Hierarchical Feedback Modules and Reaction Hubs in Cell Signaling Networks

Despite much effort, identification of modular structures and study of their organizing and functional roles remain a formidable challenge in molecular systems biology, which, however, is essential in reaching a systematic understanding of large-scale cell regulation networks and hence gaining capacity of exerting effective interference to cell activity. Combining graph theoretic methods with available dynamics information, we successfully retrieved multiple feedback modules of three important signaling networks. These feedbacks are structurally arranged in a hierarchical way and dynamically produce layered temporal profiles of output signals. We found that global and local feedbacks act in very different ways and on distinct features of the information flow conveyed by signal transduction but work highly coordinately to implement specific biological functions. The redundancy embodied with multiple signal-relaying channels and feedback controls bestow great robustness and the reaction hubs seated at junctions of different paths announce their paramount importance through exquisite parameter management. The current investigation reveals intriguing general features of the organization of cell signaling networks and their relevance to biological function, which may find interesting applications in analysis, design and control of bio-networks.     

Self-Organizing Task Modules and Explicit Coordinate Systems in a Neural Network Model for 3-D Saccades

The goal of this study was to train an artificial neural network to generate accurate saccades in Listing's plane and then determine how the hidden units performed the visuomotor transformation. A three-layer neural network was successfully trained, using back-prop, to take in oculocentric retinal error vectors and three-dimensional eye orientation and to generate the correct head-centric motor error vector within Listing's plane. Analysis of the hidden layer of trained networks showed that explicit representations of desired target direction and eye orientation were not employed. Instead, the hidden-layer units consistently divided themselves into four parallel modules: a dominant "vector-propagation" class (approximately 50% of units) with similar visual and motor tuning but negligible position sensitivity and three classes with specific spatial relations between position, visual, and motor tuning. Surprisingly, the vector-propagation units, and only these, formed a highly precise and consistent orthogonal coordinate system aligned with Listing's plane. Selective "lesions" confirmed that the vector-propagation module provided the main drive for saccade magnitude and direction, whereas a balance between activity in the other modules was required for the correct eye-position modulation. Thus, contrary to popular expectation, error-driven learning in itself was sufficient to produce a "neural" algorithm with discrete functional modules and explicit coordinate systems, much like those observed in the real saccade generator.     

Podocalyxin Is a Novel Polysialylated Neural Adhesion Protein with Multiple Roles in Neural Development and Synapse Formation

Neural development and plasticity are regulated by neural adhesion proteins, including the polysialylated form of NCAM (PSA-NCAM). Podocalyxin (PC) is a renal PSA-containing protein that has been reported to function as an anti-adhesin in kidney podocytes. Here we show that PC is widely expressed in neurons during neural development. Neural PC interacts with the ERM protein family, and with NHERF1/2 and RhoA/G. Experiments in vitro and phenotypic analyses of podxl-deficient mice indicate that PC is involved in neurite growth, branching and axonal fasciculation, and that PC loss-of-function reduces the number of synapses in the CNS and in the neuromuscular system. We also show that whereas some of the brain PC functions require PSA, others depend on PC per se. Our results show that PC, the second highly sialylated neural adhesion protein, plays multiple roles in neural development.     

Clique of Functional Hubs Orchestrates Population Bursts in Developmentally Regulated Neural Networks

It has recently been discovered that single neuron stimulation can impact network dynamics in immature and adult neuronal circuits. Here we report a novel mechanism which can explain in neuronal circuits, at an early stage of development, the peculiar role played by a few specific neurons in promoting/arresting the population activity. For this purpose, we consider a standard neuronal network model, with short-term synaptic plasticity, whose population activity is characterized by bursting behavior. The addition of developmentally inspired constraints and correlations in the distribution of the neuronal connectivities and excitabilities leads to the emergence of functional hub neurons, whose stimulation/deletion is critical for the network activity. Functional hubs form a clique, where a precise sequential activation of the neurons is essential to ignite collective events without any need for a specific topological architecture. Unsupervised time-lagged firings of supra-threshold cells, in connection with coordinated entrainments of near-threshold neurons, are the key ingredients to orchestrate population activity.     

