Sciadopitys Research Articles

The Broken Chloroplast Gene Clusters in Gymnosperms Exhibit Elevated Substitution Rates

Plant chloroplast (cp) gene clusters consist of genes arranged closely together on the cp genome. These genes are organized in operon structures and participate in cotranscription, typically exhibiting conservation. Broken gene clusters have been observed in gymnosperms. In order to investigate whether the substitution rates and selection pressure of associated genes are affected following the disruption of gene clusters, the cp genomes of 80 species (78 gymnosperms and 2 outgroups) were analyzed. A phylogenetic analysis was conducted using 58 shared genes to examine the evolutionary rates and selection pressure of genes associated with gene clusters and protein-coding genes in Sciadopitys verticillata. The results demonstrate that S. verticillata exhibited the highest number of rearrangements compared to the Cycas revoluta genome. Four gene clusters (rps2, psbB, rpoB, and petL clusters) in S. verticillata were disrupted, while rps2 in Callitris rhomboidea experienced disruption. Significantly increased evolutionary rates were observed in 12 out of 18 gene cluster-related genes in S. verticillata. Following disruption, S. verticillata and C. rhomboidea exhibited an increase in gene cluster-related genes, particularly rps2, and higher selection pressure on both rps2 and atpA genes compared to other species. Furthermore, among the 58 genes shared by S. verticillata, the evolutionary rates of 36 genes increased, and the selection pressure on 13 genes exceeded that of other species. These results indicate an increased substitution rate of gene clusters in S. verticillata and C. rhomboidea. The large-scale rearrangement and elevated substitution rates of the cp genome in S. verticillata were revealed. This study sheds light on the heterogeneity of cp genome evolution in gymnosperms.

The D14 andKAI2 Orthologs ofGymnosperms Sense Strigolactones andKL Mimics, Respectively, andthe Signals Are Transduced to Control Downstream Genes.

Strigolactones (SLs), lactone-containing carotenoid derivatives, function as signaling molecules in the rhizosphere, inducing symbiosis with arbuscular mycorrhizal. In addition, as a class of plant hormones, SLs control plant growth and development in flowering plants (angiosperms). Recent studies show that the ancestral function of SLs, which precede terrestrialization of plants, is as rhizosphere signaling molecules. SLs were then recruited as a class of plant hormones through the step-by-step acquisition of signaling components. The D14 gene encoding the SL receptor arose by gene duplication of KARRIKIN INSENSITIVE2 (KAI2), the receptor of karrikins and KAI2 ligand (KL), an unknown ligand, in the common ancestor of seed plants. KL signaling targets SMAX1, a repressor protein. On the other hand, the SL signaling targets SMXL78 subclade repressors, which arose by duplication of SMAX1 in angiosperms. Thus, gymnosperms contain the SL receptor D14 but not SMXL78, the SL signaling-specific repressor proteins. We studied two gymnosperm species, ginkgo (Ginkgo biloba) and Japanese umbrella pine (Sciadopitys verticillata), to clarify whether SLs are perceived and the signals are transduced in gymnosperms. We show that D14 and KAI2 of ginkgo and Japanese umbrella pine specifically perceive an SL analog and KL mimic, respectively. Furthermore, our results suggest that both SL signaling and KL signaling target SMAX1, and the specific localization of the receptor may result in the specificity of the signaling in gymnosperms.

A new structurally preserved fossil umbrella pine from the Jurassic of East Asia

The Sciadopityaceae are today a monotypic and relict family of the Coniferales, restricted to Japan with an endemic species of Sciadopitys verticillata Sieb. et Zucc., known as umbrella pine. Megafossil remains assigned to the Sciadopityaceae have been reported from various parts of the boreal hemisphere. However, structurally preserved fossil wood of the Sciadopityaceae is poorly documented. Here we present an anatomically preserved fossil wood of the Sciadopityaceae from the Jurassic of East Asia. A new fossil wood Zhangoxylon yanliaoense gen. et sp. nov. is proposed for the specimens, which are anatomically characterized by a heterogeneous pith, endarch primary xylem, and special secondary xylem. The anatomical structures of the new genus are similar to those of the living Sciadopitys except for the type of radial pitting. The present specimen has an araucarian radial pitting, while that of the living Sciadopitys is abietinean. Therefore, Zhangoxylon gen. nov. from the Middle to Late Jurassic (Callovian to Kimmeridgian) in western Liaoning may represent the Sciadopitys‐like petrified wood which bears pith and primary xylem. This new fossil, so far, the best‐known fossil wood of the family Sciadopityaceae, sheds light on the early evolutionary history of the family Sciadopityaceae, and contributes to a better understanding of the palaeoenvironment of the Jurassic Yanliao Biota in East Asia.

