Molecular phylogenetics and floral evolution of the Cirrhopetalum alliance (Bulbophyllum, Orchidaceae): Evolutionary transitions and phylogenetic signal variation.

The Cirrhopetalum alliance is a loosely circumscribed species-rich group within the mega-diverse genus Bulbophyllum (Orchidaceae). The monophyletic status of the alliance has been challenged by previous studies, although established sectional classifications have yet to be tested in a phylogenetic context. We used maximum likelihood and Bayesian analyses of DNA sequence data (cpDNA: matK and psbA-trnH; nrDNA: ITS and Xdh; 3509 aligned characters; 117 taxa), including all sections putatively associated with the Cirrhopetalum alliance, to reconstruct the phylogeny. We mapped 11 selected categorical floral characters onto the phylogeny to identify synapomorphies and assess potential evolutionary transitions across major clades. Our results unequivocally support the recognition of an amended Cirrhopetalum alliance as a well-supported monophyletic group characterized by clear synapomorphies, following the inclusion of sect. Desmosanthes and the exclusion of five putative Cirrhopetalum-allied sections. Most sections within the Cirrhopetalum alliance are demonstrated to be polyphyletic or paraphyletic, necessitating a new sectional classification. The inclusion of sect. Desmosanthes revolutionizes our understanding of the alliance, with significant evolutionary transitions in floral characters detected. We further investigated six continuously variable characters of the sepals and labellum, and detect phylogenetic conservatism in labellum width and the evolutionary lability of lateral sepal length, which can partly be explained by the different functional roles they play in pollination and pollinator trapping.

A symbiotic balancing act: arbuscular mycorrhizal specificity and specialist fungus gnat pollination in the mycoheterotrophic genus Thismia (Thismiaceae).

BACKGROUND AND AIMS: Mycorrhizal associations in mycoheterotrophic plants are generally more specialized than in autotrophs. Mycoheterotrophs typically bear small, inconspicuous flowers that often self-pollinate to maximize seed set, although some have structurally complex flowers indicative of xenogamy. A trade-off has previously been proposed between specialization in these above- and below-ground symbioses, although empirical data are lacking. METHODS: We used next-generation DNA sequencing to compare the mycorrhizal communities from the roots of a mycoheterotrophic species, Thismia tentaculata (Thismiaceae), and its neighbouring autotrophs. We furthermore conducted detailed assessments of floral phenology and pollination ecology, and performed artificial pollination experiments to determine the breeding system. KEY RESULTS: Thismia tentaculata maintains a symbiotic association with a single arbuscular mycorrhizal Rhizophagus species. The flowers are pollinated by a single species of fungus gnats (Corynoptera, Sciaridae), which are attracted by the yellow pigments and are temporarily restrained within the perianth chamber before departing via apertures between the anthers. The plants are self-compatible but predominantly xenogamous. CONCLUSIONS: Our findings demonstrate that T. tentaculata maintains highly specialized associations with pollinators and mycorrhizal fungi, both of which are widely distributed. We suggest that specialization in multiple symbiotic interactions is possible in mycoheterotrophs if redundant selective pressures are not exerted to further restrict an already constrained suite of life-history traits.

Gene tree discordance and coalescent methods support ancient intergeneric hybridisation between Dasymaschalon and Friesodielsia (Annonaceae).