鼠尾藻生长与生殖的权衡

2009年7月初至2009年9月中旬对烟台长岛县望福礁附近水域鼠尾藻(Sargassum thunbergii)进行了生态学调查。实验设立固定采样地点通过随机采样以及现场标记测量的方法观测了鼠尾藻的生长与生殖情况。结果表明:(1)鼠尾藻生殖始于7月初,8月初为高峰期,此间87%的藻体产生生殖托;(2)藻体长度对成熟比例和生殖力均有显著的影响,藻体成熟具有大小依赖性;(3)营养组织生物量大的鼠尾藻群体生殖组织生物量也相对较大,生殖期间营养组织生长和生殖组织生长呈异速关系;(4)成熟藻体在有性生殖高峰期过后,生长率下降,而始终未成熟的藻体则持续生长(生长率0)。以上结果表明,在鼠尾藻的生活史中存在着生长与生殖的权衡。     

Trade-off between egg mass and egg number in brown trout

Individual egg mass and fecundity increased with somatic mass in first time and repeat spawning wild anadromous and freshwater resident brown trout Salmo trutta . The egg mass was larger for similar-sized trout in south (58° N) than mid Norway (63° N), whereas fecundity was higher in mid- than in south Norway, making total gonadal investment similar in the two areas. Repeat spawners had heavier eggs than similar-sized first time spawners. The egg mass of residents was c. 10% larger than that of similar-sized first time spawning anadromous trout. Common garden experiments with offspring of wild anadromous trout showed no significant correlation between egg and somatic mass in first time spawners in two of the three populations studied. In the third population, a slight positive correlation was found. Similar results were found for repeat spawners. In the three populations, fecundity increased significantly with somatic mass in both first time and repeat spawners. Wild and hatchery-reared trout showed negative correlation between egg mass and fecundity when the effect of body size was excluded, indicating a trade-off between the two parameters. In wild trout, this was caused by variation among populations, whereas in hatchery fish, within population variation was observed in egg mass over fecundity. Furthermore, the egg mass of first time and repeat spawners were positively correlated, when adjusted for fish size. Size-specific gonadal investment was significantly higher in wild anadromous than resident trout. There was no significant difference in gonadal investment between first time and repeat spawners in wild anadromous trout. However, in the hatchery-reared trout, gonadal investment was significantly higher at repeat than first time maturation. The hatchery trout did not spawn naturally, but were artificially stripped. Among populations, a part of the variation in egg mass and fecundity is phenotypically plastic, a part appears genetically determined.     

Trade-off between flight activity and life-history components in Chrysoperla sinica

We studied the effects of flight duration on two important life-history traits, i.e., longevity and life-time egg production, of Chrysoperla sinica females, through an experimental flight manipulation bioassay, using a flight mill apparatus. In addition, we assessed the potential transgenerational effects of maternal flight durations on survival and on the development of the progeny. We found experimental evidence of a negative link between flight activity (1, 2 and 3 h) and longevity and reproductive activity in C. sinica females. Although, the development of the progeny 1st instars was significantly longer for females that flew longer, all other recorded life history traits of the progeny, including juvenile survival and overall development time and size of the pupae, were not affected. Hence, these findings suggest that the growth of new populations originated by females that flew long distances maybe be slower only for the first generation.     

Transfer of Learning between Hemifields in Multiple Object Tracking: Memory Reduces Constraints of Attention

Many tasks involve tracking multiple moving objects, or stimuli. Some require that individuals adapt to changing or unfamiliar conditions to be able to track well. This study explores processes involved in such adaptation through an investigation of the interaction of attention and memory during tracking. Previous research has shown that during tracking, attention operates independently to some degree in the left and right visual hemifields, due to putative anatomical constraints. It has been suggested that the degree of independence is related to the relative dominance of processes of attention versus processes of memory. Here we show that when individuals are trained to track a unique pattern of movement in one hemifield, that learning can be transferred to the opposite hemifield, without any evidence of hemifield independence. However, learning is not influenced by an explicit strategy of memorisation of brief periods of recognisable movement. The findings lend support to a role for implicit memory in overcoming putative anatomical constraints on the dynamic, distributed spatial allocation of attention involved in tracking multiple objects.     

