EFFECTIVE POPULATION SIZE AND GENETIC DRIFT IN TRISTYLOUS EICHHORNIA PANICULATA (PONTEDERIACEAE)

Populations of the tristylous, annual Eichhornia paniculata are markedly differentiated with respect to frequency of mating types. This variation is associated with evolutionary changes in mating system, from predominant outcrossing to high self-fertilization. To assess the potential influence of genetic drift acting on this variation, we estimated effective population size in 10 populations from northeastern Brazil using genetic and demographic methods. Effective size (N_e) was inferred from temporal changes in allele frequency at two to eight isozyme loci and also calculated using five demographic variables: 1) the number of flowering individuals (N); 2) temporal fluctuations in N; 3) variance in flower number; 4) frequency of mating types; and 5) selfing rate. Average N_e based on isozyme data was 15.8, range 3.4–70.6, and represented a fraction (mean N_e/N = 0.106) of the census number of individuals (mean N = 762.8; range: 30.5–5,040). Temporal variation in N and variance in flower number each reduced N_e to about a half of N whereas mating type frequencies and selfing rate caused only small reductions in N_e relative to N. All estimates of N_e based on demographic variables were considerably larger than those obtained from genetic data. The two kinds of estimates were in general agreement, however, when all demographic variables were combined into a single measure. Monte Carlo simulations indicated that effective size must be fewer than about 40 for drift to overcome the frequency-dependent selection that maintains the polymorphism for mating type. Applying the average N_e/N value to 167 populations censused in northeastern Brazil indicated that 72% had effective sizes below this number. This suggests that genetic drift is likely to play a dominant role in natural populations of E. paniculata.     

VARIATION IN THE MATING SYSTEM OF EICHHORNIA PANICULATA (SPRENG.) SOLMS. (PONTEDERIACEAE)

A multilocus procedure was used to estimate outcrossing rates from allozyme data in nine populations of Eichhornia paniculata from NE Brazil and Jamaica. The populations were chosen to represent stages in a proposed model of the evolutionary breakdown of tristyly to semi-homostyly; they differed in morph structure (trimorphic, dimorphic, or monomorphic) and floral traits likely to influence the mating system. The interpopulation range in outcrossing rate, t, was 0.96–0.29. Two additional populations from Jamaica, composed exclusively of self-pollinating, semi-homostylous, mid-styled plants, were invariant at 21 isozyme loci, precluding estimates of outcrossing frequency. Trimorphic populations from Brazil had uniformly high outcrossing rates of 0.96–0.88. Values for the floral morphs within populations were not significantly different. A controlled pollination experiment, comparing the competitive ability of self and cross pollen using the Got-3 marker locus, provided evidence that the maintenance of high outcrossing rates in trimorphic populations results from the prepotency of cross pollen and/or the selective abortion of selfed zygotes. Morph-dependent variation in t was detected within a dimorphic population with the L morph outcrossing with a frequency of 0.76 in comparison with 0.36 in the M morph. The difference in the mating system of floral morphs results from modifications in position of short-level stamens in flowers of the M morph resulting in automatic self-pollination. The occurrence of E. paniculata populations composed exclusively of self-pollinating, mid-styled variants is thought to be associated with the spread of genes modifying stamen position. The high level of self-fertilization demonstrated in the M morph would allow automatic selection of these genes, augmented by fertility assurance in the absence of specialized pollinators.     

Fertility differences among floral morphs following selfing in tristylous Eichhornia paniculata (Pontederiaceae): inbreeding depression or partial incompatibility?

Reduction in seed set following self- vs. cross-pollination in flowering plants can result from abortion of selfed offspring owing to inbreeding depression and/or partial self-incompatibility. Previous studies on tristylous Eichhornia paniculata (Pontederiaceae) indicate that reduced seed set following self-pollination generally occurs in the short- (S), but not the long-(L) or mid-styled (M) morphs. To determine whether this pattern results from morph-specific differences in inbreeding depression owing to the sheltering of deleterious alleles at the S locus and/or partial self-incompatibility, we conducted controlled hand-pollinations of the floral morphs and measured seed set and levels of seed abortion. There were no significant differences in fertilization success and seed set following self-, illegitimate, and legitimate pollinations in the L and M morphs. In contrast, in the S morph self-, intramorph and intermorph illegitimate pollinations resulted in significant reduction in seed set in comparison with legitimate pollination. This indicates that the reduced seed set observed in self-pollination is the result of partial incompatibility rather than inbreeding depression. Significantly reduced fertilization success and low levels of ovule abortion in illegitimate pollinations of S plants also supported this conclusion. Reduced fertility in the S morph may have implications for the observed loss of this morph from natural populations and the evolutionary breakdown of tristyly.     

