Plastome phylogenomics of Allaeanthus, Broussonetia and Malaisia (Dorstenieae, Moraceae) and the origin of B. × kazinoki.

Species of Broussonetia have been essential in the development of papermaking technology. In Japan and Korea, a hybrid between B. monoica and B. papyrifera (= B. × kazinoki) known as kozo and daknamu is still the major source of raw materials for making traditional paper washi and hanji, respectively. Despite their cultural and practical significance, however, the origin and taxonomy of kozo and daknamu remain controversial. Additionally, the long-held generic concept of Broussonetia s.l., which included Sect. Allaeanthus and Sect. Broussonetia, was challenged as phylogenetic analyses showed Malaisia is sister to the latter section. To re-examine the taxonomic proposition that recognizes Allaeanthus, Broussonetia, and Malaisia (i.e., Broussonetia alliance), plastome and nuclear ribosomal DNA (nrDNA) sequences of six species of the alliance were assembled. Characterized by the canonical quadripartite structure, genome alignments and contents of the six plastomes (160,121-162,594 bp) are highly conserved, except for the pseudogenization and/or loss of the rpl22 gene. Relationships of the Broussonetia alliance are identical between plastome and nrDNA trees, supporting the maintenance of Malaisia and the resurrection of Allaeanthus. The phylogenomic relationships also indicate that the monoecy in B. monoica is a derived state, possibly resulting from hybridization between the dioecious B. kaempferi (♀) and B. papyrifera (♂). Based on the hypervariable ndhF-rpl32 intergenic spacer selected by sliding window analysis, phylogeographic analysis indicates that B. monoica is the sole maternal parent of B. × kazinoki and that daknamu carries multiple haplotypes, while only one haplotype was detected in kozo. Because hybridizations between B. monoica and B. papyrifera are unidirectional and have occurred rarely in nature, our data suggest that daknamu might have originated via deliberate hybrid breeding selected for making hanji in Korea. On the contrary, kozo appears to have a single origin and the possibility of a Korean origin cannot be ruled out.

Identification of plastid genomic regions inferring species identity from de novo plastid genome assembly of 14 Korean-native Iris species (Iridaceae).

Iris is one of the largest genera in the family Iridaceae, comprising hundreds of species, including numerous economically important horticultural plants used in landscape gardening and herbal medicine. Improved taxonomic classification of Iris species, particularly the endangered Korean-native Iris, is needed for correct species delineation. To this end, identification of diverse genetic markers from Iris genomes would facilitate molecular identification and resolve ambiguous classifications from molecular analyses; however, only two Iris plastid genomes, from Iris gatesii and Iris sanguinea, have been sequenced. Here, we used high-throughput next-generation sequencing, combined with Sanger sequencing, to construct the plastid genomes of 14 Korean-native Iris species with one outgroup and predict their gene content. Using these data, combined with previously published plastid genomes from Iris and one outgroup (Sisyrinchium angustifolium), we constructed a Bayesian phylogenetic tree showing clear speciation among the samples. We further identified sub-genomic regions that have undergone neutral evolution and accurately recapitulate Bayesian-inferred speciation. These contain key markers that could be used to identify and classify Iris samples into taxonomic clades. Our results confirm previously reported speciation patterns and resolve questionable relationships within the Iris genus. These data also provide a valuable resource for studying genetic diversity and refining phylogenetic relationships between Iris species.

Sonerilacardamomensis (Melastomataceae), a new species from Cambodia.

Sonerilacardamomensis, a new species of family Melastomataceae from the Central Cardamom Protected Area in Koh Kong province of southwestern Cambodia, is described and illustrated. The species is similar to S.violifolia Hook.f. ex Triana, but is readily distinguished by its cordate leaf base, lack of setae at the nodes, longer pedicels, smaller petals, smaller hypanthiums and smaller capsules.

Two new genera of Podostemaceae from northern Central Laos: saltational evolution and enigmatic morphology.

