Genome sequences and population genomics reveal climatic adaptation and genomic divergence between two closely related sweetgum species.

Understanding the genetic basis of population divergence and adaptation is an important goal in population genetics and evolutionary biology. However, the relative roles of demographic history, gene flow, and/or selective regime in driving genomic divergence, climatic adaptation, and speciation in non-model tree species are not yet fully understood. To address this issue, we generated whole-genome resequencing data of Liquidambar formosana and L. acalycina, which are broadly sympatric but altitudinally segregated in the Tertiary relict forests of subtropical China. We integrated genomic and environmental data to investigate the demographic history, genomic divergence, and climatic adaptation of these two sister species. We inferred a scenario of allopatric species divergence during the late Miocene, followed by secondary contact during the Holocene. We identified multiple genomic islands of elevated divergence that mainly evolved through divergence hitchhiking and recombination rate variation, likely fostered by long-term refugial isolation and recent differential introgression in low-recombination genomic regions. We also found some candidate genes with divergent selection signatures potentially involved in climatic adaptation and reproductive isolation. Our results contribute to a better understanding of how late Tertiary/Quaternary climatic change influenced speciation, genomic divergence, climatic adaptation, and introgressive hybridization in East Asia's Tertiary relict flora. In addition, they should facilitate future evolutionary, conservation genomics, and molecular breeding studies in Liquidambar, a genus of important medicinal and ornamental values.

Genome sequences and population genomics provide insights into the demographic history, inbreeding, and mutation load of two 'living fossil' tree species of Dipteronia.

'Living fossils', that is, ancient lineages of low taxonomic diversity, represent an exceptional evolutionary heritage, yet we know little about how demographic history and deleterious mutation load have affected their long-term survival and extinction risk. We performed whole-genome sequencing and population genomic analyses on Dipteronia sinensis and D. dyeriana, two East Asian Tertiary relict trees. We found large-scale genome reorganizations and identified species-specific genes under positive selection that are likely involved in adaptation. Our demographic analyses suggest that the wider-ranged D. sinensis repeatedly recovered from population bottlenecks over late Tertiary/Quaternary periods of adverse climate conditions, while the population size of the narrow-ranged D. dyeriana steadily decreased since the late Miocene, especially after the Last Glacial Maximum (LGM). We conclude that the efficient purging of deleterious mutations in D. sinensis facilitated its survival and repeated demographic recovery. By contrast, in D. dyeriana, increased genetic drift and reduced selection efficacy, due to recent severe population bottlenecks and a likely preponderance of vegetative propagation, resulted in fixation of strongly deleterious mutations, reduced fitness, and continuous population decline, with likely detrimental consequences for the species' future viability and adaptive potential. Overall, our findings highlight the significant impact of demographic history on levels of accumulation and purging of putatively deleterious mutations that likely determine the long-term survival and extinction risk of Tertiary relict trees.

Pangenome of water caltrop reveals structural variations and asymmetric subgenome divergence after allopolyploidization.

Water caltrop (Trapa spp., Lythraceae) is a traditional but currently underutilized non-cereal crop. Here, we generated chromosome-level genome assemblies for the two diploid progenitors of allotetraploid Trapa. natans (4x, AABB), i.e., diploid T. natans (2x, AA) and Trapa incisa (2x, BB). In conjunction with four published (sub)genomes of Trapa, we used gene-based and graph-based pangenomic approaches and a pangenomic transposable element (TE) library to develop Trapa genomic resources. The pangenome displayed substantial gene-content variation with dispensable and private gene clusters occupying a large proportion (51.95%) of the total cluster sets in the six (sub)genomes. Genotyping of presence-absence variation (PAVs) identified 40 453 PAVs associated with 2570 genes specific to A- or B-lineages, of which 1428 were differentially expressed, and were enriched in organ development process, organic substance metabolic process and response to stimulus. Comparative genome analyses showed that the allotetraploid T. natans underwent asymmetric subgenome divergence, with the B-subgenome being more dominant than the A-subgenome. Multiple factors, including PAVs, asymmetrical amplification of TEs, homeologous exchanges (HEs), and homeolog expression divergence, together affected genome evolution after polyploidization. Overall, this study sheds lights on the genome architecture and evolution of Trapa, and facilitates its functional genomic studies and breeding program.

