Ant/plant symbioses

This doctoral thesis focuses on the evolution of ant/plant symbioses, a conspicuous form of mutualism involving some 113 species of ants and 684 species of vascular plants and occurring throughout the World’s tropical zones. My thesis addresses the following questions: (i) When, how often, and where did ant-plant symbioses evolve? (ii) By which steps did ant/plant symbioses evolve and which biotic or abiotic traits have favored them? (iii) How do ant/plant symbioses negotiate the tradeoff between specialization and stabilization? (iv) How often and under which conditions do ant/plant symbioses break down? (v) Are obligate epiphytic ant/plant symbioses dispersed by their ant symbionts? And (vi) how do related species of facultative and obligate ant-plants maximize benefits from the mutualism? To address these questions, I chose a clade of Australasian Rubiaceae that includes species with facultative, obligate or no ant symbioses and inferred its species relationships and geographic history, the precondition for studying the evolution of species’ interactions with ants. To answer question (i), I performed a literature survey of ant-plants and used capture-release models to estimate the expected number of ant-plants worldwide. I found that Australasia contains about 289 ant-plants, making it equally rich in ant-plants as the Neotropics (Chapter 1). Using a 1,140 species tree with ant-plants and their non-ant-plant relatives, I estimated a minimum of 158 origins of ant domatia in vascular plants (Chapter 1). I then employed molecular clock-dated phylogenies for 56% of the World’s known ant/plant lineages and found that the extant ant/plant symbioses in the Neotropics and Australasia date back to the Middle Miocene, while those in Africa only date back to 5-10 million years (Chapter 1). To answer question (ii), I used a phylogenetic framework for the ant genus with the largest number of obligate plant-ants (Pseudomyrmex) as well as phylogenies for its main plant host lineages (Chapter 2). I showed that host and symbiont broadening, meaning one partner increases the number of partners with which it interacts, is a dominant process in the evolution of ant/plant symbioses, even in the most specialized lineages such as the Central American ant/acacia mutualism (Chapter 2). Such increased host use led to the recruitment of new ant-plant lineages by plant-nesting ants; symbiont broadening in some instances appears to have resulted in complete partner replacement (Chapter 2). Another empirical finding is that parasites (i.e., ant species benefitting from plant rewards without reciprocating) originated from free-living generalists ant species, not from mutualists evolving into cheaters as predicted by theory. Host broadening apparently also was frequent in Australasian ant-gardens and seems to have favored the evolution of domatia once plants regularly ‘find themselves’ in ant-gardens (Chapter 8). Before going to the field in Fiji, I examined the relevant collections of Australasian in several herbaria (OXF, FHO, SUVA, DUB, K, L, M, BM, P), in addition to online databases and photos from other herbaria (in particular A, GH, FI, US, BISH). I discovered three new species in Fiji, resulting in now nine species of Squamellaria in the archipelago. By generating DNA sequences from relevant type material, I enlarged the (natural, monophyletic) genus Squamellaria from three species in the last revision (Jebb, 1991) to twelve species (Chapter 3). This taxonomic framework was essential to address all subsequent questions. To answer question (iii), I performed experiments and observations during eight weeks of fieldwork in September-October 2014 and March 2015 on all nine species of Squamellaria. By using DNA and morphological traits from herbarium material, I was able to place the Squamellaria data into a much larger comparative evolutionary framework (Chapter 4). Mutualism specialization requires more investment from each partner to increase levels of rewarding and partner fidelity, which increases the exploitation potential by opportunists. I showed that obligate ant-plants negotiated this tradeoff by evolving exclusive food rewards that can only be accessed by the obligate ant mutualist (Chapter 4). To answer question (iv), I generated a phylogeny for my focal clade that includes 76 of its 102 species, including several that I discovered during my fieldwork (above). Using this phylogeny and ancestral state reconstructions, I inferred ten losses of facultative symbiosis with ants, making this system well suited to study the ecological context of mutualism breakdown. In Hydnophytinae, mutualism breakdown has been driven by shifts to montane habitats (>1500 m alt.) where ants are scarce (Chapter 6). The evolution of a key mutualistic trait – entrance hole size – tightly tracked mutualistic strategies, with obligate ant-plants undergoing little evolutionary change in hole diameter, while species that lost mutualisms were free to rapidly change this trait. This indicates that mutualistic strategies, by determining the level of stabilizing selection, drive morphological evolution in mutualism-associated traits (Chapter 6; see also Discussion). To answer question (v), I used Fijian Squamellaria to study how facultative versus obligate ant-plants are dispersed, again relying on my own field observations and experiments. Facultative ant-plants are bird-dispersed, but obligate ant-plants are dispersed by their ant symbiont, the Dolichoderinae species Philidris nagasau (Chapter 5). Obligate ant-plant species of Squamellaria and P. nagasau ants engage in a type of ant-plant mutualism that is new to science, wherein the ants farm their hosts, planting the seeds inside tree bark of preferred host tree species and fertilizing the seedlings by defecating in their tiny domatia (before these are large enough to house any ant nest) (Chapter 5). To answer question (vi), I again used the Fijian Squamellaria system and designed experiments with stable isotopes (15N) to determine how ants fertilize hosts and how nitrogen uptake differs between facultative and obligate hosts. I also used Computed-Tomography Scanning to build 3D models of ant domatia. The domatia of Squamellaria attain rugby ball to pumpkin size, and their inner structure was essentially unknown. I discovered that in the obligate symbiosis, there is a single large cavity with small (ca. 2-3 mm in diameter) hyper-absorptive structures, termed ‘warts’, that are recognized by P. nagasau ants, which exclusively defecate on them, thus maximizing nitrogen benefits to the plants (Chapter 7). By contrast, facultative hosts have several unlinked cavities and lack absorptive warts. Because there is a high ant nest turnover in the facultative ant-plant species of Squamellaria, the modular domatium limits competition between inhabitants and maximizes the time any individual plant spends with nitrogen-providing ants (Chapter 7)

