Isolation and characterization of microsatellite loci in the Brazilian orchid <Emphasis Type="Italic">Epidendrum fulgens</Emphasis>

Epidendrum fulgens has a patchy distribution along the Atlantic Rainforest in the Brazilian coast, due to the destruction of its native habitat. Here, we report on both the development of nine new microsatellite markers isolated from this species and the characterization of their allele variability in two distant and unrelated populations. The number of alleles observed for each locus ranged from 2 to 17 with an average of 6.4 alleles per locus. These microsatellites should be valuable tools for studying the effect of habitat fragmentation on the genetic structure of E. fulgens populations.     

Microsatellite diversity and genetic structure of fragmented populations of the rare,fire-dependent shrub Grevillea macleayana

Recent habitat loss and fragmentation superimposed upon ancient patterns of population subdivision are likely to have produced low levels of neutral genetic diversity and marked genetic structure in many plant species. The genetic effects of habitat fragmentation may be most pronounced in species that form small populations, are fully self-compatible and have limited seed dispersal. However, long-lived seed banks, mobile pollinators and long adult lifespans may prevent or delay the accumulation of genetic effects. We studied a rare Australian shrub species, Grevillea macleayana (Proteaceae), that occurs in many small populations, is self-compatible and has restricted seed dispersal. However, it has a relatively long adult lifespan (c. 30 years), a long-lived seed bank that germinates after fire and is pollinated by birds that are numerous and highly mobile. These latter characteristics raise the possibility that populations in the past may have been effectively large and genetically homogeneous. Using six microsatellites, we found that G. macleayana may have relatively low within-population diversity (3.2-4.2 alleles/locus; Hexp = 0.420-0.530), significant population differentiation and moderate genetic structure (FST = 0.218) showing isolation by distance, consistent with historically low gene flow. The frequency distribution of allele sizes suggest that this geographical differentiation is being driven by mutation. We found a lack mutation-drift equilibrium in some populations that is indicative of population bottlenecks. Combined with evidence for large spatiotemporal variation of selfing rates, this suggests that fluctuating population sizes characterize the demography in this species, promoting genetic drift. We argue that natural patterns of pollen and seed dispersal, coupled with the patchy, fire-shaped distribution, may have restricted long-distance gene flow in the past.     

Isolation and characterization of microsatellite loci in Epidendrum puniceoluteum, an endemic orchid from the Atlantic Rainforest

Epidendrum puniceoluteum is an endemic orchid of Atlantic Rainforest, restricted to few populations only due to the destruction and fragmentation of its native habitat. Here, we report on the development of 10 microsatellite markers isolated from this orchid species. Genetic variability was characterized in two distant populations from Brazil coast. The number of alleles observed for each locus ranged from two to 12 and with an average of 6.4 alleles per locus. These microsatellites should be valuable tools for studying both fine-scale genetic structure of scattered E. puniceoluteum population and patterns will be useful genetic markers for other closely related taxa.     

Gene flow connects coastal populations of a habitat specialist,the Clapper Rail Rallus crepitans

Examining population genetic structure can reveal patterns of reproductive isolation or population mixing and inform conservation management. Some avian species are predicted to exhibit minimal genetic differentiation among populations as a result of the species high mobility, with habitat specialists tending to show greater fine‐scale genetic structure. To explore the relationship between habitat specialization and gene flow, we investigated the genetic structure of a saltmarsh specialist with high potential mobility across a wide geographical range of fragmented habitat. Little variation among mitochondrial sequences (620 bp from ND2) was observed among 149 individual Clapper Rails Rallus crepitans sampled along the Atlantic coast of the USA, with the majority of individuals at all sampling sites sharing a single haplotype. Genotyping of nine microsatellite loci across 136 individuals revealed moderate genetic diversity, no evidence of bottlenecks and a weak pattern of genetic differentiation that increased with geographical distance. Multivariate analyses, Bayesian clustering and an AMOVA all suggested a lack of genetic structuring across the Atlantic coast of the USA, with all individuals grouped into a single interbreeding population. Spatial autocorrelation analyses showed evidence of weak female philopatry and a lack of male philopatry. We conclude that high gene flow connecting populations of this habitat specialist may result from the interaction of ecological and behavioural factors that promote dispersal and limit natal philopatry and breeding‐site fidelity. As climate change threatens saltmarshes, the genetic diversity and population connectivity of Clapper Rails may promote resilience of their populations. This finding helps inform about potential fates of other similarly behaving saltmarsh specialists on the Atlantic coast.     

