Annals of Botany

O_LIIn fluctuating environments, the kinetics of stomatal opening and closing influence the balance between carbon gain and water loss. Smaller guard cells may respond faster to fluctuating environmental conditions because of their greater surface area for osmolyte flux relative to cell volume. A related hypothesis is that operational stomatal conductance (gop) is often well below its theoretical maximum (gmax) because at this stomatal aperture, guard cell volume is poised to change rapidly with small changes in turgor pressure. C_LIO_LIWe analyzed 2,124 estimates of stomatal closure kinetics in response to an abrupt increase in vapor pressure deficit (VPD) among 29 diverse wild tomato populations in the genus Solanum. C_LIO_LILeaves with small guard cells and a lower gop to gmax ratio (fgmax) closed faster, but explained variation in kinetic parameters at different levels of biological organization. Guard cell size had high phylogenetic heritability and varied relatively little within populations, whereas fgmax varied mostly among individuals and between light intensity treatments. C_LIO_LISmaller stomata can be speedier, but only if stomata are held at an aperture where they are responsive to changing turgor pressure. Selection on stomatal speed may influence not only anatomical traits like guard cell size, but also physiological controls on gop. C_LI

O_LIPlants take up nutrients from the soil while investing in absorptive root surface or mycorrhizal partners. Root hairs - a major structure for nutrient uptake and cheap to build - increase the absorptive root surface. As such they are an important component of plant resource economics but largely neglected in root economic concepts so far. C_LIO_LIThis is mainly due to data scarcity, which we set out to overcome by measuring root-hair traits on 82 European grassland species in a greenhouse experiment. Using fluorescence and light microscopy, root-hair length and incidence was measured along with mycorrhizal colonization. C_LIO_LIWe found a phylogenetically conserved trade-off between plant investment into root hairs and mycorrhiza. A similar trade-off between root-hair incidence and mycorrhiza occurred at the intraspecific level, while patterns were heterogeneous among species. Plant species with high colonization rates showed the highest variability in root-hair incidence. C_LIO_LIWe conclude that plants vary along a gradient ranging from investment into root hairs as part of a "do-it-yourself" strategy to collaboration with mycorrhizal fungi while showing intraspecific variation in root-hair incidence. These findings demonstrate that root hairs play a fundamental role in fine-root trait variation and need to be considered when studying belowground plant economic strategies. C_LI

PremiseCentaurea melitensis (Asteraceae) is a problematic invader of grasslands globally, but little is known about its genetic makeup. Here we develop a reference genome to facilitate studies of its invasion history, genetic variation, and evolution. MethodsInbred offspring of a single individual of C. melitensis from its invasion of California, USA were used for flow cytometry to estimate genome size, and for genomic DNA extraction. DNA was sequenced with PacBio HiFi technology (yield = 85.7Gb). The genome was assembled with Hifiasm and annotated with BRAKER3. GENESPACE was used to compare gene order (synteny) with three other species within the subfamily Cichorioideae. ResultsWe estimated a mean genome size of 795.0 Mbp for C. melitensis, and our assembly totaled 696.6 Mbp in 48 contigs (N50 = 55.6 Mbp; BUSCO = 98%), with annotation of 25,157 protein-encoding genes. This included four telomere-to-telomere putative chromosomes, nine additional chromosome arms terminated by telomeric repeats, and a complete chloroplast genome. Synteny varied markedly across the genus and subfamily, suggesting a dynamic history of structural variation in the lineage of C. melitensis. DiscussionWe provide a highly complete and contiguous genome assembly to facilitate the further study of genomic variation in C. melitensis.

Growth kinematics and soil mechanics are key to explain how roots overcome the mechanical resistance of soil, yet few studies are linking these two factors. Formulas for cone penetration tests are typically used to infer the friction experienced by roots, but these fail to consider how growth affects the external forces applied on the root. This study formalised how expansive growth in the root apical meristem can reduce soil friction, and applied the framework to analyse the growth strategy of 6 plant species. The results of the analysis revealed trade-offs between reducing frictions, maintaining a desired growth trajectory and elongation rate. A shorter elongation zone can reduce the fraction of the mechanical energy lost to friction, but this is done at the expense of the elongation rate. A sharper tip or increased radius can help roots maintain the elongation rate at no energetic cost, but these strategies come with the cost of growth instability (tortuous roots) and decrease in specific root length respectively. During establishment, root strategies may therefore occupy a 2-dimensional trait space in which the mechanical efficiency of growth is balanced against the explorative-exploitative trade-off. HighlightsGrowth and form of root tips explain how plants overcome mechanical resistance from the soil Trade-offs link the energy lost by friction, growth stability and elongation rate of roots Larger roots allow faster growth independently of these trade-offs New framework formalises plants strategies to acquire soil resources