THE ROLE OF INBREEDING DEPRESSION AND MATING SYSTEM IN THE EVOLUTION OF HETEROSTYLY

We investigated the role of morph‐based differences in the expression of inbreeding depression in loss of the mid‐styled morph from populations of tristylous Oxalis alpina. The extent of self‐compatibility (SC) of reproductive morphs, the degree of self‐fertilization, and the magnitude of inbreeding depression were investigated in three populations of O. alpina differing in their tristylous incompatibility relationships. All three populations exhibited significant inbreeding depression. In two populations with highly modified tristylous incompatibility, manifested as increased reciprocal compatibility between short‐ and long‐styled morphs, substantial SC and self‐fertilization of mid‐styled morphs were detected, and expected to result in expression of inbreeding depression in the progeny of mid‐styled morphs in the natural populations. In contrast, significant self‐fertility of the mid‐styled morph was absent from the population with typical tristylous incompatibility, and no self‐fertilization could be detected. Although self‐fertilization and expression of inbreeding depression should result in selection against the mid‐styled morph in the later stages of the transition from tristyly to distyly, in O. alpina selection against the mid‐styled morph in the early phases of the evolution of distyly is likely due to genic selection against mid‐alleles associated with modified tristylous incompatibility, rather than expression of inbreeding depression.     

POLLEN DISCOUNTING AND THE SPREAD OF A SELFING VARIANT IN TRISTYLOUS EICHHORNIA PANICULATA: EVIDENCE FROM EXPERIMENTAL POPULATIONS

Floral traits that increase self-fertilization are expected to spread unless countered by the effects of inbreeding depression, pollen discounting (reduced outcross pollen success by individuals with increased rates of self-fertilization), or both. Few studies have attempted to measure pollen discounting because to do so requires estimating the male outcrossing success of plants that differ in selfing rate. In natural populations of tristylous Eichhornia paniculata, selfing variants of the mid-styled morph are usually absent from populations containing all three style morphs but often predominate in nontrimorphic populations. We used experimental garden populations of genetically marked plants to investigate whether the effects of population morph structure on relative gamete transmission by unmodified (M) and selfing variants (M‘) of the mid-styled morph could explain their observed distribution. Transmission through ovules and self and outcross pollen by plants of the M and M’ morphs were compared under trimorphic, dimorphic (S morph absent), and monomorphic (L and S morphs absent) population structures. Neither population structure nor floral morphology affected female reproductive success, but both had strong effects on the relative transmission of male gametes. The frequency of self-fertilization in the M' morph was consistently higher than that of the M morph under all morph structures, and the frequency of self-fertilization by both morphs increased as morph diversity of experimental populations declined. In trimorphic populations, total transmission by the M and M' morphs did not differ. The small, nonsignificant increase in selfing by the M' relative to the M morph was balanced by decreased outcross siring success, particularly on the S morph. In populations lacking the S morph, male gamete transmission by the M' morph was approximately 1.5 times greater than that by the M morph because of both increased selfing and increased success through outcross pollen donation. Therefore, gamete transmission strongly favored the M' morph only in the absence of the S morph, a result consistent with the distribution of the M' morph in nature. This study indicates that floral traits that alter the selfing rate can have large and context-dependent influences on outcross pollen donation.     

Multiple origins of self-fertilization in tristylous Eichhornia paniculata (Pontederiaceae): Inferences from style morph and isozyme variation

