Zhi-Duan Chen

PHYLOGENETIC RELATIONSHIPS IN FAGALES BASED ON DNA SEQUENCES FROM THREE GENOMES

Nucleotide sequences of six regions from three plant genomes—trnL‐F, matK, rbcL, atpB (plastid), matR (mtDNA), and 18S rDNA (nuclear)—were used to analyze inter‐ and infrafamilial relationships of Fagales. All 31 extant genera representing eight families of the order were sampled. Congruence among data sets was assessed using the partition homogeneity test, and five different combined data sets were analyzed using maximum parsimony and the Bayesian approach. At the familial level, the same phylogenetic relationships were inferred from five different analyses of these data. Nothofagus, followed by Fagaceae, are subsequent sisters to the rest of the order. Fagaceae are then sister to the core “higher” hamamelids, which consist of two main subclades, one being Myricaceae (Rhoipteleaceae (Juglandaceae)) and the other Casuarinaceae (Ticodendraceae (Betulaceae)). The combined data sets provide the best‐supported estimate of evolutionary relationships within Fagales. Our results suggest that the combination of different sequences from several species within the same genus representing a terminal taxon has little influence on phylogenetic accuracy. Inclusion of taxa with some missing data in combined data sets also does not have a major impact on the topology.

Extensive pyrosequencing reveals frequent intra-genomic variations of internal transcribed spacer regions of nuclear ribosomal DNA.

Background Internal transcribed spacer of nuclear ribosomal DNA (nrDNA) is already one of the most popular phylogenetic and DNA barcoding markers. However, the existence of its multiple copies has complicated such usage and a detailed characterization of intra-genomic variations is critical to address such concerns. Methodology/Principal Findings In this study, we used sequence-tagged pyrosequencing and genome-wide analyses to characterize intra-genomic variations of internal transcribed spacer 2 (ITS2) regions from 178 plant species. We discovered that mutation of ITS2 is frequent, with a mean of 35 variants per species. And on average, three of the most abundant variants make up 91% of all ITS2 copies. Moreover, we found different congeneric species share identical variants in 13 genera. Interestingly, different species across different genera also share identical variants. In particular, one minor variant of ITS2 in Eleutherococcus giraldii was found identical to the ITS2 major variant of Panax ginseng, both from Araliaceae family. In addition, DNA barcoding gap analysis showed that the intra-genomic distances were markedly smaller than those of the intra-specific or inter-specific variants. When each of 5543 variants were examined for its species discrimination efficiency, a 97% success rate was obtained at the species level. Conclusions Identification of identical ITS2 variants across intra-generic or inter-generic species revealed complex species evolutionary history, possibly, horizontal gene transfer and ancestral hybridization. Although intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases. Furthermore, the inclusion of minor variants further improves the resolution of species identification.

PHYLOGENETICS AND BIOGEOGRAPHY OF ALNUS (BETULACEAE) INFERRED FROM SEQUENCES OF NUCLEAR RIBOSOMAL DNA ITS REGION

Sequences of nuclear ribosomal DNA ITS region were used to estimate phylogenetic relationships of Alnus. We recognize three major clades in Alnus, including subgenera Alnobetula, Clethropsis, and Alnus; subgenus Alnobetula is sister to subgenera Clethropsis and Alnus. The eastern Asian subgenus or section Cremastogyne is embedded in the subgenus Alnus clade. Alnus nepalensis is part of subgenus Alnus, not subgenus Clethropsis. Alnus maritima is part of subgenus Clethropsis (excl. A. nepalensis). Long and short branching pattern, sessile winter bud, and 2‐yr phenology are plesiomorphies in Alnus, while 1‐yr floral phenology and fall blooming are derived features. The simple axillary female inflorescence has evolved more than once in Alnus. Central and South American species of Alnus are derived from migrants from eastern Asia via the Bering land bridge and western North America. In subgenus Clethropsis, A. formosana of eastern Asia is more closely related to eastern North American species than to the other Asian species, A. nitida. The low sequence divergence within both the A. incana and A. viridis species complexes indicates their recent history of diversification in the circumpolar areas.