Background and AimsGroundwater-dependent ecosystems support disproportionate biodiversity in arid regions, yet the population genetics of spring-specialist plants remains poorly understood. Here, we present the first species-wide genetic dataset for crimson monkeyflower ( Mimulus verbenaceus, Phrymaceae), a spring-specialist plant distributed in seeps, springs, and associated riparian areas across desert regions of North America. MethodsUsing genome-wide reduced representation sequencing data consisting of 10,760 SNPs from 175 individuals across 17 populations, we characterized the patterns of genetic diversity using FST and Neis D. Population structure was assessed using ADMIXTURE and PCA. We examined the contributions of climate to range-wide genetic variation in crimson monkeyflower using redundancy analysis. Key ResultsPatterns of genetic differentiation were more consistent with those of spring-specialist animal taxa than those of upland plants or generalist riparian plants. We found strong population structure at both broad regional scales and at fine local scales. While riparian connectivity influenced local patterns of diversity, adaptation to local climatic variation was more influential at regional scales, with temperature, relative humidity, and a monsoon-driven climate gradient structuring genetic differentiation. ConclusionsOur findings highlight the distinctive influence of isolated perennial groundwater sources, as well as adaptation to climate, in shaping genetic variation in this spring-specialist plant. These findings suggest that spring-specialist plants deserve special consideration in ecological theory, management, and conservation.

Attracting specific pollinators can be favoured by natural selection to avoid reproductive interference between sympatric plant species. However, the ways in which fine differences in floral traits lead to the attraction of specific pollinators are diverse and unknown in many pollination interactions. We surveyed pollinators on three sympatric Aristolochia species (A. clematitis, A. pistolochia and A. rotunda) pollinated by Diptera to investigate if specific pollination occurs. To decipher if specific pollination may be mediated by different floral odours, we characterized the volatile organic compounds (VOCs) emitted by flowers and highlighted those VOCs electrophysiologically detected by pollinators in A. rotunda and A. pistolochia. Among the most abundant pollinators, Forcipomyia monilicornis was a specific pollinator of A. pistolochia while two Dasyhelea species were specific pollinators of A. clematitis. Forcipomyia aristolochiae and T. ruficeps were non-specific pollinators of A. rotunda, although they were more frequently found in A. rotunda flowers. The floral odours of A. rotunda and A. pistolochia differed significantly from each other and elicited specific electrophysiological responses in their respective pollinators. Although several pollinator species visit more than one Aristolochia species, those pollinators are preferentially found in one Aristolochia species. Selective attraction is likely mediated by specific VOCs.

(1) BackgroundSaffron crocus (Crocus sativus) is the source of saffron, the most expensive spice in the world. It evolved about 3000 years ago as a sterile triploid clone in Greece. Since then, saffron has spread across the globe, where regionally distinct practices of saffron cultivation have developed. Despite differences in morpho-physiological traits, genetic variability is low, if present at all. Here, we aim to resolve chromosomal and sequence-associated variability across saffron crocus cultivars from the crops main cultivation areas in Africa, Asia and Europe. (2) MethodsWe used genome-wide DNA polymorphisms obtained through genotyping-by-sequencing (GBS) of 33 saffron and 14 closely related Crocus accessions, which we place into a phylogenetic context. For karyotyping, we compare nine saffron accessions by multi-color fluorescent in situ hybridisation (FISH) with repetitive DNA probes. (3) Key resultsPhylogenetic analyses confirmed the single origin and clonal nature of all saffron accessions. We detected slight DNA differences among saffron crocus genotypes, which were minor compared with those in wild C. cartwrightianus populations. Still, the Iranian saffron accessions form a genetically very narrow group that differs from the other proveniences in population genetic analyses. However, chromosomes of some saffron accessions display variable FISH signals, likely resulting from gains and losses of tandemly repeated DNA. (4) Main conclusionsBased on the high genetic identity and small karyotypic differences, we confirm the clonal origin of the saffron accessions. Nevertheless, as we detected small and regional chromosomal variability, we conclude that at least four somaclonal saffron lineages emerged after saffrons origin. Societal Impact StatementFor millennia, many cultures developed cultivation practices and regional crop varieties. A notable case is saffron, the worlds most expensive spice that is harvested from stigmas of saffron crocus. This flower crop arose 3000 years ago in a singular genome triplication event and since then spread clonally across the globe. By identifying genetic and chromosomal variability in clonal saffron accessions, we highlight regional diversity, support the preservation of traditional knowledge, and underscore the risk of relying on only one clonal lineage. This informs strategies for saffron cultivation, linking cultural heritage with modern genomics to address biodiversity, evolution, and food security.

