Final Report Summary - LINARIA-SPECIATION (Integrating phylogenetics, ecology and evo-devo to understand the origin of plant species: the role of spur length evolution in speciation of the genus Linaria)
Introduction
Understanding the origin of species is one of the major challenges of modern biology, and requires the integration of ecological, evolutionary and developmental approaches. This involves the investigation of evolutionary changes in developmental control and timing in closely related species, together with their ecological implications. The number of studies using this approach is still relatively small. In this project, we aimed to understand speciation processes in plants, paying particular attention to the role of floral nectar spurs, which have been hypothesized as a key innovation promoting speciation in angiosperms as a result of pollinator interactions.
To that end, we focused on the snapdragon lineage (tribe Antirrhineae, family Plantaginaceae), a model group for plant developmental and evolutionary research. The genus Linaria (toadflaxes) is the most diverse within the Antirrhineae. It provides an ideal system to investigate pollinator-driven speciation because it displays several flower traits linked to pollinator specialization, including a spur of variable length that is formed at the base of the ventral petal. The spur collects nectar, which is offered to pollinators. We applied a multidisciplinary approach to investigate evolutionary changes in the length of nectar spurs during speciation events in a clade of eight toadflaxes (Linaria sect. Versicolores) with remarkable floral diversity, and distributed in the Mediterranean biodiversity hotspot (Iberian Peninsula).
Objectives
(1) To test the role of nectar spurs as a key innovation promoting speciation in the Antirrhineae at a macroevolutionary scale.
(2) To estimate phylogenetic relationships within the Iberian clade of Linaria sect. Versicolores using a phylogenomic approach and to reconstruct evolutionary changes in floral traits (including nectar spur length) in the course of speciation.
(3) To investigate the components of reproductive isolation among species of the study clade, including prezygotic (pollinators, environmental niche) and postzygotic breeding barriers.
(4) To understand the developmental and genetic basis of spur length variation.
Work performed and key results
(1) Nectar spurs as a key innovation in Antirrhineae
To evaluate the role of nectar spurs as a key innovation promoting speciation at a macroevolutionary scale, we assembled a DNA sequence dataset that allowed us to obtain the most complete phylogeny of the Antirrhineae available to date and to test the key innovation hypothesis (higher speciation rates in lineages with nectar spurs than in lineages lacking them) using recently developed analytical tools. Analyses supported three to four independent origins of nectar spurs in the course of Antirrhineae diversification, and significantly higher speciation rates in lineages with nectar spurs than in those lacking them, according to a BiSSE (binary state speciation and speciation) model. This would be in agreement with the key innovation hypothesis. We are currently exploring the performance of alternative methodologies to test the robustness of this result.
(2) Phylogenetic relationships in the Iberian clade of Linaria sect. Versicolores
Given the recent and fast speciation of the study clade, conventional phylogenetic markers did not provide enough information to resolve phylogenetic relationships. Therefore, a phylogenomic approach using next-generation sequencing was implemented. Specifically, a genotyping-by-sequencing library was prepared from genomic DNA of c.90 specimens representing the eight morphologically-defined taxa of the clade and eight additional outgroup species. After data assembly, a wide range of methodologies was applied to resolve species delimitation and phylogenetic relationships: genetic structure analyses (including Bayesian clustering), phylogenetic networks, concatenated analyses (using maximum likelihood and Bayesian inference) and coalescent-based methods. Analyses confirmed the single origin of the group, revealed the genetic consistency of eight species (L. clementei, L. salzmannii, L. spicata, L. viscosa, L. onubensis, L. algarviana, L. spartea and L. incarnata), and supported four nodes of the species phylogeny, while the phylogenetic position of two species remained relatively uncertain. Ancestral state reconstructions, taking into account phylogenetic uncertainty, allowed us to reconstruct morphological changes in the course of speciation, revealing widespread convergence of reproductive characters, including nectar spur length, corolla colour, flower tube width and inflorescence density. This is consistent with a role of pollination mechanisms in speciation. The short-spurred flower found in L. clementei was inferred to have evolved from a long-spurred flower similar to that of L. salzmannii.
