Sexual and reproductive behaviour of Drosophila melanogaster from a microclimatically interslope differentiated population of "Evolution Canyon" (Mount Carmel, Israel).

The strong microscale interslope environmental differences in "Evolution Canyon" provide an excellent natural model for sympatric speciation. Our previous studies revealed significant slope-specific differences for a fitness complex of Drosophila. This complex involved either adaptation traits (tolerance to high temperature, different viability and longevity pattern) or behavioural differentiation, manifested in habitat choice and non-random mating. This remarkable differentiation has evolved despite a very small interslope distance (a few hundred metres only). Our hypothesis is that strong interslope microclimatic contrast caused differential selection for fitness-related traits accompanied by behavioural differentiation and reinforced by some sexual isolation, which started incipient speciation. Here we describe the results of a systematic analysis of sexual behaviour in a non-choice situation and several reproductive parameters of D. melanogaster populations from the opposite slopes of "Evolution Canyon". The evidence indicates that: (i) mate choice derives from differences in mating propensity and discrimination; (ii) females from the milder north-facing slope discriminate strongly against males of the opposite slope; (iii) both sexes of the south-facing slope display distinct reproductive and behavioural patterns with females showing increased fecundity, shorter time before remating and relatively higher receptivity, and males showing higher mating propensity. These patterns represent adaptive life strategies contributing to higher fitness.     

Microsatellite analysis of Drosophila melanogaster populations along a microclimatic contrast at lower Nahel Oren canyon, Mount Carmel, Israel

Drosophila melanogaster populations collected at the south-facing slope (SFS) and north-facing slope (NFS) of lower Nahel Oren canyon, Mount Carmel, Israel display significant differences in survival and longevity at temperature, drought, and starvation stresses. Furthermore, significant assortative mating was previously observed between populations of the two slopes. We used a set of 48 microsatellite markers to analyze patterns of genetic differentiation between D. melanogaster populations from both slopes and D. simulans. Consistent with previous reports, we found D. simulans to be well differentiated from D. melanogaster. Genetic differentiation between SFS and NFS D. melanogaster populations was low (F(ST) = 0.0012). Also a tree of individuals based on the proportion of shared alleles and a model-based clustering method provided no evidence for population substructuring.     

Population genetics of accessory gland proteins and sexual behavior in Drosophila melanogaster populations from Evolution Canyon

Evolution Canyon in Lower Nahal Oren, Mount Carmel, Israel has been identified as a location promoting sympatric speciation. Several previous studies on Drosophila melanogaster populations from the two disparate slopes of the canyon suggest that these two populations are experiencing incipient speciation. However, recent microsatellite data did not reveal the expected level of population differentiation. Given the importance of this system for studying speciation, we set out to test two predictions of the incipient speciation hypothesis--genetic differentiation and sexual isolation. We sequenced six different Acp genes from isofemale lines from the south-facing slope (11 lines) and north-facing slope (nine lines) of Evolution Canyon. We found no evidence of genetic differentiation between the two slopes (F(ST) = -0.03). We also conducted mate choice tests, using intraslope F1 hybrids between different isofemale lines. There was no significant departure from random mating in mixtures of flies from the two slopes. Our results provide further indication that it is unlikely these two populations are experiencing incipient speciation. We discuss our results in light of the discrepancies that have been published on this enigmatic D. melanogaster system from Evolution Canyon.     

Adaptive differentiation of thermotolerance in Drosophila along a microclimatic gradient

We examined whether a remarkable occurrence - the physiological evolution of two Drosophila melanogaster populations, despite a spatial separation of only 100-400 m, was idiosyncratic and temporary, or persisted over multiple years. We ascertained the high-temperature tolerance of Drosophila descended from populations on the north-facing slope (NFS) and south-facing slope (SFS) of 'Evolution Canyon' (Lower Nahal Oren, Mt Carmel, Israel), which were collected in 1997, 1999, and 2000. Results for these Drosophila uniformly resembled other studies in many respects: an inverse relationship between survival and heat-shock temperature, male-female differences in thermotolerance, and inducible thermotolerance. Importantly, for all years of collection, SFS flies consistently exceeded NFS flies in basal and inducible thermotolerance after diverse heat shocks, with and without thermal pretreatment, and whether isofemale lines, synthetic populations, or inbred lines were compared. Inbred lines, however, had lower thermotolerance than outbred lines. Several nonexclusive processes may explain the evolution of such physiological differentiation.     