Podostemaceae, the river-weeds, are characterized by remarkable differences between species and genera, which resulted from saltational evolution. This paper presents additional cases of such two genera, which are described here from the Phou Khao Khouay National Protected Area in northern Central Laos. Molecular phylogenetic data show that Ctenobryum mangkonense (gen. & sp. nov.) is sister to Hydrodiscus koyamae, while Laosia ramosa (gen. & sp. nov.) is isolated from all Asian genera of subfamily Podostemoideae. Ctenobryum mangkonense is distinct from Hydrodiscus koyamae in the crustose roots (versus rootless in the latter), scattered flowers on the root (versus alternate on the shoot), and pectinate bracts (versus simple, sheath-like). Laosia ramosa is distinct from all the genera in the columnar, endogenously branched axes and single style-stigma complex. The axis is an enigmatic organ with combined characteristics of root, stem and leaf, pending further study. Laos, together with Thailand, is a center of diversity of the Southeast and East Asian Podostemoideae. The three monotypic genera, i.e., Ctenobryum, Hydrodiscus and Laosia, occur in neighboring and closely similar aquatic habitats within the Area. The new taxa are formally described.

Polyploidy and introgression in invasive giant knotweed (Fallopia sachalinensis) during the colonization of remote volcanic islands.

Invasive giant knotweed (Fallopia sachalinensis) is native to northeastern Asia. In Korea, F. sachalinensis is confined to two volcanic islands, Ullung and Dok islands, where it occurs as dodecaploids (2n = 132). We investigated the molecular variation in 104 accessions from 94 populations of F. sachalinensis and its relatives throughout their native range to elucidate the origin of these island populations. All F. sachalinensis plants on Ullung and Dok islands were uniquely dodecaploid, whereas other populations were tetraploid (2n = 44). Among the 39 cpDNA haplotypes identified, the accessions from these islands shared two unique haplotypes, and were resolved as a well-supported monophyletic clade. However, this clade was sister to a clade comprising F. japonica accessions from southwestern Japan and separated from the clade comprising F. sachalinensis from other areas; this relationship is inconsistent with morphological evidence. The monophyly of the F. sachalinensis populations on Ullung and Dok islands suggests a single colonization event. The progenitor was likely from Japan, where it possibly captured F. japonica var. japonica cpDNA via introgression. The Ullung Island populations subsequently differentiated through polyploidization and mutations post-introduction. Our results also indicate that giant knotweed in Europe and North America likely originated from northern Japan and/or Sakhalin Island.

Dating dispersal and radiation in the gymnosperm Gnetum (Gnetales)--clock calibration when outgroup relationships are uncertain.

Most implementations of molecular clocks require resolved topologies. However, one of the Bayesian relaxed clock approaches accepts input topologies that include polytomies. We explored the effects of resolved and polytomous input topologies in a rate-heterogeneous sequence data set for Gnetum, a member of the seed plant lineage Gnetales. Gnetum has 10 species in South America, 1 in tropical West Africa, and 20 to 25 in tropical Asia, and explanations for the ages of these disjunctions involve long-distance dispersal and/or the breakup of Gondwana. To resolve relationships within Gnetum, we sequenced most of its species for six loci from the chloroplast (rbcL, matK, and the trnT-trnF region), the nucleus (rITS/5.8S and the LEAFY gene second intron), and the mitochondrion (nad1 gene second intron). Because Gnetum has no fossil record, we relied on fossils from other Gnetales and from the seed plant lineages conifers, Ginkgo, cycads, and angiosperms to constrain a molecular clock and obtain absolute times for within-Gnetum divergence events. Relationships among Gnetales and the other seed plant lineages are still unresolved, and we therefore used differently resolved topologies, including one that contained a basal polytomy among gymnosperms. For a small set of Gnetales exemplars (n = 13) in which rbcL and matK satisfied the clock assumption, we also obtained time estimates from a strict clock, calibrated with one outgroup fossil. The changing hierarchical relationships among seed plants (and accordingly changing placements of distant fossils) resulted in small changes of within-Gnetum estimates because topologically closest constraints overrode more distant constraints. Regardless of the seed plant topology assumed, relaxed clock estimates suggest that the extant clades of Gnetum began diverging from each other during the Upper Oligocene. Strict clock estimates imply a mid-Miocene divergence. These estimates, together with the phylogeny for Gnetum from the six combined data sets, imply that the single African species of Gnetum is not a remnant of a once Gondwanan distribution. Miocene and Pliocene range expansions are inferred for the Asian subclades of Gnetum, which stem from an ancestor that arrived from Africa. These findings fit with seed dispersal by water in several species of Gnetum, morphological similarities among apparently young species, and incomplete concerted evolution in the nuclear ITS region.

