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Evolutionary studies of the Gnetales
Stockholm University, Faculty of Science, Department of Ecology, Environment and Plant Sciences. (Catarina Rydin)ORCID iD: 0000-0002-8558-5655
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The Gnetales consist of three distinct genera, Ephedra, Gnetum and Welwitschia with considerable divergence among them regarding morphological, ecological and molecular characters. A longstanding debate of the similarity between the Gnetales and angiosperms and the unresolved seed plant phylogeny intrigues plant scientists to further investigate the evolutionary history of the Gnetales. The presented projects deal with interdisciplinary questions on proteomics, chloroplast genomes, phylogenetic relationships, gross morphology and taxonomy. The thesis aims to summarize general problems encountered in previous studies, and to provide new insights and future perspectives based on the results of completed and ongoing projects.   

In Ephedra, the Mediterranean species E. foeminea has been shown to be entomophilous and it possesses an important phylogenetic status as the sister of the remaining genus. Therefore, the chloroplast genome of E. foeminea was assembled and compared to that previously presented (of the anemophilous Asian species E. equisetina, nested in the core clade of Ephedra). The genome has a quadripartite structure and comprises 118 genes and 109,584 base pairs. A pairwise genome comparison was conducted between E. foeminea and E. equisetina, resulting in the detection of 2,352 variable sites, the obtained data can be used for prospective phylogenetic studies. A proteomic study was also conducted on E. foeminea along with three anemophilous Ephedra species, in order to investigate the biochemistry of the pollination drops. The results show that detected proteins in the pollination drops of Ephedra vary dramatically among species but always occur in very low amounts. The majority of the detected proteins are degradome proteins, i.e., waste products from degrading cells of the nucellus. Some secretome proteins were also found, which are putatively functional, but also these proteins occur in very low amounts. The repeatability of the proteomic studies can, however, be questioned. The sampling methods and proteomic analyses are probably problematic although some suggestions for improvement are provided. Thus I chose to continue with other projects.

In Gnetum, reconstruction of the genus phylogeny and assessments of divergence times of clades were performed using an extensive sampling of ingroup and outgroup accessions. The results show that the South American lineage separated from the remaining genus in the Late Cretaceous. The continued diversification event gave rise to an African lineage and an Asian lineage. The crown age of the Asian clade, which comprises two arborescent species sister to the remaining liaonid species, was estimated to the Cretaceous-Paleogene (K-Pg) boundary. In light of the genus phylogeny and estimated node ages, we suggest that the breakup of Gondwana influenced diversification patterns in Gnetum. Later dispersal events also contributed to the current distribution of Gnetum, and to the phylogenetic patterns within each of the major clades. From my results, it is however clear that taxonomy and species delimitations are poorly defined, and needs to be further studied for all subclades of Gnetum. I have initiated this task by studying the Chinese lianoid clade of Gnetum more in depth. Eleven chloroplast genomes were generated, aligned and compared. Based on the information, four chloroplast markers were designed and applied to further resolve the species relationships with an extensive sampling. The results show, with strong support, that G. parvifolium is sister to all the remaining species of the Chinese linaoid clade. Another five lianoid species are confirmed using both morphological and molecular data, but several names are represented by type material that cannot be considered separate species. Modified keys for identification of male and female plants are presented, based on vegetative and reproductive structures. A subsequent dating analysis indicates that diversification in the Chinese lianoid Gnetum clade took place mainly in the Neogene, during which environmental changes probably facilitated diversification in the lineage.

