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.

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.

Long-term isolation of European steppe outposts boosts the biome's conservation value.

The European steppes and their biota have been hypothesized to be either young remnants of the Pleistocene steppe belt or, alternatively, to represent relicts of long-term persisting populations; both scenarios directly bear on nature conservation priorities. Here, we evaluate the conservation value of threatened disjunct steppic grassland habitats in Europe in the context of the Eurasian steppe biome. We use genomic data and ecological niche modelling to assess pre-defined, biome-specific criteria for three plant and three arthropod species. We show that the evolutionary history of Eurasian steppe biota is strikingly congruent across species. The biota of European steppe outposts were long-term isolated from the Asian steppes, and European steppes emerged as disproportionally conservation relevant, harbouring regionally endemic genetic lineages, large genetic diversity, and a mosaic of stable refugia. We emphasize that conserving what is left of Europe's steppes is crucial for conserving the biological diversity of the entire Eurasian steppe biome.

Genomic Signature of Shifts in Selection in a Subalpine Ant and Its Physiological Adaptations.

Understanding how organisms adapt to extreme environments is fundamental and can provide insightful case studies for both evolutionary biology and climate-change biology. Here, we take advantage of the vast diversity of lifestyles in ants to identify genomic signatures of adaptation to extreme habitats such as high altitude. We hypothesized two parallel patterns would occur in a genome adapting to an extreme habitat: 1) strong positive selection on genes related to adaptation and 2) a relaxation of previous purifying selection. We tested this hypothesis by sequencing the high-elevation specialist Tetramorium alpestre and four other phylogenetically related species. In support of our hypothesis, we recorded a strong shift of selective forces in T. alpestre, in particular a stronger magnitude of diversifying and relaxed selection when compared with all other ants. We further disentangled candidate molecular adaptations in both gene expression and protein-coding sequence that were identified by our genome-wide analyses. In particular, we demonstrate that T. alpestre has 1) a higher level of expression for stv and other heat-shock proteins in chill-shock tests and 2) enzymatic enhancement of Hex-T1, a rate-limiting regulatory enzyme that controls the entry of glucose into the glycolytic pathway. Together, our analyses highlight the adaptive molecular changes that support colonization of high-altitude environments.

Technical comment on Condamine et al. (2019): a cautionary note for users of linear diversification dependencies.

Condamine et al. (2019; 22: 1900-1912) fitted linear and exponential functions of time-dependent, diversity-dependent and temperature-dependent diversification to investigate diversification dynamics of tetrapod families. Here I highlight potential misinterpretations when using linear diversification dependencies and provide some clarifications.

Oscillayers: A dataset for the study of climatic oscillations over Plio-Pleistocene time-scales at high spatial-temporal resolution.

MOTIVATION: In order to understand how species evolutionarily responded to Plio-Pleistocene climate oscillations (e.g. in terms of speciation, extinction, migration and adaptation), it is first important to have a good understanding of those past climate changes per se. This, however, is currently limited due to the lack of global-scale climatic datasets with high temporal resolution spanning the Plio-Pleistocene. To fill this gap, I here present Oscillayers, a global-scale and region-specific bioclim dataset, facilitating the study of climatic oscillations during the last 5.4 million years at high spatial (2.5 arc-minutes) and temporal (10 kyr time periods) resolution. This dataset builds upon interpolated anomalies (Δ layers) between bioclim layers of the present and the Last Glacial Maximum (LGM) that are scaled relative to the Plio-Pleistocene global mean temperature curve, derived from benthic stable oxygen isotope ratios, to generate bioclim variables for 539 time periods. Evaluation of the scaled, interpolated estimates of palaeo-climates generated for the Holocene, Last Interglacial and Pliocene showed good agreement with independent general circulation models (GCMs) for respective time periods in terms of pattern correlation and absolute differences. Oscillayers thus provides a new tool for studying spatial-temporal patterns of evolutionary and ecological processes at high temporal and spatial resolution. MAIN TYPES OF VARIABLE CONTAINED: Nineteen bioclim variables for time periods throughout the Plio-Pleistocene. Input data and R script to recreate all 19 bioclim variables. SPATIAL LOCATION AND GRAIN: Global at 2.5 arc-minutes (4.65 x 4.65 = 21.62 km2 at the equator). TIME PERIOD AND GRAIN: The last 5.4 million years. The grain is 10 kyr (= 539 time periods). LEVEL OF MEASUREMENT: Data are for terrestrial climates (excluding Antarctica) taking sea level changes into account. SOFTWARE FORMAT: All data are available as ASCII grid files.

