Evolution and development of fruits of Erycina pusilla and other orchid species.

Fruits play a crucial role in seed dispersal. They open along dehiscence zones. Fruit dehiscence zone formation has been intensively studied in Arabidopsis thaliana. However, little is known about the mechanisms and genes involved in the formation of fruit dehiscence zones in species outside the Brassicaceae. The dehiscence zone of A. thaliana contains a lignified layer, while dehiscence zone tissues of the emerging orchid model Erycina pusilla include a lipid layer. Here we present an analysis of evolution and development of fruit dehiscence zones in orchids. We performed ancestral state reconstructions across the five orchid subfamilies to study the evolution of selected fruit traits and explored dehiscence zone developmental genes using RNA-seq and qPCR. We found that erect dehiscent fruits with non-lignified dehiscence zones and a short ripening period are ancestral characters in orchids. Lignified dehiscence zones in orchid fruits evolved multiple times from non-lignified zones. Furthermore, we carried out gene expression analysis of tissues from different developmental stages of E. pusilla fruits. We found that fruit dehiscence genes from the MADS-box gene family and other important regulators in E. pusilla differed in their expression pattern from their homologs in A. thaliana. This suggests that the current A. thaliana fruit dehiscence model requires adjustment for orchids. Additionally, we discovered that homologs of A. thaliana genes involved in the development of carpel, gynoecium and ovules, and genes involved in lipid biosynthesis were expressed in the fruit valves of E. pusilla, implying that these genes may play a novel role in formation of dehiscence zone tissues in orchids. Future functional analysis of developmental regulators, lipid identification and quantification can shed more light on lipid-layer based dehiscence of orchid fruits.

Structure and Development of Flowers and Inflorescences in Burmannia (Burmanniaceae, Dioscoreales).

Species of the genus Burmannia possess distinctive and highly elaborated flowers with prominent floral tubes that often bear large longitudinal wings. Complicated floral structure of Burmannia hampers understanding its floral evolutionary morphology and biology of the genus. In addition, information on structural features believed to be taxonomically important is lacking for some species. Here we provide an investigation of flowers and inflorescences of Burmannia based on a comprehensive sampling that included eight species with various lifestyles (autotrophic, partially mycoheterotrophic and mycoheterotrophic). We describe the diversity of inflorescence architecture in the genus: a basic (most likely, ancestral) inflorescence type is a thyrsoid comprising two cincinni, which is transformed into a botryoid in some species via reduction of the lateral cymes to single flowers. Burmannia oblonga differs from all the other studied species in having an adaxial (vs. transversal) floral prophyll. For the first time, we describe in detail early floral development in Burmannia. We report presence of the inner tepal lobes in B. oblonga, a species with reportedly absent inner tepals; the growth of the inner tepal lobes is arrested after the middle stage of floral development of this species, and therefore they are undetectable in a mature flower. Floral vasculature in Burmannia varies to reflect the variation of the size of the inner tepal lobes; in B. oblonga with the most reduced inner tepals their vascular supply is completely lost. The gynoecium consists of synascidiate, symplicate, and asymplicate zones. The symplicate zone is secondarily trilocular (except for its distal portion in some of the species) without visible traces of postgenital fusion, which prevented earlier researchers to correctly identify the zones within a definitive ovary. The placentas occupy the entire symplicate zone and a short distal portion of the synascidiate zone. Finally, we revealed an unexpected diversity of stamen-style interactions in Burmannia. In all species studied, the stamens are tightly arranged around the common style to occlude the flower entrance. However, in some species the stamens are free from the common style, whereas in the others the stamen connectives are postgenitally fused with the common style, which results in formation of a gynostegium.

Biogeography of the monocotyledon astelioid clade (Asparagales): A history of long-distance dispersal and diversification with emerging habitats.