Although hybridisation between closely related species is common and known to be important in plant evolution, hybridisation at the generic level or above is comparatively rare. We address ancient intergeneric hybridisation in the early-divergent angiosperm family Annonaceae by phylogenetic reconstruction, divergence time estimation and coalescent simulation of the genus Dasymaschalon using a multi-locus approach based on molecular data from five chloroplast (matK, psbA-trnH, ndhF, rbcL, and trnL-F) and five nuclear (ITS, ETS, AP3, PhyA, and PhyC) DNA markers. We demonstrate incongruence among different gene trees: Dasymaschalon is retrieved as monophyletic in the nuclear ribosomal tree (based on ITS and ETS), but is non-monophyletic in the chloroplast and Phy-gene trees (with poor resolution in the AP3 tree), with the majority of species assigned to a strongly supported clade but three species (D. filipes, D. longiflorum and D. tibetense) more closely related to the sister genus Friesodielsia. Three contrasting approaches-a coalescent method based on molecular dating, incongruence pattern comparison, and a multi-accession phylogenetic reconstruction-are used to assess the patterns of this gene tree incongruence and test hypotheses of ancient hybridisation and incomplete lineage sorting. Our results support a late Miocene intergeneric hybridisation between members of the Dasymaschalon and Friesodielsia lineages in continental Asia-west Malesia.

Historical biogeography and ecological niche modelling of the Asimina-Disepalum clade (Annonaceae): role of ecological differentiation in Neotropical-Asian disjunctions and diversification in Asia.

BACKGROUND: The Asimina-Disepalum clade (Annonaceae subfam. Annonoideae tribe Annoneae) includes a major Neotropical-Asian biogeographical disjunction. We evaluate whether this disjunction can be explained by the Eocene boreotropics hypothesis, which relies on the existence of extensive boreotropical forests during the Late Palaeocene-Early Eocene thermal maximum (52-50 Ma), followed by disruption of boreotropical vegetation during post-Eocene cooling. Molecular dating using an uncorrelated relaxed molecular clock (UCLD) model with two fossil calibrations, ancestral range estimation, and ecological niche modelling across evolutionary time were performed. Our focus was the geographical origin of Disepalum and general biogeographic patterns within this genus. Comparison of ecological tolerance among extant species and niche reconstructions at ancestral nodes within the clade enabled insights in likely migration routes of lineages, as well as evaluating the role of bioclimatic ecological differentiation in the diversification of Disepalum within Southeast Asia. RESULTS: The inferred vicariance event associated with the Asimina-Disepalum disjunction is estimated to have originated ca. 40 Mya [95% highest posterior density (HPD): 44.3-35.5 Mya]. The Disepalum crown lineage is estimated to have originated ca. 9 Mya (95% HPD: 10.6-7.6), either in western Malesia and continental Southeast Asia, or exclusively in western Malesia. Ecological niche modelling shows that seasonality of temperature and precipitation are major contributors determining the geographical range of species. Ancestral niche modelling furthermore indicates that the ancestor of the Asimina-Disepalum clade likely had bioclimatic preferences close to conditions found in current tropical and subtropical climates across Asia, whereas the ancestors of the Asimina and Disepalum crown groups are projected onto the more subtropical and tropical regions, respectively. CONCLUSIONS: The vicariance event associated with the Neotropical-Asian disjunction within the Asimina-Disepalum clade likely coincided with climatic deterioration at the Eocene-Oligocene boundary. Although detrended component analyses (DCA) indicate that altitude and seasonality of temperature and precipitation have the greatest influence in determining the geographical range of species, isolation due to palaeogeographic and palaeoclimatic events appears to be of greater significance than climate niche differentiation in driving diversification in Disepalum.

Preponderance of clonality triggers loss of sex in Bulbophyllum bicolor, an obligately outcrossing epiphytic orchid.

