Reproductive biology of Trichocentrum pumilum: an orchid pollinated by oil-collecting bees

The reproductive biology, reward production and pollination mechanism of Trichocentrum pumilum were studied in a gallery forest in the interior of the State of São Paulo, southeast Brazil. The floral visitors and pollination mechanism were recorded, and experimental pollinations were carried out in order to determine the breeding system of this species. Trichocentrum pumilum blooms in spring. Each paniculate inflorescence bears an average of 85 flowers that present a central yellow callus and finger‐like trichomes on the lateral lobes of the lip. A lipoidal substance is produced and stored among these trichomes. In the studied population, T. pumilum is exclusively visited and pollinated by two bee species (Tetrapedia diversipes and Lophopedia nigrispinis). Pollinaria are deposited on mouthparts of bees during collection of the lipoidal substance from the lateral lobes of the labellum. Trichocentrum pumilum is self‐incompatible and pollinator‐limited. Natural fruit set was low (9%, compared to 45% in experimentally cross‐pollinated flowers). Potentially viable seed exceed 97% in fruits obtained through cross‐pollination and in natural conditions (open pollination).     

Species‐level phylogeographical history of the endemic species Calligonum roborovskii and its close relatives in Calligonum section Medusa (Polygonaceae) in arid north‐western China

Quaternary climatic oscillations appear to have influenced the genetic diversity and evolutionary history of arid‐adapted plants. To understand the processes involved and reveal evolutionary relationships, haplotypes were examined from Calligonum roborovskii, an endemic species occurring in the arid zones across the desert regions of north‐western China, and seven other species also from Calligonum section Medusa, including C. gobicum, C. mongolicum and the narrow endemic species C. ebi‐nuricum, C. pumilum, C. taklimakanense, C. trifarium and C. yengisaricum. Forty‐three haplotypes were identified in 422 individuals from 51 natural populations, from variation of two plastid DNA intergenic spacers (rpl32‐trnL and ycf6‐psbM). A high level of total genetic diversity was found across species for which more than two populations were examined, including C. gobicum, C. mongolicum, C. pumilum and C. roborovskii. A distinct isolation‐by‐distance pattern in each of these species was suggested by the Mantel test, indicating that restricted gene flow caused high genetic differentiation among populations. Three haplotypes were shared by two or three species each, but the other 40 haplotypes were species‐specific. The 43 haplotypes split into three major clades, but not species‐specific lineages; most of the Calligonum species were not reciprocally monophyletic, probably due to incomplete lineage sorting or introgression. The identified haplotypes were dated to 1.97 Mya (95% highest posterior density: 2.95–0.99 Mya) and diverged until the late Pleistocene, possibly linked to aridification and enlargement of deserts caused by climate changes. Variation of desert habitats during the Pleistocene might play a key role in causing the divergence.     

Protection of the photosynthetic apparatus against dehydration stress in the resurrection plant Craterostigma pumilum

The group of homoiochlorophyllous resurrection plants evolved the unique capability to survive severe drought stress without dismantling the photosynthetic machinery. This implies that they developed efficient strategies to protect the leaves from reactive oxygen species (ROS) generated by photosynthetic side reactions. These strategies, however, are poorly understood. Here, we performed a detailed study of the photosynthetic machinery in the homoiochlorophyllous resurrection plant Craterostigma pumilum during dehydration and upon recovery from desiccation. During dehydration and rehydration, C. pumilum deactivates and activates partial components of the photosynthetic machinery in a specific order, allowing for coordinated shutdown and subsequent reinstatement of photosynthesis. Early responses to dehydration are the closure of stomata and activation of electron transfer to oxygen accompanied by inactivation of the cytochrome b₆f complex leading to attenuation of the photosynthetic linear electron flux (LEF). The decline in LEF is paralleled by a gradual increase in cyclic electron transport to maintain ATP production. At low water contents, inactivation and supramolecular reorganization of photosystem II becomes apparent, accompanied by functional detachment of light‐harvesting complexes and interrupted access to plastoquinone. This well‐ordered sequence of alterations in the photosynthetic thylakoid membranes helps prepare the plant for the desiccated state and minimize ROS production.     