This investigation aimed at compiling all phylogenetic lineages within and around genus Cyanoboletus. The evolutionary inference obtained from the nuclear ribosomal genes internal transcribed spacer region (ITS) suggests that part of the species currently classified in Cyanoboletus belong in lineages separate from the genus, thus suggesting a narrower boundary that includes only the species that develop a strong staining reaction to touch and to air exposure of the context. The separate lineages are the monotypic Cupreoboletus genus and a few species that do not develop such reaction, which are part of a clade together with genera Cacaoporus and Acyanoboletus, thus broadening the concept of Cacaoporus to encompass all of them. The emerging 3C perspective of Cupreoboletus, Cacaoporus and Cyanoboletus offers a remarkably consistent morphological diagnosis, overcoming the problems of a too broad concept for Cyanoboletus. This work reveals that Boletus neotropicus, B. novae-zelandiae and B. sensibilis belong respectively in Cyanoboletus, Cacaoporus and Lanmaoa, and by studying multigene alignment concatenates it identifies lineages that probably represent undescribed species: at least four in Cacaoporus and at least five in Cyanoboletus. Diagnostic tables and dichotomic keys are presented by geographic region. The present work also includes a study of the phylogenetic position of Neoboletus flavosanguineus, a species once classified in Cyanoboletus. The complexity of assigning species epithets in some lineages is addressed, namely for the boundaries between Cacaoporus instabilis and Ca. fagaceophilus as well as the diversity under the names Cyanoboletus sinopulverulentus and Cy. pulverulentus. The overall picture of evolutionary lineages sets a framework for the choice of reference data that can provide, in future phylogenetic studies that involve the 3C, a balanced and efficient coverage. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/724631v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@7f618corg.highwire.dtl.DTLVardef@dd6a14org.highwire.dtl.DTLVardef@5f7399org.highwire.dtl.DTLVardef@9e7443_HPS_FORMAT_FIGEXP M_FIG C_FIG

How plants allocate carbon determines their productivity, responses to stress, and interactions with other organisms. A substantial amount of plant carbon is stored as non-structural carbohydrates (NSC), which sustain turgor via osmoregulation and fuel metabolism when carbon is limited. NSC also support root-colonizing mycorrhizal fungi, thus we hypothesized that under carbon-limiting conditions such as drought, a trade-off between feeding mycorrhizal fungi and maintaining turgor may arise. We reduced carbon allocation to ectomycorrhizal (EcM) networks by girdling Pinus ponderosa trees exposed to drought or ambient conditions and manipulated putative fungal connections between trees by trenching. We show that, in droughted plots, trees putatively connected to girdled trees by EcM networks had 33 % less needle NSC and >10% less turgor than those connected to ungirdled trees. Trees disconnected from the mycorrhizal network by trenching had increased NSC likely from the increased water availability with girdling, but these gains were offset in the presence of networks. Our results demonstrate that the increased carbon demand by EcM fungi in response to reduced carbon inputs from some trees can deplete NSC in neighboring trees via shared mycorrhizal networks. At least in the short term, allocation trade-offs under carbon-limiting conditions may expose networked trees to carbon deficits. This may increase vulnerability to drought, which may be particularly acute given shifts in climate.

Belemia belongs to Nyctaginaceae and comprises two species of delicate vines. Both species are endemic to Brazil. Belemia fucsioides, the type species, described in 1981, occurs in a restricted area of the Atlantic Forest in southeastern Brazil. Belemia cordata, described in 2020, is known from only two records from the same area in the Cerrado of northern Brazil. Here, we describe the taxonomic history of Belemia and provide the first synopsis for the genus. We include species description, distribution map, identification key, and anatomical data. We used field observations over the past decade and modeled anthropogenic changes in the species range to conduct a conservation assessment in accordance with the IUCN Red List criteria. Conservation assessments indicate significant concerns for Belemia, classified as either endangered (B. fucsioides) or critically endangered (B. cordata). The species are threatened primarily by habitat loss to land used for agriculture, forestry, and livestock production. This study contributes to ongoing initiatives exploring plant diversity in the Neotropics and supports efforts to identify threats to biodiversity.