Vegetative propagation (clonal growth) conveys several evolutionary advantages that positively affect life history fitness and is a widespread phenomenon among angiosperms that also reproduce sexually. However, a bias towards clonality can interfere with sexual reproduction and lead to sexual extinction, although a dearth of effective genetic tools and mathematical models for clonal plants has hampered assessment of these impacts. Using the endangered tropical epiphytic or lithophytic orchid Bulbophyllum bicolor as a model, we integrated an examination of breeding system with 12 microsatellite loci and models valid for clonal species to test for the "loss of sex" and infer likely consequences for long-term reproductive dynamics. Bagging experiments and field observations revealed B. bicolor to be self-incompatible and pollinator-dependent, with an absence of fruit-set over 4 years. Challenging the assumptions that clonal populations can be as genotypically diverse as sexually reproducing ones and that clonality does not greatly influence genetic structure, just 22 multilocus genotypes were confirmed among all 15 extant natural populations, 12 of the populations were found to be monoclonal, and all three multiclonal ones exhibited a distinct phalanx clonal architecture. Our results suggest that all B. bicolor populations depend overwhelmingly on clonal growth for persistence, with a concomitant loss of sex due to an absence of pollinators and a lack of mating opportunities at virtually all sites, both of which are further entrenched by habitat fragmentation. Such cryptic life history impacts, potentially contributing to extinction debt, could be widespread among similarly fragmented, outcrossing tropical epiphytes, demanding urgent conservation attention.

Stigmatic exudate in the Annonaceae: Pollinator reward, pollen germination medium or extragynoecial compitum?

Although "dry-type" stigmas are widely regarded as ancestral in angiosperms, the early-divergent family Annonaceae has copious stigmatic exudate. We evaluate three putative functions for this exudate: as a nutritive reward for pollinators; as a pollen germination medium; and as an extragynoecial compitum that enables pollen tube growth between carpels. Stigmatic exudate is fructose dominated (72.2%), but with high levels of glucose and sucrose; the dominance of hexose sugars and the diversity of amino acids observed, including many that are essential for insects, support a nutritive role for pollinators. Sugar concentration in pre-receptive flowers is high (28.2%), falling during the peak period of stigmatic receptivity (17.4%), and then rising again toward the end of the pistillate phase (32.9%). Pollen germination was highest in sugar concentrations <20%. Sugar concentrations during the peak pistillate phase therefore provide optimal osmolarity for pollen hydration and germination; subsequent changes in sugar concentration during anthesis reinforce protogyny (in which carpels mature before stamens), enabling the retention of concentrated exudate into the staminate phase as a pollinator food reward without the possibility of pollen germination. Intercarpellary growth of pollen tubes was confirmed: the exudate therefore also functions as a suprastylar extragynoecial compitum, overcoming the limitations of apocarpy.

Molecular phylogenetics of the species-rich angiosperm genus Goniothalamus (Annonaceae) inferred from nine chloroplast DNA regions: Synapomorphies and putative correlated evolutionary changes in fruit and seed morphology.

A phylogenetic study of the genus Goniothalamus (Annonaceae) is presented using maximum parsimony, maximum likelihood and Bayesian approaches, with 65 species sampled (48.5% of the genus) based on sequences of nine chloroplast DNA regions (11,214 aligned positions). The resultant phylogeny clearly indicates that Goniothalamus is monophyletic. Preliminary research initially focused on identifying synapomorphies and estimating the phylogenetic signal of selected morphological characters based on parsimony and likelihood ancestral character state reconstructions. This prescreening of characters enabled 40 to be selected for further study, and of these 15 are shown here to demonstrate significant phylogenetic signal and to provide clear synapomorphies for several infrageneric clades. Although floral structure in Goniothalamus is comparatively uniform, suggesting a common basic pattern of pollination ecology, fruit and seed morphology in the genus is very diverse and is presumably associated with different patterns of frugivory. The present study assesses correlations amongst fruit and seed characters which are putatively of functional importance with regard to frugivory and dispersal. One-way phylogenetic ANOVA indicates significant phylogenetically independent correlation between the following fruit and seed characters: fruits borne on older branches and/or on the main trunk have larger monocarps than fruits borne on young branches; and monocarps that contain seeds with a hairy testa are larger than those with glabrous seeds. We discuss fruit morphologies and potential explanations for the inferred correlations, and suggest that they may be the result of adaptation to different frugivores (birds, larger non-volant animal and primate seed dispersers, respectively).

Climatic and biogeographic processes underlying the diversification of the pantropical flowering plant family Annonaceae.