The occurrence of pollen of Sciadopityaceae Luerss. through time

Here we present LM and SEM data of pollen of extant Sciadopitys verticillata, fossil Cerebropollenites from Aptian/Albian strata (Austria, Mongolia) and Sciadopityspollenites from Campanian/Maastrichtian and Oligocene/Miocene strata (Siberia, Germany). Measurements and image comparisons show that the investigated fossil pollen taxa range from somewhat comparable to very similar to extant Sciadopitys verticillata, and that a previous affiliation of Cerebropollenites taxa with Tsuga cannot be corroborated. Additionally, it can be speculated that either the Rhaetian to Lower Cretaceous Cerebropollenites taxa are the pollen equivalent of the Eurasian Miroviaceae macrofossils that have unresolved relationships with Sciadopityaceae, or that they might belong to a basal group in the Sciadopityaceae, which is quite recognizable due to the similarity of the pollen morphology of Cerebropollenites thiergartii and Sciadopitys verticillata. However, until in situ pollen within cones attributed to the Miroviaceae are found, we will not know for sure.

The separation of softwood and hardwood in historical wooden statues of the Nazenji-temple in Japan using NIR spectroscopy

Abstract The applicability of near-infrared (NIR) spectroscopy to the identification of wood species of archaeologically/historically valuable wooden artifacts in a non-invasive manner was investigated using reference wood samples from the xylarium of the Forestry and Forest Products Research Institute (TWTw) and applied to several wooden statues carved about 1000 years ago. Diffuse-reflectance NIR spectra were obtained from five standard wood samples each of five softwood species (Chamaecyparis obtusa, Cryptomeria japonica, Sciadopitys verticillata, Thujopsis dolabrata, Torreya nucifera) and five hardwood species (Aesculus turbinata, Cercidiphyllum japonicum, Cinnamomum camphora, Prunus jamasakura, Zelkova serrata). A principal component analysis (PCA) model was developed from the second derivative spectra. The score plot of the first two components clearly showed separation of the wood sample data into softwood and hardwood clusters. The developed PCA model was applied to 370 spectra collected from 21 wooden statues preserved in the Nazenji-temple in Shizuoka Prefecture in Japan, including 14 made from Torreya spp. and 7 made from Cinnamomum spp. In the score plot, the statue spectra were also divided into two clusters, corresponding to either softwood (Torreya spp.) or hardwood (Cinnnamomum spp.) species. These results show that NIR spectroscopy combined with PCA is a powerful technique for determining whether archaeologically/historically valuable wooden artifacts are made of softwood or hardwood.

The Impact of the Morphological Characteristics of Leaves on Particulate Matter Removal Efficiency of Plants

This study was conducted to find out differences in the removal efficiency of particulate matter (PM) depending on the type of plants and the morphological characteristics of leaves. A total of 12 plants were used, with three plants selected for each type of leaves (big leaf, small leaf, compound leaf, needle leaf). We measured the removed amount of PM10 and PM2.5, the structure of the abaxial leaf surface, and the weight of the wax layer of each plant. Plants with the high removal efficiency of PM included Pachira aquatica Aubl., Ardisia crenata, and Dieffenbachia ‘Marianne’, and plants with the low removal efficiency included Nandina domestica Thunb, Schefflera arboricola, and Quercus dentata. The abaxial leaf surface having a high removal efficiency of PM had many large wrinkles, and the abaxial leaf surface having a medium removal efficiency was flat and smooth. On the other hand, there were many fine hairs on the abaxial leaf surface with a low removal efficiency. According to the plant leaf type, the PM10 removal efficiency of plants with needle leaves was about three times higher than that of other plants. In particular, the wax layer of conifers weighed 6–24 times higher than those of other plants. The stomata of conifers were evenly distributed on the adaxial and abaxial leaf surfaces; however, the stomata of Sciadopitys verticillata appeared in the form of papillae unlike general stomata. Therefore, the removal efficiency of PM varied depending on the macro-, and micro-morphological characteristics of plant leaves such as the structure of the abaxial leaf surface, and the weight of the wax layer. Based on this research, selecting plants that are effective in reducing PM in consideration of the plant type and leaf characteristics will improve indoor air quality and decrease exposure of PM to human body. Key words: conifer, leaf surface structure, leaf type, wax layer weight

