Concordance among stream assemblages and spatial autocorrelation along a fragmented gradient

Patterns of spatial autocorrelation of biota and distributional similarity (concordance) between assemblages of different organism groups have important implications in both theoretical ecology and biodiversity conservation. Here we report environmental gradients and spatial distribution patterns of taxonomic composition among stream fish, benthic macroinvertebrate, and diatom assemblages along a fragmented stream in south‐western France. We quantified spatial patterns of lotic assemblage structure along this stream, and we tested for concordance in distribution patterns among the three taxonomic groups. Our results showed that both environmental characteristics and stream assemblages were spatially autocorrelated. For stream fish and diatom assemblages, these patterns reflected assemblage changes along the longitudinal stream gradient, whereas environmental variables and benthic macroinvertebrates exhibited a more patchy spatial pattern. Cross‐taxa concordance was significant between stream fish and diatoms, and between stream fish and benthic macroinvertebrates. The assemblage concordance between stream fish and diatoms could be attributed to similar responses along the longitudinal gradient, whereas those between stream fish and benthic macroinvertebrates may result from biotic interactions. Based on potential dispersal capacities of taxa, our results validated the hypotheses that weakly dispersing taxa exhibit greater concordance than highly dispersing ones and that dispersal capacities affect how taxonomic groups respond to their local environment. Both diatoms and highly dispersing stream fish were affected by stream fragmentation (i.e. the number of dams between sites), while the effect of fragmentation was not significant for invertebrates that fly well in their adult stage, thus emphasizing the importance of the way of dispersal. These results suggest that addressing the effects of dispersal capacity on stream assemblage patterns is crucial to identifying mechanisms behind patterns and to better understanding the determinants of stream biodiversity.     

Implications of community concordance for assessing stream integrity at three nested spatial scales in Minnesota,U.S.A.

1. Fish and invertebrate assemblage data collected from 670 stream sites in Minnesota (U.S.A.) were used to calculate concordance across three nested spatial scales (statewide, ecoregion and catchment). Predictive taxa richness models, calibrated using the same data, were used to evaluate whether concordant communities exhibited similar trends in human‐induced taxa loss across all three scales. Finally, we evaluated the strength of the relationship between selected environmental variables and the composition of both assemblages at all three spatial scales. 2. Significant concordance between fish and invertebrate communities occurred at the statewide scale as well as in six of seven ecoregions and 17 of the 21 major catchments. However, concordance was not consistently indicative of significant relationships between rates of fish and invertebrate taxa loss at those same scales. 3. Fish and invertebrate communities were largely associated with different environmental variables, although the composition of both communities was strongly correlated with stream size across all three scales. 4. Predictive taxa‐loss models for fish assemblages were less sensitive and precise than models for invertebrate assemblages, likely because of the relatively low number of common fish taxa in our data set. Both models, however, distinguished reference from non‐reference sites. 5. The importance of concordance, geographic context and scale are discussed in relation to the design and interpretation of stream integrity indicators. In particular, our findings suggest that community concordance should not be viewed as a substitute for an evaluation of how assemblages respond to environmental stressors.     

Zooplankton assemblage concordance patterns in Brazilian reservoirs

The main goal of this study was to evaluate the assemblage concordance among three zooplankton groups (Rotifera, Cladocera and Copepoda) in 30 Brazilian reservoirs. According to Mantel tests and Procrustean analyses, there was a high level of assemblage concordance. Highest assemblage concordance was observed between cladocerans and copepods, while the lowest level of concordance was detected between copepods and rotifers. Based on environmental or biotic data, patterns of among-reservoir dissimilarities were fairly stable across the two seasonal periods analyzed in this study. Multiple Mantel tests were used to model the between-reservoir dissimilarities (Bray–Curtis distance) in zooplankton assemblage structure as a function of the limnological, geographical and morphological distances between pairs of reservoirs. The best predictor of faunistic dissimilarities was the matrix containing the limnological distances among the reservoirs. In general, these results are important for monitoring purposes because they supported the use of surrogate taxa and indicate that community concordance analysis may be a powerful tool for enhancing the efficiency of monitoring programs, ensuring their long-term sustainability. Handling editor: S. I. Dodson     

Relationship of fish and macroinvertebrate assemblages to environmental factors: implications for community concordance

