Tetsuo Ohi-Toma

Molecular Phylogeny of Aristolochia sensu lato (Aristolochiaceae) based on Sequences of rbcL, matK, and phyA Genes, with Special Reference to Differentiation of Chromosome Numbers

Abstract The genus Aristolochia sensu lato contains over 400 species from warm temperate to tropical regions worldwide. Taxonomic treatments of Aristolochia have been ambiguous and controversial. In a recent cladistic analysis based on morphological characters, it was proposed that the genus should be divided into four genera in two subtribes. To reconsider the systematics of Aristolochia sensu lato, we reconstructed its phylogeny based on nucleotide sequences of the chloroplast rbcL gene and the nuclear-encoded phytochrome A (phyA) gene for 19 representative species and the chloroplast matK gene of over 80 species. All phylogenetic trees produced with the three genes indicate that Aristolochia sensu lato is a monophyletic group, consisting of two lineages that correspond to the subtribes Aristolochiinae and Isotrematinae. The matK phylogeny shows that each of the lineages includes two sublineages. The Aristolochiinae clade is composed of the Aristolochia sensu stricto and Pararistolochia clades, and the Isotrematinae clade of the Isotrema and Endodeca clades. Chromosome numbers, including newly reported counts for 30 species, are predominantly congruent with the phylogeny: the Aristolochiinae clade shows chromosome numbers of 2n = 6, 12, 14, or 16, while the Isotrematinae clade is characterized by 2n = 32. In the Isotrematinae clade, the paralogous relationships of the phyA gene suggest that polyploidization might have occurred.

Hybridization and polyploidy of an aquatic plant, Ruppia (Ruppiaceae), inferred from plastid and nuclear DNA phylogenies

UNLABELLED PREMISE OF THE STUDY The monogeneric family Ruppiaceae is found primarily in brackish water and is widely distributed on all continents, many islands, and from subartic to tropical zones. Ruppia taxonomy has been confusing because of its simplified morphology yet high phenotypic plasticity and the existence of polyploidy and putative hybrids. This study addresses the current classification of species in the genus, the origin of putative hybrids and polyploids, and the distribution of Ruppia species. • METHODS Separate molecular phylogenetic analyses using plastid DNA and nuclear-encoded PHYB data sets were performed after chromosome observations. • KEY RESULTS The resultant trees were largely congruent between genomes, but were incongruent in two respects: the first incongruence may be caused by long outgroup branches and their effect on ingroup rooting, and the second is caused by the existence of heterogeneous PHYB sequences for several accessions that may reflect several independent hybridization events. Several morphological species recognized in previous taxonomic revisions appear paraphyletic in plastid DNA and PHYB trees. • CONCLUSIONS Given the molecular phylogenies, and considering chromosome number and morphology, three species and one species complex comprising six lineages were discerned. A putative allotriploid, an allotetraploid, and a lineage of hybrid origin were identified within the species complex, and a hybrid was found outside the species complex, and their respective putative parental taxa were inferred. With respect to biogeography, a remarkably discontinuous distribution was identified in two cases, for which bird-mediated seed dispersal may be a reasonable explanation.

Origin and diversification of Hibiscus glaber , species endemic to the oceanic Bonin Islands, revealed by chloroplast DNA polymorphism