Genome Evolution of the Cyanobacterium Nostoc linckia under Sharp Microclimatic Divergence at "Evolution Canyon," Israel

We describe the genomic DNA diversity and divergence of the cyanobacterium Nostoc linckia from "Evolution Canyon," a microsite consisting of ecologically contrasting slopes, south-facing slope (SFS) and north-facing slope (NFS), at lower Nahal Oren, Mt. Carmel, Israel. The opposing slopes share their limestone lithology but vary greatly in their ecology, primarily because of different levels of solar radiation (which is six times higher on the SFS than on the NFS). The warm and xeric SFS displays a tropical African savanna, whereas the cool and mesic NFS displays a temperate South European Mediterranean live-oak maquis shrub forest. The cyanobacterium Nostoc linckia tested here is a sessile microorganism, growing as a carpet on rock surfaces and constantly exposed to environmental fluctuations of solar radiation, temperature, and desiccation. We demonstrate remarkable interslope and intraslope genetic divergence of the genome (including both coding and noncoding regions) of Nostoc linckia, by using 211 AFLP (amplified fragment length polymorphism) DNA molecular marker loci. Genetic polymorphism of N. linckia subpopulations on the ecologically harsher SFS was significantly (p <0.05) higher (p = 99.53%) than was that of the subpopulations on the climatically milder nfs (p = 85.78%). genetic polymorphism (p) and gene diversity (he) were significantly correlated with variables influencing aridity stress: solar radiation (sr) (rp = 0.956; p = 0.046), temperature (tm) (rp = 0.993; p = 0.0068), and day-night temperature difference (tdd) (rp = 0.975; p = 0.025). as in other tested organisms from "evolution canyon", but even more exceptionally because of its completely sedentary nature, we suggest that the climatically stressed sfs environment is responsible for this marked increase of genetic polymorphism, which is maintained by the combined evolutionary forces of diversifying and balancing selection. This could highlight the importance of ecological stress and selection in evolution and its remarkable effect on the genetic system across the prokaryotic genome.     

Do slope orientation and sampling location determine soil biota composition?

"Evolution Canyon" is a typical Mediterra-nean-basin canyon with a summer dry stream at the bottom and large differences in vegetation cover and solar radiation between the north-facing slope (NFS) and the south-facing slope (SFS). It is known to act as an abiotic mediator influencing the community structure of soil fauna. The aim of this study was to determine the spatial dispersion of soil microbial and free-living nematode communities in the open sites (between shrubs) in the upper (0-10 cm) soil layer at four altitudes on both slopes. The combination of relative soil moisture availability and temperature, known to be one of the main triggers for soil biota activity, was explained by slope orientation. The above-mentioned differences were found to significantly affect microbial biomass and CO2 evolution resulting in temporary stress, corresponding to higher values in metabolic quotient (qCO2) values. These differences may represent microbial investment in energy in order to overcome stress resulting from the microclimatic differ-ences between the two slopes. Moreover, the degree of substrate limitation (primary production due to the differences in plant cover) of microbial activity was explained by the difference in microbial functional groups. The total abundance of soil free-living nematodes was found to be 2-fold higher on the SFS than on the NFS. Thirty-nine genera, including 12 bacteria-feeders, 5 fungi-feeders, 12 plant-parasites, and 10 omnivore-predators, were found at the study site, with 34 genera on the NFS and 29 on the SFS. The generic diversity of the bacteria-feeding nematodes was higher on the SFS than on the NFS. This study elucidates the importance of slope orientation and its effect on the structural levels of soil microbial and nematode communities.     