Climate and symbioses with ants modulate leaf/stem scaling in epiphytes

In most seed plants, leaf size is isometrically related to stem cross-sectional area, a relationship referred to as Corner's rule. When stems or leaves acquire a new function, for instance in ant-plant species with hollow stems occupied by ants, their scaling is expected to change. Here we use a lineage of epiphytic ant-plants to test how the evolution of ant-nesting structures in species with different levels of symbiotic dependence has impacted leaf/stem scaling. We expected that leaf size would correlate mostly with climate, while stem diameter would change with domatium evolution. Using a trait dataset from 286 herbarium specimens, field and greenhouse observations, climatic data, and a range of phylogenetic-comparative analyses, we detected significant shifts in leaf/stem scaling, mirroring the evolution of specialized symbioses. Our analyses support both predictions, namely that stem diameter change is tied to symbiosis evolution (ant-nesting structures), while leaf size is independently correlated with rainfall variables. Our study highlights how independent and divergent selective pressures can alter allometry. Because shifts in scaling relationships can impact the costs and benefits of mutualisms, studying allometry in mutualistic interactions may shed unexpected light on the stability of cooperation among species

Evolutionary Relationships and Biogeography of the Ant-Epiphytic Genus Squamellaria (Rubiaceae: Psychotrieae) and Their Taxonomic Implications

Ecological research on ant/plant symbioses in Fiji, combined with molecular phylogenetics, has brought to light four new species of Squamellaria in the subtribe Hydnophytinae of the Rubiaceae tribe Psychotrieae and revealed that four other species, previously in Hydnophytum, need to be transferred to Squamellaria. The diagnoses of the new species are based on morphological and DNA traits, with further insights from microCT scanning of flowers and leaf delta C-13 ratios (associated with Crassulacean acid metabolism). Our field and phylogenetic work results in a new circumscription of the genus Squamellaria, which now contains 12 species (to which we also provide a taxonomic key), not 3 as in the last revision. A clock-dated phylogeny and a model-testing biogeographic framework were used to infer the broader geographic history of rubiaceous ant plants in the Pacific, specifically the successive expansion of Squamellaria to Vanuatu, the Solomon Islands, and Fiji. The colonization of Vanuatu may have occurred from Fiji, when these islands were still in the same insular arc, while the colonization of the Solomon islands may have occurred after the separation of this island from the Fiji/Vanuatu arc. Some of these ant-housing epiphytes must have dispersed with their specialized ants, for instance attached to floating timber. Others acquired new ant symbionts on different islands

