Long noncoding RNA-mediated epigenetic regulation of auxin-related genes controls shade avoidance syndrome in Arabidopsis.

The long noncoding RNA (lncRNA) AUXIN-REGULATED PROMOTER LOOP (APOLO) recognizes a subset of target loci across the Arabidopsis thaliana genome by forming RNA-DNA hybrids (R-loops) and modulating local three-dimensional chromatin conformation. Here, we show that APOLO regulates shade avoidance syndrome by dynamically modulating expression of key factors. In response to far-red (FR) light, expression of APOLO anti-correlates with that of its target BRANCHED1 (BRC1), a master regulator of shoot branching in Arabidopsis thaliana. APOLO deregulation results in BRC1 transcriptional repression and an increase in the number of branches. Accumulation of APOLO transcription fine-tunes the formation of a repressive chromatin loop encompassing the BRC1 promoter, which normally occurs only in leaves and in a late response to far-red light treatment in axillary buds. In addition, our data reveal that APOLO participates in leaf hyponasty, in agreement with its previously reported role in the control of auxin homeostasis through direct modulation of auxin synthesis gene YUCCA2, and auxin efflux genes PID and WAG2. We show that direct application of APOLO RNA to leaves results in a rapid increase in auxin signaling that is associated with changes in the plant response to far-red light. Collectively, our data support the view that lncRNAs coordinate shade avoidance syndrome in A. thaliana, and reveal their potential as exogenous bioactive molecules. Deploying exogenous RNAs that modulate plant-environment interactions may therefore become a new tool for sustainable agriculture.

Shade-induced lncRNA PUAR promotes shade response by repressing PHYA expression.

Shade avoidance syndrome (SAS) commonly occurs in plants experiencing vegetative shade, triggering a series of morphological and physiological changes for the plants to reach more light. A number of positive regulators, such as PHYTOCHROME-INTERACTING 7 (PIF7), and negative regulators, such as PHYTOCHROMES, are known to ensure appropriate SAS. Here, we identify 211 shade-regulated long non-coding RNAs (lncRNAs) in Arabidopsis. We further characterize PUAR (PHYA UTR Antisense RNA), a lncRNA produced from the intron of the 5' UTR of the PHYTOCHROME A (PHYA) locus. PUAR is induced by shade and promotes shade-induced hypocotyl elongation. PUAR physically associates with PIF7 and represses the shade-mediated induction of PHYA by blocking the binding of PIF7 to the 5' UTR of PHYA. Our findings highlight a role for lncRNAs in SAS and provide insight into the mechanism of PUAR in regulating PHYA gene expression and SAS.

HISTONE DEACETYLASE 9 promotes hypocotyl-specific auxin response under shade.

Vegetative shade causes an array of morphological changes in plants called shade avoidance syndrome, which includes hypocotyl and petiole elongation, leaf hyponasty, reduced leaf growth, early flowering and rapid senescence. Here, we show that loss-of-function mutations in HISTONE DEACETYLASE 9 (HDA9) attenuated the shade-induced hypocotyl elongation in Arabidopsis. However, the hda9 cotyledons and petioles under shade were not significantly different from those in wild-type, suggesting a specific function of HDA9 in hypocotyl elongation in response to shade. HDA9 expression levels were stable under shade and its protein was ubiquitously detected in cotyledon, hypocotyl and root. Organ-specific transcriptome analysis unraveled that shade induced a set of auxin-responsive genes, such as SMALL AUXIN UPREGULATED RNAs (SAURs) and AUXIN/INDOLE-3-ACETIC ACIDs (AUX/IAAs) and their induction was impaired in hda9-1 hypocotyls. In addition, HDA9 binding to loci of SAUR15/65, IAA5/6/19 and ACS4 was increased under shade. The genetic and organ-specific gene expression analyses further revealed that HDA9 may cooperate with PHYTOCHROME-INTERACTING FACTOR 4/7 in the regulation of shade-induced hypocotyl elongation. Furthermore, HDA9 and PIF7 proteins were found to interact together and thus it is suggested that PIF7 may recruit HDA9 to regulate the shade/auxin responsive genes in response to shade. Overall, our study unravels that HDA9 can work as one component of a hypocotyl-specific transcriptional regulatory machinery that activates the auxin response at the hypocotyl leading to the elongation of this organ under shade.

lncRNAs involved in the Shade Avoidance Syndrome (SAS) in Arabidopsis thaliana.