Two woody Hibiscus species co‐occur in the Bonin Islands of the northwestern Pacific Ocean: Hibiscus glaber Matsum. is endemic to the islands, and its putative ancestral species, Hibiscus tiliaceus L., is widely distributed in coastal areas of the tropics and subtropics. To infer isolating mechanisms that led to speciation of H. glaber and the processes that resulted in co‐occurrence of the two closely related species on the Bonin Islands, we conducted molecular phylogenetic analyses on chloroplast DNA (cpDNA) sequences. Materials collected from a wide area of the Pacific and Indian Oceans were used, and two closely related species, Hibiscus hamabo Siebold Zucc. and Hibiscus macrophyllus Roxb., were also included in the analyses. The constructed tree suggested that H. glaber has been derived from H. tiliaceus, and that most of the modern Bonin populations of H. tiliaceus did not share most recent ancestry with H. glaber. Geographic isolation appears to be the most important mechanism in the speciation of H. glaber. The co‐occurrence of the two species can be attributed to multiple migrations of different lineages into the islands. While a wide and overlapping geographical distribution of haplotypes was found in H. tiliaceus, localized geographical distribution of haplotypes was detected in H. glaber. It is hypothesized that a shift to inland habitats may have affected the mode of seed dispersal from ocean currents to gravity and hence resulted in geographical structuring of H. glaber haplotypes.

Comprehensive phylogenetic analyses of the Ruppia maritima complex focusing on taxa from the Mediterranean

Recent molecular phylogenetic studies reported high diversity of Ruppia species in the Mediterranean. Multiple taxa, including apparent endemics, are known from that region, however, they have thus far not been exposed to phylogenetic analyses aimed at studying their relationships to taxa from other parts of the world. Here we present a comprehensive phylogenetic analyses of the R. maritima complex using data sets composed of DNA sequences of the plastid genome, the multi-copy nuclear ITS region, and the low-copy nuclear phyB gene with a primary focus on the Mediterranean representatives of the complex. As a result, a new lineage, “Drepanensis”, was identified as the seventh entity of the complex. This lineage is endemic to the Mediterranean. The accessions included in the former “Tetraploid” entity were reclassified into two entities: an Asia–Australia–Europe disjunct “Tetraploid_α” with a paternal “Diploid” origin, and a European “Tetraploid_γ” originating from a maternal “Drepanensis” lineage. Another entity, “Tetraploid_β”, is likely to have been originated as a result of chloroplast capture through backcrossing hybridization between paternal “Tetraploid_α” and maternal “Tetraploid_γ”. Additional discovery of multiple tetraploidizations as well as hybridization and chloroplast capture at the tetraploid level indicated that hybridization has been a significant factor in the diversification of Ruppia.

Comparative Genomics and Phylogenomics of East Asian Tulips (Amana, Liliaceae)

The genus Amana Honda (Liliaceae), when it is treated as separate from Tulipa, comprises six perennial herbaceous species that are restricted to China, Japan and the Korean Peninsula. Although all six Amana species have important medicinal and horticultural uses, studies focused on species identification and molecular phylogenetics are few. Here we report the nucleotide sequences of six complete Amana chloroplast (cp) genomes. The cp genomes of Amana range from 150,613 bp to 151,136 bp in length, all including a pair of inverted repeats (25,629–25,859 bp) separated by the large single-copy (81,482–82,218 bp) and small single-copy (17,366–17,465 bp) regions. Each cp genome equivalently contains 112 unique genes consisting of 30 transfer RNA genes, four ribosomal RNA genes, and 78 protein coding genes. Gene content, gene order, AT content, and IR/SC boundary structure are nearly identical among all Amana cp genomes. However, the relative contraction and expansion of the IR/SC borders among the six Amana cp genomes results in length variation among them. Simple sequence repeat (SSR) analyses of these Amana cp genomes indicate that the richest SSRs are A/T mononucleotides. The number of repeats among the six Amana species varies from 54 (A. anhuiensis) to 69 (Amana kuocangshanica) with palindromic (28–35) and forward repeats (23–30) as the most common types. Phylogenomic analyses based on these complete cp genomes and 74 common protein-coding genes strongly support the monophyly of the genus, and a sister relationship between Amana and Erythronium, rather than a shared common ancestor with Tulipa. Nine DNA markers (rps15–ycf1, accD–psaI, petA–psbJ, rpl32–trnL, atpH–atpI, petD–rpoA, trnS–trnG, psbM–trnD, and ycf4–cemA) with number of variable sites greater than 0.9% were identified, and these may be useful for future population genetic and phylogeographic studies of Amana species.