A Study on the Latest Research Trends in Natural products with Anti-Aging Effects

In this study, four types of retinol, retinyl palmitate, adenocin, and polytoxylate-dretinamide, which are the ingredients of the Ministry of Food and Drug Safety Notice, included in the study. also we looked at trends in research on Sciadopitys verticillata, Prunella vulgaris, Celosia cristata L., Brazilin, Persicaria hydropiper, Astragalus membranaceus Bunge, Forsythiae Fructus, Lithospermum root, Rheum undulatum L. and Cistanche deserticola Y. C. Ma a natural material that has the efficacy of antioxidant aging. The anti-aging study so far has been found to be centered mainly on collagen production and elastase synthesis inhibition mechanisms. However, given that the aging process of the skin is caused by various ageing processes, it is believed that anti-aging studies using safe and effective natural materials that can help the skin age with various mechanisms should be conducted.

Sciadopitys verticillata Resin: Volatile Components and Impact on Plant Pathogenic and Foodborne Bacteria.

Sciadopitys verticillata (Sv) produces a white, sticky, latex-like resin with antimicrobial properties. The aims of this research were to evaluate the effects of this resin (Sv resin) on bacterial populations and to determine the impact of its primary volatile components on bioactivity. The impact of sample treatment on chemical composition of Sv resin was analyzed using Fourier transform infrared spectroscopy (FTIR) coupled with principal component analysis. The presence and concentration of volatiles in lyophilized resin were determined using gas chromatography/mass spectrometry (GC/MS). Changes in bacterial population counts due to treatment with resin or its primary volatile components were monitored. Autoclaving of the samples did not affect the FTIR spectra of Sv resin; however, lyophilization altered spectra, mainly in the CH and C=O regions. Three primary bioactive compounds that constituted >90% of volatiles (1R-α-pinene, tricyclene, and β-pinene) were identified in Sv resin. Autoclaved resin impacted bacterial growth. The resin was stimulatory for some plant and foodborne pathogens (Pseudomonas fluorescens, P. syringae, and Xanthomonas perforans) and antimicrobial for others (Escherichia coli, Bacillus cereus, Agrobacterium tumefaciens, and Erwinia amylovora). Treatment with either 1R-α-pinene or β-pinene reduced B. cereus population growth less than did autoclaved resin. The complex resin likely contains additional antimicrobial compounds that act synergistically to inhibit bacterial growth.

Vegetation and endemic tree response to orbital-scale climate changes in the Japanese archipelago during the last glacial–interglacial cycle based on pollen records from Lake Biwa, western Japan

This study examined the orbital-scale vegetation response to seasonal climate changes related to the East Asian monsoon and ocean currents during the last glacial–interglacial cycle based on a new continuous pollen record from Lake Biwa, western Japan. During MIS 6, 4, and 2, pinaceous conifer forests were present in both inland and Japan Sea coastal areas around Lake Biwa, influenced by cold and dry conditions in both summer and winter. In contrast, deciduous broadleaved forests and evergreen forests grew during interglacial periods in MIS 5e and 1 under relatively warm and humid conditions in summer, as well as in winter. Fagus crenata, a tree endemic to the Japanese archipelago, was especially widely distributed during MIS 5e due in part to winter snowfall caused by warm current inflows in the Sea of Japan. During MIS 5 and 3, temperate conifer trees were dominant. During periods of low summer insolation in MIS 5, the endemic tree Cryptomeria japonica became dominant in both inland and coastal areas of the region, likely as a result of high precipitation in early summer related to southward shifts of the summer monsoon front and moderate winter climate conditions. In contrast, Sciadopitys verticillata, which is also an endemic tree, had own habitat around inland areas of western Japan during the periods of high summer insolation in MIS 5. Climate fluctuations during the glacial–interglacial cycles created several ecological niches in time and space for each endemic tree in the Japanese archipelago.