Serpentine and polyploid differentiation withinGalium pumilum agg. (Rubiaceae) in Eastern C. Europe

The local differentiation of a model group,Galium pumilum agg., was studied in serpentine versus non-serpentine habitats in the Czech Republic and adjacent regions in Austria, Germany and Poland. Serpentine habitats form ecologically distinct islands within the landscape; local micro-evolutionary processes may be important there, both because of small population sizes and the strong selection effects of the metal-rich soils. TheGalium pumilum agg. is a group of taxa showing typical karyological and ecological differentiation; the following species and cytotypes were studied:Galium pumilum Murray (2n=8x=88),G. valdepilosum Heinr. Braun (2n=2x=22, 2n=4x=44),G. sudeticum Tausch (2n=4x=44) andG. austriacum Jacq. (2n=4x=44). The karyological and taxonomic investigation showed that serpentine versus non-serpentine habitats do not play an important role in the differentiation of the species studied. (1) The examined populations ofG. pumilum Murray were homogeneous in chromosome numbers, belonging to the octoploid (2n=88) cytotype only. (2)G. valdepilosum consists of two cytotypes. Both of them occur on and off serpentine sites, but they show pronounced East-West distributional difference. (3)G. sudeticum occupies special localities on serpentine in western Bohemia, which are geographically isolated from its nearest localities in the Krkono?e Mts. The plants coming from both localities belong to the tetraploid cytotype. (4) A highly polyploid plant (2n=12x=ca. 132) from theG. pumilum group was found on serpentine. From the methodological point of view, pollen size turned out to be reliable only for distinguishing octoploids from diploids and tetraploids, but it is less suitable for separating the latter two ploidy levels. The occurrence ofG. austriacum is reported for the Czech Republic for the first time with certainty. This record extends the known distribution area ofG. austriacum in the NW direction.     

In vitro propagation and characterization of phenolic content along with antioxidant and antimicrobial activities of Cichorium pumilum Jacq.

Cichorium pumilum, a member of Asteraceae, is widely used as a traditional medicinal herb. An efficient protocol for callus formation and whole plant propagation has been developed. Callus cultures were induced from leaf explants on Murashige and Skoog (MS) medium supplemented with 1.5?mg?l^?1 6-Benzyladenine (BA) and 0.5?mg?l^?1 Naphthalene acetic acid (NAA). Maximum numbers of shoots were obtained from calli transferred to shoot regeneration medium containing MS basal medium with 1.5?mg?l^?1 BA or Kinetin (Kin). The shoots were effectively rooted on MS medium supplemented with different concentrations of Indole-3-butyric acid. In the present study, the antibacterial activity of C. pumilum extracts was assayed in vitro by agar disc diffusion and agar well diffusion methods against 10 different bacterial species. The results showed effect on the growth of 50 and 70% of the tested bacterial species using methanol and ethanol extracts respectively. Klebsiella pneumoniae was susceptible to the ethanolic and methanolic extracts of wild plants and in vitro tissues, whereas Enterococcus faecalis was resistant to all the extracts. This study verified that the methanol extracts have strong antioxidant activities with high levels of phenolic compounds. The antioxidant activity and total phenol content of callus cultures and in vitro plantlets were lower than those of the wild plants. The results obtained confirm the therapeutic potency of Cichorium used in the traditional medicine, in addition, the efficient in vitro production system developed in this study provide sterile and consistent tissues for the investigation of phytochemical and pharmacological effects and germplasm conservation of C. pumilum.     

3种百合鳞茎中多酚类物质的抗氧化活性分析

以野生百合渥丹、山丹和传统食用的兰州百合为研究对象,对其鳞茎中多酚类物质、11种单体酚的含量及抗氧化活性(ABTS自由基、超氧阴离子、羟自由基的清除能力,铜离子还原能力以及抑制脂质过氧化活性)进行了分析。结果表明:两种野生百合鳞茎中的多酚类物质含量及抗氧化活性均显著高于兰州百合。3种百合鳞茎中单体酚的种类也有所不同,但均含有没食子酸、矢车菊素-3-芸香糖苷、儿茶素、表儿茶素、杨梅酮、芦丁、对香豆酸、山奈酚。相关性分析显示,除对羟自由基的清除力外,各酚类物质总量与不同抗氧化指标之间呈显著正相关关系。试验结果认为,野生百合鳞茎可作为天然抗氧化资源应用于食品和医药业,具有一定的开发应用前景。     

