Kanae Nishii

A complex case of simple leaves: indeterminate leaves co-express ARP and KNOX1 genes

The mutually exclusive relationship between ARP and KNOX1 genes in the shoot apical meristem and leaf primordia in simple leaved plants such as Arabidopsis has been well characterized. Overlapping expression domains of these genes in leaf primordia have been described for many compound leaved plants such as Solanum lycopersicum and Cardamine hirsuta and are regarded as a characteristic of compound leaved plants. Here, we present several datasets illustrating the co-expression of ARP and KNOX1 genes in the shoot apical meristem, leaf primordia, and developing leaves in plants with simple leaves and simple primordia. Streptocarpus plants produce unequal cotyledons due to the continued activity of a basal meristem and produce foliar leaves termed “phyllomorphs” from the groove meristem in the acaulescent species Streptocarpus rexii and leaves from a shoot apical meristem in the caulescent Streptocarpus glandulosissimus. We demonstrate that the simple leaves in both species possess a greatly extended basal meristematic activity that persists over most of the leaf’s growth. The area of basal meristem activity coincides with the co-expression domain of ARP and KNOX1 genes. We suggest that the co-expression of ARP and KNOX1 genes is not exclusive to compound leaved plants but is associated with foci of meristematic activity in leaves.

Taxonomic status, phylogenetic affinities and genetic diversity of a presumed extinct genus, Paraisometrum W.T. Wang (gesneriaceae) from the karst regions of Southwest China.

Background The karst regions in South China have an abundance of endemic plants that face high extinction risks. The Chinese Gesneriaceae endemic Paraisometrum mileense ( = Oreocharis mileensis), was presumed extinct for 100 years. After its re-discovery, the species has become one of five key plants selected by the Chinese forestry government to establish a new conservation category for plants with extremely small populations. For conservation purposes, we studied the phylogenetic and population genetic status of P. mileense at the three only known localities in Guangxi, Guizhou and Yunnan. Methodology/Principal Findings We collected 64 samples (52 species) of Oreocharis and 8 samples from three provinces of P. mileense and generated molecular phylogenies, and inferred that P. mileense represents a relatively isolated and derived taxonomic unit within Oreocharis. Phylogeographic results of 104 samples of 12 populations of P. mileense indicated that the populations in Yunnan have derived from those in Guangxi and Guizhou. Based on AFLP data, the populations were found to harbor low levels of genetic diversity (He = 0.118), with no apparent gradient across the species’ range, a restricted gene flow and significant isolation-by-distance with limited genetic differentiation among the populations across the three provinces (F ST = 0.207, P<0.001). The 10 populations in Yunnan were found to represent two distinct lineages residing at different altitudes and distances from villages. Conclusion/Significance The low levels of genetic diversity found in P. mileense are perhaps a consequence of severe bottlenecks in the recent past. The distribution of the genetic diversity suggests that all populations are significant for conservation. Current in situ and ex situ measures are discussed. Further conservation actions are apparently needed to fully safeguard this conservation flagship species. Our work provides a model of an integrated study for the numerous endemic species in the karst regions with extremely small populations.

The European Paleoendemic Haberlea rhodopensis (Gesneriaceae) Has an Oligocene Origin and a Pleistocene Diversification and Occurs in a Long-Persisting Refugial Area in Southeastern Europe