Chromosome-level reference genome of Tetrastigma hemsleyanum (Vitaceae) provides insights into genomic evolution and the biosynthesis of phenylpropanoids and flavonoids.

Here, we present a high-quality chromosome-scale genome assembly (2.19 Gb) and annotation of Tetrastigma hemsleyanum, a perennial herbaceous liana native to subtropical China with diverse medicinal applications. Approximately 73% of the genome was comprised of transposable elements (TEs), of which long terminal repeat retrotransposons (LTR-RTs) were a predominant group (69% of the genome). The genome size increase of T. hemsleyanum (relative to Vitis species) was mostly due to the proliferation of LTR-RTs. Of the different modes of gene duplication identified, transposed duplication (TRD) and dispersed duplication (DSD) were the predominant ones. Genes, particularly those involved in the phenylpropanoid-flavonoid (PF) pathway and those associated with therapeutic properties and environmental stress resistance, were significantly amplified through recent tandem duplications. We dated the divergence of two intraspecific lineages in Southwest (SW) versus Central-South-East (CSE) China to the late Miocene (approximately 5.2 million years ago). Of those, the former showed more upregulated genes and metabolites. Based on resequencing data of 38 individuals representing both lineages, we identified various candidate genes related to 'response to stimulus' and 'biosynthetic process', including ThFLS11, which is putatively involved in flavonoid accumulation. Overall, this study provides abundant genomic resources for future evolutionary, ecological, and functional genomics studies in T. hemsleyanum and related species.

Plastome evolution and phylogenomics of Impatiens (Balsaminaceae).

MAIN CONCLUSION: This study reported seven new plastomes from Impatiens and observed three highly variable regions for phylogeny and DNA barcoding, which resolved the relationships among sections of subgenus Impatiens. Impatiens L. (Balsaminaceae, Ericales) is one of the largest and most diverse genera of angiosperms, widely known for its taxonomic difficulty. In this study, we reevaluated the infrageneric relationships within the genus Impatiens, using complete plastome sequence data. Seven complete plastomes of Impatiens (representing 6 species) were newly sequenced and characterized along with 20 previously published plastomes of other Impatiens species, plus 2 plastomes of outgroups (Hydrocera triflora, Balsaminaceae; Marcgravia coriacea, Marcgraviaceae). The total size of these 29 plastomes ranged from 151,538 bp to 152,917 bp, except 2 samples of Impatiens morsei, which exhibited a shorter length and lost some genes encoding NADH dehydrogenase subunits. Moreover, the number of simple sequence repeats (SSRs) ranged from 51 to 113, and the number of long repeats from 17 to 26. In addition, three highly variable regions were identified (trnG-GCC (The previous one), ndhF-rpl32-trnL-UGA-ccsA, and ycf1). Our phylogenomic analysis based on 80 plastome-derived protein-coding genes strongly supported the monophyly of Impatiens and its two subgenera (Clavicarpa and Impatiens), and fully resolved relationships among the six (out of seven) sampled sections of subgenus Impatiens. Overall, the plastome DNA markers and phylogenetic results reported in this study will facilitate future identification, taxonomic and DNA barcoding studies in Impatiens as well as evolutionary studies in Balsaminaceae.

Plio-Pleistocene climatic change drives allopatric speciation and population divergence within the Scrophularia incisa complex (Scrophulariaceae) of desert and steppe subshrubs in Northwest China.