Ant/plant symbioses

This doctoral thesis focuses on the evolution of ant/plant symbioses, a conspicuous form of mutualism involving some 113 species of ants and 684 species of vascular plants and occurring throughout the World’s tropical zones. My thesis addresses the following questions: (i) When, how often, and where did ant-plant symbioses evolve? (ii) By which steps did ant/plant symbioses evolve and which biotic or abiotic traits have favored them? (iii) How do ant/plant symbioses negotiate the tradeoff between specialization and stabilization? (iv) How often and under which conditions do ant/plant symbioses break down? (v) Are obligate epiphytic ant/plant symbioses dispersed by their ant symbionts? And (vi) how do related species of facultative and obligate ant-plants maximize benefits from the mutualism? To address these questions, I chose a clade of Australasian Rubiaceae that includes species with facultative, obligate or no ant symbioses and inferred its species relationships and geographic history, the precondition for studying the evolution of species’ interactions with ants. To answer question (i), I performed a literature survey of ant-plants and used capture-release models to estimate the expected number of ant-plants worldwide. I found that Australasia contains about 289 ant-plants, making it equally rich in ant-plants as the Neotropics (Chapter 1). Using a 1,140 species tree with ant-plants and their non-ant-plant relatives, I estimated a minimum of 158 origins of ant domatia in vascular plants (Chapter 1). I then employed molecular clock-dated phylogenies for 56% of the World’s known ant/plant lineages and found that the extant ant/plant symbioses in the Neotropics and Australasia date back to the Middle Miocene, while those in Africa only date back to 5-10 million years (Chapter 1). To answer question (ii), I used a phylogenetic framework for the ant genus with the largest number of obligate plant-ants (Pseudomyrmex) as well as phylogenies for its main plant host lineages (Chapter 2). I showed that host and symbiont broadening, meaning one partner increases the number of partners with which it interacts, is a dominant process in the evolution of ant/plant symbioses, even in the most specialized lineages such as the Central American ant/acacia mutualism (Chapter 2). Such increased host use led to the recruitment of new ant-plant lineages by plant-nesting ants; symbiont broadening in some instances appears to have resulted in complete partner replacement (Chapter 2). Another empirical finding is that parasites (i.e., ant species benefitting from plant rewards without reciprocating) originated from free-living generalists ant species, not from mutualists evolving into cheaters as predicted by theory. Host broadening apparently also was frequent in Australasian ant-gardens and seems to have favored the evolution of domatia once plants regularly ‘find themselves’ in ant-gardens (Chapter 8). Before going to the field in Fiji, I examined the relevant collections of Australasian in several herbaria (OXF, FHO, SUVA, DUB, K, L, M, BM, P), in addition to online databases and photos from other herbaria (in particular A, GH, FI, US, BISH). I discovered three new species in Fiji, resulting in now nine species of Squamellaria in the archipelago. By generating DNA sequences from relevant type material, I enlarged the (natural, monophyletic) genus Squamellaria from three species in the last revision (Jebb, 1991) to twelve species (Chapter 3). This taxonomic framework was essential to address all subsequent questions. To answer question (iii), I performed experiments and observations during eight weeks of fieldwork in September-October 2014 and March 2015 on all nine species of Squamellaria. By using DNA and morphological traits from herbarium material, I was able to place the Squamellaria data into a much larger comparative evolutionary framework (Chapter 4). Mutualism specialization requires more investment from each partner to increase levels of rewarding and partner fidelity, which increases the exploitation potential by opportunists. I showed that obligate ant-plants negotiated this tradeoff by evolving exclusive food rewards that can only be accessed by the obligate ant mutualist (Chapter 4). To answer question (iv), I generated a phylogeny for my focal clade that includes 76 of its 102 species, including several that I discovered during my fieldwork (above). Using this phylogeny and ancestral state reconstructions, I inferred ten losses of facultative symbiosis with ants, making this system well suited to study the ecological context of mutualism breakdown. In Hydnophytinae, mutualism breakdown has been driven by shifts to montane habitats (>1500 m alt.) where ants are scarce (Chapter 6). The evolution of a key mutualistic trait – entrance hole size – tightly tracked mutualistic strategies, with obligate ant-plants undergoing little evolutionary change in hole diameter, while species that lost mutualisms were free to rapidly change this trait. This indicates that mutualistic strategies, by determining the level of stabilizing selection, drive morphological evolution in mutualism-associated traits (Chapter 6; see also Discussion). To answer question (v), I used Fijian Squamellaria to study how facultative versus obligate ant-plants are dispersed, again relying on my own field observations and experiments. Facultative ant-plants are bird-dispersed, but obligate ant-plants are dispersed by their ant symbiont, the Dolichoderinae species Philidris nagasau (Chapter 5). Obligate ant-plant species of Squamellaria and P. nagasau ants engage in a type of ant-plant mutualism that is new to science, wherein the ants farm their hosts, planting the seeds inside tree bark of preferred host tree species and fertilizing the seedlings by defecating in their tiny domatia (before these are large enough to house any ant nest) (Chapter 5). To answer question (vi), I again used the Fijian Squamellaria system and designed experiments with stable isotopes (15N) to determine how ants fertilize hosts and how nitrogen uptake differs between facultative and obligate hosts. I also used Computed-Tomography Scanning to build 3D models of ant domatia. The domatia of Squamellaria attain rugby ball to pumpkin size, and their inner structure was essentially unknown. I discovered that in the obligate symbiosis, there is a single large cavity with small (ca. 2-3 mm in diameter) hyper-absorptive structures, termed ‘warts’, that are recognized by P. nagasau ants, which exclusively defecate on them, thus maximizing nitrogen benefits to the plants (Chapter 7). By contrast, facultative hosts have several unlinked cavities and lack absorptive warts. Because there is a high ant nest turnover in the facultative ant-plant species of Squamellaria, the modular domatium limits competition between inhabitants and maximizes the time any individual plant spends with nitrogen-providing ants (Chapter 7)