Population genetic response to microsite ecological stress in wild barley, Hordeum spontaneum

Genetic diversity was studied in six subpopulations (a total of 60 individuals) of wild barley, Hordeum spontaneum , the progenitor of cultivated barley, sampled from six stations located along a transect of 300 m across the two opposing slopes of 'Evolution Canyon', a Mediterranean microsite at Lower Nahal Oren, Mt Carmel. The two opposing slopes are separated by between 100 and 400 m and designated SFS (South-Facing Slope) and NFS (North-Facing Slope) with each having three equidistant test stations. The SFS, which receives up to 300% more solar radiation, is drier, ecologically more heterogeneous, fluctuating, and more stressful than the NFS. Analysis of 12 RAPD primers, representing a total of 51 putative loci, revealed a significant inter- and intraslope variation in RAPD band polymorphism. A significantly higher proportion of polymorphic RAPD loci was found amongst the subpopulations on the SFS (mean P = 0.909) than on the NFS (mean P = 0.682), on the basis of the presence and absence of 22 strong bands. Polymorphism generally increased upwards from the bottom to the top of the SFS (0.636, 0.773, 0.955) and NFS (0.409, 0.500, 0.545), respectively. Gametic phase disequilibria estimates, D, revealed SFS and NFS unique predominant combinations which sharply differentiated the two slopes and indicated that there is differential interslope selection favouring slope-specific multilocus combinations of alleles, or blocks of genes over tens to hundreds of meters. This suggests that selection overrides migration. RAPD polymorphism appears to parallel allozyme diversity which is climatically adaptive and driven by natural selection in the same subpopulations at the microsite.     

Colony growth rates of microfungal isolates from the north and south facing slopes of Evolution Canyon, Mount Carmel, Israel, after irradiation at 10(6) rads 60Co

Species of microfungi were isolated from both the north facing slope (NFS) and the south facing slope (SFS) of Evolution Canyon, Mount Carmel, Israel. They were examined for growth rates before and after exposure to 10(6) rads of cobalt 60 irradiation. Above 10(6) rads all growth ceased after 1 day following exposure.     

Biodiversity and microclimatic divergence of chrysomelid beetles at 'Evolution Canyon', Lower Nahal Oren, Mt Carmel, Israel

Ninety species of leaf beetles (Chrysomelidae, Coleoptera, Insecta) were recorded from die 'Evolution Canyon' microsite (sample area 7000 m²), Lower Nahal Oren, Mt Carmel, Israel. Species richness was significandy ( P =0.018) higher on the drier, climatically more fluctuating savanna-like, soudi-facing slope (SFS, 60 species, sample area 3000 m²) than on the cooler, wetter and climatically less variable north-facing slope (NFS, 44 species, sample area 3000 m²), as in other terrestrial taxa. More species were found common to the local xeric SFS at die Canyon and the regional southern desert areas (Judean and Negev Deserts), than to the NFS and Galilee Mountains, respectively, suggesting that local patterns mirror regional patterns. Interslope differences in microclimate variability range indicate that this factor might be determinant of interslope species richness differences. Most species were collected in the spring (April; 47%) and least in the autumn (September; 3%) over the collecting period from March 1995 to June 1997.     

Do slope orientation and sampling location determine soil biota composition?

“Evolution Canyon” is a typical Mediterranean-basin canyon with a summer dry stream at the bottom and large differences in vegetation cover and solar radiation between the north-facing slope (NFS) and the south-facing slope (SFS). It is known to act as an abiotic mediator influencing the community structure of soil fauna. The aim of this study was to determine the spatial dispersion of soil microbial and free-living nematode communities in the open sites (between shrubs) in the upper (0–10 cm) soil layer at four altitudes on both slopes. The combination of relative soil moisture availability and temperature, known to be one of the main triggers for soil biota activity, was explained by slope orientation. The above-mentioned differences were found to significantly affect microbial biomass and CO₂ evolution resulting in temporary stress, corresponding to higher values in metabolic quotient (qCO₂) values. These differences may represent microbial investment in energy in order to overcome stress resulting from the microclimatic differences between the two slopes. Moreover, the degree of substrate limitation (primary production due to the differences in plant cover) of microbial activity was explained by the difference in microbial functional groups. The total abundance of soil free-living nematodes was found to be 2-fold higher on the SFS than on the NFS. Thirty-nine genera, including 12 bacteria-feeders, 5 fungi-feeders, 12 plant-parasites, and 10 omnivore-predators, were found at the study site, with 34 genera on the NFS and 29 on the SFS. The generic diversity of the bacteriafeeding nematodes was higher on the SFS than on the NFS. This study elucidates the importance of slope orientation and its effect on the structural levels of soil microbial and nematode communities.     