BACKGROUND: Plant long non-coding RNAs (lncRNAs) have important regulatory roles in responses to various biotic and abiotic stresses, including light quality. However, no lncRNAs have been specifically linked to the Shade Avoidance Response (SAS). RESULTS: To better understand the involvement of lncRNAs in shade avoidance, we examined RNA-seq libraries for lncRNAs with the potential to function in the neighbor proximity phenomenon in Arabidopsis thaliana (A. thaliana). Using transcriptomes generated from seedlings exposed to high and low red/far-red (R/FR) light conditions, we identified 13 lncRNA genes differentially expressed in cotyledons and 138 in hypocotyls. To infer possible functions for these lncRNAs, we used a 'guilt-by-association' approach to identify genes co-expressed with lncRNAs in a weighted gene co-expression network. Of 34 co-expression modules, 10 showed biological functions related to differential growth. We identified three potential lncRNAs co-regulated with genes related to SAS. T-DNA insertions in two of these lncRNAs were correlated with morphological differences in seedling responses to increased FR light, supporting our strategy for computational identification of lncRNAs involved in SAS. CONCLUSIONS: Using a computational approach, we identified multiple lncRNAs in Arabidopsis involved in SAS. T-DNA insertions caused altered phenotypes under low R/FR light, suggesting functional roles in shade avoidance. Further experiments are needed to determine the specific mechanisms of these lncRNAs in SAS.

Class I TCP transcription factors TCP14 and TCP15 promote axillary branching in Arabidopsis by counteracting the action of Class II TCP BRANCHED1.

Shoot branching is determined by a balance between factors that promote axillary bud dormancy and factors that release buds from the quiescent state. The TCP family of transcription factors is classified into two classes, Class I and Class II, which usually play different roles. While the role of the Class II TCP BRANCHED1 (BRC1) in suppressing axillary bud development in Arabidopsis thaliana has been widely explored, the function of Class I TCPs in this process remains unknown. We analyzed the role of Class I TCP14 and TCP15 in axillary branch development in Arabidopsis through a series of genetic and molecular studies. In contrast to the increased branch number shown by brc1 mutants, tcp14 tcp15 plants exhibit a reduced number of branches compared with wild-type. Our findings provide evidence that TCP14 and TCP15 act by counteracting BRC1 function through two distinct mechanisms. First, they indirectly reduce BRC1 expression levels. Additionally, TCP15 directly interacts with BRC1 decoying it from chromatin and thereby preventing the transcriptional activation of a set of BRC1-dependent genes. We describe a molecular mechanism by which Class I TCPs physically antagonize the action of the Class II TCP BRC1, aligning with their opposite roles in axillary bud development.

Sextuple knockouts of a highly conserved and coexpressed AUXIN/INDOLE-3-ACETIC ACID gene set confer shade avoidance-like responses in Arabidopsis.

AUXIN/INDOLE-3-ACETIC ACIDs are transcriptional repressors for auxin signalling. Aux/IAAs of Arabidopsis thaliana display some functional redundancy. The IAA3/SHY2 clade (IAA1, IAA2, IAA3 and IAA4) show strong sequence similarity, but no higher-order mutants have been reported. Here, through CRISPR/Cas9 genome editing, we generated loss-of-function iaa1/2/3/4 mutants. The quadruple mutants only exhibited a weak phenotype. Thus, we additionally knocked out IAA7/AXR2 and IAA16, which are coexpressed with IAA1/2/3/4. Remarkably, under white light control conditions, the iaa1/2/3/4/7/16 mutants exhibited a shade avoidance-like phenotype with over-elongated hypocotyls and petioles and hyponastic leaves. The sextuple mutants were highly sensitive to low light intensity, and the hypocotyl cells of the mutants were excessively elongated. Transcriptome profiling and qRT-PCR analyses revealed that the sextuple mutation upregulated IAA19/MSG2 and IAA29, two shared shade/auxin signalling targets. Besides, genes encoding cell wall-remodelling proteins and shade-responsive transcription regulators were upregulated. Using dual-luciferase reporter assays, we verified that IAA2/IAA7 targeted the promoters of cell wall-remodelling genes to inhibit their transcription. Our work indicates that the IAA1/2/3/4/7/16 gene set is required for the optimal integration of auxin and shade signalling. The mutants generated here should be valuable for exploring the complex interactions among signal sensors, transcription activators and transcription repressors during hormone/environmental responses.