The astelioid families (Asteliaceae, Blandfordiaceae, Boryaceae, Hypoxidaceae, and Lanariaceae) have centers of diversity in Australasia and temperate Africa, with secondary centers of diversity in Afromontane Africa, Asia, and Pacific Islands. The global distribution of these families makes this an excellent lineage to test if current distribution patterns are the result of vicariance or long-distance dispersal and to evaluate the roles of Tertiary climatic and geological drivers in lineage diversification. Sequence data were generated from five chloroplast regions (petL-psbE, rbcL, rps16-trnK, trnL-trnLF, trnS-trnSG) for 104 ingroup species sampled across global diversity. The astelioid phylogeny was inferred using maximum parsimony, maximum likelihood, and Bayesian inference methods. Divergence dates were estimated with a relaxed clock applied in BEAST. Ancestral ranges were reconstructed in the R package 'BioGeoBEARS' applying the corrected Akaike information criterion to test for the best-fit biogeographic model. Diversification rates were estimated in Bayesian Analysis of Macroevolutionary Mixtures (BAMM). Astelioid relationships were inferred as Boryaceae(Blandfordiaceae(Asteliaceae(Hypoxidaceae plus Lanariaceae))). The crown astelioid node was dated to the Late Cretaceous (75.2 million years; 95% highest posterior density interval 61.0-90.0 million years) and an Antarctic-Australasian origin was inferred. Astelioid speciation events have not been shaped by Gondwanan vicariance. Rather long-distance dispersal since the Eocene is inferred to account for current distributions. Crown Asteliaceae and Boryaceae have Australian ancestral ranges and diversified since the Eocene. In Hypoxidaceae, Empodium, Hypoxis, and Pauridia have African ancestral ranges, while Curculigo and Molineria have an Asian ancestral range. Diversification of Pauridia and the Curculigo clade occurred steadily, while diversification of Astelia and Hypoxis was punctuated over time. Diversification of Hypoxis and Astelia coincided temporally with the expansion of the habitat types occupied by extant taxa, e.g., grassland habitat in Africa during the Late Miocene and alpine habitat in New Zealand during the Pliocene, respectively.

Molecular phylogenetics of Hypoxidaceae--evidence from plastid DNA data and inferences on morphology and biogeography.

Phylogenetic relationships of the monocot family Hypoxidaceae (Asparagales), which occurs mainly in the Southern Hemisphere, were reconstructed using four plastid DNA regions (rbcL, trnL intron, trnL-F intergenic spacer, and trnS-G intergenic spacer) for 56 ingroup taxa including all currently accepted genera and seven species of the closely related families Asteliaceae, Blandfordiaceae, and Lanariaceae. Data were analyzed by applying parsimony, maximum likelihood and Bayesian methods. The intergenic spacer trnS-G--only rarely used in monocot research--contributed a substantial number of potentially parsimony informative characters. Hypoxidaceae consist of three well-supported major clades, but their interrelationships remain unresolved. Our data indicate that in the Pauridia clade one long-distance dispersal event occurred from southern Africa to Australia. Long-distance dispersal scenarios may also be likely for the current distribution of Hypoxis, which occurs on four continents. In the Curculigo clade, the present distribution of Curculigo s.s. on four continents could support a Gondwanan origin, but the level of divergence is too low for this hypothesis to be likely. The main clades correspond well with some floral characters, habit and palynological data, whereas chromosomal data exhibit plasticity and probably result from polyploidization and subsequent dysploidy and/or aneuploidy. Evolutionary flexibility is also suggested by the number of reported pollination syndromes: melittophily, myophily, sapromyophily, and cantharophily. Based on our phylogenetic results, we suggest cautious nomenclatural reorganization to generate monophyly at the generic level.

Molecular phylogeny of Aerides (Orchidaceae) based on one nuclear and two plastid markers: a step forward in understanding the evolution of the Aeridinae.