Plant receptor-like kinases (RLKs) are involved in diverse processes, ranging from growth and reproduction to interactions with microbes. Variation in the extracellular domains delineates several RLKs subfamilies, including the malectin-like domain leucine-rich repeat receptor-like kinases (MLD-LRR-RLKs). Symbiosis Receptor-like Kinase (SymRK) is the prototypical member of MLD-LRR-RLKs and is required for microbial accommodation in host roots during root endosymbiosis. Yet, comparative phylogenetic analysis of SymRK orthologs in the broader context of MLD-LRR-RLK subfamily evolution remains limited. In this study, we examined the inventory, phylogeny and clade-specific evolutionary and transcriptional characteristics of this receptor group. SymRK and its closest homologs are present in most land plant lineages and group into four major clades and six additional species-specific clades. These clades can be distinguished by their evolutionary characteristics as either conserved with reduced gene copy number changes (including SymRK) or expanded and diversified, as observed in clade IV. Clade IV dynamics are largely driven by tandem gene duplications, which often arise within gene clusters. We further analysed the evolutionary characteristics of MLD-LRR-RLKs at the population level in Arabidopsis thaliana accessions. We found that some genes are conserved across accessions and are therefore likely to be functionally important, whereas a subset of genes, often located within tandem clusters, are highly diverse and likely contribute to accession-specific adaptations. Finally, most MLD-LRR-RLKs in the A. thaliana Col-0 accession are expressed in roots and respond broadly to biotic stimuli at the transcriptional level. Notably, clustered genes frequently exhibited divergent expression profiles, suggesting transcriptional diversification. Together, we revealed two contrasting evolutionary characteristics among members of the MLD-LRR-RLK subfamily, potentially associated with their functions in plants.

Flowering time and monocarpic senescence are tightly environmentally and genetically controlled. Typically, early flowering and staygreen traits are associated with opposing life-history strategies; stress avoidance versus adaptation; with flowering time an overarching regulator of crop cycle length. We developed RIL populations segregating for Ppd-1 and NAM-1 variation, which are otherwise isogenic. Multi-year field experiments enabled exploration and uncoupling of the relationship between heading and staygreen traits. Heading date manipulation enabled introduction of staygreen traits to their target breeding environments, characterised by a hot-finish. Under moderate stress, we report a 2.9% and 1.9% increase in grain width (P<0.0001), and 5.8% and 3.7% increase in TGW (P<0.0001), plus significantly greater yield (P<0.1) for late heading staygreen RILs homozygous for NAM-A1, and NAM-D1 missense variants, respectively. Grain yield increases were proportionate to the delay in senescence, being greater for the NAM-A1 than the NAM-D1 variant. For RIL populations segregating for both traits, senescence variation was observed relative to heading-date. Regarding grain yield, the staygreen trait-associated increase in source size could not compensate for the Ppd-1a associated pleiotropic reduction in sink size, even under hypothesised continental target breeding environments, with trait competition identified. Therefore, to maximise the benefits associated with staygreen traits, especially in early-heading favouring environments required targeted manipulation of source-sink dynamics, and we propose multiple strategies. HighlightStaygreen traits were associated with extending grain fill duration, increasing grain width, TGW and grain yield. There appears an antagonist relationship between earlier heading and staygreen traits.

The transformation of the free nuclear syncytium into cellular endosperm tissue with starch and protein accumulation is a well-established phenomenon, at least in the fruits of cereals of the Triticeae tribe. The present article demonstrates that there is considerable diversity inherent in this type of caryopsis morphogenesis. By examining various taxa (species, varieties, and cultivars) of wheat, oats, and some wild grasses, this research reveals significant deviations in endosperm morphogenesis from the typical state. A new developmental pattern of endosperm was identified, characterized by several distinctive features such as incomplete cellularization of the syncytium and starch accumulation within the acellular endosperm domains and the endosperm cavity. A large number of plastids were observed in the syncytium stage, which served as the basis for the later amyloplast stage. The acellular endosperm domains and the cavity domain exhibited connections at specific discontinuities in the modified aleurone layer surrounding the cavity. The peripheral parts of the caryopsis received fewer assimilates necessary for starch synthesis, which was attributed to their increased distance from the transfer system and a likely reduction in the efficiency of assimilate transport through the apoplast in these areas. The starch cavity volume constituted a few percent of the overall caryopsis volume, which could serve as a foundation for potential breeding improvements to enhance starch yields across different varieties.