Trade-off between thermal sensitivity,hypoxia tolerance and growth in fish

One outcome of contemporary climate trends is that the involvement of hypoxia and heat tolerance in determining individual fitness will increase in many fish populations. Large fish are believed to be more tolerant to hypoxia than small fish (Nilsson and Östlund-Nilsson, 2008) whereas thermal sensitivity is thought to decrease with body size (Clark et al., 2008). To better understand the bases of inter-individual variation in environmental adaptation performance, the current study examined hypoxia and heat tolerance in a fast growing (FGS; 288.3 ±14.4 g, 26.04±0.49 cm) and a slow growing (SGS; 119.95±6.41 g; 20.98±0.41 cm) strain of 1-year old rainbow trout (Oncorhynchus mykiss). This examination was conducted using two standardized challenge tests aimed at assessing individual incipient lethal oxygen saturation and incipient upper lethal temperature. Results to these tests were then cross-correlated with swim tests during which individual basal and active metabolic rate values were also measured. Measurements of permeabilized ventricular myofibers oxygen consumption were also conducted, as well as various organ-to-body-mass ratios. Experimental data showed that FGS was more hypoxia tolerant than SGS (13.4 to 16.7% air sat versus 14.7 to 18.9% air sat respectively). On the other hand, FGS was found less tolerant to heat than SGS (24.7–27.6 °C versus 28.5 to 29.7 °C respectively). Adding to the body size effect, another source of inter-individual variation in environmental tolerance was found. Residual analysis highlighted that whereas none of the individual morphometric and energetic traits correlated with hypoxia tolerance, permeabilized ventricular myofibers maximal oxygen consumption correlated well with individual tolerance to heat.     

Trade-off between immunocompetence and growth in magpies: an experimental study

A trade-off between immunity and growth has repeatedly been suggested, mainly based on laboratory and poultry science, but also from experiments where parasitism intensity was manipulated in field bird populations. However, as resource allocation to different activities (or organs) during growth is difficult to manipulate, this trade-off has only been experimentally tested by studying the effects of non-pathogenic antigens. By providing some nestling magpies (Pica pica) with methionine, a sulphur amino acid that specifically enhances T-cell immune response in chickens, we investigated this trade-off by directly affecting allocation of limited resources during growth. Results were in accordance with the hypothetical trade-off because nestlings fed with methionine showed a lower growth rate during the four days of methionine administration, but a larger response when fledglings were challenged with phytohaemagglutinin (a measure of the intensity of T-lymphocyte-mediated immune responsiveness) than control nestlings. Surprisingly, we found that control and experimental nestlings fledged with similar body mass, size and condition, but experimental nestlings suffered less from blood parasites (Haemoproteus) and had fewer lymphocytes (a widely used measure of health status) than control nestlings, suggesting a negative effect of blood parasites or other pathogens on nestling growth.     

Trade-off between floor level and floor material in farmed silver foxes

Farmed silver foxes (Vulpes vulpes) were allowed to balance their known preference for an elevated floor against their presumed preference for a sand floor. In Experiment 1, foxes had to choose between two identical cages, connected with an opening. One cage had a wire floor and the other had a sand floor, but the cages either were on the same (low or elevated) or on different levels (one cage 40 cm higher than the other). In Experiment 2, the cage pairs were connected with a 1.2 m long wire-mesh tunnel, one cage was always on a higher level (50 cm) than the other. In Experiment 1, foxes always preferred the sand floor during their active time. They also preferred the sand floor for resting, if it was on the same level as wire floor, but did not show any genuine preference if the floors were on different levels. In Experiment 2, foxes never preferred the lower floor. They preferred the elevated sand floor for activity and the elevated wire floor for lying. When two floors were identical they preferred the elevated one. Their rest consisted of 11-22 bouts, a major part of these being spent in the preferred cage. They also preferred a previous lying site to a new one, often exclusively and independently of floor material. In Experiment 1 foxes preferred the sand floor whereas in Experiment 2 they preferred the elevated floor indicating that the ability of a trade-off situation to rank resources depends on the method it is inflicted.     

Trade-off between age of first reproduction and survival in a female primate

Trade-offs are central to life-history theory but difficult to document. Patterns of phenotypic and genetic correlations in rhesus macaques, Macaca mulatta—a long-lived, slow-reproducing primate—are used to test for a trade-off between female age of first reproduction and adult survival. A strong positive genetic correlation indicates that female macaques suffer reduced adult survival when they mature relatively early and implies primate senescence can be explained, in part, by antagonistic pleiotropy. Contrasts with a similar human study implicate the extension of parental effects to later ages as a potential mechanism for circumventing female life-history trade-offs in human evolution.     