Global population genetic structure of the starlet anemone <Emphasis Type="Italic">Nematostella vectensis</Emphasis>: multiple introductions and implications for conservation policy

Distinguishing natural versus anthropogenic dispersal of organisms is essential for determining the native range of a species and implementing an effective conservation strategy. For cryptogenic species with limited historical records, molecular data can help to identify introductions. Nematostella vectensis is a small, burrowing estuarine sea anemone found in tidally restricted salt marsh pools. This species’ current distribution extends over three coast lines: (i) the Atlantic coast of North America from Nova Scotia to Georgia, (ii) the Pacific coast of North America from Washington to central California, and (iii) the southeast coast of England. The 1996 IUCN Red List designates N. vectensis as “vulnerable” in England. Amplified fragment length polymorphism (AFLP) fingerprinting of 516 individuals from 24 N. vectensis populations throughout its range and mtDNA sequencing of a subsample of these individuals strongly suggest that anthropogenic dispersal has played a significant role in its current distribution. Certain western Atlantic populations of N. vectensis exhibit greater genetic similarity to Pacific populations or English populations than to other western Atlantic populations. At the same time, F-statistics showing high degrees of genetic differentiation between geographically proximate populations support a low likelihood for natural dispersal between salt marshes. Furthermore, the western Atlantic harbors greater genetic diversity than either England or the eastern Pacific. Collectively, these data clearly imply that N. vectensis is native to the Atlantic coast of North America and that populations along the Pacific coast and in England are cases of successful introduction.     

High Levels of Genetic Connectivity among Populations of Yellowtail Snapper,Ocyurus chrysurus (Lutjanidae – Perciformes), in the Western South Atlantic Revealed through Multilocus Analysis

In the present study, five loci (mitochondrial and nuclear) were sequenced to determine the genetic diversity, population structure, and demographic history of populations of the yellowtail snapper, Ocyurus chrysurus, found along the coast of the western South Atlantic. O. chrysurus is a lutjanid species that is commonly associated with coral reefs and exhibits an ample geographic distribution, and it can therefore be considered a good model for the investigation of phylogeographic patterns and genetic connectivity in marine environments. The results reflected a marked congruence between the mitochondrial and nuclear markers as well as intense gene flow among the analyzed populations, which represent a single genetic stock along the entire coast of Brazil between the states of Pará and Espírito Santo. Our data also showed high levels of genetic diversity in the species (mainly mtDNA), as well a major historic population expansion, which most likely coincided with the sea level oscillations at the end of the Pleistocene. In addition, this species is intensively exploited by commercial fisheries, and data on the genetic structure of its populations will be essential for the development of effective conservation and management plans.     

Geographical variation in genetic structure of an Atlantic Coastal Forest frog reveals regional differences in habitat stability

Climatic oscillations throughout the Pleistocene combined with geological and topographic complexity resulted in extreme habitat heterogeneity along the Atlantic coast of Brazil. Inferring how these historic landscape patterns have structured the current diversity of the region's biota is important both for our understanding of the factors promoting diversification, as well as the conservation of this biodiversity hotspot. Here we evaluate potential historical scenarios of diversification in the Atlantic Coastal Forest of Brazil by investigating the population genetic structure of a frog endemic to the region. Using mitochondrial and nuclear sequences, we generated a Bayesian population-level phylogeny of the Thoropa miliaris species complex. We found deep genetic divergences among five geographically distinct clades. Southern clades were monophyletic and nested within paraphyletic northern clades. Analyses of historical demographic patterns suggest an overall north to south population expansion, likely associated with regional differences in habitat stability during the Pliocene and early Pleistocene. However, genetic structure among southern populations is less pronounced and likely represents more recent vicariant events resulting from Holocenic sea-level oscillations. Our analyses corroborate that the Atlantic Coastal Forest has been a biogeographically dynamic landscape and suggest that the high diversity of its fauna and flora resulted from a combination of climatic and geologic events from the Pliocene to the present.     