Numerous temperate plants and animals on the Qinghai-Tibet Plateau (QTP) are hypothesized to have differentiated due to vicariant allopatric speciation associated with the geologic uplifts. However, this hypothesis has rarely been tested through a phylogeographic study of relative species in a broader geographic context, including the QTP, Tianshan Mountains, Mongolian Plateau, and surrounding regions. To understand the speciation and diversification process of plants across this wide area, phylogeographic analysis were examined from Scrophularia incisa and two other closely relative species comprising S. kiriloviana and S. dentata. Thirty-two populations of the three close relatives were genotyped using chloroplast DNA fragments and nuclear microsatellite loci to assess population structure and diversity, supplemented by phylogenetic dating, ancestral area reconstructions and species distribution modelings, as well as niche identity tests. Our chloroplast DNA (cpDNA) phylogeny showed that this monophyletic group of desert and steppe semi-shrub is derived from a Middle Pliocene ancestor of the Central Asia. Lineages in Central Asia vs. China diverged through climate/tectonic-induced vicariance during Middle Pliocene. Genetic and ENM data in conjunction with niche differentiation analyses support that the divergence of S. incisa, S. dentata and S. kiriloviana in China lineage proceeded through allopatric speciation, might triggered by early Pleistocene climate change of increase of aridification and enlargement of deserts, while subsequent climate-induced cycles of range contractions/expansions enhanced the geographical isolation and habit fragmentation of these taxa. These findings highlight the importance of the Plio-Pleistocene climate change in shaping genetic diversity and driving speciation in temperate steppes and deserts of Northwestern China.

Evidence for an evo-devo-derived hypothesis on three-dimensional flower shape modularity in a tropical orchid clade.

Covarying suites of phenotypic traits, or modules, are increasingly recognized to promote morphological evolution. However, information on how modularity influences flower diversity is rare and lacking for Orchidaceae. Here, we combine high-resolution X-ray computed tomography scanning with three-dimensional geometric morphometrics and phylogenetic comparative methods to test various hypotheses about three-dimensional patterns of flower evolutionary modularity in Malagasy Bulbophyllum orchids and examine rates and modes of module evolution. Based on the four evolutionary modules identified (i.e., sepals, lateral petals, labellum + column-foot, and column-part), our data support the hypothesis that both genetic-developmental and functional adaptive factors shaped evolutionary flower trait covariation in these tropical orchids. In line with "evo-devo" studies, we also find that the labellum evolved independently from the rest of the petal whorl. Finally, we show that modules evolved with different rates, and either in a neutral fashion (only column-part) or under selective constraints, as likely imposed by pollinators. Overall, this study supports current views that modular units can enhance the range and rate of morphological evolution.

A Comparative Study of Genetic Responses to Short- and Long-Term Habitat Fragmentation in a Distylous Herb Hedyotis chyrsotricha (Rubiaceae).

The genetic effects of habitat fragmentation are complex and are influenced by both species traits and landscape features. For plants with strong seed or pollen dispersal capabilities, the question of whether the genetic erosion of an isolated population becomes stronger or is counterbalanced by sufficient gene flow across landscapes as the timescales of fragmentation increase has been less studied. In this study, we compared the population structure and genetic diversity of a distylous herb, Hedyotis chyrsotricha (Rubiaceae), in two contrasting island systems of southeast China. Based on RAD-Seq data, our results showed that populations from the artificially created Thousand-Island Lake (TIL) harbored significantly higher levels of genetic diversity than those from the Holocene-dated Zhoushan Archipelago (ZA) (π = 0.247 vs. 0.208, HO = 0.307 vs. 0.256, HE = 0.228 vs. 0.190), while genetic differences between island and mainland populations were significant in neither the TIL region nor the ZA region. A certain level of population substructure was found in TIL populations, and the level of gene flow among TIL populations was also lower than in ZA populations (m = 0.019 vs. 0.027). Overall, our comparative study revealed that genetic erosion has not become much stronger for the island populations of either the TIL or ZA regions. Our results emphasized that the matrix of water in the island system may facilitate the seed (fruit) dispersal of H. chrysotricha, thus maintaining population connectivity and providing ongoing resilience to the effects of habitat fragmentation over thousands of years.

Plastid Phylogenomics and Plastome Evolution of Nandinoideae (Berberidaceae).