Desiccation resistance and mating behaviour in laboratory populations of Drosophila simulans originating from the opposing slopes of Lower Nahal Oren (Israel)

Lower Nahal Oren in Northern Israel, often referred to as 'Evolution Canyon', has been proposed as a microscale model site for ecological evolution. However, conflicting stress resistance and mating assay results contribute to controversy over the Nahal Oren model. In this study, we further tested the Nahal Oren model, while extending its focus from Drosophila melanogaster to its sister species, Drosophila simulans. Using fly populations derived from the opposing canyon slopes and acclimated to laboratory conditions for 11-22 generations, we did not find a significant slope effect on desiccation resistance (P = 0.96) or body metabolic fuel content (P > 0.43), which would indicate a genetic basis for adaptation to local resource limitation. Multiple-choice mating assays (47-48% homotypic couples in two replicate populations) did not indicate divergence from a random mating pattern between north- and south-facing slope flies. In conclusion, our findings do not support divergence of D. simulans populations across Lower Nahal Oren.     

Genomic microsatellite adaptive divergence of wild barley by microclimatic stress in 'Evolution Canyon', Israel

We examined diversity levels and patterns of 19 nuclear microsatellites and four chloroplast microsatellites in 275 genotypes of wild barley Hordeum spontaneum, in seven stations at the ‘Evolution Canyon’ (EC) microsite, Lower Nahal Oren, Mt. Carmel, Israel. EC is sharply subdivided ecologically into a tropical savannoid, ‘African’, xeric, south‐facing slope (SFS) abutting the temperate, dense, liveoak, brushwood, ‘European’, mesic, north‐facing slope (NFS). We found the following. (i) 17 of 19 (89.5%) nuDNA simple sequence repeats (SSRs) were polymorphic across all seven subpopulations and three chDNA SSRs were polymorphic. (ii) A total of 216 nuDNA SSR alleles, with a maximum of 23 alleles in a nuclear locus, and ten chDNA SSRs, with a maximum of four alleles in a locus, were registered. (iii) There were striking and significant inter‐ and intraslope diversities, based on the 19 nuDNA SSRs, climaxing with a remarkable genetic distance between the mid‐slope stations on opposite slopes (D_A = 0.481), across a distance of 200 m. This genetic distance is as large as that between the H. spontaneum populations of Jerusalem and Sede Boqer, which are separated by 100 km (× 500 larger in transect length). (iv) Slope‐unique alleles (103 = 45.6%) were higher on the ‘European’ than on the ‘African’ slope. Slope‐specific (predominant) alleles (17) were equal on opposite slopes. (v) nuDNA SSR gene diversity was higher on the ‘European’ slope and the opposite was found for the chDNA SSR. (vi) nuDNA SSR genic differentiation was very high between opposite slopes, with Gst = 0.187; for chDNA SSR this value was 0.127. Our results are inexplicable by stochastic processes and suggest that: (i) microclimatic diversifying selection is the major evolutionary, fast‐acting, interslope force, overriding migration and drift, and (ii) ecological stress can generate local, regional and global adaptive patterns, suggesting that natural selection is a major differentiating force of both coding and noncoding SSRs linking micro‐ and macroevolutionary processes. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84 , 205–224.     

Microclimatic Divergence in a Mediterranean Canyon Affects Richness,Composition, and Body Size in Saproxylic Beetle Assemblages

Large valleys with opposing slopes may act as a model system with which the effects of strong climatic gradients on biodiversity can be evaluated. The advantage of such comparisons is that the impact of a change of climate can be studied on the same species pool without the need to consider regional differences. The aim of this study was to compare the assemblage of saproxylic beetles on such opposing slopes at Lower Nahal Oren, Mt. Carmel, Israel (also known as “Evolution Canyon”) with a 200–800% higher solar radiation on the south-facing (SFS) compared to the north-facing slope (NFS). We tested specific hypotheses of species richness patterns, assemblage structure, and body size resulting from interslope differences in microclimate. Fifteen flight-interception traps per slope were distributed over three elevation levels ranging from 50 to 100 m a.s.l. Richness of saproxylic beetles was on average 34% higher on the SFS compared with the NFS, with no detected influence of elevation levels. Both assemblage structure and average body size were determined by slope aspect, with more small-bodied beetles found on the SFS. Both the increase in species richness and the higher prevalence of small species on the SFS reflect ecological rules present on larger spatial grain (species-energy hypothesis and community body size shift hypothesis), and both can be explained by the metabolic theory of ecology. This is encouraging for the complementary use of micro- and macroclimatic gradients to study impacts of climate warming on biodiversity.     