Morphology and affinities of an Early Cretaceous Ephedra (Ephedraceae) from China

Detailed investigations on Lower Cretaceous Ephedra L. fossils (Gnetopsida) reveal morphological characters similar to those of extant Ephedra rhytidosperma Pachomova, including articulate branches with many fine longitudinal striations, a dichasial branching pattern, uni- or bi-ovulate cones with paired bracts, cones terminal on branchlets, and seeds with a short, straight micropylar tubes, covered by numerous regular and prominent transverse laminar protuberances. Fossils are similar to extant E. rhytidosperma reproductive organs but differ in some vegetative structures and are described and discussed here as Ephedra archaeorhytidosperma Y. Yang et al. Because E. rhytidosperma is currently considered one of the most specialized members in Ephedra L. section Pseudobaccatae Stapf, the occurrence of E. archaeorhytidosperma in the Yixian Formation suggests that Ephedra L. was perhaps a more diverse genus in the Lower Cretaceous. Perhaps the evolution and diversity of Ephedra L. was already in place by the Lower Cretaceous and certainly before the end of the Mesozoic.

Molecular phylogeny and biogeographic diversification of Parthenocissus (Vitaceae) disjunct between Asia and North America.

UNLABELLED PREMISE OF THE STUDY Parthenocissus is a genus of the grape family Vitaceae and has a disjunct distribution in Asia and North America with members in both tropical and temperate regions. The monophyly of Parthenocissus has not yet been tested, and the species relationships and the evolution of its intercontinental disjunction have not been investigated with extensive sampling and molecular phylogenetic methods. • METHODS Plastid (trnL-F, rps16, and atpB-rbcL) and nuclear GAI1 sequences of 56 accessions representing all 12 Parthenocissus species were analyzed with parsimony, likelihood, and Bayesian inference. Divergence times of disjunct lineages were estimated with relaxed Bayesian dating. Evolution of the leaflet number was assessed by tracing this character onto Bayesian trees using the Trace Character Over Trees option in the program Mesquite. • KEY RESULTS Parthenocissus is monophyletic and sister to the newly described segregate genus Yua. Two major clades within Parthenocissus are recognizable corresponding to their distribution in Asia and North America. The disjunction between the two continents is estimated to be at 21.64 (95% higher posterior densities 10.23-34.89) million years ago. • CONCLUSIONS Parthenocissus is likely to have derived from the Eocene boreotropical element. Its current Asian-North American disjunction is dated to the early Miocene, congruent with fossil and paleoclimatic evidence. The tropical species is nested within the temperate clade and is inferred to have dispersed from the adjacent temperate regions. Parthenocissus and Yua are best treated as distinct genera. Leaflet number in this genus has a complex history and cannot be used as a character for infrageneric classification.

Phylogenetic and Biogeographic Diversification of Berberidaceae in the Northern Hemisphere

Abstract Sequences of the chloroplast genes matK and rbcL and nuclear ribosomal ITS2 were used for phylogenetic analyses of Berberidaceae. Three major clades were recognized, corresponding to the chromosome base numbers x = 6, 7, and 8/10. Bongardia was sister to the clade containing Achlys and the Podophyllum group, which consists of Diphylleia, Sinopodophyllum, Podophyllum, and Dysosma. The estimated times of divergence of six disjunct genera between Eurasia and North America ranged from 7.5 ± 2.3 Ma to 1.0 ± 0.7 Ma. The intercontinental disjunct lineages of Berberidaceae may have originated in eastern Asia and then migrated to Europe and North America. However, long-distance dispersal may explain the distribution pattern of Achlys. Desert xerophytes of Berberidaceae in southwestern Asia originated in response to the advent of dry climate at different times; Bongardia diverged from its closest relatives at 46.5 ± 3.6 Ma, whereas Leontice differentiated from Gymnospermium at 10.3 ± 3.2 Ma.

Menispermaceae and the diversification of tropical rainforests near the Cretaceous–Paleogene boundary

• Modern tropical rainforests have the highest biodiversity of terrestrial biomes and are restricted to three low-latitude areas. However, the actual timeframe during which tropical rainforests began to appear on a global scale has been intensely disputed. Here, we used the moonseed family (Menispermaceae), an important physiognomic and structural component of tropical rainforests on a worldwide basis, to obtain new insights into the diversification of this biome. • We integrated phylogenetic, biogeographic and molecular dating methods to analyse temporal and spatial patterns of global diversification in Menispermaceae. • Importantly, a burst of moonseed diversification occurred in a narrow window of time, which coincides with the Cretaceous-Paleogene (K-Pg) boundary. Our data also suggest multiple independent migrations from a putative ancestral area of Indo-Malay into other tropical regions. • Our data for Menispermaceae suggest that modern tropical rainforests may have appeared almost synchronously throughout the three major tropical land areas close to, or immediately following, the K-Pg mass extinction.

