Molecular diversity and biogeography of benthic ciliates in the Bohai Sea and Yellow Sea

This study explored the molecular diversity and biogeography of benthic ciliates in Chinese marginal seas, the Bohai Sea(BHS), North Yellow Sea(NYS) and South Yellow Sea(SYS). From a previous 18S rRNA gene pyrosequencing dataset of the benthic microeukaryotes, we retrieved the sequences affiliated with phylum Ciliophora and analyzed alpha and beta diversities of ciliate communities. We found that BHS had the highest ciliate operational taxonomic unit(OTU) richness than NYS and SYS, whereas the richness was not significantly different between summer and winter. Among all the measured environmental variables, water depth showed consistently the strongest correlations with alpha diversities. Overall, the class Spirotrichea(mostly Choreotrichia and unassigned lineages within the class) dominated the communities in terms of both relative proportion of sequences(77.0%) and OTU richness(66.5%). OTU-level ciliate community structure was significant different among the three basins, but not between the seasons. Structurally, significant differences in relative proportion among the basins were detected for the class Litostomatea, but not for other classes. Partial Mantel tests demonstrated that water depth difference was more important than geographic and environment distances in shaping the community structure of benthic ciliates in the studied area. About 60% OTUs were not assigned at a class or order level and at least 45% OTUs shared a sequence similarity no more than 97% with the described species, indicating a great potential for ciliate species discovery in the offshore sediments. Compared with previous morphology-based surveys, the spatial pattern of ciliate diversity(decreasing from NYS to SYS) is also identified in the present study. Nevertheless, structurally, the dominant class appeared to be Spirotrichea in the sequencing dataset, which differs from previous morphology-based results(dominance of classes Prostomatea and Karyorelictea in biomass). The potential causes for the discrepancies between molecular and morphological findings are also discussed.

Spatiotemporal variation in community structure of marine benthic ciliates in the Yellow Sea during and after macroalgal and giant jellyfish blooms

The annual bloom of the green macroalgal Ulva prolifera from May through July since 2008 and another of giant jellyfi sh Nemopilema nomurai from June through September have been frequent events in the Yellow Sea. However, the patterns of benthic ciliate communities during and after the blooms are still not known. In combination with analyses of benthic environmental factors, we investigated the distribution and community composition of benthic ciliates in the Yellow Sea in July and November 2011. In July, ciliates had high standing crops and diversity in the northern Yellow Sea, and in the inshore area off the southern Shandong Peninsula, where large numbers of green macroalgae accumulated. In November, the abundance, biomass and diversity of ciliates were high in the sea areas off the Shandong Peninsula and Changjiang estuary, where a large quantity of jellyfi sh occurred in August. Neither the abundance nor the biomass had signifi cant diff erence between seasons, or between diff erent compartments of the Yellow Sea. The species number, and both Margalef and Shannon-Wiener indices of ciliates were all signifi cantly higher in November than in July. In both seasons, prostomateans and karyorelicteans consistently constituted the fi rst and second most important ciliate groups in biomass; and carnivorous ciliates constituted the primary feeding type in terms of biomass as well as species richness, followed by bacterivores, algivores and omnivores. Compared with that in June 2007 when no macroalgae occurred, the percentage of small-sized bacterivores(e.g. Metacystis spp., Euplotes spp. and scuticociliates) increased in July 2011. The proportion of carnivorous ciliates increased in November, and this increased dominance of carnivorous ciliates may be a response to the increase in predominance of heterotrophic nanofl agellates, which might in turn be ascribed to an eff ect of green macroalgal and giant jellyfi sh blooms in the Yellow Sea.

Composition and Distribution of Planktonic Ciliates in the Southern South China Sea During Late Summer： Comparison Between Surface and 75 m Deep Layer

Ciliates are very important components in most marine ecosystem. They are trophic link between the microbial food web and grazing food chain. In this study, ciliates were collected from 11 sites in the southern South China Sea （SCS） during August 25 to September 28, 2011. Their composition and distribution at the surface and 75m deep depth of the ocean were studied. A total of 30 species belonging to 22 genera were identified, and 22 species of 15 genera were Tintinnids. Eutintinnusfraknoii and E. stramentus were the most common species. The other dominants were strombidiids ciliates including Strombidium conicum and S. globosaneum, which were followed by the tide form, Mesodinium pulex. Ciliates abundance ranged from 46 indL-1 to 368 indL-1 in the open sites, 46-368indL-1 at surface and 73-198indL-1 at 75m deep layer. In the Yongshu reef, ciliates abundance ranged from 167indL-1 to 365 ind L-1 in the water colunm, similar to that in Sanya coral reef waters. Ciliates composition showed obvious difference between surface and 75m deep layer at station S2 （P〈0.05）, while no similar result was observed at other sites. At 75m deep layer, salinity was negatively related to mixed layer depth （P〈 0.05）, but positively to chlorophyll a concentration （P〈 0.05）, indicating that the change of vertical mixing in water column influenced vertical distribution of ciliates in the southern SCS.