Populations of Eichhornia paniculata (Pontederiaceae) exhibit a wide range of mating systems, from predominant outcrossing to high levels of self-fertilization. The origin of self-fertilization in this tristylous species is associated with the loss of style-length morphs from populations and the spread of self-pollinating, floral variants. We examined geographic variation in style morph and allozyme frequencies to determine whether the loss of style morphs and transition to selfing could have multiple origins in E. paniculata. Surveys of floral variation in 167 populations from six states in northeastern Brazil revealed that at least one style morph was absent from 29.3%. Non-trimorphic populations occurred in all states and ranged in frequency from 9% in Ceará to 68% in Alagoas. Selfing variants occurred in 8.5% and 55% of trimorphic and non-trimorphic populations, respectively, and were distributed among five of six states with primary concentrations in Alagoas and Pernambuco. A comparison of electrophoretic variation at 24 isozyme loci in 28 trimorphic, 13 dimorphic and 3 monomorphic populations indicated that non-trimorphic populations contained 84% of the allelic variation present in trimorphic populations and were markedly differentiated from one another. Analyses of genetic distance and the distribution of rare alleles indicated that non-trimorphic populations were often more similar to neighbouring trimorphic populations than to one another. Populations with selfing variants occurred at low frequency in three genetically distinct parts of the range. These results, in combination with genetic and morphological evidence suggest that style morphs are lost repeatedly from populations of E. paniculata and that selfing variants may have originated on at least three separate occasions in northeastern Brazil.     

THE CONTRIBUTION OF MATING SYSTEM VARIATION TO REPRODUCTIVE ISOLATION IN TWO CLOSELY RELATED CENTAURIUM SPECIES (GENTIANACEAE) WITH A GENERALIZED FLOWER MORPHOLOGY

In closely related plant species that display strong similarities in phenology and pollinator communities, differences in breeding system and associated shifts in floral traits may have important effects on the magnitude and direction of heterospecific pollen flow and hybridization. Here, we quantified the strength of several pre‐ and postzygotic barriers acting between the facultatively outcrossing Centaurium erythraea and the predominantly selfing C. littorale via a suite of experiments, and estimated the frequency of hybridization in the field using molecular markers. The reproductive barriers primarily responsible for preventing hybridization were essentially prezygotic and these acted asymmetrically. Due to differences in floral display, pollen production, and pollen transfer rates, heterospecific pollen flow occurred predominantly from C. erythraea to C. littorale. In C. littorale, on the other hand, close anther–stigma positioning and resulting higher capacity for autonomous selfing functioned as an efficient barrier to counterbalance the higher risk for hybrid mating. In both species the action of all reproductive barriers resulted in a small opportunity for hybrid establishment, which was confirmed by the occurrence of only ～1% putative hybrids in the field. Our findings confirm that differences in breeding system affect heterospecific pollen transfer patterns and that autonomous selfing may efficiently prevent hybridization.     

凤眼莲对无锡电影胶片厂含银废水净化生产性应用实例

 本文报道了凤眼莲对无锡电影胶片厂含银废水的净化生产性应用试验结果：(1)污水停留时间为49h时,氧化沟出水中的银净化率为98.0%到100%(未检出);(2)COD的去除率为54.58%;(3)混浊度的去除率为68.9%;(4)NH4+–N和PO43–的去除率分别为45.55%和34.3%。该法已被该厂采纳应用,并取代了原来的活性炭吸附和O3氧化三级处理,年节约运转费用12.5万元。     

入侵植物凤眼莲研究现状及存在的问题

 凤眼莲(Eichhornia crassipes)原产南美洲,被列为世界十大恶性杂草之一,现已入侵了非洲、亚洲、北美洲、大洋州、甚至欧洲等5个大洲,至少62个国家和地区都受到了凤眼莲入侵的危害。凤眼莲的入侵已经引起了一系列的生态、经济、社会问题：首先,它改变了当地水体生态系统的物理、化学环境,进而影响水体生态系统的生物多样性,破坏食物链、物质循环等生态过程的正常运行;其次,凤眼莲造成当地经济的重大损失,航运、渔业、水利等都受到了危害;再次,凤眼莲的入侵爆发也对当地居民饮水、健康等造成威胁。目前,对于凤眼莲的控制及其治理主要有物理的、化学的、以及生物的等3种方法。利用天敌、病菌、以及化感作用等的生物控制被许多专家和学者推崇,同时,利用生物控制凤眼莲入侵也日益成为研究的热点。但是,综合目前对于凤眼莲的认识和研究,仍然具有片面性,需要从生物特性、种群生态、生态系统等方面深入研究凤眼莲入侵机制。而利用生物控制凤眼莲的研究和技术尚不完善,需要进行种间竞争、捕食及遗传变异等方面的探讨和研究。通过总结控制凤眼莲各种方法的长处和不足,最后指出利用生物的方法,并结合污水治理、水系宏观调控及监测等方法,综合治理凤眼莲,是十分必要的,而且也是最具有前景的。     