Premise of research. Even though the Balkan Peninsula is a biodiversity hot spot in southeastern Europe harboring many endemic plants, very little is known about the temporal extent of the phylogeographic history and the contemporary genetic diversity of the endemics there. Haberlea rhodopensis is one of the European Gesneriaceae species occurring in this region and represents appropriate study material to address these questions. Methodology. We generated fossil-dated molecular phylogenies (atpB-rbcL, trnH-psbA, trnLF, ITS) across the Lamiales to determine the origin and age of H. rhodopensis and conducted phylogeographic (trnH-psbA, ITS) and population genetic (ISSRs) analyses on 17 populations from Bulgaria and Greece, covering the entire distribution range of the species, to investigate their biogeographic history, present-day genetic diversity, and differentiation levels. Pivotal results. The European Gesneriaceae genera have a Tertiary origin in the early Oligocene, while the Haberlea lineage emerged in the late Oligocene. Extinctions appear to have marked the history of the genus for a long period of time, and the extant populations diverged in the late Pleistocene. A significant differentiation was apparent between populations from Bulgaria, that is, the Balkan Mountains in the north and the Bulgarian side of the Rhodopi massif in the south, but there was an even stronger differentiation between the latter and populations from Greece. This might be explained by the Rhodopi massif representing a barrier to gene flow, enforced during the Last Glacial Maximum, during which populations descended on opposing north- and south-facing mountain slopes. Conclusions. Haberlea represents an ancient lineage with recent diversification. The extant populations are of recent origin and indicate glacial refugial areas in Bulgaria and Greece. This study sheds light on historic and current phylogenetic and phylogeographic events that shaped the flora of the Balkan Peninsula, an area that has long been recognized for its species diversity and richness. Our data suggest that it may have acted as a persistent refugial area in southeast Europe since the mid-Tertiary.

Streptocarpus redefined to include all Afro-Malagasy Gesneriaceae: Molecular phylogenies prove congruent with geographical distribution and basic chromosome numbers and uncover remarkable morphological homoplasies

The taxonomy of the African, Madagascan and Comoro Island (Afro-Malagasy) Gesneriaceae attracts a large amount of interest given the horticultural importance of Cape Primroses (Streptocarpus) and African Violets (Saintpaulia). Earlier studies indicated that the Afro-Malagasy genera form a strongly supported clade, and recent classifications have included some of the genera within an expanded Streptocarpus. Given the global importance of this group, we carried out a comprehensive molecular phylogenetic analysis of all Afro-Malagasy genera in subfamily Didymocarpoideae, tribe Trichosporeae, subtribe Streptocarpinae, to investigate species relationships in these genera as the basis for a new classification. Phylogenetic analyses of the nuclear ribosomal spacer (ITS, 5S NTS) and chloroplast intron and spacer regions (rpl20-rps12 spacer, trnL intron, trnLF spacer) of 226 samples were performed, including all Streptocarpinae genera, except the monotypic Nodonema. The molecular phylogenies demonstrate that the genera with non-twisted fruits are nested within Streptocarpus which has twisted fruits. Two main clades were found, one comprising herbaceous caulescent Streptocarpus that also included Saintpaulia, the caulescents Hovanella and Schizoboea, and the unifoliates Acanthonema and Trachystigma. The second clade comprises the woody caulescents and acaulescent Streptocarpus, Colpogyne and Linnaeopsis. Altogether, twelve well-supported subclades can be recognized, each with a combination of distinct morphological characteristics. A new classification of tribe Streptocarpinae, de facto Streptocarpus, is presented, retaining the two subgenera, Streptocarpus and Streptocarpella, and dividing them into five and seven sections respectively. Nodonema is attributed to subg. Streptocarpus for morphological reasons. The former genus Saintpaulia is classified as Streptocarpus subg. Streptocarpella sect. Saintpaulia with ten species recognized.

GA2 and GA20-oxidase expressions are associated with the meristem position in Streptocarpus rexii (Gesneriaceae)