Complementary DNA sequencing (cDNA): an eff ective approach for assessing the diversity and distribution of marine benthic ciliates along hydrographic gradients

The Yellow Sea Cold Water Mass(YSCWM)is a distinct hydrographic phenomenon of the Yellow Sea,and the distribution pattern of meio-and macrobenthos diff ers inside and outside of the YSCWM.However,such a pattern has never been observed in the microbenthic ciliate communities.Therefore,we hypothesized that benthic ciliates followed a similar distribution pattern as meio-and macrobenthos,but this pattern has not been uncovered by morphological methods.We evaluated the diversity and distribution of benthic ciliates at fi ve stations along hydrographic gradients across the YSCWM and adjacent shallow water by using morphology and DNA and complementary DNA(cDNA)high-throughput sequencing of the V4 region of 18S rRNA gene.Results showed that the diversity of benthic ciliates detected by DNA(303 OTUs),and the cDNA(611 OTUs)sequencing was much higher than that detected by the morphological method(79 species).Morphological method detected roughly diff erent ciliate communities inside and outside of the YSCWM,but without statistical signifi cance.No clear pattern was obtained by DNA sequencing.In contrast,cDNA sequencing revealed a distinct distribution pattern of benthic ciliate communities like meioand macrobenthos,which coincided well with the results of the environmental parameter analysis.More than half of the total sequences detected by DNA sequencing belonged to planktonic ciliates,most(if not all)of which were recovered from historic DNA originating through the sedimentation of pelagic forms because none of them were observed morphologically.The irrelevant historic DNA greatly infl uenced the recovery of rare species and thus limited the understanding of the benthic ciliate diversity and distribution.Our research indicates that the methods used have signifi cant eff ects on the investigation of benthic ciliate communities and highlights that cDNA sequencing has great advantages in estimating the diversity and distribution of benthic ciliates,as well as the potential for benthic environmental assessments.

Notes on Two Marine Ciliates from the Yellow Sea,China:Placus salinus and Strombidium apolatum(Protozoa,Ciliophora)

The living morphology and infraciliature of two rare marine ciliates, Placussalinus Dietz, 1964 and Strombidium apolatum Wilbert and Song, 2005, collected from the coastalwaters near Qingdao, China, were investigated by in vivo observation and protargol impregnationtechnique. The improved diagnosis for Placus salinus is as follows: medium-sized marine Placus, invivo (50-60) μm X (30-40) μm; cell elliptical to barrel-shaped; 28-31 somatic kineties; singlemacronucleus usually ellipsoid and one micronucleus located in the indention of the macronucleus;one contractile vacuole posteriorly positioned. Strombidium apolatum is characterized by: marinestrombidium (40-60) μm X (30-45) μm in vivo, cordiform in shape with somewhat pointed posteriorend and conspicuous apical protrusion; extrusomes prominent, about 15 μm in length and evenlyarranged along the circle kinety; about 16 collar and 5-6 buccal membranelles; one elongatemacronucleus and one micronucleus; circle and ventral kineties consisting of about 53 and 45dikinetids respectively.

Morphological Studies on Four Brackish Water Ciliates of the Class Spirotrichea(Protista, Ciliophora)

We reinvestigate the morphology of two oligotrich and two hypotrich ciliates collected from the coasts of north and south China, viz., Parallelostrombidium obesum Liu et al., 2015, Spirostrombidium apourceolare Liu et al., 2013, Protogastrostyla pulchra(Pereyaslawzewa, 1886) Gong et al., 2007, and Uncinata bradburyae(Gong et al., 2001) Luo et al., 2015. All the populations match well with the original samples identified in previous studies. Supplemental taxonomic data were supplied for these four species of the new populations. The species Parallelostrombidium obesum is characterised by its dorsal-ventrally flattened body shape with anterior and posterior ends transversely truncating, as well as two thigmotactic membranelles and girdle kinety that spirals around the cell one and a half times. Spirostrombidium apourceolare is characterised by its elongate ellipsoidal and dorsal-ventrally flattened body shape, two thigmotactic membranelles, about 11-27 ovoid macronuclear nodules, and girdle kinety that spirals around the cell twice with two undulations. Protogastrostyla pulchra is characterised by its elongate body shape and unique, caudally located food vacuole. Based on the new populations, we described the smaller cortical granules, clustered around dorsal bristles for the first time. The new population of Uncinata bradburyae shares the diagnostic features with the type population, which include prominent beak-like projection in anterior region, the conspicuous gap of adoral zone, proximalmost adoral membranelles distinctly elongated, and infraciliature.