EVOLUTIONARY HISTORY OF THE MATING SYSTEM IN AMSINCKIA (BORAGINACEAE)

A survey of restriction site variation in the chloroplast genome of the annual plant genus Amsinckia, together with estimation of outcrossing rates, was conducted to analyze the evolutionary history of the mating system. Species, and in some cases populations within species, differ markedly in their mating system. Five taxa are distylous and predominantly outcrossing, or show mixed mating systems, while the remaining taxa are homostylous and predominantly self-fertilizing. Reconstruction of the molecular phylogeny of the group places different distylous and homostylous taxa at four separate branch tips. When distyly is treated as ancestral in the group, or when the loss of distyly is assumed to be more common than its gain, the results of the phylogenetic analysis support the hypothesis that the self-fertilizing taxa are of recent origin from outcrossing relatives. These findings are discussed with respect to theory for the evolution and breakdown of distyly and the probability of extinction of selfing lineages.     

FUNCTIONAL INCOMPATIBILITY BETWEEN THE FERTILIZATION SYSTEMS OF TWO ALLOPATRIC POPULATIONS OF CALLOSOBRUCHUS MACULATUS (COLEOPTERA: BRUCHIDAE)

Abstract Recent studies indicate that postcopulatory sexual selection may represent an important component of the speciation process by initiating reproductive isolation via the evolutionary divergence of fertilization systems. Using two geographically isolated populations of the polyandrous beetle Callosobruchus maculatus, we investigated divergence in fertilization systems by determining the extent of postcopulatory functional incompatibility. Through reciprocal, cross‐population matings we were able to separately estimate the effects of male and female population origin and their interaction on the extent of last‐male sperm precedence, female receptivity to further copulation and female oviposition. Our results indicate partial incompatibility between the fertilization systems of the two populations at all three functional levels. Males derived from the same population as females outcompete rival, allopatric males with respect to sperm preemption, sperm protection, and ability to stimulate female oviposition. This pattern is reciprocated in both populations indicating that postcopulatory, prezygotic events represent important mechanisms by which between‐population gene flow is reduced. We suggest the partial gametic isolation observed is a by‐product of the coevolution of male and female fertilization systems by a process of cryptic female choice. Our results are consistent with a mechanism akin to conventional mate choice models although they do not allow us to reject antagonistic sexual coevolution as the mechanism of cryptic female choice.     

THE INFLUENCE OF DISPERSAL PATTERNS AND MATING SYSTEM ON GENETIC DIFFERENTIATION WITHIN AND BETWEEN POPULATIONS OF THE RED HOWLER MONKEY (ALOUATTA SENICULUS)

The relationship between social structure and partitioning of genetic variance was examined in two red howler monkey populations (W and G) in Venezuela, one of which (G) was undergoing rapid growth through colonization by new troops. Rates and patterns of gene flow had been determined through radiotelemetry and direct observation data on solitary migrants, and 10 years of troop censusing. Standard electrophoresis techniques were used to examine 29 loci in blood samples taken from 137 of the study animals. Analysis of genetic variance demonstrated: (1) a significantly high level of genetic variation among troops within populations (F_ST = 0.225 for W and 0.142 for G), and (2) a significant excess of heterozygosity within troops relative to expected (F_IS = -0.136 for W and -0.064 for G), despite relatively high levels of observed and inferred inbreeding in W. Differences between the populations in F_ST values conformed to those predicted based on differences in colonization rate. Comparison of partitioning of genetic variance among different genealogical subsets of troops demonstrated that the pattern of genetic differentiation observed among troops within populations was promoted by an essentially single-male harem breeding structure, a very low rate of random exchange of breeding males among troops, and a high degree of relatedness among troop females. Between-troop genetic differentiation (F_ST) was thereby increased relative to that expected from other types of social organization, while the correlation between uniting gametes within troops (F_IS) was decreased. Genetic differentiation between populations (2%) corresponded to that predicted from migration rates. Such a mosaic of genetic variation, combined with differences in reproductive success observed among troops and a high troop failure rate, create conditions in which interdemic selection could result in more rapid spread of advantageous gene combinations than would be expected in a panmictic population, particularly in a colonizing situation in which the founder population is small.     