Chemotaxonomy of fossil woods from the Lower Rhine Embayment, Germany

In the Lower Rhine Embayment (western Germany), the Neogene lignite bearing sequence is rich in large fossil wood trunks. Woods collected from sand-filled channels of a meandering river system (Pliocene: Reuver series) and from coal seams (Miocene: seams Garzweiler, Frimmersdorf and Morken) were investigated by Curie-point pyrolysis coupled with gas chromatography–mass spectrometry in order to determine their palaeobotanical classification. In comparison with reference data of individual pyrograms of modern woods, it became possible to classify 15 fossil wood trunks chemotaxonomically as follows: eleven samples were identified as Taxodioxylon sp. by reference Sequoia sempervirens, three samples were identified as Populoxylon sp. by reference Populus nigra and one sample was identified as Sciadopityoxylon sp. by two references of Sciadopitys verticillata. Microanatomical crosschecks describe the equivalent samples taxonomically as Taxodioxylon gypsaceum (GÖPPERT) KRÄUSEL, Taxodioxylon germanicum (GREGUSS) VAN DER BURGH, Populus nigra L. and Sciadopityoxylon wettsteinii JURASKY, and confirm the chemical classifications. For both fossil and reference woods, our relative quantifications of guaicyl and syringyl moieties are in precise accordance with discriminations of gymnosperms versus angiosperms. This means that not only unknown recent woods can chemotaxonomically accurately be determined by Curie-point pyrolysis gas chromatography–mass spectrometry and their equivalent modern wood reference from our data base, but also the taxonomy of botanically related wood types reaching back to the geological past up to a Miocene age at least.

Birth of Four Chimeric Plastid Gene Clusters in Japanese Umbrella Pine.

Many genes in the plastid genomes (plastomes) of plants are organized as gene clusters, in which genes are co-transcribed, resembling bacterial operons. These plastid operons are highly conserved, even among conifers, whose plastomes are highly rearranged relative to other seed plants. We have determined the complete plastome sequence of Sciadopitys verticillata (Japanese umbrella pine), the sole member of Sciadopityaceae. The Sciadopitys plastome is characterized by extensive inversions, pseudogenization of four tRNA genes after tandem duplications, and a unique pair of 370-bp inverted repeats involved in the formation of isomeric plastomes. We showed that plastomic inversions in Sciadopitys have led to shuffling of the remote conserved operons, resulting in the birth of four chimeric gene clusters. Our data also demonstrated that the relocated genes can be co-transcribed in these chimeric gene clusters. The plastome of Sciadopitys advances our current understanding of how the conifer plastomes have evolved toward increased diversity and complexity.

Pollen morphology in selected Cupressaceae Gray. and Sciadopityaceae Luerss. species in an experimental culture

Pollen grains of Cunninghamia lanceolata, Cryptomeria japonica and Sciadopitys verticillata were exam- ined by light and scanning electron microscopy. The pollen grains were non-saccate, generally oblate-sphe- roidal, spheroidal, prolate-spheroidal or subprolate. The pollen grains had papillae of different sizes and shapes on the distal face. The papillae were more conspicuous in C. japonica than in C. lanceolata. The pollen exine sculpture in the proximal view was verrucate-granulate and verrcucate with microechinate supras- culpture in SEM. The surface of the exine was covered with echinate orbicules in Cupressaceae species. There were some differences in the size of the verrucae, perforation on the proximal surface and distal face between S. verticillata pollen grains and other species. This study of the pollen grain morphology provided some important new data which have not been reported in literature so far.

Evolution of short inverted repeat in cupressophytes, transfer of accD to nucleus in Sciadopitys verticillata and phylogenetic position of Sciadopityaceae.