Genetic diversity and structure of Lilium pumilum DC. in southeast of Qinghai–Tibet plateau

Lilium pumilum DC. is a valuable species not only for its showy flowers but also for its edible and medicinal values. As one of the distribution areas of L. pumilum, Qinghai–Tibet plateau has unique environmental features which have high impact on the evolution of the species. No population genetic studies have been done for L. pumilum so far. To provide the first reference data for evolutionary study and understanding the influence of eco-geographic factors on the distribution of genetic variation in L. pumilum, interspecific simple sequence repeat markers were used to investigate genetic diversity and population structure of 28 populations sampled from southeast of Qinghai–Tibet plateau. Fifteen selected primers generated a total of 147 polymorphic bands. The genetic diversity was low within populations (average He = 0.173), but higher at the species level (He = 0.392). A clear population structure and high level of genetic differentiation (F _ST = 0.518) were detected by unweighted pair group method for arithmetic averages, principle coordinate analysis and Bayesian clustering. All clustering approaches supported a division of the 28 populations into 4 major groups for which analysis of molecular variance confirmed a significant variation among groups (34.3 %). These population genetic parameters suggest limited gene flow among populations and evidence for isolation by distance (r = 0.272, P < 0.0001) was found in this study. Altitude, AMT and AMP explained 9.5, 11.5 and 14.0 % of the total variance among populations indicating that eco-geographic factors have a significant effect. Considering the low within-population genetic diversity, high differentiation among populations and the increasing anthropogenic pressure on the species, in situ conservation measures were recommended to preserve L. pumilum in Qinghai–Tibet plateau.     

Water deficit induces chlorophyll degradation via the ‘PAO/phyllobilin’ pathway in leaves of homoio‐ (Craterostigma pumilum) and poikilochlorophyllous (Xerophyta viscosa) resurrection plants

Angiosperm resurrection plants exhibit poikilo‐ or homoiochlorophylly as a response to water deficit. Both strategies are generally considered as effective mechanisms to reduce oxidative stress associated with photosynthetic activity under water deficiency. The mechanism of water deficit‐induced chlorophyll (Chl) degradation in resurrection plants is unknown but has previously been suggested to occur as a result of non‐enzymatic photooxidation. We investigated Chl degradation during dehydration in both poikilochlorophyllous (Xerophyta viscosa) and homoiochlorophyllous (Craterostigma pumilum) species. We demonstrate an increase in the abundance of PHEOPHORBIDE a OXYGENASE (PAO), a key enzyme of Chl breakdown, together with an accumulation of phyllobilins, that is, products of PAO‐dependent Chl breakdown, in both species. Phyllobilins and PAO levels diminished again in leaves from rehydrated plants. We conclude that water deficit‐induced poikilochlorophylly occurs via the well‐characterized PAO/phyllobilin pathway of Chl breakdown and that this mechanism also appears conserved in a resurrection species displaying homoiochlorophylly. The roles of the PAO/phyllobilin pathway during different plant developmental processes that involve Chl breakdown, such as leaf senescence and desiccation, fruit ripening and seed maturation, are discussed.     

Characterization and PCR multiplexing of polymorphic microsatellite loci in cashew (Anacardium occidentale L.) and their cross‐species utilization

Cashew (Anacardium occidentale L.) is the most economically important tropical nut crop in the world, and yet there are no sequence tagged site (STS) markers available for its study. Here we use an automated, high‐throughput system to isolate cashew microsatellites from a non‐enriched genomic library blotted onto membranes at high density for screening. Sixty‐five sequences contained a microsatellite array, of which 21 proved polymorphic among a closely related seed garden population of 49 genotypes. Twelve markers were suitable for multiplex analysis. Of these, 10 amplified in all three related tropical tree species tested: Anacardium microcarpum, Anacardium pumilum and Anacardium nanum.     

