Molecular Phylogeny and Biogeography of the Genus Ilex L. (Aquifoliaceae)

The chloroplast atpB-rbcL spacer was sequenced for 115 speciesof Ilex and forNemopanthus mucronatus (Aquifoliaceae). In addition,rbcL sequences were obtained for 47 selected species. Takentogether with fossil records in the literature, the data indicatethat: (1) the lineage Ilex was already cosmopolitan long beforethe end of the Cretaceous. A relative test of the rate of nucleotidesubstitution indicates that extant species do not representthe entire lineage because of the extinction of its basal branches.The common ancestor of the extant species probably appearedat the upper Tertiary. (2) Several Asian/North American disjunctionsare observed, as well as North American/South American relationships.The directions of these relationships are yet to be determined.The African species Ilex mitis is closely related to Asian species.Ilex anomala (Hawaii and Tahiti) is related to American species.Ilex perado and Ilex canariensis (both in Macaronesia) havetotally different relationships, the former being related toEurasian species, while relationships of the latter remain obscure.(3) Gene trees substantially contradict the systematics of Loesener.Nemopanthus,closely related to Ilex amelanchier, is nested in the genusIlex. The family Aquifoliaceae is organized in four groups,each of them having a geographic or ecological peculiarity.Copyright 2000 Annals of Botany Company Aquifoliaceae, chloroplast DNA, fossil record, historical biogeography, Ilex (holly tree), molecular phylogeny.     

Ilex Canariensis Poir. (Aquifoliaceae) Post-dispersal Seed Predation in the Canary Islands

Alien species have many negative effects on insular ecosystems worldwide. We investigated Ilex canariensis post-dispersal seed predation by introduced rats (Rattus spp.) in relict forests of the Canary Islands at different spatial scales: among microhabitats within the same forest, among forest types within the same island, and among different islands of the archipelago. Seed predation intensity was very high (>70%) in all cases considered, irrespective of the spatial scale. We did not find significant differences between forest interior, edges or gaps, as well as between different forest types in four islands of the archipelago. Comparatively low predation intensity was found in El Hierro island, where more than 50% of the seeds survived at the end of the experiment, while highest seed predation was observed in Tenerife island. It is concluded that post-dispersal seed predation by rats, due to its extent and intensity, could have an important effect on Ilex canariensis recruitment, especially in successional areas where this light tolerant tree can naturally establish.     

Pollenkitt formation in Ilex paraguariensis A.St.Hil. (Aquifoliaceae)

 We investigated the cellular and organelle transformations during the formation of the pollenkitt in the secretory tapetum of Ilex paraguariensis. After the dissolution of the callose surrounding the young microspores, the elaioplasts of the tapetum produce many globules of saturated and unsaturated lipids (plastoglobules). Further on, oleosomes with unsaturated lipids, synthesized in the endoplasmic reticulum, accumulate in the tapetal cytoplasm. In contrast to other species, the plastoglobule production seems to precede the oleosome synthesis. The tapetum shows signs of cellular maturation in the late vacuolated microspore stage, when the plastoglobules and oleosomes coalesce and form the pollenkitt mass. In mature stages of the tapetum the pollenkitt is released into the loculus. Finally, it is mainly deposited on the exine, according to the entomophilous character of this species. The mode of pollenkitt formation in Ilex para guariensis and its transfer to the pollen surface is slightly dissimilar to other Angiosperms. Received October 24, 2002; accepted December 2, 2002 Published online: March 20, 2003     

Postglacial migration and the loss of allozyme variation in northern populations of Asclepias exaltata (Asclepiadaceae)