Sciadopitys verticillata is an evergreen conifer and an economically valuable tree used in construction, which is the only member of the family Sciadopityaceae. Acquisition of the S. verticillata chloroplast (cp) genome will be useful for understanding the evolutionary mechanism of conifers and phylogenetic relationships among gymnosperm. In this study, we have first reported the complete chloroplast genome of S. verticillata. The total genome is 138,284 bp in length, consisting of 118 unique genes. The S. verticillata cp genome has lost one copy of the canonical inverted repeats and shown distinctive genomic structure comparing with other cupressophytes. Fifty-three simple sequence repeat loci and 18 forward tandem repeats were identified in the S. verticillata cp genome. According to the rearrangement of cupressophyte cp genome, we proposed one mechanism for the formation of inverted repeat: tandem repeat occured first, then rearrangement divided the tandem repeat into inverted repeats located at different regions. Phylogenetic estimates inferred from 59-gene sequences and cpDNA organizations have both shown that S. verticillata was sister to the clade consisting of Cupressaceae, Taxaceae, and Cephalotaxaceae. Moreover, accD gene was found to be lost in the S. verticillata cp genome, and a nucleus copy was identified from two transcriptome data.

Outcrossing rates and organelle inheritance estimated from two natural populations of the Japanese endemic conifer Sciadopitys verticillata

The Japanese endemic conifer Sciadopitys verticillata is one of the most phylogenetically isolated species of all plants. Occurring in small and scattered populations, the species is currently classified as Near Threatened by the International Union for Conservation of Nature and Natural Resources (IUCN) and as Vulnerable in three prefectures of Japan. This study investigated two major factors that should impact the genetic structure of the species at both the nuclear and organelle DNA level, the mating system and the inheritance of both the chloroplast and mitochondrial genomes. The mating system is crucial to determining the degree of outcrossing of plant species and thus should have a key role in shaping the species' population level genetic diversity and gene flow between populations but as yet has not been studied in S. verticillata. Nine mother trees and their seedling progeny from two natural populations were genotyped using genetic markers from three plant genomes (eight nuclear microsatellites and DNA sequence for the chloroplast and mitochondria). Using a maximum likelihood method implemented in the software MLTR, the study found an outcrossing rate in the seedling stage of 0.49 and 0.79 for Aburazaka and Mount Shirotori populations, respectively, and an average of 0.66 at the species level. These outcrossing rates were low for conifers and therefore may have potential deleterious implications for the conservation of the species. The test of organelle inheritance supported paternal transmission of both the chloroplast and mitochondria consistent with previous microscopic evidence.

Conflict in outcomes for conservation based on population genetic diversity and genetic divergence approaches: a case study in the Japanese relictual conifer Sciadopitys verticillata (Sciadopityaceae)

A major goal of conservation genetics is to determine which specific populations are most crucial for in situ or ex situ conservation. Genetic divergence and diversity are the two foundations by which priorities for conservation are typically determined. However, these measures may be confounded when past bottlenecks reduce genetic diversity of populations but also lead to their divergence. This study examines the potential conflicts in population prioritization for a relictual Japanese endemic conifer, Sciadopitys verticillata using nuclear microsatellites. High genetic structuring at the nuclear level compared to many other conifers ( Fst = 0.129) was observed across the species range along with significant differences in genetic diversity between southern and northern populations. Conflict among genetic diversity and divergence population prioritization methods was observed in populations at the southwestern range edge of Kyushu and Chugoku, which were the most genetically distinct but also harboured the lowest diversity (Kyushu, He = 0.288, Ar = 2.172, and Chugoku, He = 0.222, Ar = 2.010). These populations contained only a subset of the genetic diversity found in Central Honshu and the Kii Peninsula (Central Honshu, He = 0.347, Ar = 2.707 and the Kii Peninsula, He = 0.337, Ar = 2.683), suggesting a reduction in genetic diversity as a result of bottlenecks. To determine if these highly bottlenecked populations in southwestern Japan are on the trajectory to extinction, or, conversely, if they harbour important genetic variation that has been fixed at the southwestern edge of the species range, common garden experiments are recommended in the future.

