, & To probe if the genus Caulobacter co-exists with the cyanobacterial strains isolated from cycads, we exploited the occurrence of specialized metabolites produced by these microorganisms. Please refer to the appropriate style manual or other sources if you have any questions. To overcome technical difficulties in characterizing the breadth of microbial diversity in these environmental samples, we used EcoMining, a combined sub-community co-culture, metagenomics and phylogenomics strategy to detect and measure taxonomic diversity and phylogenetic relationships in the endophytes of the cycad coralloid root (Cibrin-Jaramillo and Barona-Gmez 2016) (fig. Stanberg, L. C. Liang, F. F. 2012) and >98% sequence identity. , & have become extremely desirable plants to collect. However, only the seeds of C.panzhihuaensis are gathered in this bank. Noticeably, nucleotide diversity of two coastal or island species, C.taitungensis and C.revoluta, is an order of magnitude larger than the other 11 inland species (Chiang etal., 2009; Huang, Chiang, Schaal, Chou, & Chiang, 2001). Thus, even in the absence of a pending detailed taxonomic description of our cyanobacterial isolates, we can conclude that 1) these cycad endophytes belong to the nitrogen-fixing endophytic Nostocales clade; 2) they are closely related despite their different Dioon host species; and 3) within Dioon, there are two phylogenetic groups that include divergent microorganisms. The N and C terminal modifications on this peptide would resemble small peptide aldehyde protease inhibitors, which have been previously reported on cyanobacteria (Fewer etal. , 2012). Eleven species of Cycas are listed as PSESPsthere are only 120 species listed as PSESP in China (Sun, 2013). Science Biology Botany ch. We made a standard Kraken database using NCBI taxonomic information for all bacteria, as well as the bacterial, archaeal, and viral complete genomes in RefSeq (October 2016). Phylogeny, systematics, and ecology are among the disciplines that are most heavily represented in recent cycad literature. Taxonomic identification was made using BlastN with an initial cut-off e-value of 1e5 against the SILVA database (Quast etal. (2007). 2013), Streptomyces aureofaciens (Novakova etal. , & To isolate bacteria from the original epiphyte-free field samples (t0) and after enrichment (t1), coralloid roots were macerated and the macerate or co-culture broth were used as inoculant. Plants of this division are known to have . , 2018 ) but less negativ e than that of conifer species (e.g. 2010), and the Dioon cyanobacterial endophyte strains, is also remarkable. Translocation, that is, movement of plant material to a seemingly suitable area with no documented past history of its existence, is the conservation action used to prevent species extinction when there is no remaining area left within a species historic range able to sustain viable populations (HoeghGuldberg etal., 2008). One species of the African cycads, genus Encephalartos, has evolved to withstand the stress of living in desert environments. , & What to know about deadly bacteria found in Gulf-area soil, water Genetic diversity and genetic differentiation of Cycas are analyzed to find out the potential influence of Quaternary climate changes and topographical events on this lineage. However, considering the fact that nearly 70% of Chinese Cycas taxa are listed as threatened, most of them lack applied research which hindering the conservation efforts. , The quality of the predicted BGCs in both cyanobacteria and Caulobacter strains (248 and 7 potential BGCs, respectively) was confirmed (only 49 cyanobacterial antiSMASH outputs did not pass this filter), leading to 77 and 7 nonredundant BGCs used as references for subsequent searches in 41 Nostocales and 21 Caulobacter selected genomes, respectively. Wei grow in steep cliffs or open stone crevices. (2015). Results of molecular phylogeography of C.taiwaniana Carruth. We estimated taxonomic diversity in single isolates using their 16S rRNA gene sequences obtained after PCR amplification and Sanger sequencing of our entire bacterial endophyte collection (t0 and t1). The final Caulobacter matrix included 82 core protein sequences with 20902 amino acids (supplementary table s2, Supplementary Material online). The genome sequence of the cyanobacterial isolate TPV09 was obtained from coralloid roots of D. caputoi. , & A. There is only a single species included in section Panzhihuaenses settling in Sichuan and Yunnan Province of China, namely C.panzhihuaensis. D5 and Cyanobacterium sp. Water properly. Connectivity of the global network of protected areas, Pollen analysis of the Cretaceous sediments in Taiwan, Global reintroduction perspectives: 2016. Fragnire, Y. , (2008). Description Cycads have a rosette of pinnate leaves around a cylindrical trunk Cycads have a cylindrical trunk which usually does not branch. Moore, S. J. 2018). This database contains a mapping of every k-mer in Kraken's genomic library to the lowest common ancestor in a taxonomic tree of all genomes that contain that k-mer. 2012), or a mixture of both (Costa etal. While the pollen is produced in male cones, the seeds are borne along the margins of modified leaves, rather than in compact cones like other cycads. Feng, X. Y. , & Cycas, genus of 115 species of palmlike cycads in the family Cycadaceae native to tropical and subtropical areas of the Old World. 