A project on nymphaeid-dominated systems

Summary In the present paper the general line of the research project on nymphaeid-dominated systems of the Laboratory of Aquatic Ecology (Catholic University, Nijmegen) is explained and a list of publications is given. In the nymphaeid system 16 structural elements could be distinguished which are spatially distributed over 15 compartments. As the nymphaeid system is very complex compared with other macrophyte-dominated systems, a team is working now on the project.The author is much indebted to other members of the nymphaeid team for their critical remarks.     

In situ andin vivo studies on the breakdown ofNymphaea alba L. (Nymphaeaceae)

Summary The breakdown of floating leaves ofNymphaea alba was studied with litterbags in two aquatic systems,viz., an alkaline, eutrophic former riverbed (Oude Waal) and an acid, oligotrophic pool (Voorste Choorven). It was found that the floating leaves from the Oude Waal decomposed at a faster rate than those from the Voorste Choorven when they were incubated in their natural system. In order to get insight in the parameters which determine the observed differences in the field, laboratory experiments were performed. Leaf discs ofN.alba were incubated in staged concentration gradients of some important chemical parameters (bicarbonate, ammonium, nitrate, orthophosphate, sulphate, pH, combinations of nitrate and phosphate; nitrate and bicarbonate, and bicarbonate and sulphate). It appeared that the differences in decomposition rate as observed in the field could be largely explained by the pH and alkalinity of the system-water, and the chemical composition and physiological state of the plant material.Contribution nr. 36 of the nymphaeid project.     

The effect of fetch on periphyton spatial variation

The marked spatial variation in periphyton could reflect differences in exposure, grazing or substratum. To determine if any of these factors was significant, I studied the relationship between the degree of exposure to waves (measured as fetch), grazing intensity (measured as invertebrate biomass) and spatial variation in periphyton biomass in sites of similar substratum along an island in the central mesotrophic basin of Lake Memphremagog (Québec). In spring, there was a positive relationship between fetch and periphyton biomass both on stones and on artificial substrata. This effect was especially strong for diatoms, for algae between 100–1000 µm³, for planktonic forms and for filamentous and long-stalked species. In spring, water renewal encouraged growth of these forms, but the effect disappeared in summer, coincident with silica depletion. Grazers, which were not important in the spatial variation observed in spring, probably contributed to the sudden decline of periphyton in the exposed sites in summer. Observations in nearby lakes indicate that these patterns may be general.Contribution of the Lake Memphremagog Project, Limnology Research Centre.     

Temporal changes in periphyton standing crop during an unusually dry winter in streams of the Western Cascades,Oregon

The effects of light and discharge on standing crops of periphyton in adjacent shaded and open reaches of first to fourth order streams were examined during winter in three streams of the Western Cascades, Oregon. Standing crops were measured in terms of chlorophylla and periphyton biomass at each site on 8 occasions. Open sites supported higher standing crops of periphyton than shaded sites and increases in standing crop were shown to be related to light input at each site. Biomass increased throughout winter until scouring associated with an unusually late winter freshet reduced periphyton standing crops to their lowest observed levels. It is concluded that periphyton levels are affected by a combination of factors of which light levels, and the periodicity of storm events are of major importance.     

Adapting molecular-genetic methods for studying the taxonomic diversity of microbial communities associated with macrophytes

The combination of molecular-genetic techniques used in the study is applied to investigate microorganisms associated with macrophytes. The method of enzymatic lysis with phenol-chloroform extraction is optimal for the total bacterial DNA isolation from both periphyton organisms and enriched cultures. Amplifying the total DNA on conservative primers at the two-step PCR regime is recommended. An analysis of the taxonomic diversity of the microbial community from biofilm on the reed grass and in the enriched cultures propagated on various growth media has been carried out. The results have revealed a high diversity of periphyton microorganisms associated with reed grass, including representatives of such phylogenetic lines as proteobacteria (α, β, γ, and δ subgroups), Bacteroidetes/Chlorobi, Chlamydiae/Verrucomicrobia, and cyanobacteria. The low diversity of sequences in enriched cultures is represented by dominating genotypes of Cellvibrio with a high percentage of homology and uncultivated bacilla.     

