Effect of random sample size on the accuracy of nucleotide diversity estimation

Mathematical simulation has been used to analyze how the sample size affects the accuracy of the estimation of molecular variation in a population. The sample size was varied from 1/200 to 1/4 of the total size of the simulated population. The possible effect of the length of the nucleotide sequences compared has also been estimated; it was varied from 500 to 15 000 bp. A tendency towards underestimation of the mean nucleotide diversity (??) by about 25% of the expected value has been found. The sample size and/or the length of the nucleotide sequence used have been shown to affect more the scatter of the ?? values than the accuracy of its measurement (the proportion of correct estimates of ?? is about 14%). The assumption is made that the sample size affects the probability of accepting a false null hypothesis in analysis of the demographic history of a species.     

Evaluating the mean frequency of responding cells in limiting dilution assays

Summary The validity of limiting dilution assays can be compromised or negated by the use of statistical methodology which does not consider all issues surrounding the biological process. This study critically evaluates statistical methods for estimating the mean frequency of responding cells in multiple sample limiting dilution assays. We show that methods that pool limiting dilution assay data, or samples, are unable to estimate the variance appropriately. In addition, we use Monte Carlo simulations to evaluate an unweighted mean of the maximum likelihood estimator, an unweighted mean based on the jackknife estimator, and a log transform of the maximum likelihood estimator. For small culture replicate size, the log transform outperforms both unweighted mean procedures. For moderate culture replicate size, the unweighted mean based on the jackknife produces the most acceptable results. This study also addresses the important issue of experimental design in multiple sample limiting dilution assays. In particular, we demonstrate that optimization of multiple sample limiting dilution assays is achieved by increasing the number of biological samples at the expense of repeat cultures.     

Assessing the Effect of Sequencing Depth and Sample Size in Population Genetics Inferences

Next-Generation Sequencing (NGS) technologies have dramatically revolutionised research in many fields of genetics. The ability to sequence many individuals from one or multiple populations at a genomic scale has greatly enhanced population genetics studies and made it a data-driven discipline. Recently, researchers have proposed statistical modelling to address genotyping uncertainty associated with NGS data. However, an ongoing debate is whether it is more beneficial to increase the number of sequenced individuals or the per-sample sequencing depth for estimating genetic variation. Through extensive simulations, I assessed the accuracy of estimating nucleotide diversity, detecting polymorphic sites, and predicting population structure under different experimental scenarios. Results show that the greatest accuracy for estimating population genetics parameters is achieved by employing a large sample size, despite single individuals being sequenced at low depth. Under some circumstances, the minimum sequencing depth for obtaining accurate estimates of allele frequencies and to identify polymorphic sites is , where both alleles are more likely to have been sequenced. On the other hand, inferences of population structure are more accurate at very large sample sizes, even with extremely low sequencing depth. This all points to the conclusion that under various experimental scenarios, in cost-limited population genetics studies, large sample sizes at low sequencing depth are desirable to achieve high accuracy. These findings will help researchers design their experimental set-ups and guide further investigation on the effect of protocol design for genetic research.     

Historical DNA reveals the demographic history of Atlantic cod (Gadus morhua) in medieval and early modern Iceland

Atlantic cod (Gadus morhua) vertebrae from archaeological sites were used to study the history of the Icelandic Atlantic cod population in the time period of 1500–1990. Specifically, we used coalescence modelling to estimate population size and fluctuations from the sequence diversity at the cytochrome b (cytb) and Pantophysin I (PanI) loci. The models are consistent with an expanding population during the warm medieval period, large historical effective population size (N_E), a marked bottleneck event at 1400–1500 and a decrease in N_E in early modern times. The model results are corroborated by the reduction of haplotype and nucleotide variation over time and pairwise population distance as a significant portion of nucleotide variation partitioned across the 1550 time mark. The mean age of the historical fished stock is high in medieval times with a truncation in age in early modern times. The population size crash coincides with a period of known cooling in the North Atlantic, and we conclude that the collapse may be related to climate or climate-induced ecosystem change.     