Taxonomy and phylogenetic position of Fomitiporia torreyae, a causal agent of trunk rot on Sanbu-sugi, a cultivar of Japanese cedar (Cryptomeria japonica) in Japan

Trunk rot poses a substantial threat to Sanbu-sugi, one of the most economically important cultivars of Japanese cedar (Cryptomeria japonica). The etiology of this disease, including its main agents, is incompletely known. This trunk rot was attributed to Fomitiporia (Phellinus) hartigii or F. (Phellinus) punctata. Here we phylogenetically analyzed DNA sequences of four markers from a set of strains isolated from trunk-rot symptoms and recovered a single, monophyletic clade, indicating that a single taxon is involved. This clade was identified as Fomitiporia torreyae, a species described from eastern China. This analysis also proved that trunk rot and/or dieback of other conifers and broadleaf trees, including Sawara cypress (Chamaecyparis pisifera), Japanese umbrella pine (Sciadopitys verticillata) and Japanese pear (Pyrus pyrifolia var. culta), were caused by the same species. The phylogenetic approach to Fomitiporia revealed that the F. torreyae clade was closely related to F. bannaensis but clearly distinct from F. punctata, which originally was thought to be the cause of trunk rot in Sanbu-sugi. Fomitiporia torreyae is redescribed on the basis of more than 40 specimens from multiple hosts from Japan and China. Fomitiporia juniperina comb. nov. is proposed.

Northern richness and southern poverty: contrasting genetic footprints of glacial refugia in the relictual treeSciadopitys verticillata(Coniferales: Sciadopityaceae)

Sciadopitys verticillata is amongst the most relictual of all plants, being the last living member of an ancient conifer lineage, the Sciadopityaceae, and is distributed in small and disjunct populations in high rainfall regions of Japan. Although mega-fossils indicate the persistence of the species within Japan through the Pleistocene glacial– interglacial cycles, how the species withstood the colder and drier climates of the glacials is not well known. The present study utilized phylogeography and palaeodistribution modelling to test whether the species survived within pollen-based coastal temperate forest glacial refugia or within previously unidentified refugia close to its current range. Sixteen chloroplast haplotypes were found that displayed significant geographical structuring. Unexpectedly, northern populations in central Honshu most distant from coastal refugia had the highest chloroplast diversity and were differentiated from the south, a legacy of glacial populations possibly in inland river valleys close to its current northern range. By contrast, populations near putative coastal refugia in southern Japan, harboured the lower chloroplast diversity and were dominated by a single haplotype. Fragment size polymorphism at a highly variable and homoplasious mononucleotide repeat region in the trn T-,i>trn L intergenic spacer reinforced the contrasting patterns of diversity observed between northern and southern populations. The divergent histories of northern and southern populations revealed in the present study will inform the management of this globally significant conifer.

First Notice of Phytophthora Root and Stem Base Rot of Sciadopitys Verticillata in Polish Ornamental Nurseries

Severe root and stem base rot was noticed for the first time on Sciadopitys verticillata in 3 hardy ornamental nursery stocks. Phytophthora citrophthora was isolated from about 4/5 of the analysed plants. In the laboratory trials this species caused stem and root rot. On stem parts and needles inoculated by isolates from the host plant and Pinus sylvestris, necrosis was observed to have spread at a similar rate. The needles, however, were colonized significantly quicker than the stem parts. Sciadopitys verticillata (Thunb.) Siebold. & Zucc., known as Japanese umbrella pine, is a slow-growing ornamental tree found in Polish nurseries over the last 10 years. Small plants grow to about 30 cm under cov- er, and later they are put in outside container nurseries. Lambe and Wills (1983) did not notice disease symptoms on plants in the landscape whereas in containers hav- ing excessive moisture, the trees are highly susceptible to root rot incited by Phytophhtora cinnamomi Rands. Af- fected plants showed wilting and an off-green color, with needles turning yellow. The reason for such changes were the rotting of the fibrous roots and the discoloration in the wood at the stem base. The first time yellowing of the needles was noticed on small S. verticillata growing in plastic tunnels and older plants as tall as 50-70 cm was in 2008-2010. This observa- tion took place in two hardy ornamental nursery stocks in the south-east part of Poland and in one nursery in the north of Poland. The purpose of this study was to evaluate a causal agent of dieback of S. verticillata, and its pathogenicity to a host plant. Nurseries were surveyed from July to September at one month intervals. On plants as tall as 10-25 cm nee- dles changed color from green to light green and finally to brown (Fig. 1). On the base of the plants, up to 5-10 cm of brown or dark brown discoloration of wood was observed. Most of the roots were dark brown and dead. On older trees growing at outside container nurseries, yellowing and browning of needles was noticed, usually on one part of the plants (Fig. 2). Base rot even extended 15 cm up the stem. Plants showing disease symptoms were placed in plastic bags together with substratum, and transported to the laboratory. The procedure of Or- likowski and Szkuta (2001) was used for isolating micro- organisms from diseased plant tissues. Over the span of 3 years, 50 diseased plants were analysed. Rhododen- dron leaf baits and the procedure described by Themann and Werres (1998) was used for the detection of Phytoph- thora spp. from the substratum. Obtained isolates were grouped by growth pattern and their morphology. Repre- sentative cultures were identified to species on the base of morphology features and confirmed by molecular meth-