Arabidopsis transcription factor TCP13 promotes shade avoidance syndrome-like responses by directly targeting a subset of shade-responsive gene promoters.

TCP13 belongs to a subgroup of TCP transcription factors implicated in the shade avoidance syndrome (SAS), but its exact role remains unclear. Here, we show that TCP13 promotes the SAS-like response by enhancing hypocotyl elongation and suppressing flavonoid biosynthesis as a part of the incoherent feed-forward loop in light signaling. Shade is known to promote the SAS by activating PHYTOCHROME-INTERACTING FACTOR (PIF)-auxin signaling in plants, but we found no evidence in a transcriptome analysis that TCP13 activates PIF-auxin signaling. Instead, TCP13 mimics shade by activating the expression of a subset of shade-inducible and cell elongation-promoting SAUR genes including SAUR19, by direct targeting of their promoters. We also found that TCP13 and PIF4, a molecular proxy for shade, repress the expression of flavonoid biosynthetic genes by directly targeting both shared and distinct sets of biosynthetic gene promoters. Together, our results indicate that TCP13 promotes the SAS-like response by directly targeting a subset of shade-responsive genes without activating the PIF-auxin signaling pathway.

BBX24 Interacts with JAZ3 to Promote Growth by Reducing DELLA Activity in Shade Avoidance.

Shade avoidance syndrome (SAS) is a strategy of major adaptive significance and typically includes elongation of the stem and petiole, leaf hyponasty, reduced branching and phototropic orientation of the plant shoot toward canopy gaps. Both cryptochrome 1 and phytochrome B (phyB) are the major photoreceptors that sense the reduction in the blue light fluence rate and the low red:far-red ratio, respectively, and both light signals are associated with plant density and the resource reallocation when SAS responses are triggered. The B-box (BBX)-containing zinc finger transcription factor BBX24 has been implicated in the SAS as a regulator of DELLA activity, but this interaction does not explain all the observed BBX24-dependent regulation in shade light. Here, through a combination of transcriptional meta-analysis and large-scale identification of BBX24-interacting transcription factors, we found that JAZ3, a jasmonic acid signaling component, is a direct target of BBX24. Furthermore, we demonstrated that joint loss of BBX24 and JAZ3 function causes insensitivity to DELLA accumulation, and the defective shade-induced elongation in this mutant is rescued by loss of DELLA or phyB function. Therefore, we propose that JAZ3 is part of the regulatory network that controls the plant growth in response to shade, through a mechanism in which BBX24 and JAZ3 jointly regulate DELLA activity. Our results provide new insights into the participation of BBX24 and JA signaling in the hypocotyl shade avoidance response in Arabidopsis.

LONG HYPOCOTYL IN FAR-RED 1 mediates a trade-off between growth and defence under shade in Arabidopsis.

Plants respond to vegetative shade with developmental and physiological changes that are collectively known as shade avoidance syndrome (SAS). Although LONG HYPOCOTYL IN FAR-RED 1 (HFR1) is known to be a negative regulator of SAS by forming heterodimers with other basic helix-loop-helix (bHLH) transcription factors to inhibit them, its function in genome-wide transcriptional regulation has not been fully elucidated. Here, we performed RNA-sequencing analyses of Arabidopsis thaliana hfr1-5 mutant and HFR1 overexpression line [HFR1(ΔN)-OE] to comprehensively identify HFR1-regulated genes at different time points of shade treatment. We found that HFR1 mediates the trade-off between shade-induced growth and shade-repressed defence, by regulating the expression of relevant genes in the shade. Genes involved in promoting growth, such as auxin biosynthesis, transport, signalling and response were induced by shade but suppressed by HFR1 under both short and long durations of shade. Likewise, most ethylene-related genes were shade-induced and HFR1-repressed. However, shade suppressed defence-related genes, while HFR1 induced their expression, especially under long durations of shade treatment. We demonstrated that HFR1 confers increased resistance to bacterial infection under shade.

Shade avoidance syndrome in soybean and ideotype toward shade tolerance.