Subfamily Nandinoideae Heintze (Berberidaceae), comprising four genera and ca. 19 species, is disjunctively distributed in eastern North America vs. Eurasia (eastern Asia, Central Asia, Middle East, and southeastern Europe), and represents an ideal taxon to explore plastid phylogenomics and plastome evolution in Berberidaceae. Many species of this subfamily have been listed as national or international rare and endangered plants. In this study, we sequenced and assembled 20 complete plastomes, representing three genera and 13 species of Nandinoideae. Together with six plastomes from GenBank, a total of 26 plastomes, representing all four genera and 16 species of Nandinoideae, were used for comparative genomic and phylogenomic analyses. These plastomes showed significant differences in overall size (156,626-161,406 bp), which is mainly due to the expansion in inverted repeat (IR) regions and/or insertion/deletion (indel) events in intergenic spacer (IGS) regions. A 75-bp deletion in the ndhF gene occurred in Leontice and Gymnospermium when compared with Nandina and Caulophyllum. We found a severe truncation at the 5' end of ycf1 in three G. altaicum plastomes, and a premature termination of ropC1 in G. microrrhynchum. Our phylogenomic results support the topology of {Nandina, [Caulophyllum, (Leontice, Gymnospermium)]}. Within the core genus Gymnospermium, we identified G. microrrhynchum from northeastern Asia (Clade A) as the earliest diverging species, followed by G. kiangnanense from eastern China (Clade B), while the rest species clustered into the two sister clades (C and D). Clade C included three species from West Tianshan (G. albertii, G. darwasicum, G. vitellinum). Clade D consisted of G. altaicum from northern Central Asia, plus one species from the Caucasus Mountains (G. smirnovii) and three from southeastern Europe (G. odessanum, G. peloponnesiacum, G. scipetarum). Overall, we identified 21 highly variable plastome regions, including two coding genes (rpl22, ycf1) and 19 intergenic spacer (IGS) regions, all with nucleotide diversity (Pi) values > 0.02. These molecular markers should serve as powerful tools (including DNA barcodes) for future phylogenetic, phylogeographic and conservation genetic studies.

Phylogeography and Population Genetics Analyses Reveal Evolutionary History of the Desert Resource Plant Lycium ruthenicum (Solanaceae).

Climactic oscillations during the Quaternary played a significant role in the formation of genetic diversity and historical demography of numerous plant species in northwestern China. In this study, we used 11 simple sequence repeats derived from expressed sequence tag (EST-SSR), two chloroplast DNA (cpDNA) fragments, and ecological niche modeling (ENM) to investigate the population structure and the phylogeographic history of Lycium ruthenicum, a plant species adapted to the climate in northwestern China. We identified 20 chloroplast haplotypes of which two were dominant and widely distributed in almost all populations. The species has high haplotype diversity and low nucleotide diversity based on the cpDNA data. The EST-SSR results showed a high percentage of total genetic variation within populations. Both the cpDNA and EST-SSR results indicated no significant differentiation among populations. By combining the evidence from ENM and demographic analysis, we confirmed that both the last interglacial (LIG) and late-glacial maximum (LGM) climatic fluctuations, aridification might have substantially narrowed the distribution range of this desert species, the southern parts of the Junggar Basin, the Tarim Basin, and the eastern Pamir Plateau were the potential glacial refugia for L. ruthenicum during the late middle Pleistocene to late Pleistocene Period. During the early Holocene, the warm, and humid climate promoted its demographic expansion in northwestern China. This work may provide new insights into the mechanism of formation of plant diversity in this arid region.

An overlooked dispersal route of Cardueae (Asteraceae) from the Mediterranean to East Asia revealed by phylogenomic and biogeographical analyses of Atractylodes.