Trade-off between travel distance and prioritization of high-reward sites in traplining bumblebees

1.Animals exploiting renewable resource patches are faced with complex multi-location routing problems. In many species, individuals visit foraging patches in predictable sequences called traplines. However, whether and how they optimize their routes remains poorly understood.2.In this study, we demonstrate that traplining bumblebees (Bombus terrestris) make a trade-off between minimizing travel distance and prioritizing the most rewarding feeding locations.3.Individual bees trained to forage on five artificial flowers of equal reward value selected the shortest possible route as a trapline. After introducing a single highly rewarding flower to the array, they re-adjusted their routes visiting the most rewarding flower first provided the departure distance from the shortest possible route remained small (18%). When routes optimizing the initial rate of reward intake were much longer (42%), bees prioritized short travel distances.4.Under natural conditions, in which individual flowers vary in nectar productivity and replenish continuously, it might pay bees to prioritize highly rewarding locations, both to minimize the overall number of flowers to visit and to beat competitors.5.We discuss how combined memories of location and quality of resource patches could allow bees and other traplining animals to optimize their routing decisions in heterogeneous environments.     

Multiple Reaction Monitoring Enables Precise Quantification of 97 Proteins in Dried Blood Spots

The dried blood spot (DBS) methodology provides a minimally invasive approach to sample collection and enables room-temperature storage for most analytes. DBS samples have successfully been analyzed by liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM-MS) to quantify a large range of small molecule biomarkers and drugs; however, this strategy has only recently been explored for MS-based proteomics applications. Here we report the development of a highly multiplexed MRM assay to quantify endogenous proteins in human DBS samples. This assay uses matching stable isotope-labeled standard peptides for precise, relative quantification, and standard curves to characterize the analytical performance. A total of 169 peptides, corresponding to 97 proteins, were quantified in the final assay with an average linear dynamic range of 207-fold and an average R² value of 0.987. The total range of this assay spanned almost 5 orders of magnitude from serum albumin ({"type":"entrez-protein","attrs":{"text":"P02768","term_id":"113576","term_text":"P02768"}}P02768) at 18.0 mg/ml down to cholinesterase ({"type":"entrez-protein","attrs":{"text":"P06276","term_id":"116353","term_text":"P06276"}}P06276) at 190 ng/ml. The average intra-assay and inter-assay precision for 6 biological samples ranged from 6.1–7.5% CV and 9.5–11.0% CV, respectively. The majority of peptide targets were stable after 154 days at storage temperatures from −20 °C to 37 °C. Furthermore, protein concentration ratios between matching DBS and whole blood samples were largely constant (<20% CV) across six biological samples. This assay represents the highest multiplexing yet achieved for targeted protein quantification in DBS samples and is suitable for biomedical research applications.The dried blood spot (DBS)¹ methodology provides several advantages over traditional plasma or serum samples throughout the entire pre-analytical workflow including sample collection, transportation, and storage (1, 2) These blood samples are typically generated using a small sterile lancet to prick the skin and then spotting a drop onto a collection card. Therefore, DBS sampling is less invasive than venipuncture and does not require a trained phlebotomist. This sampling approach also does not require time-sensitive centrifugation, which is crucial for plasma and serum samples to prevent degradation. Many analytes have been determined to be stable in the DBS format at room temperature, eliminating the cost associated with cold-chain logistics for sample transportation and storage. These considerations are also important for sample collection in remote locations that may be without reliable access to a centrifuge and/or a freezer designated for biohazardous materials. Quantitative bioanalytical methods using the DBS methodology have been developed for genomic, metabolomic, and proteomic applications including newborn screening (3, 4), therapeutic drug monitoring (5, 6), toxicology and drugs of abuse (7, 8), viral disease management (9, 10), and many others (2, 11).Targeted MS, in particular selected/multiple reaction monitoring (SRM/MRM) using internal standards, enables the rapid development of quantitative assays with high specificity, precision, and robustness (12–15). The integration of DBS sampling with MRM is well-established for quantifying a wide range of small molecules (16–18). This is now the standard analytical approach for population-wide screening of newborns for errors in metabolism by targeting amino acids, fatty acid acylcarnitines, and organic acid acylcarnitines (3, 4). DBS with MRM is also emerging as an important analytical tool throughout pre-clinical and clinical small-molecule drug development and monitoring (16, 17, 19–21). Furthermore, Zukunft et al. recently demonstrated the high multiplexing capabilities of MRM by using 2 methods to quantify 188 metabolites in DBS samples, including acylcarnitines, amino acids, biogenic amines, free carnitine, glycerophospholipids, hexoses, lysophosphatidylcholines, phosphatidylcholines, and sphingolipids (22).Although DBS with MRM is well-established in small molecule applications, there are only a handful of reports showing the use of this approach to quantify endogenous proteins (23). Daniel et al. measured the ratio between hemoglobin δ and β to screen for β-thalassemia (24). Boemer et al. measured the relative ratios of several hemoglobin variants (including HbS, HbC, HbE, and others) to help diagnose Sickle Cell disease and other clinically relevant hemoglobinopathies (25). The same group then screened >40,000 newborns in Belgium and successfully detected 16 patients with severe hemoglobin disorders (26). Moats et al. used a similar approach to screen >13,000 newborns in the UK for Sickle Cell disease and correctly identified all seven disease occurrences (27). Because hemoglobin is the most abundant protein in whole blood (∼150 mg/ml), these four studies achieved adequate sensitivity by simply infusing the trypsin digested samples into a triple-quadrupole MS. To move beyond hemoglobin, additional sensitivity can be provided by using liquid chromatography (LC) separations coupled online with MRM. deWilde et al. used LC/MRM-MS to quantify ceruloplasmin as a screen for Wilson''s disease (28). Recently, Cox et al. reported LC/MRM-MS methods for quantifying insulin-like growth facter-1 for the detection of human growth hormone abuse in sports (29, 30).Our group reported the first LC/MRM-MS assay to quantify multiple endogenous proteins in DBS samples (31). In that exploratory study, we selected a small test panel of 60 high-abundance proteins and were ultimately able to quantify 37 proteins using stable isotope-labeled standard (SIS) peptides and standard curves. In this work, we describe method refinement and further evaluation of LC/MRM-MS for quantifying endogenous proteins in human DBS samples. A more comprehensive approach has now been taken to evaluate sensitivity and suitability, as the initial target panel has been increased to 393 proteins. The protocol has also been modified so that all liquid handling steps in the sample preparation protocol are now automated in a 96-well format for improved sample throughput. Standard curves using SIS peptides were produced using a pooled patient sample, and assay precision was determined in biological samples from six different individuals. In addition, we have provided a detailed discussion of the quantification results from multiple peptides per protein, a comparison to measured protein concentrations in whole blood, an analyte stability assessment at various storage temperatures, and an evaluation of volumetric spotting devices. Ultimately, we have developed a multiplexed LC/MRM-MS assay to quantify 97 proteins in DBS samples that is suitable for biomedical research applications.     