Climatic and biogeographic variables are often used as a proxy for tree genetic diversity, but local factors can also influence it. We propose that woodland age, presence of ancient trees, and population size could impact genetic diversity. Using the RBG Kew UK National Tree Seed Project as a study case, we examined how these factors are accounted for during seed collection. We found 42% of tree seed collections come from ancient woodlands and that 8.4% overlap with ancient trees. Sampled forest patches size ranges from few individuals to several thousand. We then carried out a pilot to examine the role of population size on functional traits variation, testing the relationship between population size and seed germination and seedling thermal stress sensitivity in three populations of the Betula pubescens Ehrh. complex. We found that seeds and seedlings from larger populations showed higher fitness and stress resistance. Our results highlight the importance of local factors to predict variation in functional traits, relevant for tree resilience. Existing seed collections of native species stored in conservation seed banks offer a valuable resource to explore these factors and improve our understanding of genetic diversity in tree populations, with implications for biodiversity conservation and forestry production.

Heat tolerance determines the vitality of tree species under climate change independently of drought tolerance, but has been much less studied than tree water relations. We studied species-specific differences and the capacity for seasonal heat acclimation in Central Europes naturally most important tree species, Fagus sylvatica, in comparison with two exotic tree species (Fagus orientalis, Pseudotsuga menziesii) that are considered for silvicultural climate change adaptation in managed forests. Foliage of mature trees was incubated at temperatures from 35-50 {degrees}C for up to 4 h to simulate daily heat maxima during heat waves. The maximum quantum yield (Fv/Fm) of photosystem II (PS II) of dark-adapted leaves was measured, because the PS II is particularly sensitive to heat and its functionality can decide on plant survival under heat. Fagus sylvatica was much more tolerant to heat than Pseudotsuga menziesii, but weakly (albeit significantly) less tolerant than Fagus orientalis. Within its limits, Pseudotsuga menziesii showed high seasonal heat acclimation with constantly increasing tolerance during the growing season. Fagus orientalis, but practically not Fagus sylvatica, also acclimated to heat. This makes Fagus orientalis slightly superior over Fagus sylvatica in terms of heat tolerance, whereas the suitability of Pseudotsuga menziesii for silvicultural climate change adaptation is questionable. Strong heat acclimation, but also overall low heat tolerance, in Pseudotsuga menziesii might be the result of evergreenness, which requires the generation of both cold and heat tolerance during the year.

Urban pollinator gardens can provide refugia and support diverse populations of native bees amid threats from habitat destruction, pesticides, and potential ecological pressures from the introduced honey bee (Apis mellifera (Linnaeus, 1748)). The University of California, Berkeley, maintained a native bee garden at the Oxford Tract research facility to study the biodiversity, phenology, and foraging habits of urban bees from 2003 to 2009. That garden was decommissioned, and a new garden was re-established in 2019. Using diversity observations from the early 2000s garden and non-lethal sampling techniques, we characterized plant-pollinator interactions between flowers and urban bees in the newer bee garden with a bipartite interaction network. Across 12 flower species, we observed two non-native pollinators, the honey bee (A. mellifera) and the alfalfa leafcutter (Megachile rotundata (Fabricius, 1793)), along with at least ten native bee species across three families (Apidae, Halictidae, Megachilidae). We found that, despite the garden being created for native bees, honey bees accounted for 84% of all pollination interactions. The most abundant native bees were sweat bees (Family: Halictidae). Generalist interactions dominated the network, as both honey and sweat bees foraged on most available flowers. Honey bees showed a significant positive correlation with floral abundance, visiting flowers with the highest number of inflorescences, whereas native bees did not show this preference. These results indicate that native bee garden stewardship could benefit from greater floral diversity, while avoiding the dominance of any single species with high floral abundance, thereby reducing the likelihood of direct competition with honey bees.