Pollen wall development in Sciadopitys verticillata (Sciadopityaceae)

Pollen wall development of Sciadopitys verticillata was observed by transmission electron microscopy. The pollen of S. verticillata is non-saccate and spherical, and the exine consists of the outer thick, sculptured ectexine and the inner lamellated endexine. At the early tetrad stage, the initial ectexine and lamellae of the initial endexine begin to form on the microspore plasma membrane. The ectexine granules gradually swell. Deposition of sporopollenin materials on the ectexine granules then results it their becoming partially connected to each other. Identification of the original small ectexine granules then becomes difficult, and, finally, the ectexine appears as a homogeneous, partially discontinuous layer. The granules of the early ectexine cannot be identified. At maturity, there are four to five endexine lamellae. Recent molecular data have shown that Sciadopitys first branches off from the Cupressaceae plus Taxaceae clade, which is characterized by granular exine. Although the ectexine of Sciadopitys is similar to that of the Cupressaceae during initial development, the morphology of the ectexine is significantly different in the mature pollen. The initial stage of pollen development clearly shows the structural homology of the granular ectexine. Divergence of the exine structure occurs in the later stages.

Loss of different inverted repeat copies from the chloroplast genomes of Pinaceae and cupressophytes and influence of heterotachy on the evaluation of gymnosperm phylogeny.

The relationships among the extant five gymnosperm groups—gnetophytes, Pinaceae, non-Pinaceae conifers (cupressophytes), Ginkgo, and cycads—remain equivocal. To clarify this issue, we sequenced the chloroplast genomes (cpDNAs) from two cupressophytes, Cephalotaxus wilsoniana and Taiwania cryptomerioides, and 53 common chloroplast protein-coding genes from another three cupressophytes, Agathis dammara, Nageia nagi, and Sciadopitys verticillata, and a non-Cycadaceae cycad, Bowenia serrulata. Comparative analyses of 11 conifer cpDNAs revealed that Pinaceae and cupressophytes each lost a different copy of inverted repeats (IRs), which contrasts with the view that the same IR has been lost in all conifers. Based on our structural finding, the character of an IR loss no longer conflicts with the “gnepines” hypothesis (gnetophytes sister to Pinaceae). Chloroplast phylogenomic analyses of amino acid sequences recovered incongruent topologies using different tree-building methods; however, we demonstrated that high heterotachous genes (genes that have highly different rates in different lineages) contributed to the long-branch attraction (LBA) artifact, resulting in incongruence of phylogenomic estimates. Additionally, amino acid compositions appear more heterogeneous in high than low heterotachous genes among the five gymnosperm groups. Removal of high heterotachous genes alleviated the LBA artifact and yielded congruent and robust tree topologies in which gnetophytes and Pinaceae formed a sister clade to cupressophytes (the gnepines hypothesis) and Ginkgo clustered with cycads. Adding more cupressophyte taxa could not improve the accuracy of chloroplast phylogenomics for the five gymnosperm groups. In contrast, removal of high heterotachous genes from data sets is simple and can increase confidence in evaluating the phylogeny of gymnosperms.