Trade-off between resource seasonality and predation risk explains reproductive chronology in impala

We investigated the variation in birth synchrony displayed by impala Aepyceros melampus populations across their distribution from southern to eastern Africa. Our analysis was based on field data from Chobe National Park in Botswana and Mala Mala Private Game Reserve in South Africa (4 and 13 years of monitoring, respectively). We compared our results with those from other studies conducted across the impala species range. Impala lambing was highly synchronized in Chobe with 90% of lambs born within 2 weeks in mid-November. Variation in rainfall in the preceding wet season explained 74% of variation in the dates of the first lamb observation in Mala Mala. In Chobe, the earliest birth peak occurred after the highest rainfall and the body condition of lambs in that cohort was also best for both males and females. No association was found between the lunar cycle and the estimated onset of the conception period, despite previous studies having found an association between the lunar cycle and the rutting behaviour in males. On a regional scale, impalas in areas with a marked dry season (several months with no rain) tend to synchronize births with the onset of the rains, when grass quality is highest. Number of months with rain explained 78% of the regional variation in birth synchrony. Neither latitude nor total rainfall contributed significantly to a stepwise multiple regression model. These data support the theory that impalas synchronize births in areas with a highly seasonal food supply, and temporally space births in less seasonal (equatorial) areas to reduce predation risks.     