Mitochondrial DNA phylogeography and population history of the grey wolf canis lupus

The grey wolf (Canis lupus) and coyote (C. latrans) are highly mobile carnivores that disperse over great distances in search of territories and mates. Previous genetic studies have shown little geographical structure in either species. However, population genetic structure is also influenced by past isolation events and population fluctuations during glacial periods. In this study, control region sequence data from a worldwide sample of grey wolves and a more limited sample of coyotes were analysed. The results suggest that fluctuating population sizes during the late Pleistocene have left a genetic signature on levels of variation in both species. Genealogical measures of nucleotide diversity suggest that historical population sizes were much larger in both species and grey wolves were more numerous than coyotes. Currently, about 300 000 wolves and 7 million coyotes exist. In grey wolves, genetic diversity is greater than that predicted from census population size, reflecting recent historical population declines. By contrast, nucleotide diversity in coyotes is smaller than that predicted by census population size, reflecting a recent population expansion following the extirpation of wolves from much of North America. Both species show little partitioning of haplotypes on continental or regional scales. However, a statistical parsimony analysis indicates local genetic structure that suggests recent restricted gene flow.     

High nucleotide polymorphism and rapid decay of linkage disequilibrium in wild populations of Caenorhabditis remanei

The common ancestor of the self-fertilizing nematodes Caenorhabditis elegans and C. briggsae must have reproduced by obligate outcrossing, like most species in this genus. However, we have only a limited understanding about how genetic variation is patterned in such male-female (gonochoristic) Caenorhabditis species. Here, we report results from surveying nucleotide variation of six nuclear loci in a broad geographic sample of wild isolates of the gonochoristic C. remanei. We find high levels of diversity in this species, with silent-site diversity averaging 4.7%, implying an effective population size close to 1 million. Additionally, the pattern of polymorphisms reveals little evidence for population structure or deviation from neutral expectations, suggesting that the sampled C. remanei populations approximate panmixis and demographic equilibrium. Combined with the observation that linkage disequilibrium between pairs of polymorphic sites decays rapidly with distance, this suggests that C. remanei will provide an excellent system for identifying the genetic targets of natural selection from deviant patterns of polymorphism and linkage disequilibrium. The patterns revealed in this obligately outcrossing species may provide a useful model of the evolutionary circumstances in C. elegans' gonochoristic progenitor. This will be especially important if self-fertilization evolved recently in C. elegans history, because most of the evolutionary time separating C. elegans from its known relatives would have occurred in a state of obligate outcrossing.     

Genetic diversity of the Andean tuber-bearing species, oca (Oxalis tuberosa Mol.), investigated by inter-simple sequence repeats.

The Andean tuber-bearing species, Oxalis tuberosa Mol., is a vegetatively propagated crop cultivated in the uplands of the Andes. Its genetic diversity was investigated in the present study using the inter-simple sequence repeat (ISSR) technique. Thirty-two accessions originating from South America (Argentina, Bolivia, Chile, and Peru) and maintained in vitro were chosen to represent the ecogeographic diversity of its cultivation area. Twenty-two primers were tested and 9 were selected according to fingerprinting quality and reproducibility. Genetic diversity analysis was performed with 90 markers. Jaccard's genetic distance between accessions ranged from 0 to 0.49 with an average of 0.28 +/- 0.08 (mean +/- SD). Dendrogram (UPGMA (unweighted pair-group method with arithmetic averaging)) and factorial correspondence analysis (FCA) showed that the genetic structure was influenced by the collection site. The two most distant clusters contained all of the Peruvian accessions, one from Bolivia, none from Argentina or Chile. Analysis by country revealed that Peru presented the greatest genetic distances from the other countries and possessed the highest intra-country genetic distance (0.30 +/- 0.08). This suggests that the Peruvian oca accessions form a distinct genetic group. The relatively low level of genetic diversity in the oca species may be related to its predominating reproduction strategy, i.e., vegetative propagation. The extent and structure of the genetic diversity of the species detailed here should help the establishment of conservation strategies.     

Genetic Variation and Breeding Signature in Mass Selection Lines of the Pacific Oyster (Crassostrea gigas) Assessed by SNP Markers

In breeding industries, a challenging problem is how to keep genetic diversity over generations. To investigate genetic variation and identify breeding signatures in mass selected lines of Pacific oyster (Crassostrea gigas), three sixth-generation selected lines and four wild populations were assessed using 103 single nucleotide polymorphism (SNP) markers. The genetic diversity data indicated that the selected lines exhibited a significant reduction in the observed heterozygosity and observed number of alleles per locus compared with the wild populations (P≤0.05), indicating the selected lines tended to lose genetic diversity contrasted with the wild populations. The unweighted pair-group method with arithmetic mean (UPGMA) analysis showed that the wild populations and selected lines were not separated into two groups. Using four outlier tests, a total of 17 loci were found under selection at two levels. The global outlier detection suggested that 4 common outlier loci were subject to selection using both the hierarchical island model and Bayesian likelihood approaches. At regional level, 3 SNPs were detected as outlier using at least two outlier tests and one outlier SNP (CgSNP309) was overlapped in the two wild-selected population comparisons. The candidate outlier SNPs provide valuable resources for future association studies in C. gigas.     