(3) Components of reproductive isolation
(a) Pollination interactions
Flower visitor censuses were conducted in L. clementei and L. salzmannii populations. Several species of bees with varying proboscis lengths were identified as the main pollinators. Flower traits involved in plant-pollinator interactions were characterized, including nectar quantity and concentration, distance from the start of the spur to the nectar, and length of pollinator proboscis. The results revealed that L. clementei is not only the species with the shortest spur, but also the only species of the study clade lacking nectar production. It is pollinated by bees with a wide range of proboscis lengths both shorter and longer than the spur. This species seems to offer pollen instead of nectar as the main reward for pollinators. On the other hand, L. salzmannii displays long nectar spurs and high nectar production. However, comparison between distance to nectar and proboscis length of the two main pollinators revealed that only one of them is able to feed on the nectar. This suggests that both nectar and pollen play a role as reward in this species.
(b) Species distribution models
Two types of data are required to build species distribution models: geo-referenced species localities and environmental data. Coordinates of species localities for the eight species were collected in the field or obtained from herbarium specimens and online databases. Environmental data at different geographic scales, including climatic and lithological variables, were obtained from available databases. Distribution models were built for the eight species using the maximum entropy algorithm (software Maxent). These models revealed lithology as the main variable determining the distribution of L. salzmannii and L. clementei at a local scale and their ecological isolation, therefore supporting a role in their speciation. Evolutionary changes in climatic niche were also detected.
(c) Experimental hybridizations
Post-pollination barriers between L. clementei and L. salzmannii were assessed through experimental hybridizations. These revealed high fruit and seed set when pollinating L. salzmannii flowers with L. clementei pollen (but not in the opposite direction). In addition, intra-specific crossings were performed to evaluate differences in breeding system (selfing vs. outcrosser) as a potential isolating barrier between L. clementei and L. salzmannii. The results revealed that both species are essentially outcrossers with a low level of selfing.
(4) Developmental and genetic basis of spur length variation
Differences in nectar spur ontogeny among the eight study species were assessed, with particular focus on the two species with extreme spur length: L. salzmannii (c.13 mm) and L. clementei (c.3 mm). An analysis of spur growth timing revealed that length differences are explained mostly by differences in growth rate (but not in growth duration). This indicates that heterochronic changes in spur length in the study group are instances of neoteny/acceleration. Ontogenetic differences at the cellular level were assessed through Scanning Electron Microscopy (SEM). The observations indicated that variation in spur growth rate between L. salzmannii and L. clementei is not the result of a difference in the rate of anisotropic cell expansion rate, but of differences in the number of cells undergoing expansion. This suggested that a difference in the initial phase of cell division may drive the contrasting spur lengths of these species. Previous evidence suggests that the initial cell division phase is regulated by the activity of genes encoding KNOX transcription factors (Hirzina and Invaginata). Therefore, KNOX genes were considered as candidates to regulate the developmental difference between these species. Preliminary results showed increased expression of the Hirzina gene early in nectar spur development in L. salzmannii, but not in L. clementei.
Final results and potential impact
Integration of results from all detailed objectives provides a comprehensive view of speciation processes in Linaria, and particularly of the role of nectar spurs in it. Nectar spurs may play a role in promoting speciation, as suggested by macroevolutionary analyses. However, our detailed analysis of the Iberian clade of Linaria sect. Versicolores indicates that the specific mechanisms involved in spur length changes in the course of speciation may not always fit the conventional “pollinator shift” model in which changes in spur length are associated with changes between pollinators with different proboscis lengths. Instead, the evolution of a short spur in L. clementei from a long-spurred ancestor is linked to a loss of nectar production, and apparently to a change in floral resource use by the main pollinators, from nectar feeding and pollen collection to exclusively pollen collection. Nectar spurs may not necessarily play a role as a pre-zygotic barrier between the short-spurred L. clementei and the long-spurred, geographically and phylogenetically close L. salzmannii. Habitat isolation based on contrasting soil preferences plays a more important role as a pre-zygotic barrier, and possibly as a driver of speciation at least in some of our study species. Interestingly, evolutionary developmental changes involved in spur length variation across species in Linaria seem to be completely different to those described for a different system, the genus Aquilegia (family Ranunculaceae). While differences in the duration of cell elongation, and therefore of spur elongation, give rise to striking differences in spur length in Aquilegia, the contrasting lengths of L. salzmannii and L. clementei spurs seem to be the result of differences in the initial phase of cell division, which then results in contrasting rates of spur elongation under similar rates of cell elongation. The molecular mechanisms involved in this evolutionary change are only starting to be understood.
Plant-pollinator interactions are crucial for ecosystem function and crop management. Given the current biodiversity and pollinator crisis, research projects (such as this one) aimed at understanding pollination in an evolutionary context provide useful information for species conservation, ecosystem and crop management.