Place, publisher, year, edition, pages
Stockholm: Department of Ecology, Environment and Plant Sciences, Stockholm University , 2016. , 44 p.
National Category
Botany
Research subject
Plant Systematics
Identifiers
URN: urn:nbn:se:su:diva-128439ISBN: 978-91-7649-371-7 (print)OAI: oai:DiVA.org:su-128439DiVA: diva2:915009
Public defence
2016-06-03, Vivi Täckholmsalen (Q-salen), NPQ-huset, Frescativägen 20 A, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

Available from: 2016-05-11 Created: 2016-03-28 Last updated: 2017-02-17Bibliographically approved
List of papers
1. The chloroplast genome of Ephedra foeminea (Ephedraceae, Gnetales), an entomophilous gymnosperm endemic to the Mediterranean area
Open this publication in new window or tab >>The chloroplast genome of Ephedra foeminea (Ephedraceae, Gnetales), an entomophilous gymnosperm endemic to the Mediterranean area
2015 (English)In: Mitochondrial DNA, ISSN 1940-1736, E-ISSN 1940-1744, Vol. 28, no 3, 330-331 p.Article in journal (Refereed) Epub ahead of print
Abstract [en]

This study presents the chloroplast genome of Ephedra foeminea, an entomophilous gymnosperm, sister to the remaining (wind-pollinated) species of Ephedra (Ephedraceae, Gnetales). Based on the reference-guided assembly, the length of the chloroplast genome was estimated to be 109 584 bp, comprising a large single copy region of 60 027 bp, a small single copy 8079 bp, and inverted repeat regions of 20 739 bp. In total, 118 genes were detected, including 73 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. The gene density is 1.076 (genes/kb) and the GC content is 36.7%. The genomic sequence of the entomophilous, Mediterranean species E. foeminea, differs from that of the anemophilous, Asian species E. equisetina by 1018 point mutations and 1334 indels. The detected variation is useful for future development of new plastid markers for phylogenetic purposes. Our phylogenetic analysis based on 55 protein-coding chloroplast genes resolve Ephedra as monophyletic and sister to a Gnetum-Welwitschia clade. The Gnetales are sister to Cupressophytes.

Keyword
Chloroplast genome, Ephedra foeminea, Illumina sequencing, phylogeny
National Category
Biochemistry and Molecular Biology
Research subject
Plant Systematics
Identifiers
urn:nbn:se:su:diva-128435 (URN)10.3109/19401736.2015.1122768 (DOI)000397858400016 ()26713841 (PubMedID)
Available from: 2016-03-28 Created: 2016-03-28 Last updated: 2017-11-30Bibliographically approved
2. New insights into the evolutionary history of Gnetum (Gnetales)
Open this publication in new window or tab >>New insights into the evolutionary history of Gnetum (Gnetales)
2015 (English)In: Taxon, ISSN 0040-0262, E-ISSN 1996-8175, Vol. 64, no 2, 239-253 p.Article in journal (Refereed) Published
Abstract [en]

Gnetum (Gnetales: Gnetaceae) constitutes an evolutionarily isolated gymnosperm clade, comprising about 40 species that inhabit tropical areas of the world. While its closest living relative, the monotypic Welwitschia, has a well-documented fossil record from the Early Cretaceous, Gnetum-like fossils are rare and poorly understood. The phylogeny of Gnetum has been studied previously but the distant relationship to outgroups and the difficulty of obtaining plant material mean it is not yet fully resolved. Most species are tropical lianas with an angiospermous vegetative habit that are difficult to find and identify. Here a new phylogeny is presented based on nuclear and chloroplast data from 58 Gnetum accessions, representing 27 putative species, and outgroup information from other seed plants. The results provide support for South American species being sister to the remaining species. The two African species constitute a monophyletic group, sister to an Asian clade, within which the two arborescent species of the genus are the earliest diverging. Estimated divergence times indicate, in contrast with previous results, that the major lineages of Gnetum diverged in the Late Cretaceous. This result is obtained regardless of tree prior used in the BEAST analyses (Yule or birth-death). Together these findings suggest a correlation between early divergence events in extant Gnetum and the breakup of Gondwana in the Cretaceous. Compared to the old stem ages of major subclades of Gnetum, crown nodes date to the Cenozoic: the Asian crown group dates to the Cretaceous-Paleogene (K-Pg) boundary, the African crown group to the mid-Paleogene, and the South American crown group to the Paleogene-Neogene boundary. Although dispersal must have contributed to the current distribution of Gnetum, e.g., within South America and from Southeast Asian islands to the East Asian mainland, dispersal has apparently not occurred across major oceans, at least not during the Cenozoic.