Diversity of oligotrich ciliates(Ciliophora, Spirotrichea) in the northern coast of South China Sea as revealed in LSU rDNA sequences

Although the taxonomy of oligotrich ciliates has been widely investigated,yet the species diversity remains poorly known.We newly designed a pair of oligotrich-specific LSU rDNA primers covering the 600-bp D1/D2 region,and it was effective for detecting oligotrich species.Using the primers,we constructed the cloning libraries to investigate the species diversity of oligotrichs in the northern coastal waters of the South China Sea.In total,165 oligotrich sequences were obtained from five widely separated sampling sites.Sixty operational taxonomic units(OTUs)were obtained at 99%similarity threshold,and low-abundance OTUs with no more than two sequences contributed most of these(about 78%).Our findings are consistent with previous morphological studies,Strombidium was found the most abundant and widely distributed genus in this area.In addition,the BLAST search in the NCBI database resulted in 95%OTUs matching with named oligotrich species in similarity below 99%.Therefore,oligotrich morphospecies diversity has been underestimated as low-abundance species,and the LSU rDNA oligotrich sequence database needs to be better promoted.

Morphological studies of six free-living spirotrichean ciliates (Protozoa： Ciliophora) with three new records from the coastal South China Sea

The living morphology and infraciliature of six spirotrichous ciliates collected from the coastal South China Sea were investigated using live observation and protargol impregnation. These are Euplotes woodruffi Gaw, 1939, Hemigastrostyla enigmatica （Dragesco and Dragesco-Kerneis, 1986） Song and Wilbert, 1997, Neourostylopsis flavicana （Wang et al., 2011） Chen et al., 2013, Protogastrostyla pulchra （Perejaslawzewa, 1886） Gong et al., 2007, Pseudoamphisiella alveolata （Kahl, 1932） Song and Warren, 2000, and PseudokahlieUa marina （Foissner et al., 1982） Berger et al., 1985. Among these, Protogastrostyla pulchra, Pseudoamphisiella alveolata and PseudokahlieUa marina are reported from the South China Sea for the first time, which manifest obvious differences apart from other Chinese populations. As new contribution, the detailed description of isolates from mangrove habitat of E. woodruffi and 1t. enigmatica, and aquaculture pond isolate of N.flavicana, are present.

Insights into the phylogeny of sporadotrichid ciliates (Protozoa, Ciliophora: Hypotricha) based on genealogical analyses of multiple molecular markers

The sporadotrichid ciliates are an especially diverse group. A number of investigators have studied the morphological, morphogenetic, and molecular relationships among members of this group. Despite this, a consistent classification is still lacking and several important questions about the phylogenetic relationships within this group remain unsolved. To improve our understanding of these relationships, we constructed phylogenetic trees using the nucleotide sequences of the small-subunit rRNA (SSrRNA) gene and amino acid sequences of actin I and α-tubulin. Analyses of SSrRNA gene sequences indicated that: 1) the Sporadotrichida sensu Lynn (2008) and the Oxytrichidae are polyphyletic; 2) the Uroleptus species, which are classified to urostylids, formed a sister group with the oxytrichids; 3) Halteria grandinella, which is grouped morphologically with oligotrich species, clustered within the oxytrichids. These results are congruent with previous studies based on SSrRNA gene sequences. However, the amino acid sequences of actin I and α-tubulin yielded different topologies. The main results are: 1) in all phylogenetic trees, the genus Oxytricha was paraphyletic; 2) Uroleptus was sister to a subset of Urostyla and Holosticha, albeit with low supporting values; 3) Halteria grandinella was separated distantly from the Oxytrichidae in trees inferred from actin I amino acid sequences but clustered with oligotrichids in the α-tubulin analysis. The inconsistency among the trees inferred from these different molecular markers may be caused by rapidly accumulated genetic characterizations of ciliates. Further studies with additional molecular markers and sampling of more taxa are expected to better address the relationships among sporadotrichids.