Population size and fragmentation thresholds for the maintenance of genetic diversity in the herbaceous endemic Scutellaria montana (Lamiaceae)

Abstract.-The level and distribution of genetic variation is thought to be affected primarily by the size of individual populations and by gene flow among populations. Although the effects of population size have frequently been examined, the contributions of regional gene flow to levels of genetic variation are less well known. Here I examine the effects of population size and the number of neighboring populations (metapopulation density) on the distribution and maintenance of genetic diversity in an endemic herbaceous perennial. Reductions in the proportion of polymorphic loci and the effective number of alleles per locus were apparent for many populations with a census size of less than 100 individuals, but no effects of population size on levels of inbreeding were detected. I assess the effects of regional population density on levels of diversity and inbreeding using stepwise regression analysis of metapopulation diameter (i.e., the size of a circle within which population density is estimated). This procedure provides a spatially explicit evaluation of the effects of metapopulation size on population genetic parameters and indicates the critical number of neighboring populations (fragmentation threshold) for the regional maintenance of genetic diversity. Stepwise regression analyses revealed fragmentation thresholds at two levels; at a scale of 2 km, where small metapopulations resulted in greater levels of selfing or sibling mating, and at a scale of 8 km, where metapopulation size was positively associated with higher levels of genetic diversity. I hypothesize that the smaller fragmentation threshold may reflect higher levels of selfing in isolated populations because of the absence of pollinators. The larger threshold probably indicates the maximum distance over which pollen dispersal rates are high enough to counteract genetic drift. This study demonstrates that the regional distribution of populations can be an important factor for the long-term maintenance of genetic variation.     

Molecules,morphology and minute hooded beetles: a phylogenetic study with implications for the evolution and classification of Corylophidae (Coleoptera: Cucujoidea)

Eight genes (nuclear: 18S, 28S, H3, CAD; mitochondrial: 12S, 16S, COI, COII) and morphology were used to infer the evolutionary history of Corylophidae, some of the smallest free‐living insects. The study included 36 corylophid exemplars, representing approximately 60% of the known generic diversity of the family and 16 cucujoid outgroup taxa. Multiple partitioning strategies, molecular datasets, combined datasets and different taxon sampling regimes using maximum likelihood and mixed‐model Bayesian inference were utilized to analyse these data. Most results were highly concordant across analyses. There was strong agreement across (i) partitioning strategies, (ii) maximum likelihood and Bayesian inference analyses of the molecular data, and (iii) Bayesian inference of the molecular data alone and Bayesian inference of the combined morphological and molecular data when all terminal taxa were included. When a strict taxon sampling protocol was employed so that only single generic exemplars were included, deep relationships were affected in the resulting phylogenetic hypotheses. Under such narrow sampling strategies, deep phylogenetic relationships were also sensitive to the choice of generic exemplars. Although it is often challenging to obtain single representatives for many taxa in higher‐level phylogenetic analyses, these results indicate the importance of using denser taxon sampling approaches even at the specific level for genera included in such studies. Molecular data alone support Anamorphinae (Endomychidae) strongly as the sister group of Corylophidae. In combined data analyses, Coccinellidae is recovered as the sister group to Corylophidae. In all analyses, Corylophidae and the subfamily Corylophinae are recovered as monophyletic. The monophyly of Periptyctinae was untested, as only a single species was included. All included corylophine tribes were recovered as monophyletic with the exception of Aenigmaticini; Aenigmaticum Matthews forms the sister group to Orthoperus Stephens and Stanus?lipiński et al. is recovered as the sister group of Sericoderus Stephens. Stanus tasmanicus?lipiński et al. is transferred to a new genus, Pseudostanus Robertson, ?lipiński & McHugh gen.n. incertae sedis. We propose a new tribe, Stanini Robertson, ?lipiński & McHugh trib.n. for Stanus bowesteadi?lipiński et al. and a new concept of Aenigmaticini sensu.n. to include only the nominate genus. Anatomical transitions associated with corylophid miniaturization are highlighted. Key phenotypic modifications and elevated rates of substitution in nuclear rRNA genes are evident in a subgroup of Corylophinae that includes the most diminutive species. Other taxonomic and evolutionary implications are discussed in light of the results.     