Tropical forests harbor the richest biodiversity among terrestrial ecosystems, but few studies have addressed the underlying processes of species diversification in these ecosystems. We use the pantropical flowering plant family Annonaceae as a study system to investigate how climate and biogeographic events contribute to diversification. A super-matrix phylogeny comprising 835 taxa (34% of Annonaceae species) based on eight chloroplast regions was used in this study. We show that global temperature may better explain the recent rapid diversification in Annonaceae than time and constant models. Accelerated accumulation of niche divergence (around 15 Ma) lags behind the increase of diversification rate (around 25 Ma), reflecting a heterogeneous transition to recent diversity increases. Biogeographic events are related to only two of the five diversification rate shifts detected. Shifts in niche evolution nevertheless appear to be associated with increasingly seasonal environments. Our results do not support the direct correlation of any particular climatic niche shifts or historical biogeographical event with shifts in diversification rate. Instead, we suggest that Annonaceae diversification can lead to later niche divergence as a result of increasing interspecific competition arising from species accumulation. Shifts in niche evolution appear to be associated with increasingly seasonal environments. Our results highlight the complexity of diversification in taxa with long evolutionary histories.

Three new species of Mitrephora (Annonaceae) from Thailand.

Three new Mitrephora species (Annonaceae), M.langsuanensis sp. nov., M.sirindhorniae sp. nov. and M.sukhothaiensis sp. nov., are described from Thailand. Mitrephoralangsuanensis resembles M.macclurei, but its leaves have more secondary veins, and its flowers have more carpels, with yellow-and-pink striped outer petals with a margin that becomes undulate with age. Mitrephorasirindhorniae resembles M.tomentosa, but has larger sepals and petals, longer flowering pedicels, and shorter monocarp stipes, with monocarps that have a longitudinal ridge. Mitrephorasukhothaiensis is distinct from its Thai congeners in having outer petals that reflex at maturity and inner petals with a pair of appendages on the margin adaxially. The addition of these three new taxa raises the total number of Mitrephora species in Thailand to 14. An identification key for Thai species is provided.

Diversification Slowdown in the Cirrhopetalum Alliance (Bulbophyllum, Orchidaceae): Insights From the Evolutionary Dynamics of Crassulacean Acid Metabolism.

Evolutionary slowdowns in diversification have been inferred in various plant and animal lineages. Investigation based on diversification models integrated with environmental factors and key characters could provide critical insights into this diversification trend. We evaluate diversification rates in the Cirrhopetalum alliance (Bulbophyllum, Orchidaceae subfam. Epidendroideae) using a time-calibrated phylogeny and assess the role of Crassulacean acid metabolism (CAM) as a hypothesised key innovation promoting the spectacular diversity of orchids, especially those with an epiphytic habit. An explosive early speciation in the Cirrhopetalum alliance is evident, with the origin of CAM providing a short-term advantage under the low atmospheric CO2 concentrations (pCO2) associated with cooling and aridification in the late Miocene. A subsequent slowdown of diversification in the Cirrhopetalum alliance is possibly explained by a failure to keep pace with pCO2 dynamics. We further demonstrate that extinction rates in strong CAM lineages are ten times higher than those of C3 lineages, with CAM not as evolutionarily labile as previously assumed. These results challenge the role of CAM as a "key innovation" in the diversification of epiphytic orchids.

The rice acyl-CoA-binding protein gene family: phylogeny, expression and functional analysis.

