Molecular phylogeny of Juglans (Juglandaceae): a biogeographic perspective

The eastern Asian and eastern North American disjunction in Juglans offers an opportunity to estimate the time since divergence of the Eurasian and American lineages and to compare it with paleobotanical evidence. Five chloroplast DNA noncoding spacer (NCS) sequences: trnT−trnF, psbA−trnH, atpB−rbcL, trnV-16S rRNA, and trnS-trnfM and data from earlier studies (matK, ITS, and nuclear RFLP) were used to reconstruct phylogeny and to estimate the divergence time of major lineages. Seventeen taxa from four sections of Juglans and two outgroup taxa, Pterocarya stenoptera and Carya illinoiensis were included. NCS data was congruent only with matK data. Both maximum parsimony (MP) and maximum likelihood (ML) cladograms were concordant at the sectional level and revealed three well-supported monophyletic clades corresponding to sections Juglans, Cardiocaryon, and Rhysocaryon in both NCS and combined analyses. The single extant American butternut, Juglans cinerea was placed within the poorly resolved, but well-supported Rhysocaryon. Placement of taxa within Rhysocaryon and Cardiocaryon were inconsistent between NCS and combined analyses. Overall, the results suggest that: (1) the NCS sequence divergence observed within and between sections of Juglans is low and the addition of matK data only marginally improved resolution within Rhysocaryon; (2) the early divergence of section Juglans in both MP and ML analyses of NCS and combined data implies its ancient origin in contrast to fossil evidence, which suggests the earliest divergence of sections Rhysocaryon and Cardiocaryon; and (3) the extant taxa may not hold the footprints to unravel the evolutionary history of the genus.     

Glandular trichomes of Ceratotheca triloba (Pedaliaceae): morphology, histochemistry and ultrastructure

This study was initiated to characterize the distribution, morphology, secretion mode, histochemistry and ultrastructure of the glandular trichomes of Ceratotheca triloba using light and electron microscopy. Its leaves bear two morphologically distinct glandular trichomes. The first type has long trichome with 8-12 basal cells of pedestal, 3-14 stalk cells, a neck cell and a head of four cells in one layer. The second type has short trichome comprising one or two basal epidermal cells, a unicellular or bicellular stalk and a multicellular head of two to eight cells. There is a marked circular area in the upper part of each head cell of the long trichome. This area is provided with micropores to exudate directly the secretory product onto the leaf surface by an eccrine pathway. The secretory product has copious amount of dark microbodies arising from plastids which are positive to Sudan tests and osmium tetroxide for unsaturated lipids. The secretion mode of short trichomes is granulocrine and involves two morphologically and histochemically distinct vesicle types: small Golgi-derived vesicles which are positive to Ruthenium Red test for mucilaginous polysaccharides; the second type is dark large microbodies similar to that of long trichomes with low quantity. These two types are stored in numerous peripheral vacuoles and discharge their contents accompanied by the formation of irregular invaginations of the plasmalemma inside the vacuoles via reverse pinocytosis. These two secretion modes of long and short trichomes are reported for the first time in the family Pedaliaceae. The long trichomes have more unsaturated lipids, while the short trichomes contain more mucilaginous polysaccharides.     

Flowering phenology and wind-pollination efficacy of heterodichogamous Juglans mandshurica (Juglandaceae)

BACKGROUND AND AIMS: Heterodichogamy differs from normal dichogamy, in that it involves two mating types (protogyny and protandry) that occur at a 1 : 1 ratio in a population. Flowering phases of the two mating types are synchronized and reciprocal, which was considered to ensure between-type outcrossing. This study aims to quantify the flowering pattern and pollination efficacy in Juglans mandshurica, a wind-pollinated heterodichogamous tree. METHODS: The pattern of flowering phenology was monitored within individual trees and pollen traps were used to measure air-borne pollen loads during the spring in 2003 and 2004. Pollen longevity was determined by staining technique. Also a pollen supplementation experiment was performed in 2004 to assess pollen limitation of fruit production. KEY RESULTS: There was no overlap between sexual functions within individual trees. Flowering periods of the two mating types were reciprocal and synchronous in both 2003 and 2004. Air-borne pollen loads were large, and protogynous and protandrous individuals each produced a high pollination peak, consistent with the two blooming periods. Maximum pollen longevity was about 4 h for protandrous individuals, and 3 h for protogynous individuals. Pollen supplementation did not increase fruit production in either protogynous or protandrous individuals. CONCLUSIONS: Heterodichogamous flowering in Juglans mandshurica effectively avoids selfing, promotes between-type outcrossing, and leads to efficient pollination in a natural population.     