Museomics reveals extensive cryptic diversity of Australian prionine longhorn beetles with implications for their classification and conservation

We combined museum collection genomics and morphology to identify new genera and species, and to otherwise revise the taxonomy of the poorly understood Australian longhorn beetles in the subfamily Prioninae. Moreover, we produced a comprehensive DNA reference library for molecular species identification and a first comprehensive molecular phylogeny for all Australian Prioninae, using mitogenomic data. The Prioninae include some of the world's largest beetles, and their long-lived larvae are major recyclers of solid dead timber, yet these longhorn beetles are rarely seen in nature due to their concealed habits, short adult lifespans and nocturnal habits. Furthermore, many species are difficult to identify using morphological characters. We applied Whole Genome Shotgun (WGS) sequencing to 213 historical museum specimens, representing all morphological types of Australian Prioninae. Complete mitochondrial genomes were obtained for all genera, and at least one mitochondrial protein-coding gene (PCG) was acquired for an additional 141 specimens of Australian Prioninae. Using molecular species delimitation tools, we identified 48 new species of Australian Prioninae, nearly doubling the number of currently recognized Australian species of Prioninae. We assign generic status to Catypnes Pascoe, 1864 stat.r. and Hermerius Newman, 1844 stat.r. , propose a new synonymy for Cnemoplites Newman, 1842 (= Paroplites Lameere, 1903 syn.n. ), describe six new genera, including Paulhutchinsonia Jin, de Keyzer & Ślipiński gen.n. , Papunya Jin, de Keyzer & Ślipiński gen.n. , Dunmorium Jin, de Keyzer & Ślipiński gen.n. , Hagrides Jin, de Keyzer & Ślipiński gen.n. , Cryptipus Jin, de Keyzer & Ślipiński gen.n. , Geoffmonteithia Jin, de Keyzer & Ślipiński gen.n. , and revise the tribal composition of the Australian Macrotomini. Moreover, our phylogeny of Prioninae provides valuable new insights into the evolutionary origins and nearest relatives of several Australian endemic taxa. Our results emphasize the importance of natural history museums as a repository for information about biodiversity, and highlight the opportunities associated with using museum specimens as a source for molecular data in phylogenetic studies.     

绵羊Cytb基因序列多态性及系统进化研究

以8个中国地方绵羊品种和1个外来品种的20个个体为研究对象,通过对Cytb基因的全序列测定,结果表明：绵羊的单倍型多样性为97．1％。所有序列的平均碱基组成为27．1％T,28．5％C,31．4％A及13．0％G,G＋C含量为41．5％,核苷酸多样性为0．602％。在所有序列中共检测到43个变异位点,其中包括40处转换和3处颠换。Fu’S中性检验表明差异不显著（0．10〉P〉0．05）,说明绵羊群体未发生群体扩张事件。NJ、ME及UPGMA聚类结果均表明,我国绵羊可分为3个单倍型组,这提示我国绵羊有3个母系起源。     

Two patterns of variation among MHC class I loci in Tuatara (Sphenodon punctatus)

The genes of the major histocompatibility complex (MHC) are a central component of the immune system in vertebrates and have become important markers of functional, fitness-related genetic variation. We have investigated the evolutionary processes that generate diversity at MHC class I genes in a large population of an archaic reptile species, the tuatara (Sphenodon punctatus), found on Stephens Island, Cook Strait, New Zealand. We identified at least 2 highly polymorphic (UA type) loci and one locus (UZ) exhibiting low polymorphism. The UZ locus is characterized by low nucleotide diversity and weak balancing selection and may be either a nonclassical class I gene or a pseudogene. In contrast, the UA-type alleles have high nucleotide diversity and show evidence of balancing selection at putative peptide-binding sites. Twenty-one different UA-type genotypes were identified among 26 individuals, suggesting that the Stephens Island population has high levels of MHC class I variation. UA-type allelic diversity is generated by a mixture of point mutation and gene conversion. As has been found in birds and fish, gene conversion obscures the genealogical relationships among alleles and prevents the assignment of alleles to loci. Our results suggest that the molecular mechanisms that underpin MHC evolution in nonmammals make locus-specific amplification impossible in some species.     