BACKGROUND AND AIMS: The East Asian-Tethyan disjunction pattern and its mechanisms of formation have long been of interest to researchers. Here, we studied the biogeographical history of Asteraceae tribe Cardueae, with a particular focus on the temperate East Asian genus Atractylodes DC., to understand the role of tectonic and climatic events in driving the diversification and disjunctions of the genus. METHODS: A total of 76 samples of Atractylodes from 36 locations were collected for RAD-sequencing. Three single nucleotide polymorphism (SNP) datasets based on different filtering strategies were used for phylogenetic analyses. Molecular dating and ancestral distribution reconstruction were performed using both chloroplast DNA sequences (127 Cardueae samples) and SNP (36 Atractylodes samples) datasets. KEY RESULTS: Six species of Atractylodes were well resolved as individually monophyletic, although some introgression was identified among accessions of A. chinensis, A. lancea and A. koreana. Dispersal of the subtribe Carlininae from the Mediterranean to East Asia occurred after divergence between Atractylodes and Carlina L. + Atractylis L. + Thevenotia DC. at ~31.57 Ma, resulting in an East Asian-Tethyan disjunction. Diversification of Atractylodes in East Asia mainly occurred from the Late Miocene to the Early Pleistocene. CONCLUSIONS: Aridification of Asia and the closure of the Turgai Strait in the Late Oligocene promoted the dispersal of Cardueae from the Mediterranean to East China. Subsequent uplift of the Qinghai-Tibet Plateau as well as changes in Asian monsoon systems resulted in an East Asian-Tethyan disjunction between Atractylodes and Carlina + Atractylis + Thevenotia. In addition, Late Miocene to Quaternary climates and sea level fluctuations played major roles in the diversification of Atractylodes. Through this study of different taxonomic levels using genomic data, we have revealed an overlooked dispersal route between the Mediterranean and far East Asia (Japan/Korea) via Central Asia and East China.

Local Insect Availability Partly Explains Geographical Differences in Floral Visitor Assemblages of Arum maculatum L. (Araceae).

Geographical variation in abundance and composition of pollinator assemblages may result in variable selection pressures among plant populations and drive plant diversification. However, there is limited knowledge on whether differences in local visitor and pollinator assemblages are the result of site-specific strategies of plants to interact with their pollinators and/or merely reflect the pollinator availability at a given locality. To address this question, we compared locally available insect communities obtained by light-trapping with assemblages of floral visitors in populations of Arum maculatum (Araceae) from north vs. south of the Alps. We further investigated whether and how the abundance of different visitors affects plants' female reproductive success and examined the pollen loads of abundant visitors. Local insect availability explained inter-regional differences in total visitor abundance, but only partly the composition of visitor assemblages. Northern populations predominantly attracted females of Psychoda phalaenoides (Psychodidae, Diptera), reflecting the high availability of this moth fly in this region. More generalized visitor assemblages, including other psychodid and non-psychodid groups, were observed in the south, where the availability of P. phalaenoides/Psychodidae was limited. Fruit set was higher in the north than in the south but correlated positively in both regions with the abundance of total visitors and psychodids; in the north, however, this relationship disappeared when visitor abundances were too high. High pollen loads were recorded on both psychodids and other Diptera. We demonstrate for the first time that the quantitative assessment of floral visitor assemblages in relation to locally available insect communities is helpful to understand patterns of geographical variation in plant-pollinator interactions. This combined approach revealed that geographical differences in floral visitors of A. maculatum are only partly shaped by the local insect availability. Potential other factors that may contribute to the geographical pattern of visitor assemblages include the region-specific attractiveness of this plant species to flower visitors and the population-specific behavior of pollinators.

Antennae of psychodid and sphaerocerid flies respond to a high variety of floral scent compounds of deceptive Arum maculatum L.

Insect-pollinated plants often release complex mixtures of floral scents to attract their pollinators. Yet scent compounds eliciting physiological or behavioural responses in pollinators have only been identified in few plant species. The sapromyiophilous aroid Arum maculatum releases a highly diverse dung-like scent with overall more than 300 different compounds recorded so far to attract its psychodid and other fly pollinators. The volatiles' role in pollinator attraction is mostly unknown. To identify potential behaviourally active compounds, we recorded electroantennographic responses of four Psychodidae and one Sphaeroceridae species to (1) inflorescence scents of A. maculatum and (2) the scents released by cow dung, likely imitated by the plant species. Here we show that these flies are sensitive to 78 floral volatiles of various chemical classes, 18 of which were also found in cow dung. Our study, which for the first time determined physiologically active compounds in the antennae of Psychoda spp. and Sphaeroceridae, identified various volatiles not known to be biologically active in any floral visitors so far. The obtained results help deciphering the chemical basis that enables A. maculatum and other plants, pollinated by psychodids and sphaerocerids, to attract and deceive their pollinators.

Phylogenomics and diversification drivers of the Eastern Asian - Eastern North American disjunct Podophylloideae.