THE INFLUENCE OF THE MATING SYSTEM ON SEED SIZE VARIATION IN CRINUM ERUBESCENS (AMARYLLIDACEAE)

We present evidence that extreme seed size variation in fruits of Crinum erubescens (range: 0.1 to 66.5 g per seed) occurs when mating pairs are inbred, either from selfing or biparental inbreeding. Several relatively uniform seeds of intermediate size are produced when pollen from several pollen donors is applied simultaneously to a flower. Selfed fruits and some fruits pollinated with a single pollen donor produce both large and small seeds, although selfed fruits produce fewer seeds than outcrossed fruit. These results are contrary to the hypothesis that variation in seed size is attributable to either pollen competition or differential allocation of maternal resource to seeds of different genotypes.     

小檗科鬼臼亚科的地理分布与系统发育

以植物地理学资料为主,综合植物化学、细胞学、形态学及解剖学等方面的资料,分析了小檗科鬼臼亚科现代地理分布格局产生的原因及其对系统发育的影响,指出：①我国是鬼臼亚科植物的多样性中心和分布中心,鬼臼亚科植物的现代地理分布格局是由于第三纪以来替代分布和长期隔离的结果;②在鬼臼亚科植物中,以山荷叶属最为原始,它通过两条方向演化,一是保持其原来的异花授粉方向演化为足叶草属,另一方向是转向自花授粉,自山荷叶属演化为八角莲属,然后再演化为桃儿七属;③桃儿七属与足叶草属不具有直接的亲缘关系,它们在形态上的相似只是平行进化的结果。     

桃儿七繁殖生物学研究

以居群为单位研究了桃儿七的繁殖生物学,指出桃儿七为适应分布区内生长季节短暂的环境条件,花各部分的形态在开花前一年就已开始分化,并形成一休眠芽,但大小孢子还尚未进行减数分裂。到了第二年春天气温回升之后,大小孢子方才进行减数分裂,并在减数分裂之后,植株通过茎基部的居间生长将花及叶带出地面。桃儿七在开花时先花后叶。花在露出土面的第二、三天随即开放。虽然桃儿七具有一系列适应干虫煤传粉的形态结构和生物学特征,但同时也具有许多和自花授粉有关的适应机制。桃儿七在开花习性及花部形态上常表现出多态。在自然状况下,桃儿七是一种以自花授粉为主的植物,它的有性生殖良好,每株性成熟植株平均可产生60粒左右的种子,多者可达180粒。由种子萌发长成的植株大约需要5－6年的时间方才性成熟。在一般条件下,桃儿七行有性生殖,但在环境条件良好时,也可通过根茎来进行营养繁殖。桃儿七为二倍体,12条染色体组成6个联锁群,在减数分裂时,交叉发生较少。桃儿七具有较大当前适合度,亦具有一定的进化灵活度。     

SEX-SPECIFIC COSTS OF RESISTANCE TO THE FUNGAL PATHOGEN USTILAGO VIOLACEA (MICROBOTRYUM VIOLACEUM) IN SILENE ALBA

Costs of resistance are often invoked to explain the maintenance of polymorphisms for resistance to fungal pathogens in natural plant populations. To investigate such costs, 27 half-sib families of Silene alba, collected from a single host population, were grown in experimental populations in the presence and absence of the anther-smut fungus Ustilago violacea, a host-sterilizing pathogen transmitted by insects that are both pollinators and vectors of the disease. Host families differed significantly in resistance to inoculation, indicating the presence of genetic variation for mechanisms that impede fungal growth once the disease is encountered (“biochemical” resistance) within the host population. In addition, host families differed significantly in onset of flowering and in flower production in the absence of the disease. Path analysis revealed that late onset of flowering in male host families made a direct contribution to high field resistance (P < 0.01), probably due to a reduced rate of contact between hosts and vectors carrying high spore loads (avoidance, or “phenological” resistance). The contribution of low flower production to field resistance only approached significance (P < 0.10). There was a significantly positive genetic association between biochemical and phenological resistance, suggesting that delayed flowering is either a pleiotropic effect of biochemical resistance, or that genes governing these traits are in linkage disequilibrium. Path analysis revealed that biochemical resistance made both a direct contribution to field resistance (P < 0.01) and a positive indirect contribution via its association with phenology and flower production (P < 0.05) in male hosts. Costs of resistance were sex specific. Male host families with high field resistance had significantly lower reproductive success in healthy populations, indicating a fitness cost of field resistance (P < 0.01), whereas no costs were detected for female hosts. Path analysis revealed that the biochemical component of field resistance made no direct contribution to the observed fitness cost in male hosts, whereas its indirect effect through phenology was only marginally significant (P < 0.10). This finding indicates that fitness costs were mainly due to the phenological component of field resistance. Because the host population had no known history of disease, it is not clear whether the fitness costs are responsible for maintenance of the resistance polymorphism or whether the polymorphism is present for reasons unrelated to pathogen infection. Interactions between host families and pathogen strains with respect to inoculation success were not significant. Hence, there was no evidence for indirect costs of biochemical resistance, that is, reduced resistance to alternative strains. Infection rates in experimental populations with an initially patchy distribution of the pathogen were lower than in populations with a uniform pathogen distribution, suggesting that the effective pathogen pressure and hence the relative success of susceptible and resistant individuals may, in addition to fitness costs of resistance, depend on the spatial population structure of the pathogen.     