The recent Wisconsin glaciation has provided opportunities for examining the effects of postglacial recolonization on the population genetics of plant and animal communities. In this study allozyme Variation was examined in 19 populations of the herbaceous perennial Asclepias exaltata occurring in previously glaciated regions of North America. These northern populations of A. exaltata possess significantly fewer polymorphic loci (46.31 ± 2.7; mean ± 1 SD), alleles per polymorphic locus (1.84 ± 0.24), and expected heterozygosity (0.133 ± 0.031) than populations found in the Pleistocene refugium in the southern Appalachians. Population-level allozyme diversity decreased linearly from south to north and from east to west. Nineteen uncommon alleles previously observed in southern Appalachian populations were undetected in the northern region. Seven common alleles exhibited a clinal change in allele frequency. Of these, only Pgd-1a and Mnr-1c were at low-frequency in the southern Appalachians and increased significantly with increasing latitude and longitude, respectively. Despite this loss of allozyme diversity following postglacial migration, northern populations of A. exaltata have higher allozyme diversity and lower population differentiation (G" = 0.1 17) than mean values for other long-lived herbaceous perennials. Increased habitat fragmentation in northern regions and potential habitat loss in the southern Appalachians are likely to reduce the historically rich gene pool that has provided the genetic stock for postglacial recoveries.     

The role of self-pollen in the female reproductive success of common milkweed (Asclepias syriaca: Asclepiadaceae)

Follicle production from bagged hand-pollinations of the largely self-incompatible common milkweed (Asclepias syriaca) greatly exceeded that of open-pollinated flowers by seven- to 30-fold in certain clones, even though these clones received one to three natural pollinations per flower. To test the basis for this difference I performed an extensive set of cross- and self-pollinations on the clone exhibiting the lowest natural follicle production, with additional experiments on other clones for comparison. Bagged, hand cross-pollinations in the least productive clone produced seven to 20 times more follicles than unbagged, hand cross-pollinations in that clone exposed to pollinating insects. Bagged flowers of this clone supplied with simultaneous hand, cross- and hand, self-pollinations produced no follicles. One other clone that normally produced few follicles responded to bagged and unbagged, hand cross-pollinations in a similar way, but follicle production of two heavier producing clones did not differ in response to this manipulation. These results suggest that self-pollinations strongly reduce the success of cross-pollinations in clones that normally experience low follicle production. Resource limitation cannot account for the low follicle production, because the stems readily supported additional follicles following supplemental hand pollinations.     

Fine-scale clonal structure and diversity within patches of a clone-forming dioecious shrub, Ilex leucoclada (Aquifoliaceae)

BACKGROUND AND AIMS: The mode of reproduction (sexual vs. asexual) is likely to have important effects on genetic variation and its spatial distribution within plant populations. An investigation was undertaken of fine-scale clonal structure and diversity within patches of Ilex leucoclada (a clone-forming dioecious shrub). METHODS: Six patches were selected in a 1-ha plot previously established in an old-growth beech forest. Two of the selected patches were composed predominantly of stems with male flowers (male patch), and two contained stems with predominantly female flowers (female patch). The remaining two patches contained stems with male flowers and stems with female flowers in more or less equal proportions (mixed patch). Different genets were distinguished using random amplified polymorphic DNA (RAPD) markers. KEY RESULTS: One hundred and fifty-six genets with different RAPD phenotypes were identified among 1928 stems from the six patches. Among the six patches, the male patches had the lowest clonal diversity, and the mixed patches had the highest. Distribution maps of the genets showed that they extended downhill, reflecting natural layering that occurred when stems were pressed to the ground by heavy snow. In every patch, there were a few large genets with many stems and many small genets with a few stems. CONCLUSION: The differences in clonal diversity among patches may be due to differences in seedling recruitment frequencies. The skewed distribution of genet size (defined as the number of stems per genet) within patches may be due to differences in the timing of germination, or age (with early-establishing genets having clear advantages for acquiring resources) and/or intraspecific competition.     