The shade avoidance syndrome (SAS) in soybean can have destructive effects on yield, as essential carbon resources reserved for yield are diverted to the petiole and stem for exaggerated elongation, resulting in lodging and susceptibility to disease. Despite numerous attempts to reduce the unfavorable impacts of SAS for the development of cultivars suitable for high-density planting or intercropping, the genetic bases and fundamental mechanisms of SAS remain largely unclear. The extensive research conducted in the model plant Arabidopsis provides a framework for understanding the SAS in soybean. Nevertheless, recent investigations suggest that the knowledge obtained from model Arabidopsis may not be applicable to all processes in soybean. Consequently, further efforts are required to identify the genetic regulators of SAS in soybean for molecular breeding of high-yield cultivars suitable for density farming. In this review, we present an overview of the recent developments in SAS studies in soybean and suggest an ideal planting architecture for shade-tolerant soybean intended for high-yield breeding.

Effects of partial replacement of red by green light in the growth spectrum on photomorphogenesis and photosynthesis in tomato plants.

The artificial light used in growth chambers is usually devoid of green (G) light, which is considered to be less photosynthetically efficient than blue (B) or red (R) light. To verify the role of G light supplementation in the spectrum, we modified the RB spectrum by progressively replacing R light with an equal amount of G light. The tomato plants were cultivated under 100 µmol m-2 s-1 of five different combinations of R (35-75%) and G light (0-40%) in the presence of a fixed proportion of B light (25%) provided by light-emitting diodes (LEDs). Substituting G light for R altered the plant's morphology and partitioning of biomass. We observed a decrease in the dry biomass of leaves, which was associated with increased biomass accumulation and the length of the roots. Moreover, plants previously grown under the RGB spectrum more efficiently utilized the B light that was applied to assess the effective quantum yield of photosystem II, as well as the G light when estimated with CO2 fixation using RB + G light-response curves. At the same time, the inclusion of G light in the growth spectrum reduced stomatal conductance (gs), transpiration (E) and altered stomatal traits, thus improving water-use efficiency. Besides this, the increasing contribution of G light in place of R light in the growth spectrum resulted in the progressive accumulation of phytochrome interacting factor 5, along with a lowered level of chalcone synthase and anthocyanins. However, the plants grown at 40% G light exhibited a decreased net photosynthetic rate (Pn), and consequently, a reduced dry biomass accumulation, accompanied by morphological and molecular traits related to shade-avoidance syndrome.

Light participates in the auxin-dependent regulation of plant growth.

Light is the energy source for plant photosynthesis and influences plant growth and development. Through multiple photoreceptors, plant interprets light signals through various downstream phytohormones such as auxin. Recently, Chen et al. (2020) uncover a new layer of regulation in IPyA pathway of auxin biosynthesis by light. Here we highlight recent studies about how light controls plant growth through regulating auxin biosynthesis and signaling.

CRISPR/Cas9-mediated knockout and overexpression studies reveal a role of maize phytochrome C in regulating flowering time and plant height.

Maize is a major staple crop widely used for food, feedstocks and industrial products. Shade-avoidance syndrome (SAS), which is triggered when plants sense competition of light from neighbouring vegetation, is detrimental for maize yield production under high-density planting conditions. Previous studies have shown that the red and far-red photoreceptor phytochromes are responsible for perceiving the shading signals and triggering SAS in Arabidopsis; however, their roles in maize are less clear. In this study, we examined the expression patterns of ZmPHYC1 and ZmPHYC2 and found that ZmPHYC1, but not ZmPHYC2, is highly expressed in leaves and is regulated by the circadian clock. Both ZmPHYC1 and ZmPHYC2 proteins are localized to both the nucleus and cytoplasm under light conditions and both of them can interact with themselves or with ZmPHYBs. Heterologous expression of ZmPHYCs can complement the Arabidopsis phyC-2 mutant under constant red light conditions and confer an attenuated SAS in Arabidopsis in response to shading. Double knockout mutants of ZmPHYC1 and ZmPHYC2 created using the CRISPR/Cas9 technology display a moderate early-flowering phenotype under long-day conditions, whereas ZmPHYC2 overexpression plants exhibit a moderately reduced plant height and ear height. Together, these results provided new insight into the function of ZmPHYCs and guidance for breeding high-density tolerant maize cultivars.

Molecular mechanisms governing shade responses in maize.