Understanding the origin of species is one of the major challenges of modern biology, and requires the integration of ecological, evolutionary and developmental approaches. This involves the investigation of evolutionary changes in developmental control and timing in closely related species, together with their ecological implications. The number of studies using this approach is still relatively small. In this project, we aimed to understand speciation processes in plants, paying particular attention to the role of floral nectar spurs, which have been hypothesized as a key innovation promoting speciation in angiosperms as a result of pollinator interactions.
To that end, we focused on the snapdragon lineage (tribe Antirrhineae, family Plantaginaceae), a model group for plant developmental and evolutionary research. The genus Linaria (toadflaxes) is the most diverse within the Antirrhineae. It provides an ideal system to investigate pollinator-driven speciation because it displays several flower traits linked to pollinator specialization, including a spur of variable length that is formed at the base of the ventral petal. The spur collects nectar, which is offered to pollinators. We applied a multidisciplinary approach to investigate evolutionary changes in the length of nectar spurs during speciation events in a clade of eight toadflaxes (Linaria sect. Versicolores) with remarkable floral diversity, and distributed in the Mediterranean biodiversity hotspot (Iberian Peninsula).
Objectives
(1) To test the role of nectar spurs as a key innovation promoting speciation in the Antirrhineae at a macroevolutionary scale.
(2) To estimate phylogenetic relationships within the Iberian clade of Linaria sect. Versicolores using a phylogenomic approach and to reconstruct evolutionary changes in floral traits (including nectar spur length) in the course of speciation.
(3) To investigate the components of reproductive isolation among species of the study clade, including prezygotic (pollinators, environmental niche) and postzygotic breeding barriers.
(4) To understand the developmental and genetic basis of spur length variation.
Work performed and key results
(1) Nectar spurs as a key innovation in Antirrhineae
To evaluate the role of nectar spurs as a key innovation promoting speciation at a macroevolutionary scale, we assembled a DNA sequence dataset that allowed us to obtain the most complete phylogeny of the Antirrhineae available to date and to test the key innovation hypothesis (higher speciation rates in lineages with nectar spurs than in lineages lacking them) using recently developed analytical tools. Analyses supported three to four independent origins of nectar spurs in the course of Antirrhineae diversification, and significantly higher speciation rates in lineages with nectar spurs than in those lacking them, according to a BiSSE (binary state speciation and speciation) model. This would be in agreement with the key innovation hypothesis. We are currently exploring the performance of alternative methodologies to test the robustness of this result.
(2) Phylogenetic relationships in the Iberian clade of Linaria sect. Versicolores
Given the recent and fast speciation of the study clade, conventional phylogenetic markers did not provide enough information to resolve phylogenetic relationships. Therefore, a phylogenomic approach using next-generation sequencing was implemented. Specifically, a genotyping-by-sequencing library was prepared from genomic DNA of c.90 specimens representing the eight morphologically-defined taxa of the clade and eight additional outgroup species. After data assembly, a wide range of methodologies was applied to resolve species delimitation and phylogenetic relationships: genetic structure analyses (including Bayesian clustering), phylogenetic networks, concatenated analyses (using maximum likelihood and Bayesian inference) and coalescent-based methods. Analyses confirmed the single origin of the group, revealed the genetic consistency of eight species (L. clementei, L. salzmannii, L. spicata, L. viscosa, L. onubensis, L. algarviana, L. spartea and L. incarnata), and supported four nodes of the species phylogeny, while the phylogenetic position of two species remained relatively uncertain. Ancestral state reconstructions, taking into account phylogenetic uncertainty, allowed us to reconstruct morphological changes in the course of speciation, revealing widespread convergence of reproductive characters, including nectar spur length, corolla colour, flower tube width and inflorescence density. This is consistent with a role of pollination mechanisms in speciation. The short-spurred flower found in L. clementei was inferred to have evolved from a long-spurred flower similar to that of L. salzmannii.