On four closely related hypotrichous ciliates(Protozoa,Ciliophora,Spirotrichea):molecular characters,interspecific relationships and phylogeny defined with multigene sequence information

In order to clarify the phylogeny and relationships of the most confused hypotrichous ciliates,Holosticha-complex,four closely related holostichids（five populations）,Holosticha bradburyae,H.diademata,Anteholosticha sp.,and A.manca,were compared and analyzed using ITS2 secondary structures,ITS1-5.8S-ITS2 region and SSrRNA gene sequences.The ITS1-5.8S-ITS2 region sequences of these four species were first sequenced,and they shared sequence identities ranging from 68.0% to 90.1%,while two populations of Anteholosticha sp.differed in three nucleotides（sequence identity 99.8%）.There were several minor differences among ITS2 secondary structures of these species,while two populations of Anteholosticha sp.had the identical secondary structure.Phylogenetic trees inferred from the ITS1-5.8S-ITS2 region sequences of stichotrichs using multiple algorithms（Neighbor-Joining,Maximum Parsimony and Bayesian） revealed similar topologies.The results show that：（1） Holosticha bradburyae and H.diademata firmly clustered together with strong bootstrap supports,forming a sister clade with Anteholosticha sp.,（2） Anteholosticha appeared to be a paraphyletic assemblage,in which the morphotype A.manca was more closely related to Diaxonella trimarginata than to its congener Anteholosticha sp.Phylogenetic analyses based on the SSrRNA gene and the combined sequences of SSrRNA gene and ITS1-5.8S-ITS2 region revealed the similar relationships between Holosticha and Anteholosticha,nevertheless their positions within the subclass Stichotrichia differed from each other inferred from different genes.

Spatial distribution of aloricate ciliates in the stratified water of the Cosmonaut and Cooperation Seas in the Southern Ocean

Aloricate ciliates are the main component of microzooplankton.They play important roles in the circulation of materials and flow of energy in marine pelagic ecosystems.To determine the distribution pattern and structure of the aloricate ciliate community in vertically stratified waters of the Southern Ocean,we collected data on aloricate ciliates in the Cosmonaut and Cooperation Seas during the 36th Chinese National Antarctic Research Expedition.The ranges of aloricate ciliate abundance and biomass were 5-3097 ind·L^(-1)and 0.01-11.40μg C·L^(-1),respectively.Vertically,the average abundance of aloricate ciliates decreased from the surface to 200 m,while biomass was highest in the 50-m layer.The importance of aloricate ciliates<20μm increased along the depth gradient.Their contribution to total abundance increased from 13.04%in the surface layer to 73.71% in the 200-m layer.This is the first study to explore the distribution characteristics of the aloricate ciliate community in a stratified water column of the Southern Ocean.Our results will be helpful for understanding the variation in the pelagic community in waters of the Southern Ocean with intensified stratification.

Harmful Ciliates and Dominant Genus in Culture Ponds of Young Abalone

By using the methods of qualitative and quantitative analysis, the species composition and dominant genus of harmful ciliates in young abalone culture ponds are studied. Totally 68 ,species of ciliates, belonging to 3 classes16 orders and 46 genus, are found. The dominant ciliate species are Aspidisca leptaspis, Vorticella sp. and Euplotes rariseta. During the young abalone cultivation, the numbers of young abalones increase first, and then show a trend of decreasing. The relationship between the density and composition of ciliates and the density of algae in the young abalone culture ponds is also discussed.

Findings on three endocommensal scuticociliates(Protista,Ciliophora)from freshwater mollusks,including their morphology and molecular phylogeny with descriptions of two new species