Light is one of the most important environmental factors affecting plant growth and development. Plants use shade avoidance or tolerance strategies to adjust their growth and development thus increase their success in the competition for incoming light. To investigate the mechanism of shade responses in maize (Zea mays), we examined the anatomical and transcriptional dynamics of the early shade response in seedlings of the B73 inbred line. Transcriptome analysis identified 912 differentially expressed genes, including genes involved in light signaling, auxin responses, and cell elongation pathways. Grouping transcription factor family genes and performing enrichment analysis identified multiple types of transcription factors that are differentially regulated by shade and predicted putative core genes responsible for regulating shade avoidance syndrome. For functional analyses, we ectopically over-expressed ZmHB53, a type II HD-ZIP transcription factor gene significantly induced by shade, in Arabidopsis thaliana. Transgenic Arabidopsis plants overexpressing ZmHB53 exhibited narrower leaves, earlier flowering, and enhanced expression of shade-responsive genes, suggesting that ZmHB53 might participates in the regulation of shade responses in maize. This study increases our understanding of the regulatory network of the shade response in maize and provides a useful resource for maize genetics and breeding.

DRACULA2 is a dynamic nucleoporin with a role in regulating the shade avoidance syndrome in Arabidopsis.

When plants grow in close proximity basic resources such as light can become limiting. Under such conditions plants respond to anticipate and/or adapt to the light shortage, a process known as the shade avoidance syndrome (SAS). Following genetic screening using a shade-responsive luciferase reporter line (PHYB:LUC), we identified DRACULA2 (DRA2), which encodes an Arabidopsis homolog of mammalian nucleoporin 98, a component of the nuclear pore complex (NPC). DRA2, together with other nucleoporins, participates positively in the control of the hypocotyl elongation response to plant proximity, a role that can be considered dependent on the nucleocytoplasmic transport of macromolecules (i.e. is transport dependent). In addition, our results reveal a specific role for DRA2 in controlling shade-induced gene expression. We suggest that this novel regulatory role of DRA2 is transport independent and that it might rely on its dynamic localization within and outside of the NPC. These results provide mechanistic insights in to how SAS responses are rapidly established by light conditions. They also indicate that nucleoporins have an active role in plant signaling.

Recalculating growth and defense strategies under competition: key roles of photoreceptors and jasmonates.

The growth-defense trade-off in plant biology has gained enormous traction in the last two decades, highlighting the importance of understanding how plants deal with two of the greatest challenges for their survival and reproduction. It has been well established that in response to competition signals perceived by informational photoreceptors, shade-intolerant plants typically activate the shade-avoidance syndrome (SAS). In turn, in response to signals of biotic attack, plants activate a suite of defense responses, many of which are directed to minimize the loss of plant tissue to the attacking agent (broadly defined, the defense syndrome, DS). We argue that components of the SAS, including increased elongation, apical dominance, reduced leaf mass per area (LMA), and allocation to roots, are in direct conflict with configurational changes that plants require to maximize defense. We hypothesize that these configurational trade-offs provide a functional explanation for the suppression of components of the DS in response to competition cues. Based on this premise, we discuss recent advances in the understanding of the mechanisms by which informational photoreceptors, by interacting with jasmonic acid (JA) signaling, help the plant to make intelligent allocation and developmental decisions that optimize its configuration in complex biotic contexts.

Heritable plant phenotypes track light and herbivory levels at fine spatial scales.

Organismal phenotypes often co-vary with environmental variables across broad geographic ranges. Less is known about the extent to which phenotypes match local conditions when multiple biotic and abiotic stressors vary at fine spatial scales. Bittercress (Brassicaceae: Cardamine cordifolia), a perennial forb, grows across a microgeographic mosaic of two contrasting herbivory regimes: high herbivory in meadows (sun habitats) and low herbivory in deeply shaded forest understories (shade habitats). We tested for local phenotypic differentiation in plant size, leaf morphology, and anti-herbivore defense (realized resistance and defensive chemicals, i.e., glucosinolates) across this habitat mosaic through reciprocal transplant-common garden experiments with clonally propagated rhizomes. We found habitat-specific divergence in morphological and defensive phenotypes that manifested as contrasting responses to growth in shade common gardens: weak petiole elongation and attenuated defenses in populations from shade habitats, and strong petiole elongation and elevated defenses in populations from sun habitats. These divergent phenotypes are generally consistent with reciprocal local adaptation: plants from shade habitats that naturally experience low herbivory show reduced investment in defense and an attenuated shade avoidance response, owing to its ineffectiveness within forest understories. By contrast, plants from sun habitats with high herbivory show shade-induced elongation, but no evidence of attenuated defenses canonically associated with elongation in shade-intolerant plant species. Finally, we observed differences in flowering phenology between habitat types that could potentially contribute to inter-habitat divergence by reducing gene flow. This study illuminates how clonally heritable plant phenotypes track a fine-grained mosaic of herbivore pressure and light availability in a native plant.