• Acyl-CoA-binding proteins (ACBPs) show conservation in an acyl-CoA-binding domain (ACB domain) which binds acyl-CoA esters. Previous studies on plant ACBPs focused on eudicots, Arabidopsis and Brassica. Here, we report on the phylogeny and characterization of the ACBP family from the monocot Oryza sativa (rice). • Phylogenetic analyses were conducted using 16 plant genomes. Expression profiles of rice ACBPs under normal growth, as well as biotic and abiotic stress conditions, were examined by quantitative real-time reverse-transcription polymerase chain reactions. In vitro acyl-CoA-binding assays were conducted using recombinant (His)6-tagged ACBPs. • The ACBP family diversified as land plants evolved. Classes I and IV show lineage-specific gene expansion. Classes II and III are closely related phylogenetically. As in the eudicot Arabidopsis, six genes (designated OsACBP1 to OsACBP6) encode rice ACBPs, but their distribution into various classes differed from Arabidopsis. Rice ACBP mRNAs showed ubiquitous expression and OsACBP4, OsACBP5 and OsACBP6 were stress-responsive. All recombinant rice ACBPs bind [¹4C]linolenoyl-CoA besides having specific substrates. • Phylogeny, gene expression and biochemical analyses suggest that paralogues within and across classes are not redundant proteins. In addition to performing conserved basal functions, multidomain rice ACBPs appear to be associated with stress responses.

Goniothalamusroseipetalus and G.sukhirinensis (Annonaceae): Two new species from Peninsular Thailand.

Two new Goniothalamus species (Annonaceae), G.roseipetalus sp. nov. and G.sukhirinensis sp. nov., are described from the southern limits of Peninsular Thailand (Narathiwat and Yala Provinces). Both new species resemble G.macrophyllus, G.scortechinii and G.uvarioides. The addition of these two new species brings the total number of Goniothalamus species in Thailand to 27. Separate identification keys are provided for flowering and fruiting specimens of the Thai species.

Specificity of assemblage, not fungal partner species, explains mycorrhizal partnerships of mycoheterotrophic Burmannia plants.

Mycoheterotrophic plants (MHPs) growing on arbuscular mycorrhizal fungi (AMF) usually maintain specialized mycorrhizal associations. The level of specificity varies between MHPs, although it remains largely unknown whether interactions with mycorrhizal fungi differ by plant lineage, species, and/or by population. Here, we investigate the mycorrhizal interactions among Burmannia species (Burmanniaceae) with different trophic modes using high-throughput DNA sequencing. We characterized the inter- and intraspecific dynamics of the fungal communities by assessing the composition and diversity of fungi among sites. We found that fully mycoheterotrophic species are more specialized in their fungal associations than chlorophyllous species, and that this specialization possibly results from the gradual loss of some fungal groups. In particular, although many fungal species were shared by different Burmannia species, fully MHP species typically host species-specific fungal assemblages, suggesting that they have a preference for the selected fungi. Although no apparent cophylogenetic relationship was detected between fungi and plants, we observe that evolutionarily closely related plants tend to have a greater proportion of shared or closely related fungal partners. Our findings suggest a host preference and specialization toward fungal assemblages in Burmannia, improving understanding of interactions between MHPs and fungi.

Phylogenetic relationships of 'Polyalthia' in Fiji.

The genus Polyalthia (Annonaceae) has undergone dramatic taxonomic changes in recent years. Nine Polyalthia species have historically been recognized in Fiji, all of which have subsequently been transferred to three different genera, viz. Goniothalamus, Huberantha and Meiogyne. The transfer of six of these species has received strong molecular phylogenetic support, although the other three species, Polyalthia amoena, P. capillata and P. loriformis [all transferred to Huberantha], have never previously been sampled in a phylogenetic study. We address this shortfall by sampling available herbarium specimens of all three species and integrating the data in a molecular phylogenetic analysis. The resultant phylogeny provides strong support for the transfer of these species to Huberantha. The taxonomic realignment of all nine Fijian species formerly classified in Polyalthia is also clearly demonstrated and supported by the resultant phylogeny. The updated taxonomic treatments of the nine species, a key to the three genera and a key to the Fijian Huberantha species are provided.

Correlated evolution of diaspore traits and potential frugivore-mediated selection in a fleshy-fruited tropical lineage (Artabotrys, Annonaceae).