A dispersal-dependent zone of introgressive hybridization between weakfish, Cynoscion regalis, and sand seatrout, C. arenarius, (Sciaenidae) in the Florida Atlantic

Five diagnostic codominant nuclear DNA markers and a diagnostic mitochondrial DNA marker were used to survey weakfish (Cynoscion regalis) and sand seatrout (C. arenarius), with particular focus on heretofore uncharacterized juvenile populations along the Florida (FL) Atlantic coast. Geographic and reproductive ranges of weakfish and sand seatrout were shown to overlap on the Atlantic coast along north and central FL. An active bidirectional zone of introgressive hybridization exists between these taxa, centered in the St Johns River, FL. Strong patterns of Hardy-Weinberg, linkage, and cytonuclear disequilibrium and a bimodal hybrid index distribution were observed for juvenile cohorts in the zone center, coupled with narrow (～240 km) concordant clines. Parental forms had disparate habitat preferences; hybrid forms occurred predominantly in intermediate habitats. All genetic data were consistent with the hypothesis that the C. arenarius-C. regalis hybrid zone is maintained by a dynamic equilibrium between continued interspecific gene flow and one or more opposing forces. Cytonuclear analyses indicated that parental forms mate assortatively in the zone but that mate recognition was imperfect. Ethological mating dynamics are likely stabilized by some form of endogenous or exogenous postfertilization selection against hybrids such that parental taxa will likely continue to evolve independently.     

Historical habitat connectivity affects current genetic structure in a grassland species

Many recent studies have explored the effects of present and past landscape structure on species distribution and diversity. However, we know little about the effects of past landscape structure on distribution of genetic diversity within and between populations of a single species. Here we describe the relationship between present and past landscape structure (landscape connectivity and habitat size estimated from historical maps) and current genetic structure in a perennial herb, Succisa pratensis. We used allozymes as co‐dominant markers to estimate genetic diversity and deviation from Hardy–Weinberg equilibrium in 31 populations distributed within a 5 km² agricultural landscape. The results showed that current genetic diversity of populations was related to habitat suitability, habitat age, habitat size and habitat connectivity in the past. The effects of habitat age and past connectivity on genetic diversity were in most cases also significant after taking the current landscape structure into account. Moreover, current genetic similarity between populations was affected by past connectivity after accounting for current landscape structure. In both cases, the oldest time layer (1850) was the most informative. Most populations showed heterozygote excess, indicating disequilibrium due to recent gene flow or selection against homozygotes. These results suggest that habitat age and past connectivity are important determinants of distribution of genetic diversity between populations at a scale of a few kilometres. Landscape history may significantly contribute to our understanding of distribution of current genetic structure within species and the genetic structure may be used to better understand landscape history, even at a small scale.     

Post-Glacial Expansion and Population Genetic Divergence of Mangrove Species Avicennia germinans (L.) Stearn and Rhizophora mangle L. along the Mexican Coast

Mangrove forests in the Gulf of California, Mexico represent the northernmost populations along the Pacific coast and thus they are likely to be source populations for colonization at higher latitudes as climate becomes more favorable. Today, these populations are relatively small and fragmented and prior research has indicated that they are poor in genetic diversity. Here we set out to investigate whether the low diversity in this region was a result of recent colonization, or fragmentation and genetic drift of once more extensive mangroves due to climatic changes in the recent past. By sampling the two major mangrove species, Rhizophora mangle and Avicennia germinans, along the Pacific and Atlantic coasts of Mexico, we set out to test whether concordant genetic signals could elucidate recent evolution of the ecosystem. Genetic diversity of both mangrove species showed a decreasing trend toward northern latitudes along the Pacific coast. The lowest levels of genetic diversity were found at the range limits around the Gulf of California and the outer Baja California peninsula. Lack of a strong spatial genetic structure in this area and recent northern gene flow in A. germinans suggest recent colonization by this species. On the other hand, lack of a signal of recent northern dispersal in R. mangle, despite the higher dispersal capability of this species, indicates a longer presence of populations, at least in the southern Gulf of California. We suggest that the longer history, together with higher genetic diversity of R. mangle at the range limits, likely provides a gene pool better able to colonize northwards under climate change than A. germinans.     