Trade-off between steady and unsteady swimming underlies predator-driven divergence in Gambusia affinis

Differences in predation intensity experienced by organisms can lead to divergent natural selection, driving evolutionary change. Western mosquitofish (Gambusia affinis) exhibit larger caudal regions and higher burst-swimming capabilities when coexisting with higher densities of predatory fish. It is hypothesized that a trade-off between steady (constant-speed cruising; important for acquiring resources) and unsteady (rapid bursts and turns; important for escaping predators) locomotion, combined with divergent selection on locomotor performance (favouring steady swimming in high-competition scenarios of low-predation environments, but unsteady swimming in high-predation localities) has caused such phenotypic divergence. Here, I found that morphological differences had a strong genetic basis, and low-predation fish required less hydromechanical power during steady swimming, leading to increased endurance. I further found individual-level support for cause-and-effect relationships between morphology, swimming kinematics and endurance. Results indicate that mosquitofish populations inhabiting low-predation environments have evolved increased steady-swimming abilities via stiffer bodies, larger anterior body/head regions, smaller caudal regions and greater three-dimensional streamlining.     

Trade-off between cadmium tolerance and relative growth rate in 10 grass species

Plant species exhibit great differences in heavy metal accumulation and tolerance. In this study we compared interspecific differences in responses to Cadmium (Cd) stress among 10 C₃ grass species by growing hydroponically under the conditions of different Cd treatments (0, 5, 10 and 50 μM). Responses of plant shoot dry mass to Cd treatments (resistance) were separated into avoidance (the response of shoot Cd concentration to Cd treatments) and tolerance (the response of shoot dry mass to shoot Cd concentration). The relative growth rate (RGR) and leaf structural properties of plants were measured under optimum growth condition. There were large differences in resistance, avoidance and tolerance among the species. Avoidance and tolerance were attributed independently to total Cd resistance. Resistance and tolerance were correlated negatively with RGR, leaf water content (LW), specific leaf area (SLA), leaf elongation rate (LER), and leaf length (LL), but the leaf dry matter concentration (LMDC), and nuclear DNA content showed a positive correlation with resistance and tolerance. These results indicate a trade-off between growth rate and tolerance to Cd stress. Species with higher Ca/Mg ratios showed low avoidance (r = −0.943***), suggesting uptake inhibition by Ca²⁺ to develop avoidance against Cd stress.     

Trade-off between leaf size and vein density of Achnatherum splendens in Zhangye wetland

Aims Trade-offs between leaf size and vein density are the basis of the theory of leaf economics spectrum, and are to understand the relationship between the physical build and physiological metabolism of plant leaves under different degrees of competition for resources. Our objective was to study the changes in the relationship between leaf size and vein density (leaf dry biomass and leaf area) in Achnatherum splendens populations with four plant bundle densities located in the flood plain wetland of Zhangye. Methods The study site was located at floodplain wetlands of Zhangye, Gansu Province, China. Survey and sampling were carried out in the communities that A. splendens dominated. According to the plant bundle density, the A. splendens communities were divided into four density gradients with “bundle” for the sampling units, high density (I, > 12 bundle·m^-2), medium density (II, 8-12 bundle·m^-2), medium density (III, 4-8 bundle·m^-2) and Low density (IV, <4 bundle·m^-2). According to the density of each combination, we chose seven (5 m × 5 m) A. splendens samples, resulting in a total of 28 samples (4 × 7). The soil physical and chemical properties of four density gradients were investigated and six samples of A. splendens were used to measure the leaf area, leaf dry biomass and vein density in laboratory, and biomass of different organs was measured after being dried at 85 °C in an oven. 28 plots were categorized into three groups: high, medium and low density, and the standardized major axis (SMA) estimation method was used to examine the allometric relationships between leaf area, leaf dry biomass and vein density. Important findings The results showed that with the population density changed from high, medium, to low, the soil moisture decreased, and soil electric conductivityincreased. The leaf area, leaf biomass and height of A. splendens decreased, and the vein density, specific leaf area and photosynthetically active radiation (PAR) increased gradually. In addition, leaf net photosynthetic rate (P_n), transpiration rate (T_r) and twig number firstly increased then decreased. There was a highly significantly negative correlation (p < 0.01) between the leaf size and vein density on the high- and low-level densities (I, IV), whereas less significant (p < 0.05) on the level of medium density (II, III). The SMA slope of regression equation in the scaling relationships between leaf size and vein density was significantly smaller than -1 (p < 0.05).