Mating patterns and pollen dispersal in a heterodichogamous tree, Juglans mandshurica (Juglandaceae)

Mating patterns in heterodichogamous species are generally considered to be disassortative between flowering morphs, but this hypothesis has hitherto not been vigorously tested. Here, mating patterns and pollen dispersal were studied in Juglans mandshurica, a heterodichogamous wind-pollinated species that is widely distributed in northern and north-eastern China. Paternity analyses carried out on 11 microsatellite loci were used to estimate morph-specific rates of outcrossing and disassortative mating. Pollen dispersal and genetic structure were also investigated in the population under study. The mating pattern of J. mandshurica was highly outcrossing and disassortative. Pairwise values of intramorph relatedness were much higher than those of intermorph relatedness, and a low level of biparental inbreeding was detected. There was no significant difference in outcrossing and disassortative mating rates between the two morphs. The effective pollen dispersal distribution showed an excess of near-neighbor matings, and most offspring of individual trees were sired by one or two nearby trees. These results corroborate the previous suggestion that mating in heterodichogamous plant species is mainly disassortative between morphs, which not only prevents selfing but also effectively reduces intramorph inbreeding.     

Flora Palinologica Italiana,Sezione Aeropalinologica —S 238:Juglans regia L. (Juglandaceae)

Riassunto In accordo con il programma ?Flora Palinologica Italiana, Sezione Aeropalinologica? viene presentata la scheda morfopalinologica diJunglans regia L. (noce) relativa a polline fresco ed acetolizzato.      

Morphology, ontogeny and histochemistry of secretory trichomes of Geranium robertianum (Geraniaceae)

Geranium robertianum bears three types of glandular uniseriate trichomes which originate from a single protodermal cell and develop through periclinal divisions. Type I trichomes are procumbent and have an oval apical cell, two stalk cells and a basal cell. Type II trichomes are erect and have a pear shaped apical cell, two stalk cells and a basal cell. Type III trichomes are much longer than the other two types and have an elongated apical cell, five long stalk cells and a basal cell. Type I and type II trichomes are common on leaves while III trichomes are more abundant on flower structures.
Type I and type II trichomes secrete terpenoids and phenols. Type III trichomes are characterized by the accumulation of anthocyanins in the apical cell and secrete flavonoids.     

Foliar secretory trichomes of Ocimum obovatum (Lamiaceae): micromorphological structure and histochemistry

This study characterises the micromorphology, ultrastructure and main chemical constituents of the foliar glandular trichomes of Ocimum obovatum using light and electron microscopy and a variety of histochemical tests. Two types of glandular trichomes occur on the leaves: large peltate and small capitate. The head of each peltate trichome is made up of four broad head cells in one layer. The head of each capitate trichome is composed of two broad head cells in one layer (type I) or a single oval head cell (type II, rare). In peltate heads, secretory materials are gradually transported to the subcuticular space via fracture in the four sutures at the connecting walls of the head cells. Release to the head periphery occurs through opposite fracture in the four sutures in the head cuticle. In type I capitate trichomes, release of the secretions to the subcuticular space occurs via a pore between the two head cells, and release to the head periphery occurs through the opposite pore in the head cuticle. In type II capitate trichomes, the secreted material is released from the head cell through a ruptured particular squared area at the central part of the head cuticle. These secretion modes are reported for the first time in the family Lamiaceae. Histochemical tests showed that the secretory materials in the glandular trichomes are mainly essential oils, lipophilic substances and polysaccharides. Large peltate trichomes contain a large quantity of these substances than the small capitate trichomes. Ultrastructural evidence suggests that the plastids produce numerous lipid droplets, and the numerous polysaccharide small vesicles are derived from Golgi bodies.     

Scanning electron microscopy of trichomes of cheilanthes (sinopteridaceae)

Brittonia - The trichomes of two problematic complexes in the genusCheilanthes were studied. In one group, the trichomes ofC. tomentosa are long, multicellular, uniseriate, and flat whereas those...     