Keyword
Bayesian inference, birth-death prior, classification, node ages, phylogeny, Yule prior
National Category
Biological Sciences
Research subject
Plant Systematics
Identifiers
urn:nbn:se:su:diva-118277 (URN)000354372300004 ()
Available from: 2015-06-22 Created: 2015-06-15 Last updated: 2017-12-04Bibliographically approved
3. Degradome and Secretome of Pollination Drops of Ephedra
Open this publication in new window or tab >>Degradome and Secretome of Pollination Drops of Ephedra
Show others...
2015 (English)In: The Botanical review, ISSN 0006-8101, E-ISSN 1874-9372, Vol. 81, no 1, 1-27 p.Article in journal (Refereed) Published
Abstract [en]

Although secreted proteins (a secretome) are known to occur in gymnosperm pollination drops, this study shows evidence for the presence of a protein degradome for the first time. A protein degradome is composed of protein and peptide fragments, a product of protein breakdown, whereas a secretome is composed of whole, secreted, and often biologically active extracellular proteins. Harvested Ephedra pollination drops from seven species were pooled either by collection date or, in the case of less abundant sample volumes, by species. Samples were processed by one of two methods: 1. gel electophoresis or by 2. liquid-liquid extraction, followed by chromatographic separation. Processed samples were trypsin-digested and analyzed with a Thermo Scientific LTQ Orbitrap Velos. On average, two-thirds of the detected and characterized proteins found in Ephedra spp. pollination drops were intracellular proteins, such as ubiquitin. The remaining third represent proteins known to be secreted, often involved in apoplastic processes such as defense and carbohydrate-modification, typical of known conifer pollination drop proteins. Characterized proteins detected in our comparative study of Ephedra spp drops ranged from 6 in E. monosperma to 20 in E. foeminea. We propose that the intracellular proteins detected are present as the result of nucellar tissue degeneration during pollination drop formation; previous proteomic investigations of pollination drops were in taxa that lack nucellar degeneration during drop formation Discovery of a degradome in pollination drops is novel and significant in that its presence has biological implications for pollination biology. We predict that degradomes in pollination drops are not restricted to Ephedra, but should also occur in species with nucellar tissue breakdown that coincides with pollination drop formation, such as in cycads and Ginkgo and some Pinaceae. Analysis of several collection dates of E. monosperma shows a large number of proteins that change over the course of the pollination drop secretion period, which suggests that variation in pollination drop contents over time may be important in the pollination biology of Ephdera.

Keyword
Degradome, Ephedra, Gymnosperm reproduction, Pollination drop, Proteomics, Secretome
National Category
Biological Sciences
Research subject
Plant Systematics
Identifiers
urn:nbn:se:su:diva-116638 (URN)10.1007/s12229-014-9147-x (DOI)000350897300001 ()
Note

AuthorCount:10;