Populational dynamics of mites (Acari) in the mate-tea tree (Ilex paraguariensis St. Hil.: Aquifoliaceae)

The objective of this research was to analyze the populational dynamic of the phytophagous mites, as well as that of their natural predators in the plants Ilex paraguariensis St. Hil. (Aquifoliaceae). This study was conduced in Dois Vizinhos, State of Paraná, from August 2001 to July 2002. Leaf samples from different parts of the plant were taken and the number of mites was registered. During this period, two species of phytophagous mites, Dichopelmus notus Keifer, and Oligonychus yothersi (McGregor), and three species of predator mites identified as Euseius concordis (Chant), Iphiseiodes zuluagai Denmark & Muma, and Agistemus sp. were related to the mate-tea plant. Large numbers of D. notus appeared on mature leaves and on the inferior face of leaves. The mite was more frequent in the inferior and medium strata. O. yothersi occurred mainly on mature leaves. The concentration of E. concordis e I. zuluagai was higher on the inferior face of the leaves, and on the leaves of inferior and medium strata, as well as in the internal canopy region, and on mature leaves. The highest numbers of D. notus, O. yothersi, E. concordis and I. zuluagai occurred in periods with mild temperatures and little rain precipitation. The largest population density of Agistemus sp. occurred on the inferior face of the leaves, more often in periods of high temperature and heavy rain.     

Purification and Characterization of a New Plant Endopeptidase Isolated from Latex of Asclepias fruticosa L. (Asclepiadaceae)

Asclepias fruticosa L. is a small shrub containing latex with proteolytic activity. The crude extract (latex diluted 1:250 and ultracentrifuged) contained 276 g of protein/mL and the proteolytic activity reached 1.2 caseinolytic U/mL. This enzyme preparation was very stable even after 2 hours at 45°C, but was quickly inactivated after 5 minutes at 80°C. Chromatographic purification was achieved by FPLC using a cation exchanger (SP-Sepharose FF). Thus, a unique proteolitically active fraction could be isolated, being homogeneous by bidimensional electrophoresis and mass spectrometry (M_r = 23,652). The optimum pH range was achieved at 8.5–10.5. The enzyme activity was completely inhibited by specific cysteine peptidases inhibitors. Isoelectric focusing followed by zymogram showed the enzyme had a pI greater than 9.3. The N-terminus sequence (LPDSVDWREKGVVFPIRNQGK) shows a great deal of similarity to those of the other cysteine endopeptidases isolated from latices of Asclepiadaceae even when a high degree of homology could be observed with other plant cysteine endopeptidases.     

Intersectional gene flow between insular endemics of Ilex (Aquifoliaceae) on the Bonin Islands and the Ryukyu Islands

Hybridization and introgression play important roles in plant evolution, and their occurrence on the oceanic islands provides good examples of plant speciation and diversification. Restriction fragment length polymorphisms (RFLPs) and trnL (UAA) 3'exon-trnF (GAA) intergenic spacer (IGS) sequences of chloroplast DNA (cpDNA), and the sequences of internal transcribed spacer (ITS) of nuclear ribosomal DNA were examined to investigate the occurrence of gene transfer in Ilex species on the Bonin Islands and the Ryukyu Islands in Japan. A gene phylogeny for the plastid genome is in agreement with the morphologically based taxonomy, whereas the nuclear genome phylogeny clusters putatively unrelated endemics both on the Bonin and the Ryukyu Islands. Intersectional hybridization and nuclear gene flow were independently observed in insular endemics of Ilex on both sets of islands without evidence of plastid introgression. Gene flow observed in these island systems can be explained by ecological features of insular endemics, i.e., limits of distribution range or sympatric distribution in a small land area.     

Molecular analyses of the genus Ilex (Aquifoliaceae) in southern South America, evidence from AFLP and ITS sequence data