Major range loss predicted from lack of heat adaptability in an alpine Drosophila species.

Climate warming is threatening biodiversity worldwide. Climate specialists such as alpine species are especially likely to be vulnerable. Adaptation by rapid evolution is the only long-term option for survival of many species, but the adaptive evolutionary potential of heat resistance has not been assessed in an alpine invertebrate. Here, we show that the alpine fly Drosophila nigrosparsa cannot readily adapt to heat stress. Heat-exposed flies from a regime with increased ambient temperature and a regime with increased temperature plus artificial selection for heat tolerance were less heat tolerant than the control group. Increased ambient temperature affected negatively both fitness and competitiveness. Ecological niche models predicted the loss of three quarters of the climatically habitable areas of this fly by the end of this century. Our findings suggest that, alongside with other climate specialists, species from mountainous regions are highly vulnerable to climate warming and unlikely to adapt through evolutionary genetic changes.

Clade-age-dependent diversification under high species turnover shapes species richness disparities among tropical rainforest lineages of Bulbophyllum (Orchidaceae).

BACKGROUND: Tropical rainforests (TRFs) harbour almost half of the world's vascular plant species diversity while covering only about 6-7% of land. However, why species richness varies amongst the Earth's major TRF regions remains poorly understood. Here we investigate the evolutionary processes shaping continental species richness disparities of the pantropical, epiphytic and mostly TRF-dwelling orchid mega-genus Bulbophyllum (c. 1948 spp. in total) using diversification analyses based on a time-calibrated molecular phylogeny (including c. 45-50% spp. each from Madagascar, Africa, Neotropics, and 8.4% from the Asia-Pacific region), coupled with ecological niche modelling (ENM) of geographic distributions under present and past (Last Glacial Maximum; LGM) conditions. RESULTS: Our results suggest an early-to-late Miocene scenario of 'out-of-Asia-Pacific' origin and progressive, dispersal-mediated diversification in Madagascar, Africa and the Neotropics, respectively. Species richness disparities amongst these four TRF lineages are best explained by a time-for-speciation (i.e. clade age) effect rather than differences in net diversification or diversity-dependent diversification due to present or past spatial-bioclimatic limits. For each well-sampled lineage (Madagascar, Africa, Neotropics), we inferred high rates of speciation and extinction over time (i.e. high species turnover), yet with the origin of most extant species falling into the Quaternary. In contrast to predictions of classical 'glacial refuge' theories, all four lineages experienced dramatic range expansions during the LGM. CONCLUSIONS: As the Madagascan, African and Neotropical lineages display constant-rate evolution since their origin (early-to-mid-Miocene), Quaternary environmental change might be a less important cause of their high species turnover than intrinsic features generally conferring rapid population turnover in tropical orchids (e.g., epiphytism, specialization on pollinators and mycorrhizal fungi, wind dispersal). Nonetheless, climate-induced range fluctuations during the Quaternary could still have played an influential role in the origination and extinction of Bulbophyllum species in those three, if not in all four TRF regions.

Integrating phylogenomics, phylogenetics, morphometrics, relative genome size and ecological niche modelling disentangles the diversification of Eurasian Euphorbia seguieriana s. l. (Euphorbiaceae).