Blinded sample size recalculation in multicentre trials with normally distributed outcome

The internal pilot study design enables to estimate nuisance parameters required for sample size calculation on the basis of data accumulated in an ongoing trial. By this, misspecifications made when determining the sample size in the planning phase can be corrected employing updated knowledge. According to regulatory guidelines, blindness of all personnel involved in the trial has to be preserved and the specified type I error rate has to be controlled when the internal pilot study design is applied. Especially in the late phase of drug development, most clinical studies are run in more than one centre. In these multicentre trials, one may have to deal with an unequal distribution of the patient numbers among the centres. Depending on the type of the analysis (weighted or unweighted), unequal centre sample sizes may lead to a substantial loss of power. Like the variance, the magnitude of imbalance is difficult to predict in the planning phase. We propose a blinded sample size recalculation procedure for the internal pilot study design in multicentre trials with normally distributed outcome and two balanced treatment groups that are analysed applying the weighted or the unweighted approach. The method addresses both uncertainty with respect to the variance of the endpoint and the extent of disparity of the centre sample sizes. The actual type I error rate as well as the expected power and sample size of the procedure is investigated in simulation studies. For the weighted analysis as well as for the unweighted analysis, the maximal type I error rate was not or only minimally exceeded. Furthermore, application of the proposed procedure led to an expected power that achieves the specified value in many cases and is throughout very close to it.     

Allozyme diversity in Macbridea alba (Lamiaceae), an endemic florida mint

Macbridea alba is a herbaceous perennial mint endemic to the panhandle region of Florida. We used starch gel electrophoresis to describe allozyme diversity and genetic structure in this federally threatened plant. Ten populations were analyzed, with an average sample size of 47 plants (range 41-48 plants) per population. Of the 22 loci analyzed, 11 (50%) were polymorphic, with an average of 36.2% of the loci polymorphic within populations. Gene diversity measures for the species (Hes = 0.121) and for the populations (Hep = 0.099) were slightly higher than means found for other surveyed endemic plants. Compared to nine previously analyzed woody mints, however, M. alba is genetically depauperate. Little genetic structure was evident within the species, with 92% of the total genetic variation found within populations. Genetic identities between population pairs were high (mean I = 0.98). The perennial life habit of M. alba and long-distance pollination by bumblebees may contribute to the maintenance of genetic diversity within this threatened species.     

Hierarchical Analysis of Nucleotide Diversity in Geographically Structured Populations

Existing methods for analyzing nucleotide diversity require investigators to identify relevant hierarchical levels before beginning the analysis. We describe a method that partitions diversity into hierarchical components while allowing any structure present in the data to emerge naturally. We present an unbiased version of NEI's nucleotide diversity statistics and show that our modification has the same properties as WRIGHT's F(ST). We compare its statistical properties with several other F(ST) estimators, and we describe how to use these statistics to produce a rooted tree of relationships among the sampled populations in which the mean time to coalescence of haplotypes drawn from populations belonging to the same node is smaller than the mean time to coalescence of haplotypes drawn from populations belonging to different nodes. We illustrate the method by applying it to data from a recent survey of restriction site variation in the chloroplast genome of Coreopsis grandiflora.     

Comparisons of genetic diversity in a narrow endemic, Impatiens hypophylla, and in its widespread congener, I. textori (Balsaminaceae)

Genetic diversity in two species of Impatiens in Korea was investigated using starch gel electrophoresis. Impatiens hypophylla is a rare species endemic to southwestern Japan and southeastern Korea, whereas I. rextori is widespread in East Asia. Impatiens textori has considerably higher genetic diversity than I. hypophylla . For example, mean values of genetic diversity within populations (He) of I. textori and I. hypophylla were 0.206 and 0.102, respectively. The high outcrossing rate (mean 1 = 0.89), abundance and widespread distribution, and large population size may be factors responsible for the maintenance of high levels of genetic variation in I. textori . Analyss of fixation indices indicated that considerable population substructuring within populations of I. hypophylla , partly resulting from inbreeding and/or genetic drift in small populations coupled with limited gene flow (mean F_IS= 0.699). However, the degree of population divergence observed in I. hypophylla (mean G_ST= 0.059) was considerably lower than that in other plants with similar life history traits. It is supposed that similar selection at populations of I. hypophylla examined might maintain similar gene frequencies in isolated populations.     