In order to clarify the relationships among southern South American (sSA) representatives of the genus Ilex, an amplified fragment length polymorphism (AFLP) analysis was accomplished. In addition, the phylogenetic relationships of the species were studied using ribosomal internal transcribed spacer (ITS) sequence data alone and in combination with AFLP data, taking into account the possible existence of paralogous sequences and the influence of alignment parameters. To explore stability of phylogenetic hypotheses, a sensitivity analysis was performed using 15 indel-substitution models. Within each species assayed, the AFLPs allowed the recognition of several diagnostic bands. Furthermore, the AFLP analysis revealed that individuals belonging to the same morpho-species formed coherent clades. In addition, some cases of geographical association were noted. Studies on ITS sequences revealed divergence between data obtained herein and sequence data downloaded from GenBank. The sensitivity analyses yielded different interspecific hypotheses of relationships. Notwithstanding, analyses of the ITS data alone and in combination with AFLPs, rendered clades stable to variation in the analytical parameters. Topologies obtained for the AFLPs, the ITS data alone and the combined analyses, demonstrated the existence of a group formed by I. argentina, I. brasiliensis, I. brevicuspis, I. integerrima, and I. theezans, and that I. dumosa and I. paraguariensis were distantly related to the former. Incongruence with traditional taxonomical treatments was found.     

用RAPD技术探讨冬青属苦丁茶的遗传差异、亲缘关系与分类地位

利用随机扩增多态性DNA (RAPD)技术对 2 2份冬青属苦丁茶不同种质材料的遗传变异进行了研究。 4 0个 10 bp的引物用于多态性检测 ,从中筛选出了 2 7个多态性良好的引物。用该 2 7个引物对上述 2 2份种质材料进行PCR扩增 ,共得到 4 45条DNA谱带 ,其中多态性带4 32条 ,占 97 0 8%。根据扩增结果计算了各份材料之间的遗传距离 ,并用UPGMA构建了聚类树状图。分析结果表明 :2 2份供试材料分成 4类 5组 ,第Ⅰ类为Ilexpentagona (分为A、B两组 :A组为新变种var mashanensisG .M .L .;B组为原变种var pentagona) ,第Ⅱ类为I kudingcha,第Ⅲ类为I latifolia ,第Ⅳ类为I cornuta ;第Ⅱ ,Ⅲ ,Ⅳ三类各有一组材料组成。物种的差异、亲缘关系以及同一物种内不同种质材料在起源地域上的差异均可从系统树中反映出来。RAPD分子标记的结果可作为判断冬青属苦丁茶种质资源材料的起源地域、遗传差异、亲缘关系以及种级水平和种下分类鉴定的重要参考依据     

Cytological and morphological differentiation in Caralluma burchardii (Asclepiadaceae)

Meve, U. 1995. Cytological and morphological differentiation in Carallurna burchardii (Asclepiadaceae). — Nord. J. Bot. 15: 459–467. Copenhagen. ISSN 0107–055X.
Caralluma burchardii is revised based on detailed investigations of vegetative and floral morphology, as well as chromosome numbers. The species is now divided into two taxa, the dodecaploid ssp. burchardii , an endemic of the Canary Islands, and ssp. rnaura , a hexaploid from the west coast of Morocco. The high ploidy levels involved, the highest known in the Asclepiadaceae, are discussed in conjunction with biogeographical aspects.     

Notes on quiina (Quiinaceae) and ilex species (Aquifoliaceae) in Cuba

Quiina ternatiflora C. H. Wright is the only member of the family Quiinaceae reported from Cuba. An examination of the holotype indicates that this is a species ofIlex, and the transfer is made asIlex ternatiflora (C. H. Wright) R. Howard. A study of otherIlex species from Cuba indicates that a new name,Ilex walsinghamii Howard, is required forIlex wrightii Loes. not Trel.     

The complex history of the genus Ilex L. (Aquifoliaceae): evidence from the comparison of plastid and nuclear DNA sequences and from fossil data