2006), we adopted a whole-genome inference approach to explore the phylogenetic position of endophytes genomic sequences, including JP106B, JP106C, RF15115, 33k59, 35k25, and TVP09, and two draft genomes reconstructed from metagenomic data of co-culture sub-communities, RF31YmG and 3335mG. To further characterize this observation, trees based on gene sets from the core proteome, which correspond to 16 metabolic subsystems, were reconstructed, as different phylogenetic signatures between them may contribute to long branches. Huanglian Mountain National Nature Reserve in Lvchun, Yunan, China (1), A relative healthy population benefit from early conservation and longterm monitoring. Coupling the metagenomic analysis with traditional microbiology increased our ability to accurately identify interacting endophytes in the sub-communities, and may help us characterize microbial life strategies and their phylogeny, a challenge that has received increasing attention in complex systems (Ho etal. Provan, J. Genome mining of specialized metabolites with an emphasis in all of the genomic sequences from endophythes of the coralloid roots (JP106C, TVP09, JP106B, RF15115, 35k25, 33k59, and 3335mG) plus five taxonomically closely related microorganisms identified in the previous section (KVJ20, PCC 73102, PCC 7126, PCC 7507, and PCC 7601), unveiled a total of 248 antiSMASH hits (supplementary table s8, Supplementary Material online). We outline six proposals to strengthen conservation measures for Cycas in China and anticipate that these measures will provide guidelines for further research on population genetics as well as conservation biology of not only cycads but also other endangered species worldwide. Six classification sections are now recognized in the genus Cycas (Table1): four in Hill (1995), an additional one in Lindstrom, Hill, and Stanberg (2008) and another one in Lindstrom, Hill, and Stanberg (2009). Eighty six percent of the taxa identified can be taxonomically classified as diazotrophic plant endophytes, including species belonging to the genera Bradyrhizobium, Burkholderia, Mesorhizobium, Rhizobium, and Nostoc (supplementary table s5, fig. The Author(s) 2018. In natural habitats coralloid roots appear in early life stages of the plant (Halliday and Pate 1976); and in adults mainly in habitats with poor or inaccessible nutrients, such as sand dunes, sclerophyll forests, steep rock outcrops with high exposure to salt, and lowland forests with recurrent fires (Grove etal. , & , Hill, K. D. ShermanBroyles, S. L. 2010; Zheng etal. (2012). 2002), termed BGC 16; and BGC 8 and 14, putatively directing the synthesis of a terpene and a bacteriocinlantipeptide, respectively. , 2010; Yamada etal. To further investigate into the suggested association between Caulobacter and the cyanobacteria, potentially mediated by the predicted indigoidine-like metabolite, strains D5 and 33k25 were cultivated together in plates containing BG11 and BG-110 media. Buord, S. Zou is preserved in Jinzhong Mountain Nature Reserve in Longlin, Guangxi; C.dolichophylla is scattered in Gulinqing Nature Reserve and Dawei Mountain Nature Reserve in Yunnan. The pithy stems of this and other species are a source of sago, a food starch. , Chen, J. R. High genetic diversity at the species level and strong genetic differentiation within Cycas have been observed. Wang & C.Y. Chen & S.Y. Whitfill Nursery, whitfillnursery.com. Living cycads are found in the tropical, subtropical, and warm temperate Z. with hard freezes and snow, and in areas that have no more than a couple Ma, Y. Geographic ranges of 23 Cycas species in China. Grouping together palms of the same species or with plants other than palms makes an interesting tropical landscape. Cycads (Cycadales) are ancient gymnosperms of Permian origin, currently mostly distributed in endangered low-density or isolated populations throughout New World tropical and subtropical regions (Gutirrez-Ortega etal. , One type of cycad, the Sago Palm, is a popular landscape plant. Conservation efforts for some of the endangered species are underway on most continents . Mix the fertilizer into the soil; about 2 inches down. We used Kraken (http://ccb.jhu.edu/software/kraken/; last accessed December 2018) to assign taxonomic labels to metagenomic DNA sequences based on exact alignment of k-mers (Wood and Salzberg 2014). , Scans were performed in the image acquisition mode and in the lambda mode to obtain the spectral fingerprint of each analyzed area. 4A). , In addition, experimental research that facilitating seed germination, seedling survival and vegetative propagation should also be conducted and applied in practice. Liu, N. (1996). While substantial numbers exist on the continents of Africa, Australia and South America they are also prominent in Central America (which has the greatest