The effects of season and dietary salt content on body temperature daily rhythms of common spiny mice from different micro-habitats

We compared body temperature (T(b)) daily rhythms in two populations of common spiny mice, Acomys cahirinus, during summer and winter months in relation to increasing dietary salt content. Mice were collected from the North and South facing slopes (NFS and SFS) of the same valley, that are exhibiting mesic and xeric habitats, respectively. During the summer, whilst mice were offered a water source containing 0.9% NaCl, SFS individuals had T(b) peak values at 24:00, whereas NFS individuals had peak values at 18:00. When the salinity of the water source was increased, from 0.9 to 2.5% and then 3.5%, the difference between maximal and minimal T(b) of both populations increased. In addition, with increased salinity, the T(b) daily peak of SFS mice shifted to 18:00. During the winter, the mean daily T(b) values of both populations of mice were lower than during the summer. At 0.9% salinity, the NFS mice exhibited a daily T(b) variation with a peak at the beginning of the night. However, we did not detect any significant variation in daily T(b) in the SFS mice. At 2.5% salinity, the difference between the mean daily T(b) of mice from the two slopes increased. In winter we were unable to increase the salinity to 3.5% as the animals began to lose weight rapidly. We suggest that common spiny mice that inhabit these two micro-habitats are forming two discrete populations that respond differently to the environmental pressures prevailing in each habitat, by evolving different physiological capacities.     

Incipient sexual isolation among cosmopolitan Drosophila melanogaster populations

Understanding the biological conditions and the genetic basis of early stages of sexual isolation and speciation is an outstanding question in evolutionary biology. It is unclear how much genetic and phenotypic variation for mating preferences and their phenotypic cues is segregating within widespread and human-commensal species in nature. A recent case of incipient sexual isolation between Zimbabwe and cosmopolitan populations of the human-commensal fruit fly Drosophila melanogaster indicates that such species may initiate the process of sexual isolation. However, it is still unknown whether other geographical populations have undergone evolution of mating preferences. In this study we present new data on multiple-choice mating tests revealing partial sexual isolation between the United States and Caribbean populations. We relate our findings to African populations, showing that Caribbean flies are partially sexually isolated from Zimbabwe flies, but mate randomly with West African flies, which also show partial sexual isolation from the United States and Zimbabwe flies. Thus, Caribbean and West African populations seem to exhibit distinct mating preferences relative to populations in the United States and in Zimbabwe. These results suggest that widespread and human-commensal species may harbor different types of mating preferences across their geographical ranges.     

The courtship song of African Drosophila melanogaster

For many years it was thought that Drosophila melanogaster was relatively panmictic, without differentiation in the Mate Recognition System. Recent studies have demonstrated that flies from Africa vary in pheromones and assortative mating. Strains from Zimbabwe show strong sexual isolation from others. We show that the interpulse interval (IPI) of courtship song, an important mating signal, is unusually short among African flies. Zimbabwean flies have the shortest IPI, but there is no correlation with assortative mating, suggesting little direct role in sexual isolation. Chromosome replacements show that the IPI difference is largely due to genes on chromosome III, with significant interactions involving other chromosomes. Several traits potentially influencing sexual isolation among the melanogaster group of Drosophila seem to be localized to this chromosome. A concentration of important genetic differences might mean that the interaction effects reflect secondary coadaptation of the genetic background to changes associated with chromosome III.     