Phylogeny and biogeography of Juglans (Juglandaceae) based on matK and ITS sequence data

We investigated phylogenetic and biogeographic relationships within Juglans (walnuts), a Tertiary disjunct genus, using 15 species of Juglans and related (Juglandaceae) outgroups. The relationships were analyzed using nucleotide sequences of the chloroplast gene matK and its flanking spacers and of the internal transcribed spacers (ITS) and 5.8S gene of the nuclear ribosomal DNA. The DNA sequences provided 246 informative characters for parsimony analysis. ITS data supported as monophyletic groups the four generic sections, Cardiocaryon, Dioscaryon, Rhysocaryon, and Trachycaryon. Within Rhysocaryon, the temperate black walnuts and the tropical black walnuts were supported as monophyletic groups. When the two data sets were combined, J. cinerea was nested within Cardiocaryon. Combined analysis with published nuclear DNA restriction site data placed J. cinerea in a monophyletic group with Cardiocaryon. These analyses consistently supported Juglans as a monophyletic group and as the sister group to the genus Pterocarya. The results of this work are consistent with the known geological history of Juglans. The fossil record suggests that the butternuts had evolved by the early Oligocene in North America. The presence of butternuts in Eurasia could be the result of migration from North America to Eurasia during the warming trend of the mid Oligocene.     

Morphology and histochemistry of the glandular trichomes of Lippia scaberrima (Verbenaceae)

BACKGROUND AND AIMS: Lippia scaberrima, an aromatic indigenous South African plant, with medicinal application, potentially has economic value. The production of essential oil from this plant has not been optimized, and this study of the chemico-morphological characteristics was aimed at determining the location of oil production within the plant. Furthermore, the locality of other secondary metabolites important in medicinal applications needed to be ascertained. This information would be useful in deciding the protocol required for isolation of such compounds. METHODS: The morphology of the glandular trichomes was investigated using a combination of scanning electron and light microscopy. Concurrently, the chemical content was studied by applying various chemical reagents and fluorescence microscopy. KEY RESULTS: Three types of trichomes were distinguished on the material investigated. Large, bulbous peltate glands containing compounds of terpenoid nature are probably the main site of essential oil accumulation. Small glands were found to be both peltate and capitate and fluorescent stain indicated the possible presence of phenolic compounds. The third type was a slender tapered seta with an ornamented surface and uniseriate base, and evidently secretory in nature. CONCLUSIONS: This study linking the chemical content and morphology of the glandular trichomes of L. scaberrima has contributed to the knowledge and understanding of secretory structures of Lippia spp. in general.     

Periods of organogenesis in mono- and bicyclic annual shoots of Juglans regia L. (Juglandaceae)

The organogenetic cycle of shoots on main branches of 4-year-old Juglans regia trees was studied. Mono- and bicyclic floriferous and vegetative annual shoots were analysed. Five parent annual shoot types were sampled between October 1992 and August 1993. Organogenesis of summer growth units was monitored between 16 Jun. and 3 Aug. 1993. Variations over time in the number of nodes, cataphylls and embryonic green leaves of terminal buds were studied. The number of nodes of parent shoot buds was compared with the number of nodes of shoots derived from parent shoot buds. The spring growth units of mono- and bicyclic shoots consist exclusively of preformed leaves which were differentiated, respectively, during the spring flush of growth (mid-April until mid-May) or the summer flush of growth (mid-June until early August) in the previous growing season. Thus, winter buds may consist of flower and leaf primordia differentiated in two different periods during annual shoot extension. The summer growth units of bicyclic shoots consist of preformed leaves that were differentiated in spring buds during the spring flush of growth in the current growing season. Bud morphology is compared between spring and summer shoots.     

The relationship between phenology of pistillate flower organogenesis and mode of heterodichogamy in Juglans regia L. (Juglandaceae)

 We investigated the degree of organogenesis completed at the end of the growing season in pistillate flowers of heterodichogamous Juglans regia, English or Persian walnut. Terminal buds from paired cultivars, one each protandrous and protogynous, chosen to represent early, midseason and late leafing walnuts, were examined by scanning electron microscopy. Results indicate that pistillate floral primordia in protandrous individuals had not progressed beyond involucre initiation during the season prior to bloom. In protogynous individuals, floral differentiation had progressed to the initiation of perianth primordia. These observations are compared with an earlier report on staminate flower differentiation in the same cultivars where a comparable, but opposite, relationship exists. We conclude that the degree of differentiation in both staminate and pistillate flowers that must be completed between the time growth resumes in the spring and anthesis is a developmental determinant of the mode of heterodichogamy in walnut. Received: 15 June 1996 / Revision accepted: 25 October 1996     

Light, conventional and environmental scanning electron microscopy of the trichomes of Cucurbita pepo subsp. pepo var. styriaca and histochemistry of glandular secretory products