Species of the ciliate genera Myxophyllum and Conchophthirus are found as endocommensals of terrestrial and freshwater mollusks,respectively.So far,there have been few studies of these genera and morphological data for most members are often incomplete.In the present work,two new species,Myxophyllum weishanense sp.nov.and Conchophthirus paracurtus sp.nov.,and a known species,Conchophthirus lamellidens,were isolated from hosts in Lake Weishan Wetland,China.Taxonomic studies indicate that M.weishanense sp.nov.can be recognized mainly by the combination of about 60 somatic kineties on both ventral and dorsal sides and the presence of caudal cilia.Conchophthirus paracurtus sp.nov.differs from congeners in its body shape and size,having a glabrous area on the posterior right side,and having fewer somatic kineties.In addition,differences in their ITS2(Internally Transcribed Spacer 2)secondary structures support the discrimination of the two new species from their highly similar congeners.An improved diagnosis for the poorly known species,C.lamel-lidens is also provided.Phylogenetic analyses reveal that members of the genus Myxophyllum belong to a fully supported clade that is sister to a large,poorly supported clade consisting of Hemispeiridae,Ancistridae,and several lineages of the nonmonophyletic Cyclidiidae.The Myxophyllum clade also includes Protophyra ovicola JQ956552,a possible misidenti-fication.Sequences of the two new Conchophthirus species cluster with other congeners in a fully supported clade that is unrelated to either the‘typical’thigmotrichs or to pleuronematids,thus conflicting with the traditional classification,and may represent an orphan scuticociliate lineage.

Further analyses of variation of ribosome DNA copy number and polymorphism in ciliates provide insights relevant to studies of both molecular ecology and phylogeny

Sequence-based approaches, such as analyses of ribosome DNA(rDNA) clone libraries and high-throughput amplicon sequencing, have been used extensively to infer evolutionary relationships and elucidate the biodiversity in microbial communities.However, recent studies demonstrate both r DNA copy number variation and intra-individual(intra-genomic) sequence variation in many organisms, which challenges the application of the rDNA-based surveys. In ciliates, an ecologically important clade of microbial eukaryotes, rDNA copy number and sequence variation are rarely studied. In the present study, we estimate the intraindividual small subunit rDNA(SSU r DNA) copy number and sequence variation in a wide range of taxa covering nine classes and 18 orders of the phylum Ciliophora. Our studies reveal that:(i) intra-individual sequence variation of SSU rDNA is ubiquitous in all groups of ciliates detected and the polymorphic level varies among taxa;(ii) there is a most common version of SSU rDNA sequence in each cell that is highly predominant and may represent the germline micronuclear template;(iii)compared with the most common version, other variant sequences differ in only 1–3 nucleotides, likely generated during macronuclear(somatic) amplification;(iv) the intra-cell sequence variation is unlikely to impact phylogenetic analyses;(v) the rDNA copy number in ciliates is highly variable, ranging from 103 to 106, with the highest record in Stentor roeselii. Overall,these analyses indicate the need for careful consideration of SSU r DNAvariation in analyses of the role of ciliates in ecosystems.

Morphogenesis and Molecular Phylogeny of a Marine Urostylid Ciliate Apokeronopsis wrighti(Protista, Ciliophora, Hypotrichia)

Hypotrichs are one of the highly differentiated ciliated lineages which play important roles in ecological, environmental,evolutionary and basic biological studies. In the present study, we investigated the living characteristics, infraciliature, nuclear apparatus, ontogenesis and phylogenetic position of a marine hypotrichous ciliate, Apokeronopsis wrighti Long et al., 2008, which was isolated from coastal waters in Shenzhen, China. The new isolate resembles the type population in terms of morphological characteristics, morphometrics, and SSU rRNA gene sequence that is with a 99.7% similarity. Ontogenesis of A. wrighti is characterized by oral primordium for the proter as well as marginal and dorsal kineties anlagen in both filial products formed de novo, and the cirral row arranged along the paroral and endoral arises from several anterior frontoventral-transverse cirral streaks. Phylogenetic analyses based on SSU and concatenated gene data suggest that five species of Apokeronopsis form a monophyletic clade, and the genus Apokeronopsis is closely related to Thigmokeronopsis and Metaurostylopsis.

Salinity Acclimation Induces Reduced Energy Metabolism,Osmotic Pressure Regulation and Transcriptional Reprogramming in Hypotrichida Ciliate Gastrostyla setifera