Community concordance describes similarity in distributions and abundances of organisms from different taxonomic groups across a region of interest, with highly concordant communities assumed to respond similarly to major environmental gradients, including anthropogenic stressors. While few studies have explicitly tested for concordance among stream-dwelling organisms, it frequently is assumed that both macroinvertebrates and fish respond in concert to environmental factors, an assumption that has implications for their management. We investigated concordance among fish and macroinvertebrates from tributaries of two catchments in southeastern Michigan having varied landscape characteristics. Classifications of fish and macroinvertebrate assemblages resulted in groups distinguished by differences in taxonomic characteristics, functional traits, and stressor tolerance of their respective dominant taxa. Biological groups were associated with principal landscape gradients of the study region, which ranged from forests and wetlands on coarse surficial geology to agricultural lands on finer, more impervious surficial geology. Measures of stream habitat indicated more stable stream flows and greater heterogeneity of conditions at site groups with catchments comprising forests and wetlands on the coarsest geology, but did not distinguish well among remaining site groups, suggesting that habitat degradation may not be the driving mechanism leading to differences in groups. Despite broadly similar interpretations of relationships of site groups with landscape characteristics for both fish and macroinvertebrates, examination of site representation within groups indicated weak community concordance. Our results suggest that explicit responses of fish and macroinvertebrates to landscape factors vary, due to potential differences in their susceptibility to controls or to differences in the scale at which landscape factors influence these organisms. Handling editor: Robert Bailey     

Missing data,incomplete taxa,and phylogenetic accuracy

The problem of missing data is often considered to be the most important obstacle in reconstructing the phylogeny of fossil taxa and in combining data from diverse characters and taxa for phylogenetic analysis. Empirical and theoretical studies show that including highly incomplete taxa can lead to multiple equally parsimonious trees, poorly resolved consensus trees, and decreased phylogenetic accuracy. However, the mechanisms that cause incomplete taxa to be problematic have remained unclear. It has been widely assumed that incomplete taxa are problematic because of the proportion or amount of missing data that they bear. In this study, I use simulations to show that the reduced accuracy associated with including incomplete taxa is caused by these taxa bearing too few complete characters rather than too many missing data cells. This seemingly subtle distinction has a number of important implications. First, the so-called missing data problem for incomplete taxa is, paradoxically, not directly related to their amount or proportion of missing data. Thus, the level of completeness alone should not guide the exclusion of taxa (contrary to common practice), and these results may explain why empirical studies have sometimes found little relationship between the completeness of a taxon and its impact on an analysis. These results also (1) suggest a more effective strategy for dealing with incomplete taxa, (2) call into question a justification of the controversial phylogenetic supertree approach, and (3) show the potential for the accurate phylogenetic placement of highly incomplete taxa, both when combining diverse data sets and when analyzing relationships of fossil taxa.     

Untangling associations between chironomid taxa in Neotropical streams using local and landscape filters

1. Analyses of species association have major implications for selecting indicators for freshwater biomonitoring and conservation, because they allow for the elimination of redundant information and focus on taxa that can be easily handled and identified. These analyses are particularly relevant in the debate about using speciose groups (such as the Chironomidae) as indicators in the tropics, because they require difficult and time‐consuming analysis, and their responses to environmental gradients, including anthropogenic stressors, are poorly known. 2. Our objective was to show whether chironomid assemblages in Neotropical streams include clear associations of taxa and, if so, how well these associations could be explained by a set of models containing information from different spatial scales. For this, we formulated a priori models that allowed for the influence of local, landscape and spatial factors on chironomid taxon associations (CTA). These models represented biological hypotheses capable of explaining associations between chironomid taxa. For instance, CTA could be best explained by local variables (e.g. pH, conductivity and water temperature) or by processes acting at wider landscape scales (e.g. percentage of forest cover). 3. Biological data were taken from 61 streams in Southeastern Brazil, 47 of which were in well‐preserved regions, and 14 of which drained areas severely affected by anthropogenic activities. We adopted a model selection procedure using Akaike’s information criterion to determine the most parsimonious models for explaining CTA. 4. Applying Kendall’s coefficient of concordance, seven genera (Tanytarsus/Caladomyia, Ablabesmyia, Parametriocnemus, Pentaneura, Nanocladius, Polypedilum and Rheotanytarsus) were identified as associated taxa. The best‐supported model explained 42.6% of the total variance in the abundance of associated taxa. This model combined local and landscape environmental filters and spatial variables (which were derived from eigenfunction analysis). However, the model with local filters and spatial variables also had a good chance of being selected as the best model. 5. Standardised partial regression coefficients of local and landscape filters, including spatial variables, derived from model averaging allowed an estimation of which variables were best correlated with the abundance of associated taxa. In general, the abundance of the associated genera tended to be lower in streams characterised by a high percentage of forest cover (landscape scale), lower proportion of muddy substrata and high values of pH and conductivity (local scale). 6. Overall, our main result adds to the increasing number of studies that have indicated the importance of local and landscape variables, as well as the spatial relationships among sampling sites, for explaining aquatic insect community patterns in streams. Furthermore, our findings open new possibilities for the elimination of redundant data in the assessment of anthropogenic impacts on tropical streams.     