A non-DNA-binding activity for the ATHB4 transcription factor in the control of vegetation proximity.

In plants, perception of vegetation proximity by phytochrome photoreceptors activates a transcriptional network that implements a set of responses to adapt to plant competition, including elongation of stems or hypocotyls. In Arabidopsis thaliana, the homeodomain-leucine zipper (HD-Zip) transcription factor ARABIDOPSIS THALIANA HOMEOBOX 4 (ATHB4) regulates this and other responses, such as leaf polarity. To better understand the shade regulatory transcriptional network, we have carried out structure-function analyses of ATHB4 by overexpressing a series of truncated and mutated forms and analyzing three different responses: hypocotyl response to shade, transcriptional activity and leaf polarity. Our results indicated that ATHB4 has two physically separated molecular activities: that performed by HD-Zip, which is involved in binding to DNA-regulatory elements, and that performed by the ETHYLENE-RESPONSIVE ELEMENT BINDING FACTOR-associated amphiphilic repression (EAR)-containing N-terminal region, which is involved in protein-protein interaction. Whereas both activities are required to regulate leaf polarity, DNA-binding activity is not required for the regulation of the seedling responses to plant proximity, which indicates that ATHB4 works as a transcriptional cofactor in the regulation of this response. These findings suggest that transcription factors might employ alternative mechanisms of action to regulate different developmental processes.

Differential response of Scots pine seedlings to variable intensity and ratio of red and far-red light.

We investigated the response to increasing intensity of red (R) and far-R (FR) light and to a decrease in R:FR ratio in Pinus sylvestris L. (Scots pine) seedling. The results showed that FR high-irradiance response for hypocotyl elongation may be present in Scots pine and that this response is enhanced by increasing light intensity. However, both hypocotyl inhibition and pigment accumulation were more strongly affected by the R light compared with FR light. This is in contrast to previous reports in Arabidopsis thaliana (L.) Heynh. In the angiosperm, A. thaliana R light shows an overall milder effect on inhibition of hypocotyl elongation and on pigment biosynthesis compared with FR suggesting conifers and angiosperms respond very differently to the different light regimes. Scots pine shade avoidance syndrome with longer hypocotyls, shorter cotyledons and lower chlorophyll content in response to shade conditions resembles the response observed in A. thaliana. However, anthocyanin accumulation increased with shade in Scots pine, which again differs from what is known in angiosperms. Overall, the response of seedling development and physiology to R and FR light in Scots pine indicates that the regulatory mechanism for light response may differ between gymnosperms and angiosperms.

Plasticity to simulated shade is associated with altitude in structured populations of Arabidopsis thaliana.

Plants compete for photosynthesis light and induce a shade avoidance syndrome (SAS) that confers an important advantage in asymmetric competition for light at high canopy densities. Shade plasticity was studied in a greenhouse experiment cultivating Arabidopsis thaliana plants from 15 populations spread across an altitudinal gradient in the northeast area of Spain that contain a high genetic variation into a reduced geographical range. Plants were exposed to sunlight or simulated shade to identify the range of shade plasticity. Fourteen vegetative, flowering and reproductive traits were measured throughout the life cycle. Shade plasticity in flowering time and dry mass was significantly associated with the altitude of population origin. Plants from coastal populations showed higher shade plasticity indexes than those from mountains. The altitudinal variation in flowering leaf plasticity adjusted negatively with average and minimum temperatures, whereas dry mass plasticity was better explained by negative regressions with the average, maximum and minimum temperatures, and by a positive regression with average precipitation of the population origin. The lack of an altitudinal gradient for the widest number of traits suggests that shade light could be a driver explaining the distribution pattern of individuals in smaller geographical scales than those explored here.