Next generation sequencing has revolutionised biology. Restriction-associated DNA sequencing (RADseq) has primarily been used to study infraspecific relationships but has also been applied in multi-species phylogenomic analyses. In this study, we used a combination of phylogenomic (with RADseq data) and phylogenetic (with sequences of the nuclear internal transcribed spacer, ITS) methods to explore relationships within the taxonomically intricate Euphorbia seguieriana s. l., one of the most widespread Euphorbia taxa inhabiting zonal and extrazonal steppes from Iberia to Central Asia. In the inferred phylogenies the southeastern Balkan and Anatolian populations were clearly separated, supporting the distinction of E. niciciana from E. seguieriana at the species level. Within E. seguieriana, the populations from the Caucasus, Iran, and easternmost Anatolia were sister to all other populations based on RADseq, making necessary the description of a new, morphologically divergent subspecies, E. seguieriana subsp. armeniaca. Conversely, additional studies are needed to understand the status of E. seguieriana subsp. hohenackeri, which is sympatric with E. seguieriana subsp. armeniaca. Niche analyses indicated that differences in the climatic niche between E. niciciana and E. seguieriana are relatively small compared with the climatic differences between the regions over which they are distributed. Contrary to previous believes, E. niciciana and E. seguieriana are allopatric and have likely diverged during the Pleistocene in two different glacial refugia as suggested by distribution modelling. Euphorbia niciciana nowadays has a submediterranean distribution, occupying habitats that are slightly warmer, moister, and less seasonal in temperature but more seasonal in precipitation than E. seguieriana, a characteristic species of continental steppes. Using flow cytometry, we demonstrate that the relative genome sizes of E. niciciana and E. seguieriana differ significantly. Additionally, multivariate morphometric analyses of 56 morphological characters indicated clear morphological divergence of the two species. Importantly, we also provide a revised taxonomic treatment including formal nomenclatural changes, an identification key and species descriptions. Our study demonstrates that an integrative approach, combining modern phylogenomic methods with traditional phylogenetic, cytogenetic, environmental and morphological analyses can result in satisfactorily resolved relationships in intricate groups of closely related species. Finally, phylogenetic inference using ITS sequences is still a useful tool for resolving relationships among the taxa at the species level, but the phylogenomic approach based on RADseq data certainly provides better resolution both among and within species.

Evolution of morphological crypsis in the Tetramorium caespitum ant species complex (Hymenoptera: Formicidae).

Cryptic species are morphologically very similar to each other. To what extent stasis or convergence causes crypsis and whether ecology influences the evolution of crypsis has remained unclear. The Tetramorium caespitum complex is one of the most intricate examples of cryptic species in ants. Here, we test three hypotheses concerning the evolution of its crypsis: H1: The complex is monophyletic. H2: Morphology resulted from evolutionary stasis. H3: Ecology and morphology evolved concertedly. We confirmed (H1) monophyly of the complex; (H2) a positive relation between morphological and phylogenetic distances, which indicates a very slow loss of similarity over time and thus stasis; and (H3) a positive relation between only one morphological character and a proxy of the ecological niche, which indicates concerted evolution of these two characters, as well as a negative relation between p-values of correct species identification and altitude, which suggests that species occurring in higher altitudes are more cryptic. Our data suggest that species-specific morphological adaptations to the ecological niche are exceptions in the complex, and we consider the worker morphology in this complex as an adaptive solution for various environments.

Turning one into five: Integrative taxonomy uncovers complex evolution of cryptic species in the harvester ant Messor "structor".

Seed harvesting ants are ecosystem engineers that shape vegetation, nutrient cycles, and microclimate. Progress in ecological research is, however, slowed down by poor species delimitation. For example, it has not been resolved to date, how many species the European harvester ant Messor "structor" (Latreille, 1798) represents. Since its first description, splitting into additional taxa was often proposed but not accepted later on due to inconsistent support from morphology and ecology. Here, we took an iterative integrative-taxonomy approach - comparing multiple, independent data sets of the same sample - and used traditional morphometrics, Wolbachia symbionts, mitochondrial DNA, amplified fragment length polymorphism, and ecological niche modelling. Using the complementarity of the data sets applied, we resolved multiple, strong disagreements over the number of species, ranging from four to ten, and the allocation of individuals to species. We consider most plausible a five-species hypothesis and conclude the taxonomic odyssey by redescribing Messor structor, M. ibericus Santschi, 1925, and M. muticus (Nylander, 1849) stat.rev., and by describing two new species, M. ponticus sp.n. and M. mcarthuri sp.n. The evolutionary explanations invoked in resolving the various data conflicts include pronounced morphological crypsis, incomplete lineage-sorting or ongoing cospeciation of endosymbionts, and peripatric speciation - these ants' significance to evolutionary biology parallels that to ecology. The successful solution of this particular problem illustrates the usefulness of the integrative approach to other systematic problems of comparable complexity and the importance of understanding evolution to drawing correct conclusions on species' attributes, including their ecology and biogeography.

Correction to "Notes on the Statistical Power of the Binary State Speciation and Extinction (BiSSE) Model".

[This corrects the article DOI: 10.4137/EBO.S39732.].

Notes on the Statistical Power of the Binary State Speciation and Extinction (BiSSE) Model.