Molecular Phylogeny of Typhonium sensu lato and Its Allied Genera in the Tribe Areae of the Subfamily Aroideae (Araceae) Based on Sequences of Six Chloroplast Regions

Abstract Typhonium was recognized as a monophyletic genus in the tribe Areae of the subfamily Aroideae (Araceae) until a recent molecular phylogenetic study indicated that the genus was paraphyletic respective to other Areae genera. However, that phylogenetic study did not discuss the details of infrageneric relationships due to the limited numbers of Typhonium samples. To elucidate the phylogenetic relationships in Typhonium sensu lato, we conducted phylogenetic analyses based on the combined DNA sequences of six chloroplast regions (3′trnL—trnF, rp120—5′rps12, psbB—psbH, trnG intron, rpoC2—rps2, and trnK 3′intron) from 18 representative Typhonium species and additional samples from related genera. The resultant tree strongly suggests that Typhonium is not a monophyletic group and that it comprises at least two separate lineages, with other Areae genera nested within, and that Typhonium sensu lato may be subdivided into several monophyletic groups. These groups are distinguishable based on the stem-type of shoot organization as well as other morphological characters, which mostly correspond to traditionally recognized taxa. Based on molecular phylogeny and morphology, we proposed a revision of the Areae, wherein Typhonium sensu lato is divided into four genera: Typhonium sensu stricto, Sauromatum, and three new genera, Diversiarum, Hirsutiarum, and Pedatyphonium, which are described here which results in the following combinations: Diversiarum diversifolium, Diversiarum alpinum, Pedatyphonium horsfieldii, Pedatyphonium larsenii, Pedatyphonium kunmingense, Pedatyphonium calcicolum, Pedatyphonium omeiense, Hirsutiarum hirsutum, Hirsutiarum brevipilosum, and Sauromatum giganteum.

Phylogeny of Ruppia (Ruppiaceae) revisited: molecular and morphological evidence for a new species from Western Cape, South Africa.

Abstract The aquatic plant genus Ruppia (Ruppiaceae) comprises eight species mainly in coastal brackish areas of the world. While the known taxa of Ruppia thus far generally had either four- or eight-carpelled flowers, our recent Ruppia collection from Western Cape, South Africa showed flowers with only two carpels. This characteristic morphological evidence, together with elongated coiled peduncles, implied either: i) extensive morphological variation of the cosmopolitan R. cirrhosa; or ii) the occurrence of a new species in the genus. We tested these alternative hypotheses of the bicarpellate Ruppia taxon in a phylogenetic framework. Sequence data from four plastid DNA regions and nuclear phyB were analyzed using maximum parsimony, maximum likelihood, and Bayesian inference. We obtained moderately to highly resolved phytogenies with both data sets. The collection from Western Cape showed unique DNA sequences which were, in both plastid and nuclear phylogenetic trees, placed as sister to the rest of the genus and thus clearly rejected the first hypothesis. Given the distinctive phylogenetic position and the unique morphological evidence, here we describe Ruppia bicarpa, a new species from Western Cape, South Africa. The other two Ruppia populations from the region are classified or categorized into either a tetraploid or an apparent hexaploid entity of the R. maritima complex.

Multiple hybridization in the Aristolochia kaempferi group (Aristolochiaceae): evidence from reproductive isolation and molecular phylogeny