 A plastid phylogeny of the genus Ilex based on three different loci (the atpB-rbcL spacer, trnL-trnF and rbcL) is compared with its nuclear phylogeny based on two different loci (the ribosomal ITS and the 5S RNA spacer). These two sets of molecular data are then compared to geographical and temporal data from the fossil record. The plastid phylogeny is strongly correlated with the geographic distribution of extant species. However, the nuclear phylogeny is strongly incongruent with the plastid phylogeny, suggesting frequent interlineage hybridizations. Moreover, the comparison of the ribosomal ITS tree and the 5S RNA spacer tree indicates also possible lineage sorting. Particularly interesting is the finding of two different Ilex lineages in the plastid American clade showing different biogeographic patterns in South America. One of them has a simple North American/South American biogeographical relationship. The other has complex biogeographical relationships, some species showing direct Asian/South American biogeographical relationships. During its history, the genus Ilex probably experienced frequent lineage sorting and interlineage hybridization with subsequent nuclear or cytoplasmic introgression, making the study of its history very complex. Received September 24, 2001; accepted August 19, 2002 Published online: November 28, 2002 Addresses of the authors: Jean-Fran?ois Manen (e-mail: manen@cjb.ville-ge.ch), Yamama Naciri-Graven, Conservatoire et Jardin Botaniques, Impératrice 1, CH-1292 Chambésy/Genève, Switzerland. Michael C. Boulter, Palaeobiology Research Unit, University of East London, Romford Road, London E15 4LZ, UK.     

青阳参花部特征及其传粉适应性

对青阳参花（Cynanchum otophyllum）部综合特征、访花昆虫种类、访花行为及传粉过程进行了研究,结果表明,青阳参花结构复杂,两个子房基部离生、花柱联合与雄蕊形成合蕊柱,柱头表面被邻近花药的侧翼紧密包围形成5个柱头腔。青阳参的花粉形成独特的花粉块,一次传粉过程可以转运大量的花粉。东方蜜蜂（Apis cerana）是青阳参的主要传粉昆虫,其传粉包括两个过程：（1）当蜜蜂的口器或足插入着粉腺的槽口后借助蜜蜂的力量将花粉块从花上拔起;（2）当蜜蜂再次访花时将携带的花粉块插入其中一个柱头腔。花粉块里面的花粉粒住柱头腔中萌发出花粉管,然后沿着花柱道向下生长最后进入子房。在整个花期仡粉保持有相对较高的生活力,而其柱头可授性则在7天后逐渐降低。     

Determination of Heavy Metal Concentrations and Their Potential Sources in Selected Plants: Xanthium strumarium L. (Asteraceae), Ficus exasperata Vahl (Moraceae), Persicaria attenuata (R.Br) Sojak (Polygonaceae), and Kanahia laniflora (Forssk.) R.Br. (Asclepiadaceae) from Awash River Basin,Ethiopia

The objective of this study was to determine the concentrations, potential sources and evaluate the risks of heavy metals in selected plants from Awash River Basin, Ethiopia. A total of 57 samples were analyzed from four different plant species. Microwave-assisted digestion was applied to digest the samples and the concentration of nine elements namely: Al, Cr, Ni, Cu, Zn, As, Cd, Hg, and Pb were analyzed using Inductively Coupled Plasma-Mass Spectrometry (ICP- MS) (Thermos X SERIES2). The obtained data were analyzed using Statistical Package for Social Sciences (SPSS IBM version 20). The recorded mean concentration of heavy metals in the plants were 1.934, 0.023, 0.023, 0.045, 0.129, ND, 0.025, ND, and 0.009 mg/kg in Xanthium strumarium L.(Asteraceae); 0.834, 0.036, 0.024, 0.021, 0.090, ND, 0.002, 0.001, and 0.006 mg/kg in Ficus exasperata Vahl (Moraceae); 1.603, 0.018, 0.019, 0.025, 0.133, 0.005, 0.006, 0.002, and 0.012 mg/kg in Persicaria attenuata (R.Br) Sojak (Polygonaceae); and 0.557, 0.010, 0.010, 0.024, 0.098, ND, 0.012, 0.020, and 0.004 mg/kg in Kanahia laniflora (Forssk.) R.Br. (Asclepiadaceae) for Al, Cr, Ni, Cu, Zn, As, Cd, Hg, and Pb, respectively. Pearson’s correlation analysis indicated that there was a strong positive correlation between Al–Ni (r = 0.927^**) and Zn–Cu (r = 0.764^**) at α = 0.01 significant level. Principal component analysis (PCA) indicated that the sources of heavy metals in the plants were associated with anthropogenic factors. The mean concentrations of all elements except Cd in Xanthium strumarium L. (Asteraceae) and Pb in Ficus exasperata Vahl (Moraceae) were below the permissible limit of FAO/WHO (2001 /2005).