diversity) and the Caribbean Islands. As the most widespread genus within extant cycads, Cycas occupies several distribution centers with one scattered in China, particularly in the southwestern mountainous regions (Fig. Cycads - University of Notre Dame Cycad - an overview | ScienceDirect Topics PCR products of 1.4kb in length, obtained using the F27 and R1492 primers (Lane 1991), were cloned and sequenced using the Sanger method. Poaching rare and endangered plants should not be allowed. Rondinini, C. Our goal in this study is to investigate the endophytic bacterial community of the coralloid roots of species of Dioon, which are long-lived, perennial, entomophilous, dioecious, and arborescent cycads endemic to Mexico (De Luca etal. (2015). Cycads: Ancient tropical plants produce cones in UK for first time on We focused our sequencing effort on 13 samples: 1) two D. merolae samples from two plants from JP, named JP2 and JP6, and two D. merolae samples from two individuals from RF, named RF1 and RF3; 2) one sample from D. caputoi (TVP09); and 3) eight samples from two individuals from SJD, named SJD1SJD8 (fig. , As data are gathered from more genomes of bacterial cycad symbionts isolated from different environments of the rhizosphere-coralloid root ecological niche, it will be possible to test for deeper co-evolutionary relationships. the contents by NLM or the National Institutes of Health. Notably, genome mining of the cyanobacterial strains revealed three BGCs unique to the Dioon-specific cyanobionts, in agreement with the hypothesis of a specialized community driven by metabolic adaptation regardless of the taxonomic plasticity. Gong, X. 2018). Plants for the Southwest (Tucson), plantsforthesouthwest.com , & These , Living cycads are found in the tropical, subtropical and warm temperate regions of both the north and south hemispheres. It should be noted that aside from N.punctiforme PCC 73102, the genome of a cyanobacterial strain isolated from the coralloid roots of a C.revoluta plant, termed Nostoc cycadae WK-1 (Kanesaki etal. We acknowledge Juan Palacios and Antonio Hernndez for help during field collections, as well as Flor Zamudio for technical support. Diazotrophic microorganisms are prevalent: cyanobacterial species (marked in green) and Bradyrhizobium, Burkholderia, Mesorhizobium, and Rhizobium species (marked in blue). We undertook two approaches to bacterial isolation (fig. Btrisey, S. There are no reports of this predicted indigoidine-like metabolite in other bacteria, although synthesis of related congeners with antibacterial activity has been reported (ODowd etal. Jian, S. G. Cycads are essentially living fossils, or evolutionary relicts, and are of great scientific and conservation value because of their long evolutionary history. South China Botanical Garden was the first organization to conduct ex situ conservation for cycads, with 43 species belonging to nine genera currently planted (Huang, 2014). However, the genomic sequences TVP09 from D. caputoi, plus JP106B, JP106C and RF31YmG from D. merolae, form a monophyletic clade, and group with Tolypothrix sp. Raven, P. H. In analogy with the pairwise interdependencies reported for Pleurocapsa and Calothrix (Paerl 2012), it is tempting to speculate about similar cases occurring within the Dioon coralloid roots microbiome. Yang & S.L. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (, Impacts of sex ratio meiotic drive on genome structure and function in a stalk-eyed fly, Brain gene expression of foraging behaviour and social environment in Ceratina calcarata, Extensive Copy Number Variation Explains Genome Size Variation in the Unicellular Zygnematophycean Alga, Closterium peracerosum-strigosum-littorale Complex, A PacBio Hi-Fi genome assembly of the Painter's Mussel Unio pictorum (Linnaeus, 1758), Ribosomal intergenic spacers are filled with transposon remnants, Volume 15, Issue 6, June 2023 (In Progress), About the Society for Molecular Biology and Evolution, http://tree.bio.ed.ac.uk/software/figtree/, https://www.ncbi.nlm.nih.gov/bioproject/369422, http://www.ncbi.nlm.nih.gov/bioproject/472628, https://www.ncbi.nlm.nih.gov/sra/SRP149992, http://creativecommons.org/licenses/by-nc/4.0/, Receive exclusive offers and updates from Oxford Academic, Copyright 2023 Society for Molecular Biology and Evolution. Visit cycad.org for details. At the same time, cycads are also protected in other nature reserves. Genetic diversity, genetic structure and demographic history of. , (2003). Encephalartos aemulans | PlantZAfrica - SANBI We thank R. Porter for constructive comments on manuscript writing. Simultaneously, we enriched the same samples for specific sub-communities in co-cultures grown under oligotrophic conditions specific for cyanobacteria, namely, BG-110 medium. , 2014). Bickerton, D. C. Deltoro, V. , Dao, Z. L. Forster, P. I. FOIA Huang, S. , Another general conclusion we found is that significant genetic differentiation among populations exists in Cycas from China, especially in southwest China, which is in accordance with results derived from a previous summary of six Cycadaceae species (Fst=0.254) (GonzlezAstorga etal., 2009). The presence of diazotrophic microorganisms in the sub-community cultures was confirmed by BLAST-positive hits of the NifH enzyme to selected genera, such as Bradyrhizobium and Mesorhizobium species, with 99% sequence identity. The genetic information contained in cycads is important for paleontology, paleoclimatology, and paleogeography. Moore, C. J. Li, Y. Y. Serra, L. As ancient gymnosperm and woody plants, cycads have survived through dramatic tectonic activities, climate fluctuation, and environmental variations making them of great significance in studying the origin and evolution of flora biodiversity. Likewise, various chemical, physical, and physiological processes appear to regulate the cycadbacteria interaction (Lobakova etal. (PDF) Correlations between leaf economics, mechanical resistance, and In addition, the newly published species, C.chenii X. Gong & Wei Zhou, is affiliated with section Stangerioides (Zhou, Guan, & Gong, 2015). The population size is large (>2,000 individuals) and the population structure is healthy with a third of plants are juveniles. Wang, X. The material for reinforcement must originate either from the same location or from the geographically closest populations within the same habitat, as well as being genetically diverse. Updates? These low-growing beauties are often incorporated into landscapes for their architectural, palmlike appearance. The overall sequence identity between these BGCs, which may provide examples of specialized metabolites with functional implications within the coralloid root, is of 85%, 89%, and 87%, respectively. Both our two Caulobacter isolates are able to produce a specific specialized metabolite, which could mediate the association between either microorganisms or their host. Koch, M. A. For example, C.chenii is a newly published species with six natural populations discovered in Yunnan, China (Zhou etal., 2015). The evergreen leaves grow in a rosette directly from the top of the trunk, creating a crown of foliage as the plant ages and the older leaves fall off. Frequent mudrock flows and landslides triggered by increased rainfall have ruined habitats. 2014); but also in the distantly related Gammaproteobacteria, and plant pathogen, Erwinia chrysanthemi (Reverchon etal. 2017), and/or interactions with the host, adding to the growing notion that symbiotic relations occur under heavy influence of metabolic exchange. , 2012). Choose a variety that thrives in your climate zone. , Chase, M. W. Set of sequences were concatenated and aligned using MUSCLE v3.8.31 with default parameters (Edgar 2004), and trimmed by Gblocks (Talavera and Castresana 2007) with five positions as minimum block length, 10 as a maximum number of contiguous nonconserved positions, and only considering positions with a gap in <50% of the sequences in the final alignment. We grew and isolated bacteria from environmental samples using a diverse set of semiselective media that aim to capture as much of the cultured bacterial diversity as possible (t0). 1987), similar to the adaptive functions in legume nodules, but having appeared millions of years before them. Jr BGCs that are Dioon clade-specific (black boxes) and Dioon-specific (red boxes), with chemical structure prediction and functional annotation () are shown. Some specific conservation areas have been founded for protecting Cycas, such as the National Nature Reserve for C.panzhihuaensis in Panzhihua, Sichuan; the National Nature Reserve of Dawei Mountain for protecting C. hongheensis, C. multipinnata, and C.diannanensis in Honghe, Yunnan; and the Nature Reserve for C.taitungensis in Taitung, Taiwan (Ma, Li, Su, & Lin, 2005). outside the Hertz music building on the U.C. Key characteristics of six Cycas sections worldwide and species list of Cycas in China. (2014). Search for other works by this author on: Herbario Eizi Matuda, Laboratorio de Ecologa Evolutiva, Instituto de Ciencias Biolgicas, Universidad de Ciencias y Artes del Estado de Chiapas, Tuxtla Gutirrez, Chiapas, Mxico, The RAST Server: rapid annotations using subsystems technology, Hopwood, DA, editor. Hence, the Caulobacter-Nostocales consortium could be playing a previously unknown role inside the coralloid roots of cycads by producing few but specific natural products, making them specialized metabolites. Overexploitation with uneven age structure, Dawei Mountain Nature Reserve, Yunnan, China; Nature Reserve in Yuanjiang, State Nature Reserve in dinosaur river, Provincial Reserve of A'mu Mountain in Honghe for, This specie has almost extinct in wild with only one population discovered. , Malanson, G. P. , The successful reintroduction of C.debaoensis in Huanglian Mountain Nature Reserve has set a good example for ex situ conservation practice (Gan etal., 2013). Also, two Caulobacter strains, D5 and D4A, associated with cyanobionts from D. edule were isolated, and their genomes sequenced and mined for niche-specific metabolites. Thus, we focus on the biosynthetic systems that are Dioon-specific, which have NRPS or PKS components, facilitating the prediction of their chemical structure.