7种川产淫羊藿属植物遗传多样性及亲缘关系的ISSR分析

利用ISSR分子标记对7种12居群的川产淫羊藿属植物进行遗传多样性及亲缘关系分析。结果发现23个ISSR随机引物共扩增194条清晰条带,其中169条具多态性,平均多态性位点比率为87.11%,有效等位基因数Ne=1.534 2,Nei基因多样性指数H=0.314 4,Shannon多样性指数I=0.469 7,表明物种间遗传多样性丰富。7种川产淫羊藿属植物间的遗传相似系数为0.653 2～0.748 9,聚类分析和主成分分析直观的显示出12份供试材料间的亲缘关系,并将其分为两类。表明ISSR分子标记技术可以用于川产淫羊藿属植物的遗传多样性和亲缘关系分析。     

Evaluation of AFLPs for germplasm fingerprinting and assessment of genetic diversity in cultivars of tomato (Lycopersicon esculentum L.).

Cultivated tomato (L. esculentum L.) germplasm exhibits limited genetic variation compared with wild Lycopersicon species. Amplified fragment length polymorphism (AFLP) markers were used to evaluate genetic variation among 74 cultivars, primarily from California, and to fingerprint germplasm to determine if cultivar-specific patterns could be obtained. All 74 cultivars were genotyped using 26 AFLP primer combinations; of the 1092 bands scored, 102 AFLP bands (9.3%) were polymorphic. Pair-wise genetic similarity coefficients (Jaccard and Nei-Li) were calculated. Jaccard coefficients varied from 0.16 to 0.98 among cultivar pairs, and 72% of pair-wise comparisons exceeded 0.5. UPGMA (unweighted pair-group method with arithmetic averaging) clustering and principle component analysis revealed four main clusters, I-IV; most modern hybrid cultivars grouped in II, whereas most vintage cultivars grouped in I. Clusters III and IV contained three and two cultivars, respectively. Some groups of cultivars closely related by pedigree exhibited high bootstrap values, but lower values (<50%) were obtained for cluster II and its four subgroups. Unique fingerprints for all 74 cultivars were obtained by a minimum of seven AFLP primer pairs, despite inclusion of some closely related cultivars. This study demonstrated that AFLP markers are effective for obtaining unique fingerprints of, and assessing genetic diversity among, tomato cultivars.     

物种多度和径级尺度对于评价群落系统发育结构的影响: 以尖峰岭热带山地雨林为例

研究不同径级尺度群落系统发育多样性有助于了解不同年龄模式下物种的亲缘关系及其群落系统发育结构; 但是关于物种多度对群落系统发育结构影响的研究较少。以海南尖峰岭热带山地雨林群落为例, 首先在不同径级尺度比较物种多度加权与否分别对4个广泛采用的系统发育指数的影响, 继而利用其中2个经过标准化处理的系统发育多样性指数: 净种间亲缘关系指数(net relatedness index, NRI)和净最近种间亲缘关系指数(nearest taxon index, NTI), 结合群落的生境类型来量度不同局域生境条件下不同径级尺度木本植物系统发育关系。结果发现: (1)未考虑物种多度加权的系统发育平均成对距离(mean pairwise distance, MPD)指数比考虑物种多度加权的MPD指数显著地高估了群落整体系统发育多样性, 且这种现象在小径级尺度(1 cm≤DBH<5 cm)最为明显。因此, 在森林监测样地中对于中、小径级群落系统发育结构研究中建议考虑物种多度信息。(2) 从群落组成整体系统发育结构来看, 尖峰岭热带山地雨林在几乎所有径级尺度和生境下均倾向于系统发育发散, 且随着径级的递增发散程度趋于明显(NRI<0)。(3)从群落组成局部系统发育结构来看, 尖峰岭热带山地雨林在中、小径级倾向于系统发育聚集(NTI>0), 而在大径级(DBH≥15 cm)则倾向于系统发育发散(NTI<0)。总之, 研究群落系统发育结构时应考虑物种多度的影响以及径级尺度效应。