A simple periphyton sampler for algal biomass estimates in streams

• 1 A simple periphyton sampler, which is cheap and easy to construct, is described. The key component is a disposable scouring disc which also serves to adsorb detached material.
• 2 Field trials show that the scouring sampler is significantly more efficient at removing and retaining periphyton from stones in streams for biomass estimation than other commonly used techniques.
• 3 The scouring sampler requires only one operator. Its small size and speed of use facilitate replication of samples. Samples can be collected from stones as small as 30mm diameter.

     

Do submerged aquatic plants influence their periphyton to enhance the growth and reproduction of invertebrate mutualists?

It has been suggested that submerged aquatic plants can influence the nutritional quality of the periphyton which grows on their surfaces, making it more nutritious for grazing invertebrates, particularly snails. In return, these grazers might preferentially feed on the periphyton and clear the plants of a potential competitor, with the plants and grazers both gaining from this mutualistic relationship. A highly replicated experiment was conducted, in which the nature of the plant (isoetid and elodeid types compared with similar-shaped inert substrata), the nutrient loading, and the influence of periphyton grazers (the bladder snail, Physa fontinalis) of similar size and history were controlled. Plant growth and survival significantly increased in the presence of the periphyton grazer. Whilst the presence of the grazers had the largest influence on periphyton abundance, nutrient availability and plant type also had effects. Plant type had little influence on the nutritional quality of the periphyton measured as carbohydrate, protein and C:N. Effects of treatment on snail growth, and the timing and extent of snail reproduction disappeared when they were compared with the quantity of periphyton available. There was no evidence of enhanced grazer success in the presence of the live plants compared with inert substrata. Although submerged plants affect the growth and reproduction of the grazers which feed on their surfaces, through differences in the amount of periphyton which grows there, we found no evidence that they manipulate the periphyton to encourage such grazers. Received: 1 September 1998 / Accepted: 12 May 1999     

Dolichopodidae (Diptera) of a Nymphaeid‐Dominated system in the Netherlands: Species composition,diversity, spatial and temporal distribution

The composition of the Dolichopodid fauna was studied at various sites within nymphaeid stands during one growth season in an oxbow lake with the aid of adhesive traps; additional observations were made by means of hand-net collecting at various localities within nymphaeid stands. The first method gave the best results.

With this method the species composition, diversity, spatial and temporal distribution of the Dolichopodid fauna within nymphaeid stands could be described.

Nine species appeared to be common, viz. )in order of decreasing numbers( Hydrophorus praecox )Lehm.(, Dolichopus latilimbatus Mcq.¹(, Campsicnemus picticornis )Zett.(, Rhaphium antennatum )Carl.(, Dolichopus nubilus Mg., Hydrophorus litoreus Fall., Campsicnemus pectinulatus Lw., Campsicnemus lumbatus Lw. and Rhaphium elegantulum )Mg.(. With respect to the spatial distribution within the nymphaeid zone both Hydrophorus species were found in large numbers on sites bordering on the open water, while the largest numbers of all other species mentioned above were caught on sites near the helophyte zone. A distinct succession of Dolichopodid species with respect to their numbers could be observed during the growth season on the floating leaves in the sequence Hydrophorus, Campsicnemus, Rhaphium and Dolichopus spp.

The reasons for these distribution patterns are discussed on the basis of what is known of the ecology and behaviour of the various species.     

Snail–periphyton interactions in a prairie lacustrine wetland

This study examined the effects of nutrients and macrophytes on snail grazers and periphyton in a prairie wetland food web. Snails (Gyraulus circumstriatus) and periphyton in large enclosures in a lacustrine wetland, Delta Marsh, MB, Canada were subjected to two experimental treatments, nutrient addition (nitrogen, phosphorus) and macrophyte exclusion (using a porous geotextile carpet) during July and August. Snail biomass and periphyton biomass (on both artificial substrata and submerged macrophytes) increased over time in all treatments, representing seasonal growth. Snail biomass was three times higher on macrophytes than on artificial substrata. In response to nutrient addition, snail biomass was significantly elevated over time on macrophytes but not on artificial substrata. Conversely, periphyton biomass was higher on artificial substrata but not on macrophytes in response to nutrient addition. Snail biomass and periphyton biomass on artificial substrata showed no response to macrophyte exclusion. Snail biomass on all substrata was inversely correlated with turbidity, whereas periphyton biomass showed no relationship with turbidity. Timing of nutrient additions to wetlands may influence whether the response occurs primarily in phytoplankton or in periphyton and macrophytes.     