Two new species of <Emphasis Type="Italic">Cotingacola</Emphasis> Carriker,Phthiraptera: Ischnocera: Philopteridae) from Amazonian Brazil,with comments on host-specificity

This paper describes two new species of Cotingacola Carriker, 1956, C. lutzae n. sp. and C. engeli n. sp., from the cinereous mourner Laniocera hypopyrra (Vieillot) and black-necked red cotinga Phoenicircus nigricollis Swainson (Aves: Passeriformes: Cotingidae), respectively. These species are fully illustrated and compared with their morphologically closest relatives. With the addition of these two new species, this Neotropical chewing louse genus now includes ten species. We also present records from Brazil for two previously described species, C. stotzi Clayton & Price, 1998 and C. parmipapillae Carriker, 1956. The re-examination of a specimen identified as ‘Cotingacola sp.’ in a recent phylogenetic study confirmed that it is C. parmipapillae and that Querula purpurata (Statius Müller) regularly hosts two Cotingacola species, which means that at least three cotingid hosts carry more than one species of Cotingacola. We comment on the variability in the host-specificity of Cotingacola species.     

Cephalanthera exigua rediscovered: new insights in the taxonomy,habitat requirements and breeding system of a rare mycoheterotrophic orchid

Five mycoheterotrophic species of Cephalanthera have been proposed from tropical Asia. Until recently, all of them were only known from the type specimens, for which reason it has been difficult to judge if some of them were actually conspecific. The recent discovery of two large populations of C. exigua in Thailand made it possible to describe the range of variation of this species in much more detail, and we then found it to be well distinguished from the others. A phylogenetic analysis based on nuclear rDNA ITS sequences corroborated the systematic position of C. exigua at the generic level. Examination of the previously neglected type of C. pusilla from Myanmar lead to the conclusion that this taxon of disputed generic affinity likewise belongs to Cephalanthera, and that it is a clearly distinct species. Finally, we accept that also C. ericiflora from Laos as well as C. gracilis and C. calcarata from Yunnan are probably distinct species. Cephalanthera exigua grows in hill evergreen forest, but we found little floristic similarity between its two localities in Thailand (IS_S=22.5%). Ectomycorrhizal trees of the families Fagaceae and Ulmaceae are probably the ultimate carbon source for C. exigua at both sites, as the mycorrhizal partners of this orchid (basidiomycetes of the family Thelephoraceae) obtain their carbon nutrition through ectomycorrhizal association with forest trees. Pollination experiments demonstrated that the nectarless flowers of C. exigua are not spontaneously autogamous. Based on the pollination syndrome and previously published observations on other species of the genus, we suggest that C. exigua has an insect‐operated breeding system acting by deceit, and that the pollinators are probably solitary bees.     

Ecological germination requirements of the aggregate species Teucrium pumilum (Labiatae) endemic to Spain

Teucrium pumilum L., T. lepicephalum Pau, T. libanitis Schreber, T. turredanum Losa & Rivas Goday, and T. carolipaui Vicioso ex Pau (Labiatae) constitute an aggregate included in the Pumilum subsection (Polium section). These species are endemic and geographically restricted to central and south-eastern Spain. They grow under different climatic conditions and have different edaphic requirements, particularly the gypsum and lime affinities. The effects of some ecological factors (i.e. edaphic and climatic factors) on the germination parameters of these Teucrium species were investigated. The species under study differed in their germination behaviour. The majority of the germination parameters appeared more correlated to edaphic than to climatic factors and particularly to the water availability in the soil. The calcicolous T. carolipaui, which grows in the driest habitat, had a more rapid germination than the gypsophilous species. This could be due to the existence of a narrower optimal germination period for T. carolipaui. The different germination requirements of these species of Teucrium are important results to be considered in conservation programs of these endemic and endangered taxa. Section Editor: H. Lambers     

Host acceptance behaviour of the jack pine tip beetle, Conophthorus anksianae and the red pine cone beetle, C. resinosae

