Aiping Meng

Phylogenomic and structural analyses of 18 complete plastomes across nearly all families of early-diverging eudicots, including an angiosperm-wide analysis of IR gene content evolution

The grade of early-diverging eudicots includes five major lineages: Ranunculales, Trochodendrales, Buxales, Proteales and Sabiaceae. To examine the evolution of plastome structure in early-diverging eudicots, we determined the complete plastome sequences of eight previously unsequenced early-diverging eudicot taxa, Pachysandra terminalis (Buxaceae), Meliosma aff. cuneifolia (Sabiaceae), Sabia yunnanensis (Sabiaceae), Epimedium sagittatum (Berberidaceae), Euptelea pleiosperma (Eupteleaceae), Akebia trifoliata (Lardizabalaceae), Stephania japonica (Menispermaceae) and Papaver somniferum (Papaveraceae), and compared them to previously published plastomes of the early-diverging eudicots Buxus, Tetracentron, Trochodendron, Nelumbo, Platanus, Nandina, Megaleranthis, Ranunculus, Mahonia and Macadamia. All of the newly sequenced plastomes share the same 79 protein-coding genes, 4 rRNA genes, and 30 tRNA genes, except for that of Epimedium, in which infA is pseudogenized and clpP is highly divergent and possibly a pseudogene. The boundaries of the plastid Inverted Repeat (IR) were found to vary significantly across early-diverging eudicots; IRs ranged from 24.3 to 36.4kb in length and contained from 18 to 33 genes. Based on gene content, the IR was classified into six types, with shifts among types characterized by high levels of homoplasy. Reconstruction of ancestral IR gene content suggested that 18 genes were likely present in the IR region of the ancestor of eudicots. Maximum likelihood phylogenetic analysis of a 79-gene, 97-taxon data set that included all available early-diverging eudicots and representative sampling of remaining angiosperm diversity largely agreed with previous estimates of early-diverging eudicot relationships, but resolved Trochodendrales rather than Buxales as sister to Gunneridae, albeit with relatively weak bootstrap support, conflicting with what has been found for these three clades in most previous analyses. In addition, Proteales was resolved as sister to Sabiaceae with the highest support (bootstrap >90%) yet observed in plastome-scale phylogenetic analyses.

Complete Plastid Genome Sequencing of Trochodendraceae Reveals a Significant Expansion of the Inverted Repeat and Suggests a Paleogene Divergence between the Two Extant Species

The early-diverging eudicot order Trochodendrales contains only two monospecific genera, Tetracentron and Trochodendron. Although an extensive fossil record indicates that the clade is perhaps 100 million years old and was widespread throughout the Northern Hemisphere during the Paleogene and Neogene, the two extant genera are both narrowly distributed in eastern Asia. Recent phylogenetic analyses strongly support a clade of Trochodendrales, Buxales, and Gunneridae (core eudicots), but complete plastome analyses do not resolve the relationships among these groups with strong support. However, plastid phylogenomic analyses have not included data for Tetracentron. To better resolve basal eudicot relationships and to clarify when the two extant genera of Trochodendrales diverged, we sequenced the complete plastid genome of Tetracentron sinense using Illumina technology. The Tetracentron and Trochodendron plastomes possess the typical gene content and arrangement that characterize most angiosperm plastid genomes, but both genomes have the same unusual ∼4 kb expansion of the inverted repeat region to include five genes (rpl22, rps3, rpl16, rpl14, and rps8) that are normally found in the large single-copy region. Maximum likelihood analyses of an 83-gene, 88 taxon angiosperm data set yield an identical tree topology as previous plastid-based trees, and moderately support the sister relationship between Buxaceae and Gunneridae. Molecular dating analyses suggest that Tetracentron and Trochodendron diverged between 44-30 million years ago, which is congruent with the fossil record of Trochodendrales and with previous estimates of the divergence time of these two taxa. We also characterize 154 simple sequence repeat loci from the Tetracentron sinense and Trochodendron aralioides plastomes that will be useful in future studies of population genetic structure for these relict species, both of which are of conservation concern.

Floral organogenesis of Cocculus Orbiculatus and Stephania Dielsiana (Menispermaceae)

Floral ontogeny of two dioecious species in the Menispermaceae, Cocculus orbiculatus and Stephania dielsiana, was compared using scanning electron microscopy (SEM). In C. orbiculatus, flowers exhibit rudiments of a nonfunctional organ of the opposite sex. Female flowers have six stamens that remain primordial; however, male flowers usually have three carpellodes rather than six, and hence have a different number of carpels than female flowers. Development of carpellodes in the family was described under SEM for the first time. The male and female of C. orbiculatus are almost homomorphic in early developmental stages, with eight alternating whorls of four types of organs (sepals, petals, stamens/staminodes, and carpels/carpellodes) arising centripetally. In S. dielsiana, no vestigial sexual organs were found, and the symmetry of male and female flowers differs, with males being actinomorphic and females zygomorphic. The six stamens of C. orbiculatus are free, whereas the three or four stamens of S. dielsiana are united congenitally. There are six carpels in females of C. orbiculatus but a single one in S. dielsiana. In overview, all flowers are actinomorphic and trimerous, with the female flower of S. dielsiana being the exception. This may be the result of reduction and is yet to be determined.