Coastal and estuarine protists are frequently exposed to salinity undulation.While the tolerance and stress responses of microalgae to salinity have been extensively studied,there have been scarce studies on the physiological response of heterotrophic protists to salinity stressing.In this study,we investigated the physiological response of the heterotrophic ciliate Gastrostyla setifera to a salinity of 3,via a transcriptomic approach.The first transcriptome of genus Gastrostyla was obtained utilizing a group of manually isolated ciliate individuals(cells)and RNA-seq technique.The completeness of the transcriptome was verified.Differentially expressed gene(DEG)analysis was performed among the transcriptomes of G.setifera acclimated in saline water(salinity 3)and those cultured in fresh water.The results demonstrated a significant alternation in gene transcription,in which the ciliate exhibits a transcripttomic acclimation in responding salinity stressing.The up-regulated DEGs were enriched in the pathways of cytoskeleton proteins,membrane trafficking,protein kinases and protein phosphatases.These may represent enhanced functions of ion transport,stress response and cell protections.Pathways involved in energy metabolism and biosynthesis were markedly down-regulated,reflecting decreased cell activity.Particularly,we detected significantly down-regulated genes involved in several pathways of amino acid catabolism,which may lead to accumulation of amino acids in the ciliate cell.Amino acid could act as compatible solutes in the cytoplasm to maintain the osmotic balance in saline water.Overall,this work is an initial exploration to the molecular basis of the heterotrophic protist responding to salinity stressing.The result sheds light on the mechanisms of enhancement of cell protection,reduction of cell activity,and osmotic pressure regulation in ciliates acclimated to salinity.

Phylogenomics of non-model ciliates based on transcriptomic analyses

Ciliates are one of the oldest living eukaryotic unicellular organisms, widely distributed in the waters around the world. As a typical marine oligotrich ciliate, Strombidium sulcatum plays an important role in marine food webs and energy flow. Here we report the first deep se- quencing and analyses of RNA-Seq data from Strombidium sulcatum. We generated 42,640 unigenes with an N50 of 1,451 bp after de novo assembly and removing rRNA, mitochondrial and bacteria contaminants. We employed SPOCS to detect orthologs from S. sulcatum and 17 other ciliates, and then carried out the phyloge- nomic reconstruction using 127 single copy orthologs. In phylogenomic analyses, concatenated trees have similar topological structures with concordance tree on the class level. Together with phylogenetic networks analysis, it aroused more doubts about the placement of Protocruzia, Mesodinium and Myrionecta. While epi- plasmic proteins are known to be related to morphological characteristics, we found the potential relationship between gene expression of epiplasmic proteins and morphological characteristics. This work supports the use of high throughput approaches for phylogenomic analysis as well as correlation analysis between expression level of target genes and morphological characteristics.

Malignant transformation of pulmonary bronchiolar adenoma into mucinous adenocarcinoma:A case report

BACKGROUND Bronchiolar adenoma(BA)and ciliated muconodular papillary tumor are rare tumors that have bilayered cell proliferation and continuous expression of p40 and CK5/6 in the basal cell layer.Diagnosis is difficult because of the limited knowledge of these tumors and their morphological similarities to malignant tumors,including invasive mucinous adenocarcinoma,especially based on the histopathology of intraoperative frozen sections.These tumors are now considered to be benign neoplasms,with malignant transformation reported in only a few cases.CASE SUMMARY A 57-year-old woman presented with a 17.0 mm×7.0 mm nodule in the lower lobe of the left lung.Hematoxylin-eosin staining and immunohistochemistry of a surgical specimen were performed.The tumor consisted of a BA area and a mucinous adenocarcinoma(MA)area.In the BA area,the tumor had a bilayered structure of luminal cells and basal cells.The basal cells were positive for CK5/6 and p40,but the MA area was negative for these biomarkers.The Ki-67 proliferation index was low(1%-2%).The patient was diagnosed with BA accompanied by MA,and had a favorable outcome.CONCLUSION The present study indicated that BA may be carcinogenic,and suggests that clinicians should be aware of its potential for malignant transformation.

Cultivation Density of Toona ciliate

ln order to discuss the optimum seedling density of Toona ciliate, different seedling densities were studied. The results showed that seeding height and basal diameter decreased and quality of the seedlings declined with the increase of seedling density. The reasonable density of seedlings is key to cultivate high-quality seedlings and achieve the best seedling efficiency. The optimum density was 30 plants/m2 for the first class seedlings of biennial seedlings, and the optimum density of l and ll seedlings for production afforestation was 40 plants/m2.

Tissue Culture of Toona ciliata

In this study, a screening experiment was carried out among MS, WPM, DCR, MT, B5 and N6. The results showed that for proliferation culture of Toona ciliate, the optimum basic medium was WPM, and the optimum medium was WPM ＋ 6-BA 0.5 mg/L ＋ IBA 0.05 mg/L. The proliferation coefficient reached 4.29. The optimum rooting culture medium was 1/2 MS ＋ IBA 0.5 mg/L ＋ NAA 0.5 mg/L with rooting rate of 100%. In the optimum rooting ＇culture medium, the roots of T. ciliate developed neatly, and the seedlings grew well.