Dispersal syndromes are often defined by reference to fruit traits that are associated with distinct frugivore guilds. Studies rarely examine the relationship between seed traits and frugivores or test the alternative hypothesis that traits are shaped by climatic variables. We assess whether the evolution of seed size and physical defense are correlated with dispersal-related traits and climatic variables in Artabotrys, a fleshy-fruited tropical lineage. Diaspore traits and WorldClim bioclimatic variables were compiled for 43 species. Correlated evolution was evaluated using phylogenetic regression and model-fitting approaches. The best-fitting multioptima Ornstein-Uhlenbeck model suggests that lineages with smooth testa and thin pericarp (SP) have evolved toward smaller seeds with a thinner testa, whereas lineages with rough testa and/or thick pericarp have evolved toward larger seeds with a thicker testa. A smooth testa facilitates spitting and/or swallowing of intact seeds while fruits with thin pericarp may be preferentially consumed by frugivores with less destructive oral processing, enabling lower investment in seed physical defense in SP lineages. Moreover, small seeds are more likely to be swallowed intact with a food bolus. The effect of climate on seed size and physical defense is equivocal and warrants further investigation.

Evolutionary divergence times in the Annonaceae: evidence of a late Miocene origin of Pseuduvaria in Sundaland with subsequent diversification in New Guinea.

BACKGROUND: Phylogenetic analyses of the Annonaceae consistently identify four clades: a basal clade consisting of Anaxagorea, and a small 'ambavioid' clade that is sister to two main clades, the 'long branch clade' (LBC) and 'short branch clade' (SBC). Divergence times in the family have previously been estimated using non-parametric rate smoothing (NPRS) and penalized likelihood (PL). Here we use an uncorrelated lognormal (UCLD) relaxed molecular clock in BEAST to estimate diversification times of the main clades within the family with a focus on the Asian genus Pseuduvaria within the SBC. Two fossil calibration points are applied, including the first use of the recently discovered Annonaceae fossil Futabanthus. The taxonomy and morphology of Pseuduvaria have been well documented, although no previous dating or biogeographical studies have been undertaken. Ancestral areas at internal nodes within Pseuduvaria are determined using dispersal-vicariance analysis (DIVA) and weighted ancestral area analysis (WAAA). RESULTS: The divergence times of the main clades within the Annonaceae were found to deviate slightly from previous estimates that used different calibration points and dating methods. In particular, our estimate for the SBC crown (55.2-26.9 Mya) is much younger than previous estimates (62.5-53.1 +/- 3.6 Mya and ca. 58.76 Mya). Early diversification of Pseuduvaria was estimated to have occurred 15-8 Mya, possibly associated with the 'mid-Miocene climatic optimum.' Pseuduvaria is inferred to have originated in Sundaland in the late Miocene, ca. 8 Mya; subsequent migration events were predominantly eastwards towards New Guinea and Australia, although several migratory reversals are also postulated. Speciation of Pseuduvaria within New Guinea may have occurred after ca. 6.5 Mya, possibly coinciding with the formation of the Central Range orogeny from ca. 8 Mya. CONCLUSION: Our divergence time estimates within the Annonaceae are likely to be more precise as we used a UCLD clock model and calibrated the phylogeny using new fossil evidence. Pseuduvaria is shown to have dispersed from Sundaland after the late Miocene. The present-day paleotropical distribution of Pseuduvaria may have been achieved by long-distance dispersal, and speciation events might be explained by global climatic oscillations, sea level fluctuations, and tectonic activity.

Mitrephoramonocarpa (Annonaceae): a new species from Surat Thani Province, Peninsular Thailand.

A new species, Mitrephoramonocarpa R.M.K.Saunders & Chalermglin, sp. nov. (Annonaceae), is described from Surat Thani Province in Peninsular Thailand. It resembles two other species, M.alba Ridl. and M.keithii Ridl., with which it is broadly sympatric, but can be distinguished by its solitary flowers (not borne in thyrsoid inflorescences as in most other Mitrephora species), single carpel per flower (and hence single monocarp per fruit) and the lack of a monocarp stipe. A key to the nine Mitrephora species in Thailand is provided.