     

Host range and searching behaviour of Cricotopus lebetis (Diptera: Chironomidae), a tip miner of Hydrilla verticillata (Hydrocharitaceae)

A chironomid midge, Cricotopus lebetis Sublette (Diptera: Chironomidae), was discovered feeding on Hydrilla verticillata (L.f.) Royle (Hydrocharitaceae) in Crystal River, Citrus, Co., Florida, in the 1990s. Larvae of the midge mine the apical meristems of hydrilla, causing terminal branching and stunting of the plant. We investigated the fundamental host range of the midge by conducting a series of no-choice and paired-choice tests. No-choice developmental tests with neonate larvae revealed that the fundamental host range of C. lebetis included not only on hydrilla but also several other aquatic plants in different families, suggesting that this insect is not a hydrilla specialist. In paired-choice bioassays, larval colonisation of Elodea canadensis Michx. (Hydrocharitaceae) and Najas guadalupensis (Spreng.) Magnus (Najadaceae) was greater than colonisation of H. verticillata. Behavioural bioassays in a Y-tube olfactometer and in Petri dishes suggested that neonate larvae were not able to locate host plant material, whereas older larvae were successful in finding hosts. In paired-choice oviposition tests, adult females discriminated between potential oviposition sites, with greater numbers of eggs laid on E. canadensis and N. guadalupensis than on H. verticillata. This study is the first detailed account of host searching and oviposition behaviour of a phytophagous chironomid midge. The results will be used to assess the potential value of C. lebetis as a biological control agent of hydrilla.     

Biology and Host Range of the Hawk Moth Nephele densoi Keferstein (Lep.: Sphingidae), a Potential Agent for the Biological Control of Rubbervine Cryptostegia grandiflora R. Br. (Asclepiadaceae)

The larvae of the hawk moth, Nephele densoi Keferstein, feed on the leaves of Cryptostegia grandiflora R. Br. in Madagascar. Host-specificity tests showed that feeding by N. densoi is restricted to plants in the families Asclepiadaceae (subfamily Periplocoideae), Apocynaceae and Moraceae, but only plants in the subfamily Periplocoideae supported larval development. The comparatively wide host range contrasts with the narrow host range demonstrated for the Australian moth N. subvaria (Walker). The possible evolution of host plant specialization in this genus is discussed.     

Removal and insertion of pollinia in flowers of Oxypetalum (Asclepiadaceae) in southeastern Brazil

Pollinarium removal and pollinium insertion of seven Oxypetalum species (O. alpinum var. alpinum, O. appendiculatum, O. banksii subsp. banksii, O. jacobinae, O. mexiae, O. pachyglossum and O. subriparium) were recorded in Vi?osa. Minas Gerais. They presented a tendency of one or two pollinarium removals and one pollinium insertion (single insertion), except O. appendiculatum. In this species, mainly, two pollinia of the same pollinarium were inserted per stigmatic chamber (double insertion), resulting exceptionally in 6-10 inserted pollinia in a flower, an unusual occurrence among the Asclepiadaceae. No association between removal and insertion was found, e.g., O. subriparium and O. banksii subsp. banksii had the highest pollinarium removal (1.78 and 1.45, respectively) and one of the lowest pollinium insertions (0.02 in both species), per flower. Oxypetalum mexiae showed the lowest pollinarium removal and pollinium insertion per flower (0.09 and 0.01, respectively) among the studied species and other Asclepiadaceae. Oxypetalum subriparium, O. banksii subsp. banksii and O. mexiae might be having reproductive limitations. Pollinarium removal and pollinium insertion per flower of the studied species varied from site to site, similarly to what was recorded for other Asclepiadaceae.