Evidence of population bottleneck in Astragalus michauxii (Fabaceae), a narrow endemic of the southeastern United States

Genetic factors such as decreased genetic diversity and increased homozygosity can have detrimental effects on rare species, and may ultimately limit potential adaptation and exacerbate population declines. The Gulf and Atlantic Coastal Plain physiographic region has the second highest level of endemism in the continental USA, but habitat fragmentation and land use changes have resulted in catastrophic population declines for many species. Astragalus michauxii (Fabaceae) is an herbaceous plant endemic to the region that is considered vulnerable to extinction, with populations generally consisting of fewer than 20 individuals. We developed eight polymorphic microsatellites and genotyped 355 individuals from 24 populations. We characterized the population genetic diversity and structure, tested for evidence of past bottlenecks, and identified evidence of contemporary gene flow between populations. The mean ratios of the number of alleles to the allelic range (M ratio) across loci for A. michauxii populations were well below the threshold of 0.68 identified as indicative of a past genetic bottleneck. Genetic diversity estimates were similar across regions and populations, and comparable to other long-lived perennial species. Within-population genetic variation accounted for 92 % of the total genetic variation found in the species. Finally, there is evidence for contemporary gene flow among the populations in North Carolina. Although genetic factors can threaten rare species, maintaining habitats through prescribed burning, in concert with other interventions such as population augmentation or (re)introduction, are likely most critical to the long term survival of A. michauxii.     

Broad-scale genetic homogeneity in natural populations of common hazel (<Emphasis Type="Italic">Corylus avellana</Emphasis>) in Ireland

Hazel (Corylus avellana) has been a key species in European woodlands throughout the Holocene (10 KYA–present). Like many tree species, it is increasingly under threat from climate change, habitat loss and fragmentation, invasive species and emergent pathogens. As knowledge of the genetic structure of natural populations of trees is vital for managing these threats, as well as an essential basis for selection of material for replanting and restocking, we analysed levels and patterns of genetic diversity in the species at a range of spatial scales using high-resolution microsatellite markers. Our findings indicate that hazel populations exhibit high levels of genetic diversity along with low levels of population differentiation, suggesting extensive gene flow. Fine-scale genetic structuring was observed in some of the woodlands studied, probably resulting from restricted dispersal of the heavy nuts produced by the species. This, coupled with higher levels of pollen-mediated gene flow, resulted in a weak but significant pattern of isolation by distance. These results suggest that replanting following potential loss of hazel populations may not necessarily require the use of material from the same locality and mirror findings in other broadleaved tree species from the same area.     

Population structure and landscape genetics in the endangered subterranean rodent Ctenomys porteousi

In order to devise adequate conservation and management strategies for endangered species, it is important to incorporate a reliable understanding of its spatial population structure, detecting the existence of demographic partitions throughout its geographical range and characterizing the distribution of its genetic diversity. Moreover, in species that occupy fragmented habitats it is essential to know how landscape characteristics may affect the genetic connectivity among populations. In this study we use eight microsatellite markers to analyze population structure and gene flow patterns in the complete geographic range of the endangered rodent Ctenomys porteousi. Also, we use landscape genetics approaches to evaluate the effects of landscape configuration on the genetic connectivity among populations. In spite of geographical proximity of the sampling sites (8–27 km between the nearest sites) and the absence of marked barriers to individual movement, strong population structure and low values of gene flow were observed. Genetic differentiation among sampling sites was consistent with a simple model of isolation by distance, where peripheral areas showed higher population differentiation than those sites located in the central area of the species’ distribution. Landscape genetics analysis suggested that habitat fragmentation at regional level has affected the distribution of genetic variation among populations. The distance of sampling sites to areas of the landscape having higher habitat connectivity was the environmental factor most strongly related to population genetic structure. In general, our results indicate strong genetic structure in C. porteousi, even at a small spatial scale, and suggest that habitat fragmentation could increase the population differentiation.     

When physical oceanography meets population genetics: The case study of the genetic/evolutionary discontinuity in the endangered goliath grouper (Epinephelus itajara; Perciformes: Epinephelidae) with comments on the conservation of the species

Epinephelus itajara is one of the marine fish species most threatened for extinction and it is considered to be “critically endangered” by the IUCN. The present study evaluated the genetic diversity of the species and the genetic/evolutionary relationships of its populations along the Atlantic coast of South America. The results indicate relatively reduced genetic variation, re-emphasizing the low adaptive potential of the species. One of the populations presented relatively high degrees of genetic diversity and it is evolutionary isolated from the all other populations. The evidences indicate the existence of two Evolutionarily Significant Units comprising E. itajara in the Atlantic coast of South America and the conservation prospects for the species must take these evidences into account.     