BACKGROUND AND AIMS: In the present study, the differences between glandular and non-glandular trichomes, the secretory process and the method of secretion were studied. Previous studies on leaves of Styrian oil pumpkin (Cucurbia pepo var. styriaca) plants have shown that four morphologically and ontogenetically independent glandular and non-glandular trichome types and one bristle hair type can be distinguished. The four types of trichomes can be categorized into three glandular trichome types: type I, a short-stalked trichome with four head cells including a 'middle-cell', two stalk cells and one basal cell; type II, a long-stalked trichome with two head cells, a 'neck-cell' region and a long stalk area; type IV, a 'stipitate-capitate' trichome with a mesophyll cell basement, a short stalk and a multicellular head; type III, a non-glandular 'columnar-digit' trichome, which consists of two head cells continuous with three-celled stalk, and the basal cell. METHODS: The histochemical studies (the main classes of metabolite in secreted material of glandular trichomes) were conducted in fresh and fixed hand sections, using the following tests: Sudan black B, Nile blue A, osmium tetroxide, neutral red, Naturstoffreagent A, FSA (fuchsin-safranin-astra blue), NADI (naphthol + dimethylparaphenylenediamine) and ruthenium red. Each suggested differences in the intercalations during the ontogenetical development of each trichome during the development stage. KEY RESULTS: The histochemical reactions revealed the main components of the materials secreted by all types of trichomes, which include lipids, flavones and terpenes and the different cell wall compositions. Glandular secretions were observed during environmental scanning electron microscopy (ESEM) and the trichomes compared with those seen by conventional scanning electron microscopy (CSEM). CONCLUSIONS: Scanning electron microscopy and histochemical analysis demonstrated that each of the trichomes studied produced and released secretory products in a characteristic way.     

Leaf glandular trichomes in Empetrum nigrum: morphology,histochemistry, ultrastructure and secondary metabolites

The morphology, histochemistry and ultrastructure of the glandular trichomes on Empetrumnigrum leaves have been studied and more than a third of the metabolites were identified. Samples of the leaves were fixed and processed for light and electron microscopy. Glandular trichomes are situated on the inner surface of the rolled leaves. They have a clavate head and a short stalk. Histochemical tests and fluorescent microscopy demonstrate differentiated staining of the various cell types in the glandular trichome for proteins, pectins, lipids, tannins and phenylpropanoids. During secretion, the secretory cells contain rough and smooth endoplasmic reticulum, Golgi stacks with large vesicles, diversiform leucoplasts in contact with a reticular sheath and opaque deposits in the vacuoles. There are ultrastructural and functional differences between the secretory cells in the trichome head: synthesis of hydrophilic substances predominately occurs in the upper and middle secretory cells, whereas synthesis of lipophilic compounds takes place in the middle and lower secretory cells. Gas chromatography–mass spectrometry was used to determine the content of metabolites in the methanol extracts from the leaves. Many phenolic compounds (phenolic acids, bibenzyls, catechins, flavanones and flavan‐3‐ols) as well as several terpenoids were found. Two chalcones (2′,4′‐dimethoxydihydrochalcone and 2′,4′,6′‐trihydroxydihydrochalcone), one bibenzyl (batatasin III), one flavanone (7‐hydroxyflavanone) and 8 terpenoids (including phytol, α‐tocopherol, ß‐sitosterol, α‐amyrin, uvaol, oleanolic acid, ursolic acid and dehydroursolic acid) were identified in E. nigrum leaf extracts. The total yield of phenolic compounds is five to six times higher than the yield of terpenoids. It has been established that chalcones have no hydroxyl groups in ring B whereas bibenzyls have a hydroxyl group in the 3‐position in ring B. On the basis of the histochemistry, fluorescent microscopy, ultrastructure and chemical analysis, it may be concluded that synthesis and accumulation of phenolic substances and terpenoids takes place in the clavate glandular trichomes. Secondary metabolites synthesized in the trichomes protect leaf tissues from viruses, bacteria and pathogenic fungi.     