Montane Refugia for Endemic and Red Listed Dragonflies in the Cape Floristic Region Biodiversity Hotspot

One of the features of many endemic organisms is that they are highly spatially restricted, and habitat specialists. The Kogelberg Biosphere Reserve (KBR) is a major centre of plant endemism within a global hotspot, the Cape Floristic Region (CFR). Dragonflies in this botanical hotspot have a range of habitat specialization from narrow-range specialists to widespread generalists, with an unusually strong bias towards the specialists. A huge 53% of dragonfly individuals and 26% of taxa recorded are national endemics, and three species are Red Listed. Thus, a group of predatory insects, which are largely not dependent on plant composition, mirrors the level of habitat specialization and restricted distributions of the plants at the spatial scale of the whole reserve. Although some studies caution the use of one taxon as a surrogate for another, the results here show that at the reserve scale in this global hotspot there can be remarkable concordance, suggesting further studies on other taxa should be carried out to determine the full extent of taxonomic concordance in this irreplaceable area.     

Species‐specific responses to island connectivity cycles: refined models for testing phylogeographic concordance across a Mediterranean Pleistocene Aggregate Island Complex

The contribution of Pleistocene sea level changes to diversification patterns in archipelagos around the world, and specifically whether the repeated cycles of island connectivity and isolation acted as a ‘species pump’ is debated. The debate has been perpetuated in part because of the type of evidence used to evaluate the species‐pump hypothesis. Specifically, existing tests of the ‘Pleistocene Aggregate Island Complex’ (PAIC) model of diversification interpret the lack of concordant divergence times among multiple codistributed taxa as a rejection of the PAIC model. However, the null expectation of concordance disregards taxon‐specific ecological traits and geographic characteristics that may affect population persistence and gene flow among islands. Here, we study the factors affecting population divergence in thirteen flightless darkling beetle species (Coleoptera: Tenebrionidae) across the PAIC system of the Cycladic plateau in the Aegean archipelago. Based on isolation‐by‐resistance analyses, hierarchical amova and the degree of genealogical sorting on individual islands, we identify a major effect of bathymetry and habitat stability on the levels of genetic divergence across the PAIC, with island size and body size playing a secondary role as well. We subsequently use bathymetric maps and habitat association to generate predictions about the set of islands and group of taxa expected to show phylogeographic concordance. We test these predictions using hierarchical approximate Bayesian computation and show how our interpretations regarding the role of PAICs as drivers of divergence change when relying on a null expectation of concordance compared to a refined model that takes geography and ecological traits into account.     

The probability of topological concordance of gene trees and species trees

The concordance of gene trees and species trees is reconsidered in detail, allowing for samples of arbitrary size to be taken from the species. A sense of concordance for gene tree and species tree topologies is clarified, such that if the "collapsed gene tree" produced by a gene tree has the same topology as the species tree, the gene tree is said to be topologically concordant with the species tree. The term speciodendric is introduced to refer to genes whose trees are topologically concordant with species trees. For a given three-species topology, probabilities of each of the three possible collapsed gene tree topologies are given, as are probabilities of monophyletic concordance and concordance in the sense of N. Takahata (1989), Genetics 122, 957-966. Increasing the sample size is found to increase the probability of topological concordance, but a limit exists on how much the topological concordance probability can be increased. Suggested sample sizes beyond which this probability can be increased only minimally are given. The results are discussed in terms of implications for molecular studies of phylogenetics and speciation.     