Evolutionary and biogeographic processes determine species richness patterns of vascular plants between Eastern Asia (EA) and Eastern North America (ENA). However, the strikingly higher species richness of EA relative to ENA remains poorly understood from this perspective. Here, we studied the relative importance of biogeographical, evolutionary and ecological factors underlying differences in species richness between EA and ENA in Podophylloideae (Berberidaceae, Ranunculales; in total 10 spp. in EA vs. 2 spp. in ENA). Based on large-scale transcriptome data, our phylogenomic analyses strongly supported Podophylloideae and its two multi-species genera, i.e. Dysosma (EA) and Diphylleia (EA/ENA), as monophyletic groups. Sinopodophyllum hexandrum (EA) was identified as sister to the remainder of Podophylloideae. Dysosma (7 spp.) was recovered as sister to Podophyllum peltatum (ENA), forming an EA-ENA disjunct pair with a strong bias of species diversity in the EA counterpart. Our biogeographic analyses support the 'out-of-Tibet' hypothesis, suggesting that Podophylloideae started to diversify in the Himalaya-Hengduan Mountains (Mid-Miocene) and migrated eastward (since the Late Miocene) into Central-eastern China, Japan, and ENA (only P. peltatum and Diphylleia cymosa). Overall, we conclude that the striking species diversity anomaly between EA and ENA in Podophylloideae may be explained by a combination of (1) a longer period of time available to accumulate species in EA; and (2) a greater diversification rate in EA, which might have been promoted by greater physiographic and environmental heterogeneity in this region.

Genome sequencing and transcriptome analyses provide insights into the origin and domestication of water caltrop (Trapa spp., Lythraceae).

Humans have domesticated diverse species from across the plant kingdom; however, our current understanding of plant domestication is largely founded on major cereal crops. Here, we examine the evolutionary processes and genetic basis underlying the domestication of water caltrop (Trapa spp., Lythraceae), a traditional, yet presently underutilized non-cereal crop that sustained early Chinese agriculturalists. We generated a chromosome-level genome assembly of tetraploid T. natans, and then divided the allotetraploid genome into two subgenomes. Based on resequencing data from 57 accessions, representing cultivated diploid T. natans, wild T. natans (2x and 4x) and diploid T. incisa, we showed that water caltrop was likely first domesticated in the Yangtze River Valley as early as 6300 yr BP, and experienced a second improvement c. 800 years ago. We also provided strong support for an allotetraploid origin of T. natans within the past 230 000-310 000 years. By integrating selective sweep and transcriptome profiling analyses, we identified a number of genes potentially selected and/or differentially expressed during domestication, some of which likely contributed not only to larger fruit sizes but also to a more vigorous root system, facilitating nutrient uptake, environmental stress response and underwater photosynthesis. Our results shed light on the evolutionary and domestication history of water caltrop, one of the earliest domesticated crops in China. This study has implications for genomic-assisted breeding of this presently underutilized aquatic plant, and improves our general understanding of plant domestication.

Variation in scent amount but not in composition correlates with pollinator visits within populations of deceptive Arum maculatum L. (Araceae).

Floral scent is vital for pollinator attraction and varies among and within plant species. However, little is known about how inter-individual variation in floral scent affects the abundance and composition of floral visitor assemblages within populations. Moreover, for deceptive plants it is predicted that intra-population variation in scent can be maintained by negative frequency-dependent selection, but empirical evidence is still lacking. To investigate the ecological and evolutionary relations between inter-individual scent variation (i.e., total emission and composition) and floral visitors in deceptive plants, we studied floral scent, visitor assemblages, and fruit set in two populations of fly-pollinated (Psychodidae, Sphaeroceridae; Diptera) and deceptive Arum maculatum from Austria (JOS) and northern Italy (DAO). By correlating individual data on floral scent and visitor assemblages, we show that inter-individual variation in floral scent partly explains variation in visitor assemblages. The quantity of floral scent emitted per individual correlated positively with visitor abundance in both populations but explained visitor composition only in DAO, where strongly scented inflorescences attracted more sphaerocerid flies. However, in each population, the composition of floral scent did not correlate with the composition of floral visitors. There was also no evidence of negative frequency-dependent selection on floral scent. Instead, in JOS, more frequent scent phenotypes attracted more pollinators and were more likely to set an infructescence than rarer ones. Our results show that floral scent, despite being key in pollinator attraction in A. maculatum, only partly explains variation in pollinator abundance and composition. Overall, this study is the first to shed light on the importance of inter-individual variation in floral scent in explaining floral visitor assemblages at the population level in a deceptive plant species.