Role of trichomes and pericarp in the seed biology of the desert annual Lachnoloma lehmannii (Brassicaceae)

In many angiosperms, the fruit rather than the seed is the dispersal/germination unit, and this is the case with Lachnoloma lehmannii, a desert annual ephemeral in central southwestern Asia with indehiscent nonmucilaginuous silicles covered with trichomes. The primary aim of this study was to assess the role of trichomes and pericarp in dispersal, anchorage of diaspores, and seed germination of this species. Mature silicles are dispersed by wind and gravity, and trichomes not only significantly increased their dispersal distance, adherence to sandy soil particles, mass of water imbibed and moisture content, but also decreased the rate of water loss and moisture content of seeds. A significantly higher percentage of seeds within silicles than of isolated seeds retained viability after exposure to 60 °C for 24 h. Seed dormancy is due to the pericarp and to nondeep physiological dormancy, as shown by the increase in germination percentage of isolated seeds following dry storage and treatment with GA₃. Removal of pericarp increased germination of 6-month-old seeds from 0 to 80–90 %, and leachate from both pericarp and trichomes significantly inhibited germination of isolated seeds. Ninety-five percent of seeds within silicles buried in soil for 2 years were viable, but only 28 % of them germinated in light at 15/2 °C; thus L. lehmannii forms a persistent soil seed bank. The pericarp and its trichomes may maximize plant fitness by determining the settlement location of silicles, thus helping to ensure that seeds germinate during the cool season for seedling survival in the desert environment.     

The fluorescent probe HPTS as a phloem-mobile, symplastic tracer: an evaluation using confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) has been used to evaluatethe use of the fluorescent probe HPTS (8-hydroxypyrene-1,3,6-trisulphonicacid) as a symplastic tracer. HPTS-acetate was used to loadHPTS into the phloem and its movement was followed in threesystems where symplastic unloading has been proposed. In Arabidopsisroot tips and Abutilon nectaries the intercellular distributionof HPTS differed markedly from that observed with 5-(and 6)-carboxyfluorescein(CF)- HPTS was observed in the nuclei and cytoplasm whilst CFwas rapidly transferred into the vacuoles. In contrast, bothHPTS and CF accumulated in the vacuoles of the vascular parenchymaand nucellus cells following unloading from the phloem of thedeveloping barley caryopsis. The results indicate that HPTShas a number of advantages as a symplastic probe compared withCF. The findings are discussed in relation to the influenceof vacuolar sequestration on dye distribution. Key words: Confocal laser scanning microscopy (CLSM), HPTS, intercellular transport, phloem (unloading)     

Genetic variation in walnuts (Juglans regia and J. sigillata; Juglandaceae): Species distinctions, human impacts, and the conservation of agrobiodiversity in Yunnan, China

Walnuts are a major crop of many countries and mostly cultivated in large-scale plantations with few cultivars. Landraces provide important genetic reservoirs; thus, understanding factors influencing the geographic distribution of genetic variation in crop resources is a fundamental goal of agrobiodiversity conservation. Here, we investigated the role of human settlements and kinship on genetic variation and population structure of two walnut species: Juglans regia, an introduced species widely cultivated for its nuts, and J. sigillata, a native species cultivated locally in Yunnan. The objectives of this study were to characterize sympatric populations of J. regia and J. sigillata using 14 molecular markers and evaluate the role of Tibetan villages and kin groups (related households) on genotypic variation and population structure of J. regia and J. sigillata. Our results based on 220 walnut trees from six Tibetan villages show that although J. regia and J. sigillata are morphologically distinct, the two species are indistinguishable based on microsatellite data. Despite the lack of interspecific differences, AMOVAs partitioned among villages (5.41%, P = 0.0068) and kin groups within villages (3.34%, P = 0.0068) showed significant genetic variation. These findings suggest that village environments and familial relationships are factors contributing to the geographic structure of genetic variation in Tibetan walnuts.     

Comparative flower development of Juglans regia,Cyclocarya paliurus and Engelhardia spicata: homology of floral envelopes in Juglandaceae

Homology among reproductive structures is pivotal for understanding angiosperm evolution. In Juglandaceae, homologies of floral parts have been disputed due to morphological variability in flowers. Previous interpretations were based mainly on mature flowers and lacked developmental studies. We investigated the unisexual flower development of Juglans regia, Cyclocarya paliurus and Engelhardia spicata using scanning electron microscopy. The ‘floral envelope’ of staminate flowers in J. regia and C. paliurus consists of a bract and several tepals. Six tepals are initiated in a whorled pattern in J. regia, whereas four to six tepals are arranged in a variable pattern in C. paliurus. The three‐lobed bract of E. spicata results from the cleavage of an entire bract, rather than adnation of the bract and its bracteoles. Pistillate flowers of J. regia and C. paliurus, subtended by a bract and a horseshoe‐shaped bracteole, usually have four tepals initiating simultaneously in one whorl. The organogenesis patterns of the ‘floral envelope’ and stamens in staminate flowers show greater diversity, indicating different degrees of reduction among genera. Our floral developmental data support the division of Juglandaceae into two subfamilies and then further into three tribes.