Evidence against the use of surrogates for biomonitoring of Neotropical floodplains

1. Community concordance measures the level of association between the compositional patterns shown by two groups of organisms. If strong community concordance occurs, one group could be used as a surrogate for another in conservation planning and biodiversity monitoring. In this study, we evaluated the variability in the strength of community concordance, the likely mechanisms underlying community concordance and the degree to which one community can predict another in a set of Neotropical floodplain lakes (Upper Paraná River floodplain, Brazil). 2. We used a data set including six aquatic communities: fish, macrophytes, benthic macroinvertebrates, zooplankton, phytoplankton and periphyton. We used Mantel and PROTEST approaches to evaluate the levels of community concordance in up to four sampling periods. Also, we used partial Mantel test and information about biotic interactions to investigate reasons for observed patterns of concordance. Finally, we used co‐correspondence analysis to evaluate the performance of one taxonomic group in predicting the structures of other communities. 3. The levels of community concordance varied over time for almost all cross‐taxa comparisons. Concordance between phytoplankton and periphyton probably resulted from similar responses to environmental gradients, whereas other patterns of concordance were likely generated by interactions among groups. However, the levels of predictability were low, and no particular taxonomic group significantly predicted all other groups. 4. The low and temporally variable levels of community concordance cast doubts on the use of surrogate groups for biodiversity management in Neotropical floodplains.     

Community concordance in lotic ecosystems: How to establish unbiased congruence between macroinvertebrate and fish communities

Community concordance within aquatic biota could provide useful information for improving the methods used in bioassessment and biodiversity conservation management. The main goal of the study was to investigate the mechanism of community concordance between macroinvertebrates and fish in a single river basin (South Morava river Basin, Serbia). In order to achieve this, a Self organizing map (SOM) ordinated and classified sampling sites based on the community structures of five different taxa groups (macroinveretbrates (MIB), fish (FSH), Chironomidae (CHI), Macroinvertebrates without Chironomidae (MWC) and the Ephemeroptera Plecoptera Trichioptera group (EPT)). SOM also revealed 6 environmental gradients along the groups tested that significantly changed their community structures. Using the results of the SOM analysis as the input, the Mantel test quantified the highest community concordance between FSH and MIB (r = 0.42) followed by FSH and CHI (r = 0.29). The lowest concordance was recorded between FSH and EPT (r = 0.14). The indicator species analysis (IndVal) revealed 39 species to be responsible for the community patterns obtained. The Geo-SOM visualized the spatial distribution of the IndVal taxa, revealing the generators of community concordance. The strength of community concordance depends on the variability of the data on the aquatic biota. Thus, having an appropriate sampling and statistical design as well as high taxonomic resolution, as some of the factors which increase the variability in the data set, could present community concordance between fish and macroinvertebrates in an unbiased way.     

Review and meta‐analysis of natural selection in mitochondrial complex I in metazoans

Variation in mitochondrial DNA is often assumed to be neutral and is used to construct the genealogical relationships among populations and species. However, if extant variation is the result of episodes of positive selection, these genealogies may be incorrect, although this information itself may provide biologically and evolutionary meaningful information. In fact, positive Darwinian selection has been detected in the mitochondrial‐encoded subunits that comprise complex I from diverse taxa with seemingly dissimilar bioenergetic life histories, but the functional implications of the selected sites are unknown. Complex I produces roughly 40% of the proton flux that is used to synthesize ATP from ADP, and a functional model based on the high‐resolution structure of complex I described a unique biomechanical apparatus for proton translocation. We reported positive selection at sites in this apparatus during the evolution of Pacific salmon, and it appeared this was also the case in published reports from other taxa, but a comparison among studies was difficult because different statistical tests were used to detect selection and oftentimes, specific sites were not reported. Here we review the literature of positive selection in mitochondrial genomes, the statistical tests used to detect selection, and the structural and functional models that are currently available to study the physiological implications of selection. We then search for signatures of positive selection among the coding mitochondrial genomes of 237 species with a common set of tests and verify that the ND5 subunit of complex I is a repeated target of positive Darwinian selection in diverse taxa. We propose a novel hypothesis to explain the results based on their bioenergetic life histories and provide a guide for laboratory and field studies to test this hypothesis.     

