groundfish survey (referred to as the spring survey). This has been controversial among lumpfish fishers who insist that the behaviour and variations in the migration of lumpfish make the use of this survey inappropriate for assessment. To evaluate whether changes in the biomass index from the spring survey adequately reflect changes in the population, the spatial and depth distribution of catches of lumpfish from this survey are compared with the total coverage of the survey. The biomass index from the spring survey is also considered alongside data from two other surveys (gillnet survey and autumn groundfish survey) and catch per unit effort (CPUE) from the female lumpfish fishery. In the spring survey, female lumpfish were predominantly caught at depths shallower than 300m and within 100km of shore, while the survey extends to a depth of 500m and 227km from shore, indicating that survey coverage was fitting for this species. The changes in the biomass index from the spring survey were similar to that of the gillnet survey, and also to changes in CPUE of the fishery. A decrease in the biomass index of large lumpfish (≥45cm) caught during the survey was correlated with the decreasing use of large mesh sizes in the fishery indicating that changes in size distribution in the population could be detected using the spring survey. These results indicate that the use of the spring survey to monitor changes in lumpfish population in Iceland is justified. The use of data from bottom trawl surveys to track lumpfish populations in other regions is discussed.

Novel thermostable amine transferases from hot spring metagenomes.

Hot spring metagenomes, prepared from samples collected at temperatures ranging from 55 to 95°C, were submitted to an in silico screening aimed at the identification of novel amine transaminases (ATAs), valuable biocatalysts for the preparation of optically pure amines. Three novel (S)-selective ATAs, namely Is3-TA, It6-TA, and B3-TA, were discovered in the metagenome of samples collected from hot springs in Iceland and in Italy, cloned from the corresponding metagenomic DNAs and overexpressed in recombinant form in E. coli. Functional characterization of the novel ATAs demonstrated that they all possess a thermophilic character and are capable of performing amine transfer reactions using a broad range of donor and acceptor substrates, thus suggesting a good potential for practical synthetic applications. In particular, the enzyme B3-TA revealed to be exceptionally thermostable, retaining 85% of activity after 5days of incubation at 80°C and more than 40% after 2weeks under the same condition. These results, which were in agreement with the estimation of an apparent melting temperature around 88°C, make B3-TA, to the best of our knowledge, the most thermostable natural ATA described to date. This biocatalyst showed also a good tolerance toward different water-miscible and water-immiscible organic solvents. A detailed inspection of the homology-based structural model of B3-TA showed that the overall active site architecture of mesophilic (S)-selective ATAs was mainly conserved in this hyperthermophilic homolog. Additionally, a subfamily of B3-TA-like transaminases, mostly uncharacterized and all from thermophilic microorganisms, was identified and analyzed in terms of phylogenetic relationships and sequence conservation.

Radon in Icelandic Cold Groundwater and Low-Temperature Geothermal Water

Samples of hot and cold water for radon analysis were collected from boreholes and springs in Iceland in 2014 and 2015. The majority of the samples was collected from municipal district heating services or potable water utilities. The total number of samples was 142, covering most towns and villages in Iceland. Radon activity is generally rather low, in most cases less than 5 Bq/L. Only 12 samples had a measured radon activity higher than 5 Bq/L, with a maximum activity of 10.8 Bq/L. The hot water samples generally have a higher radon activity than cold water samples, but samples from boiling borholes have a lower radon activity as radon fractionates into the vapour phase. The geographical distribution of the samples indicates that radon activity is generally lower within the active rift zones. This is most likely due to the very low uranium content of the tholeiites typically erupted within the rift zones. Higher radon values (> 5 Bq/L) are in most cases close to extinct central volcanoes and thus it seems plausible that the water sampled has been in contact with felsic rocks.

Effect of culture conditions on hydrogen production by Thermoanaerobacter strain AK68

Thermoanaerobacter strain AK68, isolated from a hot spring in Iceland, was investigated for hydrogen production from sugars. Strain AK68 is most closely related to Thermoanaerobacter thermohydrosulfuricus (99.9% similarity). The strain has a temperature optimum between 65 °C and 70 °C and a pH optimum between pH 7.0 and 8.0. The strain degraded the majority of substrates examined including common hexoses and disaccharides as well as xylose but not arabinose or starch. Various environmental variables were investigated to gain a deeper understanding of the factors that are of importance for hydrogen production since the strain shows inhibition at relatively low initial glucose concentrations. Factors investigated included initial glucose concentration, phosphate buffer concentration, liquid–gas phase ratio, effect of various external electron acceptors, and different initial pH values. Substrate utilization was shown to be inhibited by increased partial pressure of hydrogen, as evidenced by increased utilization of glucose using electron scavenging systems and manipulation of the liquid–gas phase ratio, rather than substrate or end product inhibition.

Warming winter effects, fat store accumulation and timing of spring departure of Greenland White-fronted Geese Anser albifrons flavirostris from their winter quarters

Migratory geese accumulate energy and nutrient stores in winter to fly to refuelling spring staging areas before onward migration to breeding areas. Mean ground temperatures at two important Greenland White-fronted Geese wintering sites rose in winter and spring by 1.0–1.3°C during 1973–2007. Greenland White-fronted Geese departed the Wexford winter quarters on 3rd April 2007 for Icelandic spring staging areas, the earliest on record, representing a mean advancement of 15 days since 1973, mirrored amongst mean dates of departure amongst Scottish wintering birds that have advanced by 12 days during 1973–2007. Icelandic temperatures at critical midway staging areas en route to Greenland showed no significant change since 1973, suggesting that it is warming on the winter quarters that enable geese to depart earlier, rather than elevated temperatures at ultimate spring staging areas. However, Wexford departure date did not correlate with spring temperature. Data presented here show that Greenland White-fronted Geese have accumulated threshold body stores progressively earlier in spring migration, especially during 1995–2007. Although this did not correlate with ambient temperature, the mean degree of accumulated fat stored by 1st April in each year was a statistically significant predictor of departure date for the wintering population at Wexford. These data support the hypothesis that it is intrinsic factors (i.e. improvements in internal body state resulting from better feeding conditions) that has permitted progressively earlier departure of these geese from Wexford on spring migration, rather than amelioration of spring conditions in Iceland or solely the result of warming of the winter quarters.

Fine-scale parallel patterns in diversity of small benthic Arctic charr (Salvelinus alpinus) in relation to the ecology of lava/groundwater habitats.

It is critical to study factors that are important for origin and maintenance of biological diversity. A comparative approach involving a large number of populations is particularly useful. We use this approach to study the relationship between ecological factors and phenotypic diversity in Icelandic Arctic charr (Salvelinus alpinus). Numerous populations of small benthic charr have evolved in lava springs in Iceland. These charr appear morphologically similar, but differ in important morphological features related to feeding. We found a clear relationship between diversity in morphology, diet, and ecological factors among populations. In particular, there were clear differences in morphology and diet between fish coming from habitats where the lava spring flowed on as a stream compared to habitats where the lava spring flowed into a pond. Our study shows that ecological factors are important for the origin and maintenance of biological diversity. The relationship between phenotype and ecological factors are observed on a fine scale, when comparing numerous populations that are phenotypically similar. This strongly suggests that for understanding, managing, and conserving biological diversity important ecological variables have to be taken into the account.