Pollination Drop Proteome and Reproductive Organ Transcriptome Comparison in Gnetum Reveals Entomophilous Adaptation.

Gnetum possesses morphologically bisexual but functionally unisexual reproductive structures that exude sugary pollination drops to attract insects. Previous studies have revealed that the arborescent species (G. gnemon L.) and the lianoid species (G. luofuense C.Y.Cheng) possess different pollination syndromes. This study compared the proteome in the pollination drops of these two species using label-free quantitative techniques. The transcriptomes of fertile reproductive units (FRUs) and sterile reproductive units (SRUs) for each species were furthermore compared using Illumina Hiseq sequencing, and integrated proteomic and transcriptomic analyses were subsequently performed. Our results show that the differentially expressed proteins between FRUs and SRUs were involved in carbohydrate metabolism, the biosynthesis of amino acids and ovule defense. In addition, the differentially expressed genes between the FRUs and SRUs (e.g., MADS-box genes) were engaged in reproductive development and the formation of pollination drops. The integrated protein-transcript analyses revealed that FRUs and their exudates were relatively conservative while the SRUs and their exudates were more diverse, probably functioning as pollinator attractants. The evolution of reproductive organs appears to be synchronized with changes in the pollination drop proteome of Gnetum, suggesting that insect-pollinated adaptations are not restricted to angiosperms but also occur in gymnosperms.

Phylogeny of the basal angiosperm genus Pseuduvaria (Annonaceae) inferred from five chloroplast DNA regions, with interpretation of morphological character evolution.

Phylogenetic relationships within the magnoliid basal angiosperm genus Pseuduvaria (Annonaceae) are investigated using chloroplast DNA sequences from five regions: psbA-trnH spacer, trnL-F, matK, rbcL, and atpB-rbcL spacer. Over 4000 nucleotides from 51 species (of the total 53) were sequenced. The five cpDNA datasets were analyzed separately and in combination using maximum parsimony (MP), maximum likelihood (ML), and Bayesian methods. The phylogenetic trees constructed using all three phylogenetic methods, based on the combined data, strongly support the monophyly of Pseuduvaria following the inclusion of Craibella phuyensis. The trees generated using MP were less well resolved, but relationships are similar to those obtained using the other methods. ML and Bayesian analyses recovered trees with short branch lengths, showing five main clades. This study highlights the evolutionary changes in seven selected morphological characters (floral sex, stamen and carpel numbers, inner petal color, presence of inner petal glands, flowering peduncle length, and monocarp size). Although floral unisexuality is ancestral within the genus, several evolutionary lineages reveal reversal to bisexuality. Other phylogenetic transitions include the evolution of sapromyophily, and fruit-bat frugivory and seed dispersal, thus allowing a wide range of adaptations for species survival.

Two new species and two new records of Artabotrys (Annonaceae) from Thailand.

Two new species of Artabotrys are described from Thailand. Artabotrys tanaosriensis J.Chen, Chalermglin & R.M.K.Saunders, sp. nov., is similar to A. oblanceolatus Craib but differs in its symmetrical, cuneate or decurrent leaf base, externally distinct outer petal blades and claws, deltoid and undulate outer petal blades, rhomboid and undulate inner petal blades and shorter, subsessile and slightly beaked monocarps. Artabotrys spathulatus J.Chen, Chalermglin & R.M.K.Saunders, sp. nov., is most similar to A. tanaosriensis but differs in having flat outer petal blades, broadly rhomboid outer petal claws, broadly spathulate and strongly concave inner petal blades and strongly beaked monocarps. Two new records for the Flora of Thailand are furthermore reported here: A. punctulatus C.Y.Wu ex S.H.Yuan and A. byrsophyllus I.M.Turner & Utteridge, which were previously confused with A. aeneus Ast and A. grandifolius King, respectively. A key to Artabotrys species indigenous to Thailand is provided here.