Abstract We examined genes involved in the regulatory pathway of gibberellin (GA) in meristems of Streptocarpus rexii. The plants do not possess a typical shoot apical meristem (SAM) and form unique meristems: the basal meristem extends the lamina area of one cotyledon to produce anisocotylous seedlings; the groove meristem forms new leaves at the base of the macrocotyledon. Exogenous application of GA significantly suppresses the basal meristem activity in developing cotyledons and the seedlings remain isocotyl. To examine the role of endogenous GA on these meristems in vivo, we isolated homologs of GA2-oxidase responsible for degrading active GAs (SrGA2ox), and GA20-oxidase regulating the rate limiting step of active GA synthesis (SrGA20ox). During embryogenesis, while first partly overlapping, the expression of SrGA2ox and SrGA20ox became more differentiated and mutually exclusive, ending with SrGA2ox being expressed solely in the adaxial–proximal domain of the embryo in regions with meristem activity, whereas SrGA20ox was restricted to the fork between the two cotyledons. The latter may be responsible for suppressing the formation of an embryonic SAM in S. rexii. In developing seedlings, SrGA2ox expression also followed the centers of meristem activity, where SrGA20ox expression was excluded. Our results suggest that low levels of GA are required in S. rexii meristems for their establishment and maintenance. Thus, the meristems in S. rexii share similar regulatory pathways suggested for the SAM in model plants, but that in S. rexii evolutionary modifications involving a lateral transfer of function, from shoot to leaves, is implicated in attaining the unusual morphology of the plants.

Gibberellin as a suppressor of lateral dominance and inducer of apical growth in the unifoliate Streptocarpus wendlandii (Gesneriaceae)

Abstract We report on the effects of exogenously applied hormones on the lateral and apical dominance that governs morphogenesis in the unifoliate Streptocarpus wendlandii. In this phenotype, lateral dominance is extreme as the plants only retain a macrocotyledon that develops into a leaf-like phyllomorph by means of a basal meristem and do not show apical growth. Gibberellin applications suppressed the basal meristem activity of the macrocotyledon resulting in an isocotylous seedling with two microcotyledons and caused the formation of a primary phyllomorph, which suggests that the groove meristem, a shoot apical meristem equivalent, is released from apical suppression by the basal meristem. Interestingly, uniconazol, a gibberellin biosynthesis inhibitor, also caused a reduction in basal meristem activity, but without primary phyllomorph formation, suggesting that some gibberellin is required for proper function of the basal meristem. Co-application of gibberellin and cytokinin resulted in two macrocotyledons also without phyllomorph formation, which is similar to previous results for cytokinin-only applications. Thus, cytokinin may act downstream in the regulatory pathway of the basal meristem. Our results suggest that the balance between gibberellin and cytokinin in the cotyledons appears thus as key factor in the regulation of lateral and apical dominance in Streptocarpus. Their interplay may well be the primary explanation for the great diversity in growth form exhibited in species of this genus. Our work shows that small imbalances of hormones in early stages of plant development can have major effects on the final phenotype.

Chayamaritia (Gesneriaceae: Didymocarpoideae), a new genus from Southeast Asia

Based on a phylogenetic analysis of Asian Gesneriaceae with the most comprehensive coverage at the genus level to date, the new genus Chayamaritia is established and described in subfamily Didymocarpoideae, tribe Trichosporeae, subtribe Didymocarpinae. It contains two species, of which one, Chayamaritia smitinandii (B.L.Burtt) D.J.Middleton, was formerly placed in the genera Chirita and Henckelia. The other, Chayamaritia banksiae D.J.Middleton, is newly described. The exclusion of Chayamaritia smitinandii from Henckelia further clarifies the taxonomic and biogeographic limits of Henckelia following its considerable recircumscription during the recent remodelling and synonymisation of Chirita.

From shoot to leaf: step-wise shifts in meristem and KNOX1 activity correlate with the evolution of a unifoliate body plan in Gesneriaceae.