Available from: 2015-04-23 Created: 2015-04-22 Last updated: 2017-12-04Bibliographically approved
4. Proteome of pollination drops in Ephedra (Gnetales)
Open this publication in new window or tab >>Proteome of pollination drops in Ephedra (Gnetales)
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Proteins are important in many physiological and ecological processes. In pollination drops of gymnosperms, proteins take part in a number of pollen-ovule interactions and aid for example in ovule defense. Pollination drops in Ephedra (Gnetales) are secreted in large volumes and are known to be high in sucrose, but their proteomic profile has not been thoroughly studied. In this study, proteomic analysis of pollination drops of Ephedra was carried out based on four species: E. distachya, E. foeminea, E. likiangensis and E. minuta. Pollination drop samples were analyzed using gel electrophoresis (1D SDS-PAGE) and mass spectrometry (LC-MS analysis). Protein profile and putative function of proteins were assessed by searches against a set of proteomic databases. Results indicate that number of proteins as well as their amount was low, and most proteins belong to a degradome, i.e., they are remains of degrading tissue at the apex of the nucellus during formation of the pollination chamber. Variation among species was considerable. Ephedra foeminea, which is sister to all other species, contains more kinds of proteins than other investigated species. The closely related species E. likiangensis and E. minuta differ considerably in protein profiles. In E. distachya, no secretome was found; all detected proteins in this species are degradome. The observed differences between species may be related to pollination biology. Insect pollinated species, such as E. foeminea appear to have more proteins in their pollination drops than wind-pollinated species such as E. distachya. Many things are still uncertain regarding proteomics of pollination drops in Ephedra. Among other things, repeatability of results may be a problem; however, this study clearly shows that the amount of proteins in ephedran pollination drops is very low compared to other studied gymnosperms. Future studies of ovule defense in Ephedra should probably have another focus, and perhaps consider sugar content and carbohydrate metabolism rather than proteomics.

Keyword
pollination drops, Ephedra, proteomics, mass spectrometry, evolution of pollination, ovule defense, carbohydrate metabolism
National Category
Botany
Research subject
Plant Systematics
Identifiers
urn:nbn:se:su:diva-128437 (URN)
Available from: 2016-03-28 Created: 2016-03-28 Last updated: 2016-03-30Bibliographically approved
5. Resolving phylogenetic relationships and species delimitations in closely related gymnosperms using high-throughput NGS, Sanger sequencing and morphology
Open this publication in new window or tab >>Resolving phylogenetic relationships and species delimitations in closely related gymnosperms using high-throughput NGS, Sanger sequencing and morphology
2016 (English)In: Plant Systematics and Evolution, ISSN 0378-2697, E-ISSN 1615-6110, Vol. 302, no 9, 1345-1365 p.Article in journal (Refereed) Published
Abstract [en]

Plastid genomes have been widely applied to elucidate plant evolution at higher taxonomic levels, but have rarely been considered useful for addressing close relationships. Here, we resolve the phylogeny and taxonomy of the Chinese lianoid Gnetum clade (Gnetales), using high throughput and Sanger sequencing techniques and studies of plant morphology. Despite previous efforts, relationships among taxa and the taxonomy within the clade have remained unclear. We generated 11 plastid genomes representing one arborescent and four lianoid species. Phylogenetic analyses were conducted using (a) the entire plastid genomes and (b) the protein-coding genes only. Sequence divergence among the lianoid species was substantial, with 9345 variable sites. Four variable regions were identified, targeted and sequenced for an additional 54 specimens and analyzed together with one nuclear ribosomal marker. Results from the phylogenetic analyses corroborate G. parvifolium as sister to the remaining lianoid species and support the presence of at least five additional species in the Chinese lianoid clade: G. catasphaericum, G. formosum, G. luofuense, G. montanum and G. pendulum. Following morphological investigations, G. giganteum and G. gracilipes are included in and synonymized with G. pendulum. Gnetum hainanense is included in and synonymized with G. luofuense. Two names, G. indicum and G. cleistostachyum, remain questionable. A taxonomic revision and a key to Chinese lianoid Gnetum are presented. Internal nodes in the Chinese lianoid Gnetum clade are from the Miocene and onwards and coincide with the expansion of tropical to subtropical forests in South China, which may have facilitated speciation in the clade.

Keyword
Historical diversification, Next-generation sequencing, Phylogeny, Plant taxonomy, Plastid genomes
National Category
Botany
Research subject
Plant Systematics
Identifiers
urn:nbn:se:su:diva-128438 (URN)10.1007/s00606-016-1335-1 (DOI)000385192100012 ()
Available from: 2016-03-28 Created: 2016-03-28 Last updated: 2017-11-30Bibliographically approved

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