The jack pine tip beetle, Conophthorus banksianae McPherson (Coleoptera: Scolytidae) and the red pine cone beetle, C. resinosae Hopkins, are doubtful sibling species. However, it is possible that these two taxa are valid species that maintain reproductive isolation because they accept different hosts. In a series of laboratory and field cage experiments, the host acceptance behaviours of these two species under choice and no choice conditions were compared. The field experiments demonstrated that the two species have a similar propensity to accept jack pine cones and shoots for feeding, but differ consistently in their acceptance of red pine cones, and variably in their acceptance of red pine shoots. However, the laboratory experiments did not indicate a difference between the two species in their propensity to accept red pine cones for feeding. In field cages, C. resinosae accepted significantly more red pine cones for oviposition than C. banksianae; the situation was reversed for jack pine shoots. In comparison to C. banksianae, C. resinosae is a more generalist feeder. The results from this study suggest that host acceptance behaviour is a permeable barrier unable to ensure reproductive isolation between the two species. Although there are differences in the host acceptance behaviours between C. banksianae and C. resinosae, we conclude that these differences do not necessarily support their designation as distinct species.     

Functional consequences of morphological differentiation between populations of the Cape Dwarf Chameleon (Bradypodion pumilum)

Variation in phenotype between species or populations of the same species living in different habitats is often explained in an adaptive context with local habitat differences driving selection on morphological traits relevant in a given ecological context. Previous studies have demonstrated significant differences in limb and tail morphology between populations of the Cape Dwarf Chameleon (Bradypodion pumilum) living in closed vs. open habitats. However, the adaptive nature of the observed differences remains unclear. Here, we quantify the structural habitat use in two different populations, test whether the random habitat differs between the two sites and whether or not chameleons select perches randomly. Next, we test whether morphology is correlated with structural habitat use and test for differences in performance between populations. Our results demonstrate that habitats are structurally different, that chameleons in the two populations use perches of different diameters and that, in one of the populations, chameleons select relatively wider perches than available at random. Performance traits (hand and tail grip performance and sprint speed) are correlated with morphology (hand size, tail length and tibia length) and differ between sexes and populations. Moreover, performance is dependent on dowel size. These results suggest that differences in performance between populations are indeed adaptive and indicate the existence of true ecomorphs in chameleons of the genus Bradypodion. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 692–700.     

Studies on two species of Asellus in Rutland Water

The study comprised a comparison of the ecology of two species of Asellus (A. aquaticus and A. meridianus), and had two main aims. First the energy budget of the population of A. aquaticus, which is one of the most abundant species in the reservoir was assessed. Secondly, the two species appear to be ecologically very similar and it has been assumed by many workers that they are in competition. The study aimed to investigate how these two species might be able to coexist.The population dynamics of both species and the interaction between them is under study, as are their relations with the other benthic animals recorded in the reservoir (see Bullock et al. 1982). The sampling programme to cover variations in depth and substratum is also described by Bullock et al. 1982. A. aquaticus occurs in both arms while A. meridianus exists only in the south arm. A preliminary examination of the available data reveals that the density of A. aquaticus has increased in the south arm while the density of A. meridianus decreased drastically in February 1980. Since then low numbers of A. meridianus have been recorded every month even in the summer (breeding) season. It is noteworthy that simultaneously the number of predators, particularly Helobdella stagnalis, increased in comparison with previous years.It was therefore decided to study the predation on Asellus in the laboratory as well as in the field by using a serological technique. Antisera were produced against each species of Asellus by injections into rabbits of precipitated proteins (antigens). Thus a positive reaction obtained from a homogenised predator when tested with the specific antiserum indicates which species of Asellus had been consumed and the percentage of predators which had fed on Asellus in every sample could be calculated. The results obtained to date reveal that considerable numbers of Asellus spp. are removed from the environment by the predators (Helobdella stagnalis, Erpobdella octoculata, Polycelis tenuis. Dendrocoelum lacteum and Dugesia lugubris).Size class structures of the population of both species were constructed to study their life cycles and to estimate the population production. Comparitive respirometry of the two species was carried out at 4 °C, 10 °C and 16 °C, using a Gilson differential respirometer. This study aimed firstly to assess one of the essential parameters in the energy budget study of A. aquaticus and secondly, since A. aquaticus appears to be more active than A. meridianus, it would be expected that a comparison of their metabolic rate at different temperature would reveal marked differences between two species. It was found that there was no significant difference between the two species at 4 °C and 10 °C, but A. aquaticus had a significantly higher metabolic rate than A. meridianus at 16 °C.Consumption and assimilation of decaying oak leaves and of Cladophora glomerata (both of which are available in the environment) by two species of Asellus are being measured at present in the laboratory at three different temperatures (4 °C, 10 °C and 16 °C), and will contribute to the estimation of energy budgets.     