The Binary State Speciation and Extinction (BiSSE) method is one of the most popular tools for investigating the rates of diversification and character evolution. Yet, based on previous simulation studies, it is commonly held that the BiSSE method requires phylogenetic trees of fairly large sample sizes (>300 taxa) in order to distinguish between the different models of speciation, extinction, or transition rate asymmetry. Here, the power of the BiSSE method is reevaluated by simulating trees of both small and large sample sizes (30, 60, 90, and 300 taxa) under various asymmetry models and root state assumptions. Results show that the power of the BiSSE method can be much higher, also in trees of small sample size, for detecting differences in speciation rate asymmetry than anticipated earlier. This, however, is not a consequence of any conceptual or mathematical flaw in the method per se but rather of assumptions about the character state at the root of the simulated trees and thus the underlying macroevolutionary model, which led to biased results and conclusions in earlier power assessments. As such, these earlier simulation studies used to determine the power of BiSSE were not incorrect but biased, leading to an overestimation of type-II statistical error for detecting differences in speciation rate but not for extinction and transition rates.

Frequent but asymmetric niche shifts in Bulbophyllum orchids support environmental and climatic instability in Madagascar over Quaternary time scales.

BACKGROUND: Species or clades may retain or shift their environmental niche space over evolutionary time. Understanding these processes offers insights into the environmental processes fuelling lineage diversification and might also provide information on past range dynamics of ecosystems. However, little is known about the relative contributions of niche conservatism versus niche divergence to species diversification in the tropics. Here, we examined broad-scale patterns of niche evolution within a Pliocene-Pleistocene clade of epiphytic Bulbophyllum orchids (30 spp.) whose collective distribution covers the northwest and eastern forest ecosystems of Madagascar. RESULTS: Using species occurrence data, ecological niche models, and multivariate analyses of contributing variables, we identified a three-state niche distribution character for the entire clade, coinciding with three major forest biomes viz. phytogeographical provinces in Madagascar: A, Northwest 'Sambirano'; B, 'Eastern Lowlands'; and C, 'Central Highlands'. A time-calibrated phylogeny and Bayesian models of niche evolution were then used to detect general trends in the direction of niche change over the clade's history (≤5.3 Ma). We found highest transitions rates between lowlands (A and B) and (mostly from B) into the highland (C), with extremely low rates out of the latter. Lowland-to-highland transitions occurred frequently during the Quaternary, suggesting that climate-induced vegetational shifts promoted niche transitions and ecological speciation at this time. CONCLUSIONS: Our results reveal that niche transitions occurred frequently and asymmetrically within this Madagascan orchid clade, and in particular over Quaternary time scales. Intrinsic features germane to Bulbophyllum (e.g., high dispersal ability, drought tolerance, multiple photosynthetic pathways) as well as extrinsic factors (ecological, historical) likely interacted to generate the niche transition patterns observed. In sum, our results support the emerging idea of dramatic environmental and climatic fluctuations in Madagascar during the recent geological past, which overturns the long-held paradigm of long-term stability in tropical forest settings. The generality of the patterns and timings reported here awaits the availability of additional comparative studies in other Madagascan endemics.

Multiple independent origins of auto-pollination in tropical orchids (Bulbophyllum) in light of the hypothesis of selfing as an evolutionary dead end.

BACKGROUND: The transition from outcrossing to selfing has long been portrayed as an 'evolutionary dead end' because, first, reversals are unlikely and, second, selfing lineages suffer from higher rates of extinction owing to a reduced potential for adaptation and the accumulation of deleterious mutations. We tested these two predictions in a clade of Madagascan Bulbophyllum orchids (30 spp.), including eight species where auto-pollinating morphs (i.e., selfers, without a 'rostellum') co-exist with their pollinator-dependent conspecifics (i.e., outcrossers, possessing a rostellum). Specifically, we addressed this issue on the basis of a time-calibrated phylogeny by means of ancestral character reconstructions and within the state-dependent evolution framework of BiSSE (Binary State Speciation and Extinction), which allowed jointly estimating rates of transition, speciation, and extinction between outcrossing and selfing. RESULTS: The eight species capable of selfing occurred in scattered positions across the phylogeny, with two likely originating in the Pliocene (ca. 4.4-3.1 Ma), one in the Early Pleistocene (ca. 2.4 Ma), and five since the mid-Pleistocene (ca. ≤ 1.3 Ma). We infer that this scattered phylogenetic distribution of selfing is best described by models including up to eight independent outcrossing-to-selfing transitions and very low rates of speciation (and either moderate or zero rates of extinction) associated with selfing. CONCLUSIONS: The frequent and irreversible outcrossing-to-selfing transitions in Madagascan Bulbophyllum are clearly congruent with the first prediction of the dead end hypothesis. The inability of our study to conclusively reject or support the likewise predicted higher extinction rate in selfing lineages might be explained by a combination of methodological limitations (low statistical power of our BiSSE approach to reliably estimate extinction in small-sized trees) and evolutionary processes (insufficient time elapsed for selfers to go extinct). We suggest that, in these tropical orchids, a simple genetic basis of selfing (via loss of the 'rostellum') is needed to explain the strikingly recurrent transitions to selfing, perhaps reflecting rapid response to parallel and novel selective environments over Late Quaternary (≤ 1.3 Ma) time scales.