The legacy of ice ages in mountain species: post‐glacial colonization of mountain tops rather than current range fragmentation determines mitochondrial genetic diversity in an endemic Pyrenean rock lizard

Aim The genetic impact of Quaternary climatic fluctuations on mountain endemic species has rarely been investigated. The Pyrenean rock lizard (Iberolacerta bonnali) is restricted to alpine habitats in the Pyrenees where it exhibits a highly fragmented distribution between massifs and between habitats within massifs. Using mitochondrial DNA markers, we set out: (1) to test whether several evolutionary units exist within the species; (2) to understand how the species persisted through the Last Glacial Maximum and whether the current range fragmentation originates from distribution shifts after the Last Glacial Maximum or from more ancient events; and (3) to investigate whether current mitochondrial diversity reflects past population history or current habitat fragmentation. Location The Pyrenees in south‐western France and northern Spain. Methods We used variation in the hypervariable left domain of the mitochondrial control region of 146 lizards collected in 15 localities, supplemented by cytochrome b sequences downloaded from GenBank to cover most of the species’ distribution range. Measures of population genetic diversity were contrasted with population isolation inferred from topography. Classical (F‐statistics) and coalescence‐based methods were used to assess the level of gene flow and estimate divergence time between populations. We used coalescence‐based simulations to test the congruence of our genetic data with a scenario of simultaneous divergence of current populations. Results Coalescence‐based analyses suggested that these peripheral populations diverged simultaneously at the end of the last glacial episode when their habitats became isolated on mountain summits. High mitochondrial diversity was found in peripheral, isolated populations, while the populations from the core of the species’ range were genetically impoverished. Where mitochondrial diversity has been retained, populations within the same massif exhibited high levels of genetic differentiation. Main conclusions As suggested for many other mountain species, the Pyrenean rock lizard survived glacial maxima through short‐distance range shifts instead of migration or contraction in distant southern refugia. Most of the main Pyrenean range has apparently been re‐colonized from a single or a few source populations, resulting in a loss of genetic diversity in re‐colonized areas. As a result, current levels of intra‐population mitochondrial diversity are better explained by post‐glacial population history than by current habitat fragmentation. Genetic population differentiation within massifs implies severe reduction in female‐mediated gene flow between patches of habitats.     

Genetic assessment of the Atlantic Forest bristle porcupine, Chaetomys subspinosus (Rodentia: Erethizontidae), an endemic species threatened with extinction

The bristle-spined porcupine, Chaetomys subspinosus, an endemic rodent from Atlantic Forest, was considered to be abundant in the recent past, but population reductions due to habitat loss and expansion of human activities caused this species to be included in the "vulnerable" category of the World Conservation Union Red List. We performed the first genetic assessment in natural populations of this focal species along its geographical distribution. Thirty-five non-invasive samples (hair) were collected from three natural populations in the Brazilian States of Sergipe, Bahia and Espírito Santo. Genetic similarity obtained by Jaccard's index, based on dominant RAPD and ISSR markers, varied between 25 and 100%. Four clusters, mainly coincident with the geographical distribution of the populations, were observed. Analysis of molecular variance based on 47 polymorphic loci showed that there was 15.99% genetic variability among populations and 84.01% within populations. The estimated genetic structure among populations (Φ(ST)) was 0.16. The populations may have formed a continuum along the past distribution of the Atlantic rainforest but historical events of human occupation resulted in recent divergence among sampled populations.     

Genetic and ecological consequences of recent habitat fragmentation in a narrow endemic plant species within an urban context