Karyomorphology of Epimedium (Berberidaceae) and its phylo- genetic implications

Abstract The karyomorphology of 21 species and one variety of Epimedium was investigated in this study, among which 14 species and one variety were reported for the first time. The interphase nucleus and mitotic pro-phase chromosomes were found to be of the complex chromocenter type, and the interstitial type, respectively. The chromosome numbers of all species studied were 2n = 2x = 12. The investigated Epimedium species had a uniform karyotype with 2n = 6m + 6sm, except for three species, E. platyplatum, E. pubescens and E. stellulatum, which had a pair of chromosomes with subterminal centromeres. A secondary constriction was commonly found on the proximal side of the short arm of the chromosome pair I in these species, except for E. pseudowushanense and E. elachyphyllum in which the constriction was on chromosome pair II. The karyotype symmetry of all the investigated species was relatively primitive 2A. Pericentric inversion was observed in E. leptorrhizum, and unequal translocation occured in E. franchetii, E. sutchuenense, E. dolichostemon and E. koreanum. These chromosomal variations probably result in speciation of Epimedium to some extent. The infrageneric and intergeneric relationships of Epimedium in Berberidaceae were discussed.

Floral Development of Stephania (Menispermaceae): Impact of Organ Reduction on Symmetry

Stephania is the sole genus in the basal eudicot family Menispermaceae that possesses both actinomorphic and zygomorphic flowers. Variation in perianth merism can have an important impact on flower symmetry and thus eminent biological significance in evolution of Menispermaceae. Using SEM, we studied the floral development of four representative species, which present the two predominant floral patterns of the genus, namely, homomorphy of both male and female flowers (actinomorphy) or heteromorphy (actinomorphy/zygomorphy). The sepals of the male flowers are arranged mostly in two alternate whorls of three or four each, whereas in female flowers they are in a single whorl of three or four or there is only a single sepal. Petals of male flowers are in a whorl of three or four organs, whereas female flowers of some species have only two petals. Trimerous and tetramerous perianths can coexist in the same umbellets of some species. Variation in perianth merism and loss of perianth parts of the female flowers may result in flower symmetry switching from actinomorphy to zygomorphy. The two main floral patterns are consistent with a distinction of two subclades within Stephania. The unicarpellate genera of the Menispermaceae share a unique combination of characters, including a synandrium, unitegmic ovules, and absence of vestigial sexual organs. However, Stephania differs from its unicarpellate relatives by two features: a two-whorled arrangement of floral organs of the male flowers and a free perianth. The investigation provides new and valuable developmental information on flowers of the little-known Menispermaceae and provides a background for a discussion of the evolution of merism and unisexual flowers in the basal eudicots.

Floral development of Phyllanthus chekiangensis (Phyllanthaceae), with special reference to androecium and gynoecium

The floral development of Phyllanthus chekiangensis has been studied by scanning electron microscopy. The perianth organs are initiated in two whorls, dimerous in male flowers and trimerous in female flowers, with a longer plastochron between whorls than between the organs within a whorl. Male flowers have two stamens. The prominent connective protrusions begin development simultaneously with the floral disk. The disk is two-lobed in male flowers but continuous in female flowers. In female flowers, the developing gynoecium remains open relatively long, so the developing ovules are visible from the outside for some time. The direction of the hemitropous ovules in the carpels is antitropous (epitropous). Two small obturators are formed per carpel, one above each ovule. The prominent nucellar beak extends far beyond the “micropyle”. A micropyle in the classical sense formed by integuments closing over the nucellus apex is not present at any stage of development. Thus, it is not correct to say that the nucellar beak “grows through the micropyle”. The exposed nucellar beak continues the curvature of the antitropous (epitropous) ovule and becomes contiguous with the obturator. The unusual length of the nucellar beak may be a potential synapomorphy of the enlarged Phyllanthus clade as inferred from molecular phylogenetics.

Observation on meiotic behavior in three Mahonia species, with special reference to the intergeneric relationship of Mahonia and Berberis

Abstract Cytological characters in meiosis of three Mahonia species-Mahonia fortunei, M. bealei and M. polydonta were investigated using the method of wall degradation and hypotonic treatment for chromosomal preparations. Chromosome numbers of the three species were all 2n = 2x = 28; those of M. fortunei and M. polydonta represent the first report. The three species at metaphase I possessed the same configuration of 14II, however, the secondary association was also observed. During anaphase I, 1.5% of the cells of M. fortunei had lagging chromosomes and chromosome bridges. During anaphase II, lagging chromosomes, chromosome bridges and asynchronism in second division were observed, with abnormality rate in M. fortunei being about 9.5%. The rate of fertile pollen grains of the three species amounted to more than 90%, which was consistent with the cell abnormality rate at anaphase II. The results indicate that, overall, meiotic process of the three species is normal, which is in line with the high pollen fertility rate and high seed yield of the three species. Our results support the view that Mahonia and Berberis represent a sister group in phylogenetic evolution, and that they should be treated as two distinct genera.