The chloroplast trnT-trnF region in the seed plant lineage Gnetales.

The trnT-trnF region is located in the large single-copy region of the chloroplast genome. It consists of the trnL intron, a group I intron, and the trnT-trnL and trnL-trnF intergenic spacers. We analyzed the evolution of the region in the three genera of the gymnosperm lineage Gnetales (Gnetum, Welwitschia, and Ephedra), with especially dense sampling in Gnetum for which we sequenced 41 accessions, representing most of the 25-35 species. The trnL intron has a conserved secondary structure and contains elements that are homologous across land plants, while the spacers are so variable in length and composition that homology cannot be found even among the three genera. Palindromic sequences that form hairpin structures were detected in the trnL-trnF spacer, but neither spacer contained promoter elements for the tRNA genes. The absence of promoters, presence of hairpin structures in the trnL-trnF spacer, and high sequence variation in both spacers together suggest that trnT and trnF are independently transcribed. Our model for the expression and processing of the genes tRNA(Thr)(UGU), tRNA(Leu)(UAA), and tRNA(Phe) (GAA) therefore attributes the seemingly neutral evolution of the two spacers to their escape from functional constraints.

The internal transcribed spacer of nuclear ribosomal DNA in the gymnosperm Gnetum.

We analyze the structure of the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA in the gymnosperm Gnetum, using a phylogenetic framework derived mainly from an intron in the nuclear low-copy LEAFY gene. Gnetum comprises 25-35 species in South America, Africa, and Asia, of which we sampled 16, each with two to six clones. Criteria used to assess ITS functionality were highly divergent nucleotide substitution, GC content, secondary structure, and incongruent phylogenetic placement of presumed paralogs. The length of ITS1 ranged from 225 to 986 bp and that of ITS2 from 259 to 305 bp, the largest ranges so far reported from seed plants. Gnetum ITS1 contains two informative sequence motifs, but different from other gymnosperms, there are only few and short (7-13 bp) tandem repeats. Gnetum ITS2 contains two structural motifs, modified in different clades by shortening of stems and loops. Conspecific sequences grouped together except for two recombinant pseudogenes that had ITS1 of one clade and ITS2 of another. Most of the pseudogenic ITS copies, paralogs, and putative chimeras occurred in a clade that according to a fossil-calibrated chloroplast-DNA clock has an age of a few million years. Based on morphology and chromosome numbers, the most plausible causes of the observed high levels of ITS polymorphism are hybridization, allopolyploidy, and introgression.

Horizontal gene transfer from flowering plants to Gnetum.

Although horizontal gene transfer is well documented in microbial genomes, no case has been reported in higher plants. We discovered horizontal transfer of the mitochondrial nad1 intron 2 and adjacent exons b and c from an asterid to Gnetum (Gnetales, gymnosperms). Gnetum has two copies of intron 2, a group II intron, that differ in their exons, nucleotide composition, domain lengths, and structural characteristics. One of the copies, limited to an Asian clade of Gnetum, is almost identical to the homologous locus in angiosperms, and partial sequences of its exons b and c show characteristic substitutions unique to angiosperms. Analyses of 70 seed plant nad1 exons b and c and intron 2 sequences, including representatives of all angiosperm clades, support that this copy originated from a euasterid and was horizontally transferred to Gnetum. Molecular clock dating, using calibrations provided by gnetalean macrofossils, suggests an age of 5 to 2 million years for the Asian clade that received the horizontal transfer.