Recurrent polymorphic mating type variation in Madagascan Bulbophyllum species (Orchidaceae) exemplifies a high incidence of auto-pollination in tropical orchids.

The transition from outcrossing to self-fertilization is one of the most common evolutionary changes in angiosperms. The orchid family exemplifies this evolutionary trend but, because of a general lack of large-scale surveys on auto-pollination in orchid taxa, the incidence and modes of auto-pollination among (sub)tropical orchids remain poorly known. In the present study, we assessed the frequency and mode of auto-pollination within and among species of a largely monophyletic group of Madagascan Bulbophyllum. The capacity for autonomous fruit set was investigated by bagging experiments in the greenhouse and the field, complemented with detailed floral micromorphological studies of the gynostemium. Our survey comprises 393 accessions, representing at least 78 species, and thus approximately 37% of the species diversity of the genus in the Madagascan region. Our studies revealed that mating type is directly related to gynostemium structure, most often involving the presence or absence of a physical barrier termed 'rostellum'. As a novel and unexpected finding, we identified eight species of a single lineage of Madagascan Bulbophyllum (termed 'clade C'), in which auto-pollinating morphs (selfers), either lacking a rostellum or (rarely) possessing a stigmatic rostellum, co-exist with their pollinator-dependent conspecifics (outcrossers). We hypothesize that auto-pollination via rostellum abortion has a simple genetic basis, and probably evolved rapidly and recurrently by subtle changes in the timing of rostellum development (heterochrony). Thus, species of clade C may have an intrinsic genetic and developmental lability toward auto-pollination, allowing rapid evolutionary response under environmental, perhaps human-disturbed conditions favouring reproductive assurance. Overall, these findings should stimulate further research on the incidence, evolution, and maintenance of mating type variation in tropical orchids, as well as how they adapt(ed) to changing environmental conditions.

Histological and micro-CT evidence of stigmatic rostellum receptivity promoting auto-pollination in the madagascan orchid Bulbophyllum bicoloratum.

BACKGROUND: The rostellum, a projecting part of the gynostemium in orchid flowers, separates the anther(s) from the stigma and thus commonly prevents auto-pollination. Nonetheless, as a modified (usually distal) portion of the median stigma lobe, the rostellum has been frequently invoked of having re-gained a stigmatic function in rare cases of orchid auto-pollination. Here it is shown that a newly discovered selfing variant of Madagascan Bulbophyllumbicoloratum has evolved a modified rostellum allowing the penetration of pollen tubes from in situ pollinia. METHODS: Gynostemium micro-morphology and anatomy of selfing and outcrossing variants of B. bicoloratum was studied by using light and scanning electron microscopy and histological sections. Pollen tube growth in the selfing variant was further observed via X-ray computed microtomography (micro-CT), providing 3D reconstructions of floral tissues at a micron scale. FINDINGS: Selfing variants possess a suberect ('displaced') rostellum rather than the conventional, erect type. Very early in anthesis, the pollinia of selfers are released from the anther and slide down onto the suberect rostellum, where pollen tube growth preferentially occurs through the non-vascularized, i.e. rear (adaxial) and (semi-) lateral parts. This penetrated tissue is comprised of a thin layer of elongate and loosely arranged cells, embedded in stigmatic exudates, as also observed in the stigmatic cavity of both selfing and outcrossing variants. CONCLUSIONS: Our results provide the first solid evidence of a stigmatic function for the rostellum in orchid flowers, thereby demonstrating for the first time the feasibility of the micro-CT technique for accurately visualizing pollen tube growth in flowering plants. Rostellum receptivity in B. bicoloratum probably uniquely evolved as an adaptation for reproductive assurance from an outcrossing ancestor possessing an erect (non-receptive) rostellum. These findings open up new avenues in the investigation of an organ that apparently re-gained its 'primordial function' of being penetrated by pollen tubes.