Anthropogenic land-use stress alters community concordance at the river-riparian interface

Organisms often respond in similar ways to environmental or spatial gradients, particularly at large spatial scales. Yet, while understanding these relationships is important for both basic and applied purposes, such as identifying surrogate taxa for conservation and monitoring purposes, patterns at finer scales and across ecotones are less certain. Our central aim was to explore patterns in community concordance at the river-riparian interface and examine whether concordance was decoupled by increasing anthropogenic stress (a gradient of local land-use intensity). We explored this at 15 sites over three years within the LTER site, Rhine-Main-Observatory, a low mountain river system in central Germany, assessing concordance between four organism groups: riparian spiders and carabid beetles, benthic macroinvertebrates, and combined aquatic macrophytes and riparian plants. This represented three different linkages: (1) predator–prey, (2) direct competition, and (3) habitat associations. While there were no correlations in richness patterns, multivariate community structure was highly concordant between all groups. Anthropogenic stress strongly reduced links between riparian spiders and carabid beetles, likely resulting from their shared resource requirements. However, increasing concordance generally occurred between plants and other groups, although inconsistently between the three groups. We posit that patterns may be resulting from two processes: (1) linkages between directly competing species decouple with increasing anthropogenic stress, and (2) stronger coupling may occur between habitat providers and dependent species when overall habitat complexity is reduced. Our results highlight the complex manner in which anthropogenic stress can influence ecosystem structure, particularly at small scales. Based on these complexities, we recommend considering the full suite of community data to adequately explore biodiversity patterns or when searching for surrogate taxa.     

Phylogenetic analysis of Aspergillus species using DNA sequences from four loci

DNA sequences were determined for beta tubulin (BT2), calmodulin (CF), ITS and lsu rDNA (ID) and RNA polymerase II (RPB2) from ca. 460 Aspergillus isolates. RPB2 and rDNA sequences were combined and analyzed to determine relationships in the genus and in the family Trichocomaceae. Eupenicillium species form a statistically supported clade with origins among the Aspergillus clades. A. crystallinus, A. malodoratus and H. paradoxus are members of the Eupenicillium clade. A. zonatus, A. clavatoflvus and W. spinulosa occur in a clade along with Hamigera sp. Other than these exceptional species, Aspergillus species and sections occur on three strongly supported clades that descend from a polytomy. Section Versicolores as a monophyletic group includes only A. versicolor and A. sydowii and is superfluous. The other sections were retained but modified. All four loci were used in genealogical concordance analysis of species boundaries. Fennellia flavipes and F. nivea are not conspecific with their supposed anamorphs A. flavipes and A. nivea. Synonymies were found for some species and more than 20 undescribed taxa were identified in genealogical concordance analysis. Newly discovered taxa will be described elsewhere. Possibly paralogous gene fragments were amplified with the BT2 primers in sections Nidulantes, Usti and Nigri. Use of nonhomologous sequences in genealogical concordance analysis could lead to false conclusions and so BT2 sequences were not used in analysis of those sections.     

An ecological tardis: the implications of facilitation through evolutionary time

It is often challenging to link ecological processes to evolution because different temporal scales are studied and necessarily inferred. In a recent paper by Valiente-Banuet et al., Quaternary plant taxa are shown to 'pull' more ancient Tertiary taxa through evolutionary time by facilitation. This finding has profound implications for population and community ecology. Positive species interactions are of wider importance than was assumed previously in determining community composition by enhancing long-term biodiversity, mediating climate change and providing an interdependent set of selection processes in addition to the environment. Future experiments should consider the evolutionary history of species, manipulate species interactions explicitly to test for environmental effects and re-evaluate the adaptive significance of traits in the context of other species.     

Delimiting species boundaries and the conservation genetics of the endangered maritime ringlet butterfly (Coenonympha nipisiquit McDunnough)