Hybridization via distributional changes should be an important factor for plant speciation. Previous cpDNA analyses of the Aristolochia kaempferi group, comprising six taxa in East Asia, showed a distinct phylogeographic structure resulting from distributional changes brought about by paleoclimatic oscillations. However, the cpDNA phylogeny was incongruent with morphologically defined taxa. To explore the evolutionary processes responsible for the inconsistency between cpDNA and morphology, we made artificial crosses and performed phylogenetic analyses using multiple nuclear markers. All crosses among different taxa or cpDNA clades set fruit, if crossing direction is not considered. The five nuclear phylogenies mostly did not support either the taxa or the cpDNA clades. A combined analysis of cpDNA and the PI exon revealed the two major lineages in the group, lacking a prezygotic isolating barrier between them. However, an asymmetric prezygotic isolating barrier occurs between populations of the Japanese main islands and of other areas that belong to different cpDNA subclades. It seems reasonable to conclude that the development of a prezygotic isolating mechanism is not necessarily proportional to the degree of genetic divergence. These results suggested that species boundaries within the group are blurred due to speciational processes associated with multiple hybridization and introgression resulting from repeated contacts among differentiated populations.

Molecular phylogenetics and biogeography of the mint tribe Elsholtzieae (Nepetoideae, Lamiaceae), with an emphasis on its diversification in East Asia

Elsholtzia and its allied genera such as Collinsonia and Perilla (tribe Elsholtzieae, Lamiaceae) are an ecologically and economically important plant group consisting of ~71 species, with most species distributed in East and Southeast Asia, and several species in North America. Their phylogeny and historical biogeography resulting in a distant intercontinental disjunction are poorly understood. Here we use two nuclear (ETS, ITS) and five chloroplast (rbcL, matK, trnL-F, ycf1, ycf1-rps15) fragments to reconstruct the phylogeny, biogeographic history, and patterns of diversification of Elsholtzieae. The tribe Elsholtzieae is monophyletic and divided into five clades. The woody Elsholtzia species are nested within herbaceous ones and are inferred to have evolved from herbaceous ancestors. Molecular dating shows that the five major clades were established during the Eocene period, but most of the modern diversity did not originate until the Miocene. The divergence between the New World Collinsonia and the Old World Mosla-Keiskea-Perilla clade was dated to the mid-Miocene. Ancestral area reconstructions suggest that the tribe originated in East Asia, and then dispersed to Southeast Asia and North America. Overall, our findings highlight the important roles of the uplifts of the Qinghai-Tibetan Plateau (QTP) and climate changes from Middle Miocene onwards in promoting species diversification of Elsholtzieae.

Molecular phylogenetics and historical biogeography of Sorbus sensu stricto (Rosaceae)

Explaining how plants from eastern Asia migrated to other Northern Hemisphere regions is still challenging. The genus Sorbus sensu stricto (including c. 88 species) is considered as a good example to illuminate this scenario, due to the wide distribution in the temperate zone and high diversity in the Himalayas and Hengduan Mountains. Based on four nuclear markers (LEAFY-2, GBSSI-1, SBEI and WD) and one chloroplast marker (rps16-trnK), we reconstructed the phylogenetic relationship of Sorbus using 54 taxa (60% of the genus representing all subgenera, sections, or series and geographical areas in the previous classifications), and estimated divergence time and historical biogeography of the genus. Phylogenetic analyses supported that the subgenera Sorbus and Albo-carmesinae, as defined by traditional morphological characters (such as fruit color), are suitable. However, five clades defined by phylogenetic analysis of the genus are not consistent with traditionally defined sections or series. Molecular dating and biogeographic reconstruction showed that the age of the most recent common ancestor was estimated at 41mya (95% HPD: 49-35mya) in eastern Asia. Four dispersal events are assumed to explain the wide distribution of Sorbus in the temperate zone and diversification in the edges of Qinghai-Tibet Plateau (QTP). Species dispersed from eastern Asia to the Tianshan Mountains, North America and Europe during the Oligocene and Miocene period. We found that polyploidization occurred multiple times in the subgenus Albo-carmesinae, in the Tianshan Mountains, Himalayas, and H-D Mountains. Finally, we suggest that apomixis, polyploidization, and hybridization may have combined with the multistage uplifting of Himalayas and H-D Mountains (and the subsequent increases in geologic, ecological and climatic heterogeneity) to drive the striking species diversity of Sorbus in this region.