Floral Scents of a Deceptive Plant Are Hyperdiverse and Under Population-Specific Phenotypic Selection.

Floral scent is a key mediator in plant-pollinator interactions. However, little is known to what extent intraspecific scent variation is shaped by phenotypic selection, with no information yet in deceptive plants. In this study, we collected inflorescence scent and fruit set of the deceptive moth fly-pollinated Arum maculatum L. (Araceae) from six populations north vs. five populations south of the Alps, accumulating to 233 samples in total, and tested for differences in scent, fruit set, and phenotypic selection on scent across this geographic barrier. We recorded 289 scent compounds, the highest number so far reported in a single plant species. Most of the compounds occurred both north and south of the Alps; however, plants of the different regions emitted different absolute and relative amounts of scent. Fruit set was higher north than south of the Alps, and some, but not all differences in scent could be explained by differential phenotypic selection in northern vs. southern populations. This study is the first to provide evidence that floral scents of a deceptive plant are under phenotypic selection and that phenotypic selection is involved in shaping geographic patterns of floral scent in such plants. The hyperdiverse scent of A. maculatum might result from the imitation of various brood substrates of its pollinators.

Evidence for selectively constrained 3D flower shape evolution in a Late Miocene clade of Malagasy Bulbophyllum orchids.

Questions concerning the evolution of complex biological structures are central to the field of evolutionary biology. Yet, still little information is known about the modes and temporal dynamics of three-dimensional (3D) flower shape evolution across the history of clades. Here, we combined high-resolution X-ray computed tomography with 3D geometric morphometrics and phylogenetic comparative methods to test models of whole-flower shape evolution in the orchid family, using an early Late Miocene clade (c. 50 spp.) of Malagasy Bulbophyllum as model system. Based on landmark data of 38 species, our high-dimensional model fitting decisively rejects a purely neutral mode of evolution, suggesting instead that flower shapes evolved towards a primary adaptive optimum. Only a small number of recently evolved species/lineages attained alternative shape optima, resulting in an increased rate of phenotypic evolution. Our findings provide evidence of constrained 3D flower shape evolution in a small-sized clade of tropical orchids, resulting in low rates of phenotypic evolution and uncoupled trait-diversification rates. We hypothesise that this deep imprint of evolutionary constraint on highly complex floral structures might reflect long-term (directional and/or stabilizing) selection exerted by the group's main pollinators (flies).

Evolution of crassulacean acid metabolism (CAM) as an escape from ecological niche conservatism in Malagasy Bulbophyllum (Orchidaceae).

Despite growing evidence that niche shifts are more common in flowering plants than previously thought, little is known of whether such shifts are promoted by changes in photosynthetic pathways. Here we combine the most complete phylogeny for epiphytic Malagasy Bulbophyllum orchids (c. 210 spp.) with climatic niche and carbon isotope ratios to infer the group's spatial-temporal history, and the role of strongly expressed crassulacean acid metabolism (CAM) in facilitating niche shifts and diversification. We find that most extant species still retain niche (Central Highland) and photosynthesis (C3 ) states as present in the single mid-Miocene (c. 12.70 million yr ago (Ma)) ancestor colonizing Madagascar. However, we also infer a major transition to CAM, linked to a late Miocene (c. 7.36 Ma) invasion of species from the sub-humid highland first into the island's humid eastern coastal, and then into the seasonally dry 'Northwest Sambirano' rainforests, yet without significant effect on diversification rates. These findings indicate that CAM in tropical epiphytes may be selectively advantageous even in high rainfall habitats, rather than presenting a mere adaptation to dry environments or epiphytism per se. Overall, our study qualifies CAM as an evolutionary 'gateway' trait that considerably widened the spatial-ecological amplitude of Madagascar's most species-rich orchid genus.