云杉核型的研究兼论云杉属的进化地位

本文分析了我国特产树种云杉Picea asperata的核型,K(2n)=24=20m+4sm,属2A类型,染色体相对长度组成为2n=24=2L+12M_2+SM_1+2S。云杉属植物(22种、变种)的核型全由臂比小于2的中部和近中着丝粒染色体构成,是较为原始的核型。根据松科各属核型的比较,作者讨论了云杉属的亲缘关系和进化地位,并得到形态学、解剖学、孢粉学、植化学、生化学及古植物学等的支持。     

CONTRIBUTION OF TEMPORAL AND SPATIAL COMPONENTS TO MORPHOLOGICAL VARIATION IN THE KELP ECKLONIA (LAMINARIALES)1

Environmental conditions that are known to cause morphological variation in algae (e.g., wave exposure) often vary in both space and time and are superimposed onto the distinct seasonal growth cycles of most temperate macroalgae. We tested the hypothesis that the morphology of the small kelp Ecklonia radiata (C. Agardh) J. Agardh is the product of an interaction between site (five reefs of different wave exposure) and the time of year that sampling occurs (summer vs. winter 2004). We determined that wave exposure had a strong directional effect on kelp morphology, with “Reefs” accounting for up to 43.4% of variation in individual morphological characters. “Times” had a narrowly nonsignificant effect on overall morphology but accounted for up to 31% of variation in individual characters. Many characters were affected by wave exposure, whereas only a few were (strongly) affected by time (e.g., thallus biomass). Interactive effects between “Reefs” and “Times” were generally small, accounting for 15.8% of variation in lamina thickness, but much less for most other characters. We conclude that wave exposure exerts a strong control over the morphology of E. radiata, but that the nature of the effect depends on the magnitude of wave exposure. We also conclude that most of the effects of wave exposure are consistent through time and do not interact with cycles of growth and pruning in any major way.     

MATING BEHAVIOR OF THE CAT FLEA, CTENOCEPHALIDES FELZS BOUCHE(SIPHONAPTERA:PULICIDAE) AND MALE RESPONSE TO FEMALE EXTRACT ON AN ARTIFICIAL FEEDING SYSTEM

Abstract  The mating behavior of cat flea, Ctenocepholides felis (Bouche) was studied on an artificial feeding device. Male and female can mate repeatedly with same partner or Merent ones. In the situation of male: female ratio of 1 :5, each mating lasted an average of 6.6 min, with a mean interval between matings at 2.5 min., compared to 11. 1 min and 12.1 min respectively in a cell with 5 males and 1 female. As many as 48 mating events were observed for one male during an 8 h period. One female mated 27 times in 7 h with 5 males in the same cell. Newly emerged males and females can not mate before blood meal and about 24 h blood feeding is rewuired for successful mating. Newly emerged males can not mate with fed females (fed for 48 h), but fed males can mate with newly emerged females who are feeding the blood. Significantly more male contacts and male-male mating attempts were observed after the paper treated with female extract was introduced into the cell. The paper contacts and mating attempts were 16.75–32.25 times and 15.75–31.38 times, respectively, on average during a period of 20 min when different doses (FE) of extract were provided.