Microclimate,developmental plasticity and community structure in artificial temporary pools

Solar radiation, which influences temperature and hydroperiod, should strongly influence colonization, development rate, development strategies and survival in temporary pools. We assessed these hypotheses at two spatial microscales with varying microclimate. One scale, ca. 200 m, was opposing slopes of a canyon on MT Carmel, Israel, referred to as 'Evolution Canyon' (Nevo, 1995). Due primarily to differential solar radiation, the warmer and drier South-facing slope (SFS) supports a savanna-like open park forest while the cooler, moister North-facing slope (NFS) is heavily forested, primarily with live oak. Using artificial pools, we assessed how these close but contrasting environments may influence developmental plasticity in the frog, Hyla savignyi, and colonization of insects. As expected, hydroperiod was considerably longer on the NFS. Hyla tadpole development was significantly faster on the SFS, while size at metamorphosis was greater on the NFS. Insect species richness was higher in NFS pools. The most common species, Culex pipiens, oviposited significantly more in NFS pools than in SFS pools. In the second scale, we placed pools only meters apart, but varying in shade and thus maximum temperature at Haifa University Campus, MT Carmel. Species richness and maximum temperature among pools were not correlated during winter and early spring (cool to mild temperatures), but were strongly and negatively correlated during early summer (hot temperatures). Thermal tolerance in the laboratory of selected species matched their thermal spatial distribution in the field. These results indicate that temperature can play a strong role in affecting developmental strategies, species′ distributions and community structure in temporary pools over a very small spatial scale. This revised version was published online in July 2006 with corrections to the Cover Date.     

Disruptive selection for sternopleural bristle phenotypes in Drosophila ananassae

Disruptive selection is potentially critical in maintaining variation and initiating speciation and plays an important role in the organization of genetic variability in natural populations. It occurs when extreme phenotypes have a fitness advantage over intermediate phenotypes. Disruptive selection for high and low numbers of sternopleural bristles in Drosophila ananassae was applied for 12 generations to test its effect in induction of behavioural isolation. Pattern of mating between flies of high and low lines was tested in an Elens-Wattiaux mating chamber by using a multiple-choice technique after G(5) and G(12). Data was analyzed by calculating χ(2) under the assumption of random mating to test the difference between homoand heterogamic matings, and sexual isolation was tested by calculating the isolation estimate. The results show that there is no evidence for sexual isolation in G(5) and G(12). The realized heritability, standard error of regression coefficient, and t values suggest that disruptive selection for sternopleural bristle phenotypes was effective, but it does not lead to behavioral isolation in D. ananassae. Rather, it induces differences in mating propensity, which is influenced by sternopleural bristle phenotypes.     

Lichen Colonization on Cobbles in the Negev Desert Following 15 Years in the Field

Rock slabs were placed from 1991 through 2006 at the bottom of a north- (NFS) and a south-facing (SFS) slope of a first order drainage basin in the Negev Desert Highlands, Israel (P = 95 mm) where natural cobbles have >95% cover of endolithic and epilithic lichens. Periodic measurements of rain, dew and chlorophyll content took place. Lichen cover and the regeneration time were aspect-dependent. Lichen cover was 98% and 61% at NFS and SFS, respectively, while the lichen's recovery time was respectively ～45 and ～80 years. The fast regeneration time may have important implications regarding weathering and soil formation.     

Carbon source utilization patterns of Bacillus simplex ecotypes do not reflect their adaptation to ecologically divergent slopes in 'Evolution Canyon', Israel

The 'Evolution Canyons' I and II in Israel are model habitats to study adaptation and speciation of bacteria in the environment. These canyons represent similar ecological replicates, separated by 40 km, with a strongly sun-exposed and hot 'African' south-facing slope (SFS) vs. a cooler and mesic-lush 'European' north-facing slope (NFS). Previously, among 131 Bacillus simplex isolates, distinct genetic lineages (ecotypes), each specific for either SFS or NFS, were identified, suggesting a temperature-driven slope-specific adaptation. Here, we asked whether the ecological heterogeneity of SFS vs. NFS also affected carbon utilization abilities, as determined using the Biolog assay. Contrary to expectation, a correlation between substrate utilization patterns and the ecological origin of strains was not found. Rather, the patterns split according to the two major phylogenetic lineages each of which contain SFS and NFS ecotypes. We conclude that traits related to the general energy metabolism, as far as assessed here, are neither shaped by the major abiotic features of 'Evolution Canyon', namely solar radiation, temperature, and drought, nor by the soil characteristics. We further conclude that some traits diverge rather neutrally from each other, whereas other, more environmentally related traits are shaped by natural selection and show evolutionary convergence.