Winter warming is altering plant exposure to cold events, yet its effects on seasonal cold hardiness dynamics remain poorly understood. Here we quantified bud cold hardiness across four dormant seasons in a boreal peatland forest whole ecosystem warming experiment. Across a +0.00 to +9.00{degrees}C warming gradient, we semi-regularly measured cold hardiness in two overstory (Larix laricina and Picea mariana) and two understory species (Chamaedaphne calyculata and Rhododendron groenlandicum). Warming reduced cold hardiness in fall and spring by delaying acclimation and advancing deacclimation. However, risk was only increased in late winter and spring for three species. Warming reduced snow cover, increasing temperature variability and cold damage to understory shrubs. Together, our results show that cold damage risk depends on species traits, microclimate, and seasonal timing.

Most plants require animal pollination to reproduce, prompting concern that pollinator declines immediately threaten plant populations. This concern is warranted if pollinator-mediated seed losses cause declines in plant population growth rates ({lambda}). However, demographic trade-offs might reduce the risk of population decline if seed loss improves performance elsewhere in the life cycle. We conducted a multi-year pollination manipulation on four species and measured how demographic vital rates and {lambda} responded. Seed responses did not predict net changes in {lambda}. Reduced pollination decreased seed production, but only caused a net decrease in {lambda} in one species; in the others, improved survival buffered {lambda}. Increased pollination boosted seed production, but at a cost to survival that caused a net reduction in {lambda} in three species. Our results highlight the importance of demographic trade-offs for understanding the impacts of pollinator declines on plant biodiversity and, more broadly, the population-level impacts of changing mutualisms.

Forest trees pose numerous potential challenges to phylogenomic inference. Their large effective population sizes and relatively long generation times lead to deep allele coalescence and consequently incomplete lineage sorting (ILS), which biases inferences of divergence times toward older ages and introduces gene tree discordance. Deep phylogenetic divergences, reaching back into the Paleocene, introduce reference-mapping biases. Introgression--the movement of genes between lineages--may result in different phylogenies being inferred depending on which individuals are included in analysis, even if the plurality of the genome favors the divergence history unaffected by introgression. These factors influence phylogenetic inference across the Tree of Life but are particularly prevalent in forest trees. Oaks (Quercus) are notable for all three influences. In addition, our knowledge of the oak phylogeny is currently based strongly on restriction site associated DNA sequencing (RADseq) datasets published over the past decade, which may introduce additional sources of uncertainty. In this chapter, we analyze a 322-species RADseq dataset and genome resequencing data from across the genus to address sources of uncertainty in our understanding of the global oak phylogeny, which we hope will serve as a model for other research groups working on comparable woody plant groups.

Aimto characterize the evolution of climatic niches during the diversification of the Phyllotis darwini species group, in order to assess the extent to which divergences involved in radiation were associated with patterns of conservatism or divergence of climatic niches, and whether the differentiation found among climatic niches correlated with species phylogenetic relationships. Locationsouth-central Andes, surrounding lowlands, and Patagonia, South America. Methodsspecies climatic niches were characterized by sampling contemporaneous precipitation and temperature conditions across occurrence locations and entire distributional ranges. Climatic niches were analyzed and modeled using multivariate statistics (PCA, PERMANOVA), a maximum entropy-based algorithm, and novel methods developed to explore levels of differentiation (niche overlap test) and divergence (niche divergence test) between realized and fundamental niches. Comparative phylogenetic methods were applied using a time-calibrated phylogeny and integrating climate niche data to estimate ancestral environmental niches within geographic and environmental spaces. Resultscomparisons revealed low levels of climatic niche overlap, both among species realized niches and among their fundamental niches, suggesting high levels of niche differentiation during the diversification of Phyllotis species. Quantifications of niche overlap further showed that observed differences among species lay primarily in the multidimensional nature of climatic niches, as unidimensional quantifications exhibited higher levels of overlap. Evolved differences among species climatic niches were better fitted to a Brownian motion model of evolution, but lacked phylogenetic signal and showed no significant association with species phylogenetic distances. Main conclusionslow levels of differentiation between ancestral climatic niches suggest that the early radiation of species in the Phyllotis darwini species group was promoted by geographic isolation, whereas the more recent diversification of extant species was accompanied by climatic niche differentiation, possibly involving local adaptation to regional ecoclimatic changes associated with Quaternary glacial cycles. The spatial separation of sister species, the complete divergence of their climatic niches, and the lack of phylogenetic signal in niche differences suggest a scenario of diversification in which divergences were prompted by the spatial isolation, but also by the divergent selection exerted by regional climatic differences.