How light and nutrients affect the relationship between autotrophic and heterotrophic biomass in a tropical black water periphyton community

This study examines how nutrients and light affect the relationship between autotrophic biomass and non-autotrophic periphyton organic matter in a tropical black water lake biofilm community. We hypothesized that there is no positive correlation between autotrophic and non-autotrophic organic matter in the periphytic community of a black water humic lake, where non-algal components of periphyton can rely on carbon sources external to the periphyton matrix and where nutrient availability is low. Second, we sought to test our hypothesis that non-autotrophic periphyton organic matter will benefit from nutrient enhancement in a lake where the availability of DOC is high. We performed a field experiment using in situ lake mesocosms to manipulate nutrient concentrations and light availability in a 2 × 2 factorial design. Control treatments (no nutrient added) and nutrient treatments (N + P) were compared in different light conditions: high light (near surface water) and low light (near bottom). No positive correlation was found between autotrophic biomass and non-autotrophic periphyton organic matter, but a negative correlation was observed in high nutrient and light conditions. The low C:P and N:P ratios revealed that the non-autotrophic organic matter mostly comprised a heterotrophic microbial biofilm. High levels of light and nutrients together caused significant changes in periphyton community properties. The non-autotrophic periphyton organic matter was negatively affected by nutrient addition, whereas autotrophic biomass was positively affected, especially in high light conditions. Our results strongly suggest that non-autotrophic periphyton organic matter in a humic lake is primarily comprised of a bacterial biofilm that directly competes for nutrients with autotrophs in the periphytic community. We also observed no effect of nutrient addition on periphyton growing in light-limited conditions. These results suggest that heterotrophic periphytic organisms might experience carbon limitation despite the high availability, but usually low quality, of dissolved carbon in the water column of humic lakes.     

A phytosociological study of the macrophytic vegetation of running waters in western lower saxony (Federal Republic of Germany)

A survey was carried out of the macrophytic vegetation in running waters of Western Lower Saxony. Three hundred and eighty-two phytosociological relevées were classified by common table work to 30 vegetation types of different phytosociological validity. The vegetation types are described and floristically characterized. Most of them belong to the complex Sparganium emersum community, which is characterized by the dominant occurrence of the nymphaeid species Sparganium emersum Rehm., Nuphar lutea (L.) Sm., Sagittaria sagittifolia L. and Potamogeton natans L., but magnopotamid-, parvopotamid-, batrachiid- and pleustophyte-dominated communities also occur. Similarity between the different vegetation types was checked by multivariate techniques, one classification technique (minimum variance clustering) and one ordination technique (principal components analysis). Additionally, syndynamical relations between the types were studied by observing the vegetation changes of 46 sample points within 2 years. The relations between different vegetation types are shown by combination of the different approaches. Finally, some more general statements are made regarding the handling of such data sets, as well as the consequences of the results for the classification of macrophytic vegetation in Central European rivers.     

Periphyton responds differentially to nutrients recycled in dissolved or faecal pellet form by the snail grazer Theodoxus fluviatilis

1. We aimed to separate the effects of grazers on periphyton via grazing from that of nutrient recycling from their faecal pellets. 2. We set up three different experimental treatments (snails/no snails/faecal pellets) and sampled over 16 days. The ‘snail’ treatment contained a low density (snail biomass c. 14 g^?2) of the gastropod grazer Theodoxus fluviatilis and the ‘faecal pellet’ treatment received the same amount of faecal pellets as were produced in the ‘snail’ treatment. Whereas the ‘faecal pellet’ treatment provided extra nutrients to periphyton from the faeces, the ‘snail’ treatment provided nutrients in the form of both faeces and in excreta. There was also direct grazing on periphyton in the ‘snail’ treatment. The ‘no snail’ was not grazed and received no nutrients in faeces or excreta. 3. We measured periphyton C, N and P content, chlorophyll‐a (chl‐a), primary production, bacterial biomass, bacterial production and bacterial respiratory activity. In the water column we measured dissolved inorganic N and soluble reactive P. 4. Snails increased the amount of dissolved inorganic N in the water. On day 16, the periphyton N : P ratio in the ‘faecal pellet’ treatment was lower, and periphyton P content was higher, than in the other two treatments. N : P ratios decreased over time in the ‘faecal pellet’ treatment. Primary and bacterial production were positively correlated in all treatments. 5. Algal chl‐a and primary production of periphyton per unit area and periphyton chl‐a : C ratios increased over the 16 day in the ‘snail’ treatment, and thus excretion of dissolved N by snails had a stronger positive effect on the periphyton community than N and P in faecal pellets. 6. Our data show that excretion and egestion can have different effects on periphyton, probably because of the higher proportion of dissolved N in excreta and the higher proportion of P recycled in faecal pellets. The relative effect of nutrients recycled in egesta or in excretions, probably depends on the form of nutrient limitation of the periphyton. Further, the different components of the periphyton matrix could react differently to the different forms of nutrient recycling. 7. We conclude that direct grazing effects are less important than nutrient effects when nutrients are limiting and grazing pressure is low. Further, the spatial separation of different grazing effects can lead to differences in periphyton production and nutrient stoichiometry. This might be an explanation for the patchiness of periphyton in nature.     