Understanding the timescales that shape spatial genetic structure is pivotal to ascertain the impact of habitat fragmentation on the genetic diversity and reproductive viability of long-lived plant populations. Combining genetic and ecological information with current and past fragmentation conditions allows the identification of the main drivers important in shaping population structure and declines in reproduction, which is crucial for informing conservation strategies. Using historic aerial photographs, we defined the past fragmentation conditions for the shrub Conospermum undulatum, a species now completely embedded in an urban area. We explored the impact of current and past conditions on its genetic layout and assessed the effects of genetic and environmental factors on its reproduction. The historically high structural connectivity was evident in the genetics of the species. Despite the current intense fragmentation, we found similar levels of genetic diversity across populations and a weak spatial genetic structure. Historical connectivity was negatively associated with genetic differentiation among populations and positively related to within-population genetic diversity. Variation partitioning of reproductive performance explained?~?66% of the variance, showing significant influences for genetic (9%), environmental (15%), and combined (42%) fractions. Our study highlights the importance of considering the historical habitat dynamics when investigating fragmentation consequences in long-lived plants. A detailed characterization of fragmentation from 1953 has shown how low levels of genetic fixation are due to extensive gene flow through the non-fragmented landscape. Moreover, knowledge of the relationships between genetic and environmental variation and reproduction can help to implement effective conservation strategies, particularly in highly dynamic landscapes.

     

Stock Identification of Gag, Mycteroperca microlepis, Along the Southeast Coast of the United States

The gag grouper Mycteroperca microlepis is an important component of commercial and recreational fisheries along the South Atlantic coast of the United States and in the Gulf of Mexico. Over the past two decades, this species has experienced significant declines in abundance and an increasing skew in sex ratios. Analysis of microsatellite DNA variation in this species shows mosaic patterns of population subdivision and significant departures from Hardy-Weinberg equilibrium in all sampling locations. Given the length of the pelagic stage (egg and larvae), the prevailing current patterns, and the migratory capabilities of the adults, it is unlikely that these observations are the result of restricted gene flow among genetically differentiated populations. The apparent structure of gag populations most likely reflects inbreeding in size-limited populations. Population declines, skewed sex ratios, and perhaps variance in female fecundity appear to have acted in concert to limited the number of individuals that contribute to a given year class. These data are reinforced by studies of other fish stocks that have experienced precipitous declines over the past two decades. Received April 13, 1998; accepted September 30, 1998.     

Multilocus approach reveals an incipient differentiation process in the Stone-curlew, <Emphasis Type="Italic">Burhinus oedicnemus</Emphasis> around the Mediterranean basin

The Stone-curlew Burhinus oedicnemus, a steppe bird species, is mainly distributed in the Mediterranean and Macaronesian regions, which are considered hotspots of biodiversity with priority for animal and plant species richness conservation. In this study, we investigated the genetic diversity of the Stone-curlew in the Mediterranean basin and in the Canary Islands by applying a multilocus approach. We analysed mitochondrial and nuclear markers in order to evaluate the genetic structure and the congruence between morphological subspecies and geographic samples. We found a significant level of genetic differentiation between Mediterranean and Canary Island populations with all markers. Both in the Mediterranean basin and in the Canary Islands, we found a significant level of genetic diversity with nuclear markers only. We identified seven population groups, including insular populations. The four subspecies described for the Western Palaearctic were confirmed with some changes in distribution range. In spite of habitat fragmentation and negative population trend, the Stone-curlew showed a significant level of genetic diversity and gene flow among continental populations. However, islands constitute important reservoirs of genetic diversity and a potential for the evolution of the species.     

Population structure and dispersal of the coral‐excavating sponge Cliona delitrix

Some excavating sponges of the genus Cliona compete with live reef corals, often killing and bioeroding entire colonies. Important aspects affecting distribution of these species, such as dispersal capability and population structure, remain largely unknown. Thus, the aim of this study was to determine levels of genetic connectivity and dispersal of Cliona delitrix across the Greater Caribbean (Caribbean Sea, Bahamas and Florida), to understand current patterns and possible future trends in their distribution and effects on coral reefs. Using ten species‐specific microsatellite markers, we found high levels of genetic differentiation between six genetically distinct populations: one in the Atlantic (Florida‐Bahamas), one specific to Florida and four in the South Caribbean Sea. In Florida, two independent breeding populations are likely separated by depth. Gene flow and ecological dispersal occur among other populations in the Florida reef tract, and between some Florida locations and the Bahamas. Similarly, gene flow occurs between populations in the South Caribbean Sea, but appears restricted between the Caribbean Sea and the Atlantic (Florida‐Bahamas). Dispersal of C. delitrix was farther than expected for a marine sponge and favoured in areas where currents are strong enough to transport sponge eggs or larvae over longer distances. Our results support the influence of ocean current patterns on genetic connectivity, and constitute a baseline to monitor future C. delitrix trends under climate change.