Phylogenetic relationships of the orchid genus Aerides (Epidendroideae, Vandeae, Aeridinae) from Southeast Asia were inferred from DNA sequences of one nuclear (nrITS) and two plastid (matK, trnL-trnL-F) regions of 48 taxa (21 Aerides, 25 other Aeridinae, 2 outgroup). Analyses of the combined datasets with parsimony, maximum likelihood and Bayesian methods revealed that Aerides is monophyletic and consists of three well-supported subclades which are only partly in accordance with previous sectional delimitations based on floral characters. The two different flower types in Aerides (hidden versus open spur entrance) seem to have evolved at least twice in geographically distinct areas. The phylogeny presented here is yet another example in Orchidaceae where floral morphology cannot be relied on to reconstruct phylogenetic history but rather is the result of pollinator-driven selection. The Aerides subclades are characterized by three different length classes of the mutation-rich P8 region in the trnL intron. To our knowledge, this is the first time that the P8 region was studied in orchids. The matK gene has been assumed to be a pseudogene in orchids due to occasional occurrence of frameshift indels, low transition/transversion (ts:tv) ratios and low substitution rates at the 3rd codon position. However, matK does not appear to be a pseudogene in Aerides and a comparison with data from other angiosperms suggests that ts:tv ratios and low substitution rates have been overestimated as arguments for a pseudogene status of matK in orchids.

Phylogenetics of Cucumis (Cucurbitaceae): cucumber (C. sativus) belongs in an Asian/Australian clade far from melon (C. melo).

BACKGROUND: Melon, Cucumis melo, and cucumber, C. sativus, are among the most widely cultivated crops worldwide. Cucumis, as traditionally conceived, is geographically centered in Africa, with C. sativus and C. hystrix thought to be the only Cucumis species in Asia. This taxonomy forms the basis for all ongoing Cucumis breeding and genomics efforts. We tested relationships among Cucumis and related genera based on DNA sequences from chloroplast gene, intron, and spacer regions (rbcL, matK, rpl20-rps12, trnL, and trnL-F), adding nuclear internal transcribed spacer sequences to resolve relationships within Cucumis. RESULTS: Analyses of combined chloroplast sequences (4,375 aligned nucleotides) for 123 of the 130 genera of Cucurbitaceae indicate that the genera Cucumella, Dicaelospermum, Mukia, Myrmecosicyos, and Oreosyce are embedded within Cucumis. Phylogenetic trees from nuclear sequences for these taxa are congruent, and the combined data yield a well-supported phylogeny. The nesting of the five genera in Cucumis greatly changes the natural geographic range of the genus, extending it throughout the Malesian region and into Australia. The closest relative of Cucumis is Muellerargia, with one species in Australia and Indonesia, the other in Madagascar. Cucumber and its sister species, C. hystrix, are nested among Australian, Malaysian, and Western Indian species placed in Mukia or Dicaelospermum and in one case not yet formally described. Cucumis melo is sister to this Australian/Asian clade, rather than being close to African species as previously thought. Molecular clocks indicate that the deepest divergences in Cucumis, including the split between C. melo and its Australian/Asian sister clade, go back to the mid-Eocene. CONCLUSION: Based on congruent nuclear and chloroplast phylogenies we conclude that Cucumis comprises an old Australian/Asian component that was heretofore unsuspected. Cucumis sativus evolved within this Australian/Asian clade and is phylogenetically far more distant from C. melo than implied by the current morphological classification.

A multi-locus chloroplast phylogeny for the Cucurbitaceae and its implications for character evolution and classification.