Periphyton density and shading in relation to tidal depth and fiddler crab activity in intertidal seagrass beds of the Banc d'Arguin (Mauritania)

Periphyton development was studied on microscopic glass slides and leaves of Zostera noltii Hornem. in an intertidal area in the Banc d'Arguin (Mauritania). The effects of shading, tidal depth and grazing activities by the fiddler crab Uca tangeri Eydoux were evaluated. For all experiments, periphyton ash content was high (52–93%) and ash-free dry weight ranged between 0.10–0.63 mg cm^–2. Slides accumulated more periphyton than leaves.Artificial shading (62–99%) for 13 days had no effect on periphyton densities on leaves. Increased tidal depth resulted in higher ash-free dry weight on slides, but in lower ash-free dry weight on leaves. Significant variation along the coastline also existed. The effect of fiddler crabs was studied using exclosures. Presence of fiddler crabs reduced periphyton density on slides, whereas light transmittance was increased. On leaves, no significant fiddler crab effect was found. This difference between leaves and slides was probably caused by a storm at the day before the end of the experiment, and by the higher periphyton density on slides as compared with leaves. As visual inspection during the experiment showed clear differences in appearance of leaves inside and outside the exclosures, the storm probably sloughed off mainly the older leaves, i.e. those on which the differences in periphyton cover were the highest.It is hypothesized that periphyton accumulation is higher with increased tidal depth, whereas fiddler crab grazing pressure also increases in this direction. The result is a decreased periphyton density with increased tidal depth.The presently found light extinction coefficients (mean 0.8 m^–1) and periphyton light attenuance (up to 25%) in Banc d'Arguin are not likely to affect seagrass leaf growth.     

周丛藻类及其在水质净化中的应用

周丛藻类广泛存在于自然界水体中,具有生物量大、生态功能强、水质响应灵敏等特征.周丛藻类通过吸收代谢利用、吸附和络合等过程去除水体污染物质,用于水质净化,具有耐污能力强,N、P去除效果好,藻类细胞回收利用价值高等优点,推广应用前景广阔.近年开发的藻丛刷系统、周丛藻类 生物膜系统和周丛藻类水产养殖系统等已经成功用于畜禽水产养殖废水、生活污水处理.而周丛藻类的生存规律、对污染物浓度的生理响应机理和污染物质吸收利用的分子生物学机制仍有待进一步研究.     

Ecological responses to nutrients in streams and rivers of the Colorado mountains and foothills

1. Abundance and composition of periphyton and benthic macroinvertebrates were treated as potential nutrient response variables for 74 streams in montane Colorado. The streams ranged from unenriched to mildly enriched with nutrients (N, P). 2. The study showed no meaningful relationship between periphyton biomass accumulation and concentrations of total or dissolved forms of nitrogen or phosphorus. Nutrient concentrations were also unrelated to periphyton and macroinvertebrate richness, diversity and community composition. Macroinvertebrate communities did, however, show a strong positive relationship to periphyton abundance. 3. A positive response of periphyton biomass to increasing nutrient concentrations has been well documented over large ranges of nutrient concentrations. Our study suggests that the nutrient response is suppressed by other controlling factors on the lower limb of the nutrient response curve (i.e. at low nutrient concentrations); a quantitatively significant response occurs only in excess of a threshold beyond which nutrients become dominant over other controlling factors. This interpretation of the results is consistent with published meta‐analyses showing lack of nutrient response for a high proportion of experimentally enriched periphyton communities, and division of responses between N and P for communities that do show growth in response to enrichment. 4. Grazing probably is not the key controlling variable for periphyton in Colorado mountain streams, given that the highest chlorophyll concentrations are associated with the highest abundances of macroinvertebrates. Modelling indicates that the initial amount of periphyton biomass at the start of the growing season, in conjunction with elevation‐related length of the growing season and water temperature, explains most of the variation in periphyton accumulation among these streams, but there is a yet unexplained suppression of periphyton growth rates across all elevations.     