(3) Components of reproductive isolation
(a) Pollination interactions
Flower visitor censuses were conducted in L. clementei and L. salzmannii populations. Several species of bees with varying proboscis lengths were identified as the main pollinators. Flower traits involved in plant-pollinator interactions were characterized, including nectar quantity and concentration, distance from the start of the spur to the nectar, and length of pollinator proboscis. The results revealed that L. clementei is not only the species with the shortest spur, but also the only species of the study clade lacking nectar production. It is pollinated by bees with a wide range of proboscis lengths both shorter and longer than the spur. This species seems to offer pollen instead of nectar as the main reward for pollinators. On the other hand, L. salzmannii displays long nectar spurs and high nectar production. However, comparison between distance to nectar and proboscis length of the two main pollinators revealed that only one of them is able to feed on the nectar. This suggests that both nectar and pollen play a role as reward in this species.
(b) Species distribution models
Two types of data are required to build species distribution models: geo-referenced species localities and environmental data. Coordinates of species localities for the eight species were collected in the field or obtained from herbarium specimens and online databases. Environmental data at different geographic scales, including climatic and lithological variables, were obtained from available databases. Distribution models were built for the eight species using the maximum entropy algorithm (software Maxent). These models revealed lithology as the main variable determining the distribution of L. salzmannii and L. clementei at a local scale and their ecological isolation, therefore supporting a role in their speciation. Evolutionary changes in climatic niche were also detected.
(c) Experimental hybridizations
Post-pollination barriers between L. clementei and L. salzmannii were assessed through experimental hybridizations. These revealed high fruit and seed set when pollinating L. salzmannii flowers with L. clementei pollen (but not in the opposite direction). In addition, intra-specific crossings were performed to evaluate differences in breeding system (selfing vs. outcrosser) as a potential isolating barrier between L. clementei and L. salzmannii. The results revealed that both species are essentially outcrossers with a low level of selfing.
(4) Developmental and genetic basis of spur length variation
Differences in nectar spur ontogeny among the eight study species were assessed, with particular focus on the two species with extreme spur length: L. salzmannii (c.13 mm) and L. clementei (c.3 mm). An analysis of spur growth timing revealed that length differences are explained mostly by differences in growth rate (but not in growth duration). This indicates that heterochronic changes in spur length in the study group are instances of neoteny/acceleration. Ontogenetic differences at the cellular level were assessed through Scanning Electron Microscopy (SEM). The observations indicated that variation in spur growth rate between L. salzmannii and L. clementei is not the result of a difference in the rate of anisotropic cell expansion rate, but of differences in the number of cells undergoing expansion. This suggested that a difference in the initial phase of cell division may drive the contrasting spur lengths of these species. Previous evidence suggests that the initial cell division phase is regulated by the activity of genes encoding KNOX transcription factors (Hirzina and Invaginata). Therefore, KNOX genes were considered as candidates to regulate the developmental difference between these species. Preliminary results showed increased expression of the Hirzina gene early in nectar spur development in L. salzmannii, but not in L. clementei.
Final results and potential impact
Integration of results from all detailed objectives provides a comprehensive view of speciation processes in Linaria, and particularly of the role of nectar spurs in it. Nectar spurs may play a role in promoting speciation, as suggested by macroevolutionary analyses. However, our detailed analysis of the Iberian clade of Linaria sect. Versicolores indicates that the specific mechanisms involved in spur length changes in the course of speciation may not always fit the conventional “pollinator shift” model in which changes in spur length are associated with changes between pollinators with different proboscis lengths. Instead, the evolution of a short spur in L. clementei from a long-spurred ancestor is linked to a loss of nectar production, and apparently to a change in floral resource use by the main pollinators, from nectar feeding and pollen collection to exclusively pollen collection. Nectar spurs may not necessarily play a role as a pre-zygotic barrier between the short-spurred L. clementei and the long-spurred, geographically and phylogenetically close L. salzmannii. Habitat isolation based on contrasting soil preferences plays a more important role as a pre-zygotic barrier, and possibly as a driver of speciation at least in some of our study species. Interestingly, evolutionary developmental changes involved in spur length variation across species in Linaria seem to be completely different to those described for a different system, the genus Aquilegia (family Ranunculaceae). While differences in the duration of cell elongation, and therefore of spur elongation, give rise to striking differences in spur length in Aquilegia, the contrasting lengths of L. salzmannii and L. clementei spurs seem to be the result of differences in the initial phase of cell division, which then results in contrasting rates of spur elongation under similar rates of cell elongation. The molecular mechanisms involved in this evolutionary change are only starting to be understood.
Plant-pollinator interactions are crucial for ecosystem function and crop management. Given the current biodiversity and pollinator crisis, research projects (such as this one) aimed at understanding pollination in an evolutionary context provide useful information for species conservation, ecosystem and crop management.