A new species of Argentina (Rosaceae, Potentilleae) from Southeast Tibet, with reference to the taxonomic status of the genus

Argentina songzhuensis, a new species of tribe Potentilleae (Rosaceae) from Southeast Tibet, is described and illustrated. Placement of this species within Argentina was assessed based on morphological comparisons with related species and on phylogenetic analyses of nuclear ITS and plastid trnL-F sequences. The new species is similar to A. glabriuscula, but can be easily distinguished by its densely hairy leaflets, number of stamens (ca.10–12) and decurrent bases of the uppermost pair of leaflets. Our results support the generic status of Argentina and suggest that ventral stipular auricles may be a good synapomorphy for the genus. The recent transfer to Argentina of several Sibbaldia species as well as the genera Piletophyllum and Tylosperma is also confirmed by our molecular phylogenetic analyses. A taxonomic key to A. songzhuensis and other Argentina species in Tibet is provided.

Plastid phylogenomics resolves infrafamilial relationships of the Styracaceae and sheds light on the backbone relationships of the Ericales

Relationships among the genera of the small, woody family Styracaceae and among families of the large, diverse order Ericales have resisted complete resolution with sequences from one or a few genes. We used plastome sequencing to attempt to resolve the backbone relationships of Styracaceae and Ericales and to explore plastome structural evolution. Complete plastomes for 23 species are newly reported here, including 18 taxa of Styracaceae and five of Ericales (including species of Sapotaceae, Clethraceae, Symplocaceae, and Diapensiaceae). Combined with publicly available complete plastome data, this resulted in a data set of 60 plastomes, including 11 of the 12 genera of Styracaceae and 12 of 22 families of Ericales. Styracaceae plastomes were found to possess the quadripartite structure typical of angiosperms, with sizes ranging from 155 to 159 kb. Most of the plastomes were found to possess the full complement of typical angiosperm plastome genes. Unusual structural features were detected in plastomes of Alniphyllum and Bruinsmia, including the presence of a large 20-kb inversion (14 genes) in the Large Single-Copy region, the loss or pseudogenization of the clpP and accD genes in Bruinsmia, and the loss of the first exon of rps16 in B. styracoides. Likewise, the second intron from clpP was found to be lost in Alniphyllum and Huodendron. Phylogenomic analyses including all 79 plastid protein-coding genes provided improved resolution for relationships among the genera of Styracaceae and families of Ericales. Styracaceae was strongly supported as monophyletic, with Styrax, Huodendron, and a clade of Alniphyllum + Bruinsmia successively sister to the remainder of the family, all with strong support. All genera of Styracaceae were recovered as monophyletic, except for Halesia and Pterostyrax, which were each recovered as polyphyletic with strong support. Within Ericales, all families were recovered as monophyletic with strong support, with Balsaminaceae sister to remaining Ericales. Most relationships recovered in plastome analyses are congruent with previous analyses based on smaller data sets. Our results demonstrate the power of plastid phylogenomics to improve phylogenetic hypotheses among genera and families, and provide new insight into plastome evolution across Ericales.

Development and Application of Transcriptome-Derived Microsatellites in Actinidia eriantha (Actinidiaceae)

Actinidia eriantha Benth. is a diploid perennial woody vine native to China and is recognized as a valuable species for commercial kiwifruit improvement with high levels of ascorbic acid as well as having been used in traditional Chinese medicine. Due to the lack of genomic resources for the species, microsatellite markers for population genetics studies are scarce. In this study, RNASeq was conducted on fruit tissue of A. eriantha, yielding 5,678,129 reads with a total output of 3.41 Gb. De novo assembly yielded 69,783 non-redundant unigenes (41.3 Mb), of which 21,730 were annotated using protein databases. A total of 8,658 EST-SSR loci were identified in 7,495 unigene sequences, for which primer pairs were successfully designed for 3,842 loci (44.4%). Among these, 183 primer pairs were assayed for PCR amplification, yielding 69 with detectable polymorphism in A. eriantha. Additionally, 61 of the 69 polymorphic loci could be successfully amplified in at least one other Actinidia species. Of these, 14 polymorphic loci (mean NA = 6.07 ± 2.30) were randomly selected for assessing levels of genetic diversity and population structure within A. eriantha. Finally, a neighbor-joining tree and Bayesian clustering analysis showed distinct clustering into two groups (K = 2), agreeing with the geographical distributions of these populations. Overall, our results will facilitate further studies of genetic diversity within A. eriantha and will aid in discriminating outlier loci involved in local adaptation.