Molecular relationships between closely related strains and species of nematodes

Summary Electrophoretic comparisons have been made for 24 enzymes in theBergerac andBristol strains ofCaenorhabditis elegans and the related species,Caenorhabditis briggsae. No variation was detected between the two strains ofC. elegans. In contrast, the two species,C. elegans andC. briggsae exhibited electrophoretic differences in 22 of 24 enzymes. A consensus 5S rRNA sequence was determined forC. elegans and found to be identical to that fromC. briggsae. By analogy with other species with relatively well established fossil records it can be inferred that the time of divergence between the two nematode species is probably in the tens of millions of years.The limited anatomical evolution during a time period in which proteins undergo extensive changes supports the hypothesis that anatomical evolution is not dependent on overall protein changes.     

New evidence for the use of root foods in pre-agrarian subsistence recovered from the late Mesolithic site at Halsskov, Denmark

Roots and tubers, together with other plant storage organs such as rhizomes, bulbs, corms, etc. are known to be a source of human food. Until very recently, however, remains of root foods were rarely identified from archaeological contexts in temperate Europe. New evidence for the use of root foods has been recently recovered from the early Erteb?lle settlement at Halsskov in Denmark. Remains included charred bulbs of Allium cf. ursinum and tubers of Conopodium majus. They were accompanied by charred remains of parenchymatous tissues derived from underground parts of other plants. All parenchymatous remains were recovered from features that could be interpreted as pit-cooking depressions. A minimum of two periods of root food gathering can be assumed, spring to early summer and autumn. The presence of charred seeds of Nuphar pumilum suggests that either the seeds or rhizomes (or both) were use as food. Received September 4, 2001 / Accepted February 27, 2002     

A New Molecular Phylogeny and a New Genus,Pendulorchis, of the Aerides–Vanda Alliance (Orchidaceae: Epidendroideae)

Background

The Aerides–Vanda alliance is a complex group in the subtribe Aeridinae (subfamily Epidendroideae, Orchidaceae). Some phylogenetic systems of this alliance have been previously proposed based on molecular and morphological analyses. However, several taxonomic problems within this alliance as well as between it and its allies remain unsolved.

Methodology/Principal Findings

We utilized ITS and five plastid DNA regions in this phylogenetic analysis. Consensus trees strongly indicate that the Aerides–Vanda alliance is monophyletic, and the 14 genera of this alliance can be grouped into the following clades with 14 subclades: 1. Aerides, comprising two subclades: Rhynchostylis and Aerides; 2. Ascocentropsis; 3. Papilionanthe; 4. Vanda, comprising five subclades: Neofinetia, Christensonia, Seidenfadenia, Ascocentrum, and Vanda–Trudelia, in which Vanda and Trudelia form a subclade; 5. Tsiorchis, comprising three subclades: Chenorchis, Tsiorchis, and two species of Ascocentrum; 6. Paraholcoglossum; and 7. Holcoglossum. Among the 14 genera, only Ascocentrum is triphyletic: two species of the Ascocentrum subclade, an independent subclade Ascocentrum subclade in the Tsiorchis clade; the Ascocentrum subclade in the Vanda clade; and one species in the Holcoglossum clade. The Vanda and Trudelia species belong to the same subclade. The molecular conclusion is consistent with their morphological characteristics.

Conclusions

We elucidate the relationship among the 14 genera of the Aerides–Vanda alliance. Our phylogenetic results reveal that the Aerides–Vanda alliance is monophyletic, but it can be divided into 14 genera. The data prove that Ascocentrum is triphyletic. Plants with elongate-terete leaves and small flowers should be treated as a new genus, Pendulorchis. Saccolabium himalaicum (Ascocentrum himalaicum) should be transferred to Pendulorchis. Ascocentrum pumilum, endemic to Taiwan, should be transferred to Holcoglossum. A new combination, Holcoglossum pumilum, was also established. Trudelia should not be recognized as an independent genus. Two new species, Pendulorchis gaoligongensis and Holcoglossum singchianum, were described as well.