Deep phylogenetic incongruence in the angiosperm clade Rosidae.

Analysis of large data sets can help resolve difficult nodes in the tree of life and also reveal complex evolutionary histories. The placement of the Celastrales-Oxalidales-Malpighiales (COM) clade within Rosidae remains one of the most confounding phylogenetic questions in angiosperms, with previous analyses placing it with either Fabidae or Malvidae. To elucidate the position of COM, we assembled multi-gene matrices of chloroplast, mitochondrial, and nuclear sequences, as well as large single- and multi-copy nuclear gene data sets. Analyses of multi-gene data sets demonstrate conflict between the chloroplast and both nuclear and mitochondrial data sets, and the results are robust to various character-coding and data-exclusion treatments. Analyses of single- and multi-copy nuclear loci indicate that most loci support the placement of COM with Malvidae, fewer loci support COM with Fabidae, and almost no loci support COM outside a clade of Fabidae and Malvidae. Although incomplete lineage sorting and ancient introgressive hybridization remain as plausible explanations for the conflict among loci, more complete sampling is necessary to evaluate these hypotheses fully. Our results emphasize the importance of genomic data sets for revealing deep incongruence and complex patterns of evolution.

Conservation and divergence of candidate class B genes in Akebia trifoliata (Lardizabalaceae)

There is evidence that gene duplication and diversification within the MADS-box gene family had significant impact on floral architecture. In this study, we report the isolation of four class B homologous genes from Akebiatrifoliata, termed AktAP3_1, AktAP3_2, AktAP3_3, and AktPI. Phylogenetic analysis indicates that the three AktAP3 paralogs were produced by two gene duplication events and AktAP3_2 and AktAP3_3 are recent paralogs, which are yielded by the duplication before the origin of the genus Akebia. In situ hybridization demonstrates that these genes are mainly expressed in the stamens and carpels of A. trifoliata, but in differential patterns, similar to those in other basal eudicot and basal angiosperm species. AktAP3_3 and AktPI are expressed in the developing petaloid perianth, suggesting that the petaloidy of the perianth is caused by the expression of class B genes. Reverse transcriptase polymerase chain reaction analyses indicate that these genes are expressed in both male and female flowers, but at different levels. We explore the interaction behavior of the class B proteins in the basal eudicots using yeast two-hybrid system for the first time. The AktAP3_1/2/3 proteins and the AktPI protein can form obligate heterodimers, but at different strength. From the mRNA expression and protein interaction patterns of the duplicated copies of the AktAP3 genes, we conclude that subfunctionalization very likely contributes to the maintenance of multiple AP3-like gene copies in A. trifoliata.

The MIK region rather than the C-terminal domain of AP3-like class B floral homeotic proteins determines functional specificity in the development and evolution of petals

In core eudicots, euAP3-type MADS-box genes encode a PISTILLATA (PI)-derived motif, as well as a C-terminal euAP3 motif that originated from a paleoAP3 motif of an ancestral APETALA3 (AP3)-like protein through a translational frameshift mutation. To determine the functional and evolutionary relevance of these motifs, a series of point mutation and domain-swap constructs were generated, involving CsAP3, a paleoAP3-type gene from the basal angiosperm Chloranthus spicatus encoding a truncated paleoAP3 motif, and AtAP3, a euAP3-type gene from the core eudicot Arabidopsis thaliana. The chimeric constructs were expressed in A. thaliana under the control of the AP3 promoter or the CaMV 35S promoter in an ap3 mutant or wild-type background, respectively. Significant recovery of AP3 function was obtained in both complementation and ectopic expression experiments whenever the region upstream of the C-terminal motifs (MIK region) from A. thaliana was taken, even when the PI-derived motif and the truncated paleoAP3 motif of CsAP3 substituted for the corresponding sequences from AtAP3. However, no or very weak complementation or gain-of-function was seen when the MIK region was from CsAP3. Our data suggest that changes in the MIK region rather than mutations in the C-terminal domain were of crucial importance for the evolution of the functional specificity of euAP3-type proteins in stamen and petal development.