水产养殖水体附着生物对磺胺甲恶唑胁迫响应及其降解效应

研究通过模拟养殖水体,探究附着生物对磺胺甲恶唑(SMX)的胁迫响应及其对SMX的降解途径。结果表明,在5和10 mg/L SMX胁迫下,附着生物胞外聚合物(EPS)分泌较对照组增加31.3%和21.5%,抗氧化能力降低20.6%和31.8%,但生物量的积累没有显著差异。SMX使附着生物抗氧化系统受到损伤,并刺激其分泌更多胞外聚合物以提高耐受性。去除实验结果表明,附着生物会促进SMX的消减, 1 g/L附着生物在第14天对SMX的去除率为25.10%。主要降解中间产物有N4-乙酰基-磺胺甲恶唑(TP295)、对苯醌亚胺(TP107)、去砜基磺胺甲恶唑(TP189)和去氨基磺胺甲恶唑(TP238),键的断裂和苯环上氨基反应是SMX降解的两种主要途径。研究结果对评估SMX在养殖水体中的生态风险和利用附着生物去除SMX提供技术支撑。     

Periphytic food and predatory crayfish: relative roles in determining snail distribution

Summary In the laboratory and field, we examined how periphyton (food of snails) and predatory crayfish influenced snail distribution in Trout Lake, a permanent, northern Wisconsin lake. Laboratory experiments (with no crayfish) tested the importance of periphyton biomass in determining snail preference among rocks, and among rock, sand, and macrophyte substrates. Among rocks with four different amounts of periphyton, periphyton biomass and the number of Lymnaea emarginata, Physa spp., and Amnicola spp. were positively related. A similar, but non-significant, trend occurred for Helisoma anceps. A field experiment at a site in Trout Lake where predation risk was low confirmed the preference by snails for periphyton covered rocks; more snails colonized rocks with periphyton than rocks without. When given a choice of rock, sand, and macrophytes in the laboratory, L. emarginata preferred high periphyton biomass and rock. Laboratory and field results contrasted with the distribution of snails in Trout Lake; no snails occurred in areas with abundant periphyton-covered rocks, but snails were abundant nearby on scattered rocks with little periphyton. However, where snails were absent, crayfish were abundant (14.5 crayfish-trap^–1-day^–1), and where snails were abundant, crayfish were rare (3.2 crayfish-trap^–1-day^–1), suggesting that crayfish predation reduced snails. The hypothesis that the negative association between snail and periphyton biomass resulted from snail grazing was supported by the results of a field snail enclosure-exclosure experiment (1 m² cages; n=3). All experiments and observations therefore suggest that: 1) crayfish predation is more important than a preference for high periphyton biomass in determining snail distribution in Trout Lake; 2) periphyton biomass is negtively related to snail grazing; and 3) crayfish had a positive indirect effect on periphyton by preying on grazing snails.     