Species delimitation is a difficult problem that has implications across organismal biology, yet no single method has proved wholly satisfactory. We tested the utility of combining species-delimitation methods based on phylogeny and gene flow statistics using two parapatric members of the Coenonympha tullia group as an example: the endangered maritime ringlet butterfly (Coenonympha nipisiquit McDunnough) and the common inornate ringlet butterfly (Coenonympha inornata Edwards). We reconstructed the phylogeny of the nearctic C. tullia-group taxa from mitochondrial DNA (mtDNA) sequences (cytochrome oxidase I and mitochondrial control region) to explore the ancestry of the C. nipisiquit lineage within the group. We investigated the extent of gene flow between the two taxa with F-statistics using 587 nuclear amplified fragment length polymorphism markers, accounting for the effect of potential scoring 'collisions' where a marker may represent more than one DNA fragment. Combining species-delimitation methods was especially effective because it uncovered both historical and recent evolutionary patterns. Phylogenetic analysis of mtDNA revealed the early divergence of C. nipisiquit from other C. tullia-group taxa, including the morphologically similar C. inornata. F-statistics and gene-by-gene introgression profiles demonstrated clear isolation between the two taxa and revealed strong population structure within C. nipisiquit. C. nipisiquit is the first taxon in the nearctic C. tullia group showing strong evidence of genetic isolation. The methods we used are relatively inexpensive and can be widely used to delimit taxonomic boundaries near the species level, both generally and in particular for taxa that may be targets of conservation efforts.     

Phylogeographical patterns in the American Pacific Northwest: lessons from the arionid slug Prophysaon coeruleum

Some of the earliest studies of phylogenetic concordance involve native plants from the Pacific Northwest where many taxa showed clear genetic breaks between southern and northern populations. To test whether similar breaks also occur in invertebrate species with low dispersal capacities, genetic data from two mitochondrial genes are assembled for individuals of the arionid slug Prophysaon coeruleum throughout the species' range. Bayesian inference revealed three major clades and a total of eight subclades. It is argued that the demographic and genealogical past of P. coeruleum has resulted in a deep and shallow phylogeographical structure. The deep structure is at least 2.6-5.9 million years old and therefore clearly predates the Pleistocene period. Superimposed on this structure is a shallow structure that is most likely less than 2 million years old and probably the result of Pleistocene perturbations. Molecular analyses revealed that the three known colour traits in P. coeruleum do not represent monophyletic groups and that they do not match the patterns of genetic structure found. It is argued that the colour traits are perhaps a response to different levels of UV-radiation. The study adds to the increasing evidence that the phylogeographical structure of some taxa is more complex than previously thought. Moreover, it shows that genealogical concordance should not be deduced from phylogeographical patterns alone but should be based on an understanding of timing and causes of historical processes that lead to those patterns.     

The effect of excluding taxa with low abundances or taxa with small distribution ranges on ecological assessment

The present study aims to investigate whether taxa with a small distribution range or taxa with low abundances indicate specific habitats or a high ecological quality and what the effect is if these taxa are excluded from ecological assessment. We compared autecological features between stream dwelling taxa with a mean abundance >5 individuals per sample and a mean abundance ≤5 individuals per sample as well as between taxa with a small distribution range and taxa with a large distribution range. The number of rare taxa (either with a small distribution range or with low abundances) in a sample was related to the ecological quality classes. To test the effect of exclusion of rare taxa we constructed 8 data sets all including 142 samples of Dutch lowland streams. From each data set we stepwise excluded taxa that had low abundances or taxa that were known to be restricted in their distribution range. With help of the AQEM assessment software we calculated the final ecological quality classes and the metrics that were included in the multimetric for the original data and the 8 selected data sets. Autecological features of the taxa within the different selections showed that taxa with small distribution ranges were often running water taxa, living on stones and gravel and indicating oligosaprobic water conditions in contrast to taxa that had a large distribution range. There were only small differences between taxa with low and high abundances. However, current velocity preference was lower for taxa with abundance ≤5 individuals per sample, saprobic values were higher and scores for typical stream habitats, such as lithal, psammal and akal were lower compared to high abundant taxa. If taxa with low abundances were excluded a higher ecological quality class was achieved in most cases, while excluding taxa with a small distribution range resulted in lower ecological quality classes. In conclusion, excluding taxa with a small distribution range led to worse ecological quality classes because these taxa have special autecological features that often indicate natural streams. On the other hand, excluding taxa with low abundances resulted in higher ecological quality classes because these taxa indicate more disturbed situations and because the number of taxa per sample was strongly reduced. Although the documentation of rare taxa (either with low abundances or with small distribution ranges) is often time and cost-intensive regarding field work, laboratory work, data processing, and analyses, the indicative power of these taxa for natural circumstances is essential and therefore rare taxa should be included in ecological assessment studies.