Cucurbitaceae contain c. 800 species in 130 genera and are among the economically most important families of plants. We inferred their phylogeny based on chloroplast DNA sequences from two genes, one intron, and two spacers (rbcL, matK, trnL, trnL-trnF, rpl20-rps12) obtained for 171 species in 123 genera. Molecular data weakly support the traditional subfamilies Cucurbitoideae (111 genera) and Nhandiroboideae (19 genera, 60 species), and recover most of the eleven tribes, but almost none of the subtribes. Indofevillea khasiana is sister to all other Cucurbitoideae, and the genera of Joliffieae plus a few Trichosantheae form a grade near the base of Cucurbitoideae. A newly discovered large clade consists of the ancestrally Asian genera Nothoalsomitra, Luffa, Gymnopetalum, Hodgsonia, Trichosanthes, and the New World tribe Sicyeae. Genera that are poly- or paraphyletic include Ampelosicyos, Cucumis, Ibervillea, Neoachmandra, Psiguria, Trichosanthes, and Xerosicyos. Flower characters, especially number of free styles, fusion of filaments and/or anthers, tendril type, and pollen size, exine, and aperture number correlate well with the chloroplast phylogeny, while petal and fruit characters as well as karyotype exhibit much evolutionary flexibility.

Wax plants disentangled: a phylogeny of Hoya (Marsdenieae, Apocynaceae) inferred from nuclear and chloroplast DNA sequences.

Hoya (Marsdenieae, Apocynaceae) includes at least 200 species distributed from India to the Pacific Islands. We here infer major species groups in the genus based on combined sequences from the chloroplast atpB-rbcL spacer, the trnL region, and nuclear ribosomal DNA ITS region for 42 taxa of Hoya and close relatives. To assess levels of ITS polymorphism, ITS sequences for a third of the accessions were obtained by cloning. Most ITS clones grouped by species, indicating that speciation in Hoya usually predates ITS duplication. One ITS sequence of H. carnosa, however, grouped with a sequence of the morphologically similar H. pubicalyx, pointing to recent hybridization or the persistence of paralogous copies through a speciation event. The topology resulting from the combined chloroplast and nuclear data recovers some morphology-based sections, such as Acanthostemma and Eriostemma, as well as a well-supported Australian/New Guinean clade. The combined data also suggest that morphological adaptations for ant-symbiosis evolved at least three times within Hoya.

Phylogeny of the Cucurbitales based on DNA sequences of nine loci from three genomes: implications for morphological and sexual system evolution.

The Cucurbitales are a clade of rosids with a worldwide distribution and a striking heterogeneity in species diversity among its seven family members: the Anisophylleaceae (29-40 species), Begoniaceae (1400 spp.), Coriariaceae (15 spp.), Corynocarpaceae (6 spp.), Cucurbitaceae (800 spp.), Datiscaceae (2 spp.), and Tetramelaceae (2 spp.). Most Cucurbitales have unisexual flowers, and species are monoecious, dioecious, andromonoecious, or androdioecious. To resolve interfamilial relationships within the order and to polarize morphological character evolution, especially of flower sexual systems, we sequenced nine plastids (atpB, matK, ndhF, rbcL, the trnL-F region, and the rpl20-rps12 spacer), nuclear (18S and 26S rDNA), and mitochondrial (nad1 b/c intron) genes (together approximately 12,000 bp) of 26 representatives of the seven families plus eight outgroup taxa from six other orders of the Eurosids I. Cucurbitales are strongly supported as monophyletic and are closest to Fagales, albeit with moderate support; both together are sister to Rosales. The deepest split in the Cucurbitales is that between the Anisophylleaceae and the remaining families; next is a clade of Corynocarpaceae and Coriariaceae, followed by Cucurbitaceae, which are sister to a clade of Begoniaceae, Datiscaceae, and Tetramelaceae. Based on this topology, stipulate leaves, inferior ovaries, parietal placentation, and one-seeded fruits are inferred as ancestral in Cucurbitales; exstipulate leaves, superior ovaries, apical placentation, and many-seeded fruits evolved within the order. Bisexual flowers are reconstructed as ancestral, but dioecy appears to have evolved already in the common ancestor of Begoniaceae, Cucurbitaceae, Datiscaceae, and Tetramelaceae, and then to have been lost repeatedly in Begoniaceae and Cucurbitaceae. Both instances of androdioecy (Datisca glomerata and Schizopepon bryoniifolius) evolved from dioecious ancestors, corroborating recent hypotheses about androdioecy often evolving from dioecy.