Typical dicots possess equal-sized cotyledons and leaf-bearing shoots topped with a shoot apical meristem (SAM), the source of lateral organs, and where KNOX1 homeobox genes act as key regulators. New World Gesneriaceae show typical cotyledons, whereas Old World Gesneriaceae show anisocotyly, the unequal post-germination growth of cotyledons, and include unifoliate (one-leaf) plants. One-leaf plants show an extremely reduced body plan: the adult above-ground photosynthetic tissue consisting of a single cotyledon, a macrocotyledon enlarged by the basal meristem (BM), but lacking a SAM. To investigate the origin and evolution of the BM and one-leaf plants, the meristem activity and KNOX1 SHOOTMERISTEMLESS (STM) expression in cotyledons and leaves were systematically studied by RT-PCR and in situ hybridization across the family Gesneriaceae, Jovellana in Calceolariaceae (sister family to Gesneriaceae), and Antirrhinum in Plantaginaceae, all families of order Lamiales (asterids), in comparison to Arabidopsis (Brassicales, rosids). In all examined Lamiales samples, unlike Arabidopsis, BM activity accompanied by STM expression was found in both cotyledons in early stages. Foliage leaves of Gesneriaceae and Jovellana also showed the correlation of BM and STM expression. An extension of BM activity was found following a phylogenetic trajectory towards one-leaf plants where it is active throughout the lifetime of the macrocotyledon. Our results suggest that KNOX1 involvement in early cotyledon expansion originated early on in the diversification of Lamiales and is proposed as the prerequisite for the evolution of vegetative diversity in Gesneriaceae. Step-wise morphological shifts, driven by transfers of meristematic activity, as evidenced by shifts in KNOX1 expression, may be one mechanism by which morphological diversity evolves in plants.

Tangled history of a multigene family: The evolution of ISOPENTENYLTRANSFERASE genes

ISOPENTENYLTRANSFERASE (IPT) genes play important roles in the initial steps of cytokinin synthesis, exist in plant and pathogenic bacteria, and form a multigene family in plants. Protein domain searches revealed that bacteria and plant IPT proteins were to assigned to different protein domains families in the Pfam database, namely Pfam IPT (IPTPfam) and Pfam IPPT (IPPTPfam) families, both are closely related in the P-loop NTPase clan. To understand the origin and evolution of the genes, a species matrix was assembled across the tree of life and intensively in plant lineages. The IPTPfam domain was only found in few bacteria lineages, whereas IPPTPfam is common except in Archaea and Mycoplasma bacteria. The bacterial IPPTPfam domain miaA genes were shown as ancestral of eukaryotic IPPTPfam domain genes. Plant IPTs diversified into class I, class II tRNA-IPTs, and Adenosine-phosphate IPTs; the class I tRNA-IPTs appeared to represent direct successors of miaA genes were found in all plant genomes, whereas class II tRNA-IPTs originated from eukaryotic genes, and were found in prasinophyte algae and in euphyllophytes. Adenosine-phosphate IPTs were only found in angiosperms. Gene duplications resulted in gene redundancies with ubiquitous expression or diversification in expression. In conclusion, it is shown that IPT genes have a complex history prior to the protein family split, and might have experienced losses or HGTs, and gene duplications that are to be likely correlated with the rise in morphological complexity involved in fine tuning cytokinin production.

Rearranging Specimens on Herbarium Type Sheets of Streptocarpus betsiliensis Humbert (Gesneriaceae)

Abstract Möller, M., K. Nishii, M. W. Callmander, P. B. Phillipson & O. Poncy (2015). Rearranging specimens on herbarium type sheets of Streptocarpus betsiliensis Humbert (Gesneriaceae). Candollea 70: 145–150. In English, English and French abstracts. During recent herbarium research on the Malagasy species of Gesneriaceae, a hitherto undetected mixed collection of the type of Streptocarpus betsiliensis Humbert (Perrier de la Bâthie 12482), was observed at the Paris herbarium (P). Of the three sheets present, one, the holotype [P00088746], comprised only Streptocarpus betsiliensis specimens, while the two isotypes, [P00088747, P00088748], were found to comprise a mixture of this species and a closely related and easily confused species, Streptocarpus lanatus MacMaster. As a result of this, the specimens on the mixed sheets were physically separated and the collections were distinguished by the suffixes A and B. They were then remounted to form species-specific sheets, rescanned, and the databases at P updated accordingly.