Community assembly is determined by a combination of historical events and contemporary processes that are difficult to disentangle, but eco-evolutionary mechanisms may be uncovered by the joint analysis of species and genetic diversity across multiple sites. Mountain streams across Europe harbour highly diverse macroinvertebrate communities whose composition and turnover (replacement of taxa) among sites and regions remain poorly known. We studied whole-community biodiversity within and among six mountain regions along a latitudinal transect from Morocco to Scandinavia at three levels of taxonomic hierarchy: genus, species and haplotypes. Using DNA barcoding of four insect families (>3100 individuals, 118 species) across 62 streams, we found that measures of local and regional diversity and intraregional turnover generally declined slightly towards northern latitudes. However, at all hierarchical levels we found complete (haplotype) or high (species, genus) turnover among regions (and even among sites within regions), which counters the expectations of Pleistocene postglacial northward expansion from southern refugia. Species distributions were mostly correlated with environmental conditions, suggesting a strong role of lineage- or species-specific traits in determining local and latitudinal community composition, lineage diversification and phylogenetic community structure (e.g., loss of Coleoptera, but not Ephemeroptera, at northern sites). High intraspecific genetic structure within regions, even in northernmost sites, reflects species-specific dispersal and demographic histories and indicates postglacial migration from geographically scattered refugia, rather than from only southern areas. Overall, patterns were not strongly concordant across hierarchical levels, but consistent with the overriding influence of environmental factors determining community composition at the species and genus levels.

The European Water Framework Directive establishes the need to define stream type-specific reference conditions to identify ''high ecological status''. Methods for selecting reference sites using a priori criteria have been proposed by many authors. A review of these criteria revealed that the most relevant criteria for streams and rivers were those related to riparian vegetation, diffuse and point sources of pollution, river morphology and hydrological conditions and regulation. In this work, we propose 20 criteria that reflect the characteristics of Mediterranean streams and their most frequent disturbances for the selection of reference sites in Mediterranean streams in Spain. We studied 162 sites located in 33 Mediterranean basins belonging to five stream types. Of the locations, 57% were selected as a priori reference sites by having applied the proposed criteria. Reference sites were identified for all stream types except for ''large watercourses'' which includes the lower reaches of some rivers in this study area. This a priori selection of reference sites was subjected to validation using the macroinvertebrate community by applying of an IBMWP threshold, which is considered to be an indicator of undisturbed sites in Mediterranean streams. This approach determined that whole of this selection (100%) could be considered valid reference sites. Furthermore, we identified differences in the reference conditions for each stream type on the basis of macroinvertebrate assemblage composition.

1. According to the guidelines of the European Water Framework Directive, assessment of the ecological quality of streams and rivers should be based on ecotype-specific reference conditions. Here, we assess two approaches for establishing a typology for Mediterranean streams: a top-down approach using environmental variables and bottom-up approach using macroinvertebrate assemblages. 2. Classification of 162 sites using environmental variables resulted in five ecotypes: (i) temporary streams; (ii) evaporite calcareous streams at medium altitude; (iii) siliceous headwater streams at high altitude; (iv) calcareous headwater streams at medium to high altitude and (v) large watercourses. 3. Macroinvertebrate communities of minimally disturbed sites (n ¼ 105), grouped using UPGMA (unweighted pair-group method using arithmetic averages) on Bray-Curtis similarities, were used to validate four of the five ecotypes obtained using environmental variables; ecotype 5, large watercourses, was not included as this group had no reference sites. 4. Analysis of similarities (ANOSIM A NO S IM ) showed that macroinvertebrate assemblage composition differed among three of the four ecotypes, resulting in differences between the bottom-up and top-down classification approaches. Siliceous streams were clearly different from the other three ecotypes, evaporite and calcareous ecotypes did not show large differences in macroinvertebrate assemblages and temporary streams formed a very heterogeneous group because of large variability in salinity and hydrology. 5. This study showed that stream classification schemes based on environmental variables need to be validated using biological variables. Furthermore, our findings indicate that special attention should be given to the classification of temporary streams.

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In endotherms insects, the thermoregulatory mechanisms modulate heat transfer from the thorax to the abdomen to avoid overheating or cooling in order to obtain a prolonged flight performance. Scarabaeus sacer and S. cicatricosus, two sympatric species with the same habitat and food preferences, showed daily temporal segregation with S. cicatricosus being more active during warmer hours of the day in opposition to S. sacer who avoid it. In the case of S. sacer, their endothermy pattern suggested an adaptive capacity for thorax heat retention. In S. cicatricosus, an active ‘heat exchanger’ mechanism was suggested. However, no empirical evidence had been documented until now. Thermographic sequences recorded during flight performance showed evidence of the existence of both thermoregulatory mechanisms. In S. sacer, infrared sequences showed a possible heat insulator (passive thermal window), which prevents heat transfer from meso- and metathorax to the abdomen during flight. In S. cicatricosus, infrared sequences revealed clear and effective heat flow between the thorax and abdomen (abdominal heat transfer) that should be considered the main mechanism of thermoregulation. This was related to a subsequent increase in abdominal pumping (as a cooling mechanism) during flight. Computer microtomography scanning, anatomical dissections and internal air volume measurements showed two possible heat retention mechanisms for S. sacer; the abdominal air sacs and the development of the internal abdominal sternites that could explain the thermoregulation between thorax and abdomen. Our results suggest that interspecific interactions between sympatric species are regulated by very different mechanisms. These mechanisms create unique thermal niches for the different species, thereby preventing competition and modulating spatio-temporal distribution and the composition of dung beetle assemblages.