Fish-mediated indirect effects in a littoral food web

The strength of the direct effect by scraping cladocerans and the indirect effect of nutrient regeneration by filtering herbivorous cladocerans on periphyton growth was investigated in a littoral food web. Ten enclosures were erected in a lake in an area with artificial vegetation. Five enclosures were stocked with juvenile perch ( Perca fluviatilis ) and five lacked fish. In addition, a reference area in the artificial vegetation was sampled. The mesh size of the net surrounding the cages was chosen to allow an inflow of phytoplankton into the cages from the surrounding water. The periphyton and filtering herbivorous cladoceran biomasses were highest in the fish-free treatment. There was no difference in phytoplankton biomass between treatments despite the large difference in filtering herbivorous cladoceran biomass, suggesting that the inflow of phytoplankton into enclosures completely compensated for the loss due to filtering. The reference area and the enclosure with fish showed the same patterns in developments with respect to filtering herbivorous cladocerans and periphyton. Scraping cladoceran biomass was higher in the fish-free treatment resulting in a positive correlation between scraping cladoceran and periphyton biomass. Our results suggest that the positive indirect effect of filtering herbivorous cladoceran nutrient regeneration on periphyton was stronger than the negative direct effect of grazing by scraping cladocerans on periphyton in this semi-open system, and that pelagic production by phytoplankton may foster periphyton growth in the littoral habitat via filtering herbivorous cladocerans. Furthermore, heterogeneity within trophic levels involving primary producers of different growth forms such as phytoplankton and periphyton may enhance the potential for compensatory responses via nutrient recycling.     

The effect of periphyton stoichiometry and light on biological phosphorus immobilization and release in streams

Periphyton stoichiometry can vary substantially as a result of differences in stream nutrient availability. A decrease in the periphyton carbon to phosphorus (C:P) ratio should decrease the demand for new P to be immobilized from stream water, but no studies to our knowledge have explored the relationship between periphyton stoichiometry and net P immobilization and release by periphyton. We sought to model biological P immobilization and release (flux) in streams by measuring periphyton stoichiometry and light availability. We measured P flux to and from intact periphyton on stream cobbles (20–100 mm diameter) in 1 L microcosms incubated with streamwater under variable light conditions. Net P immobilization occurred in 75% of microcosms, net P release occurred in only 5% of microcosms, and 20% of microcosms had neither net immobilization nor net release. When normalized to stream conditions, net P immobilization was highest when light availability was high (<60% canopy attenuation) and the periphyton C:P ratio was also high. In contrast, net P release occurred only when light availability was low (>60% canopy attenuation) and the periphyton C:P ratio was also low. A multiple regression model that included both periphyton stoichiometry and light availability from the growing season only, and the interaction term of these two variables, explained 99% of the variation in daily periphyton P flux observed in the study. These results indicate that in order to predict periphyton P immobilization, periphyton stoichiometry and light availability should be considered together. Furthermore, the results indicate that net P immobilization occurs even in very P-rich periphyton, which can act as a P sink when light availability is high.     

Redundancy among three herbivorous insects across an experimental current velocity gradient

We conducted streamside experiments to determine if the ability of herbivorous insects to remove algal periphyton varies with local current velocity. We used two mayfly species (Baetis bicaudatusand Drunella grandis) and one caddisfly species (Glossosoma verdona), which differ from one another in body morphology and mobility. Periphyton was grown for 30 days on ceramic tiles in constant velocity to create similar initial forage conditions for grazers. Tiles were transferred to three velocity regimes characteristic of the natural streambed: slow (3-5 cm s(-1)), medium (15-20 cm s(-1)) and fast (32-41 cm s(-1)). Four grazer treatments (Baetis, Drunella, and Glossosoma alone, and all species combined) were repeated for each velocity treatment to isolate the effect of local current on grazer ability to crop periphyton. Grazers differed in their abilities to remove periphyton across current treatments. Glossosoma removed significantly (P<0.05) more periphyton at fast versus either slow or medium velocities; Baetis showed a similar (but non-significant) trend; and, Drunella always removed about 75% of periphyton, irrespective of current. At fast current, periphyton removal was equivalent among the species. At medium current, Drunella removed significantly more than both Baetis and Glossosoma, whereas at slow current, Drunella removed more than Baetis, which removed more than Glossosoma. Periphyton removal under the combined three-grazer treatment was similar qualitatively to the combined effects of individual grazers. More periphyton tended to be removed as current increased, with the fast versus slow contrast showing marginal significance (P=0.10). Under all current regimes, the quantity of periphyton removed did not differ from the null model expectation of simple additive effects among individual grazers (i.e., no facilitation or inhibition). These experiments show that for some species, herbivory varies with current, which suggests that the herbivore "function" of cropping periphyton may vary with the environmental context of local current. Under some local velocities, however, different herbivore species "function" similarly and are potentially redundant with respect to periphytic removal. In naturally heterogeneous streams characterized by sharp gradients in local current velocity, we expect current-dependent species interactions to be common and at least partially contribute to intra-guild co-existence of species.