Tradeoffs in the evolution of plant farming by ants

Diverse forms of cultivation have evolved across the tree of life. Efficient farming requires that the farmer deciphers and actively promotes conditions that increase crop yield. For plant cultivation, this can include evaluating tradeoffs among light, nutrients, and protection against herbivores. It is not understood if, or how, nonhuman farmers evaluate local conditions to increase payoffs. Here, we address this question using an obligate farming mutualism between the ant Philidris nagasau and epiphytic plants in the genus Squamellaria that are cultivated for their nesting sites and floral rewards. We focused on the ants’ active fertilization of their crops and their protection against herbivory. We found that ants benefited from cultivating plants in full sun, receiving 7.5-fold more floral food rewards compared to shade-cultivated plants. The higher reward levels correlated with higher levels of crop protection provided by the ants. However, while high-light planting yielded the greatest immediate food rewards, sun-grown crops contained less nitrogen compared to shade-grown crops. This was due to lower nitrogen input from ants feeding on floral rewards instead of insect protein gained from predation. Despite this tradeoff, farming ants optimize crop yield by selectively planting their crops in full sun. Ancestral state reconstructions across this ant–plant clade show that a full-sun farming strategy has existed for millions of years, suggesting that nonhuman farmers have evolved the means to evaluate and balance conflicting crop needs to their own benefit

Mutualisms drive plant trait evolution beyond interaction‐related traits

Mutualisms have driven the evolution of extraordinary structures and behavioural traits, but their impact on traits beyond those directly involved in the interaction remains unclear. We addressed this gap using a highly evolutionarily replicated system – epiphytes in the Rubiaceae forming symbioses with ants. We employed models that allow us to test the influence of discrete mutualistic traits on continuous non‐mutualistic traits. Our findings are consistent with mutualism shaping the pace of morphological evolution, strength of selection and long‐term mean of non‐mutualistic traits in function of mutualistic dependency. While specialised and obligate mutualisms are associated with slower trait change, less intimate, facultative and generalist mutualistic interactions – which are the most common – have a greater impact on non‐mutualistic trait evolution. These results challenge the prevailing notion that mutualisms solely affect the evolution of interaction‐related traits via stabilizing selection and instead demonstrate a broader role for mutualisms in shaping trait evolution

Convergence in carnivorous pitcher plants reveals a mechanism for composite trait evolution.

Composite traits involve multiple components that, only when combined, gain a new synergistic function. Thus, how they evolve remains a puzzle. We combined field experiments, microscopy, chemical analyses, and laser Doppler vibrometry with comparative phylogenetic analyses to show that two carnivorous pitcher plant species independently evolved similar adaptations in three distinct traits to acquire a new, composite trapping mechanism. Comparative analyses suggest that this new trait arose convergently through "spontaneous coincidence" of the required trait combination, rather than directional selection in the component traits. Our results indicate a plausible mechanism for composite trait evolution and highlight the importance of stochastic phenotypic variation as a facilitator of evolutionary novelty

Recent origin and rapid speciation of Neotropical orchids in the world's richest plant biodiversity hotspot

The Andean mountains of South America are the most species-rich biodiversity hotspot worldwide with c. 15% of the world's plant species, in only 1% of the world's land surface. Orchids are a key element of the Andean flora, and one of the most prominent components of the Neotropical epiphyte diversity, yet very little is known about their origin and diversification. We address this knowledge gap by inferring the biogeographical history and diversification dynamics of the two largest Neotropical orchid groups (Cymbidieae and Pleurothallidinae), using two unparalleled, densely sampled orchid phylogenies (including more than 400 newly generated DNA sequences), comparative phylogenetic methods, geological and biological datasets. We find that the majority of Andean orchid lineages only originated in the last 20–15 million yr. Andean lineages are derived from lowland Amazonian ancestors, with additional contributions from Central America and the Antilles. Species diversification is correlated with Andean orogeny, and multiple migrations and recolonizations across the Andes indicate that mountains do not constrain orchid dispersal over long timescales. Our study sheds new light on the timing and geography of a major Neotropical diversification, and suggests that mountain uplift promotes species diversification across all elevational zones.O.A.P-E. is supported by a Colombian National Science Foundation (COLCIENCIAS) scholarship and G.C. is supported by a German Science Foundation grant (RE 603/20). F.L.C. is supported by a Marie Curie grant (BIOMME project, IOF627684) and has benefited from an ‘Investissements d’Avenir’ grant managed by Agence Nationale de la Recherche (CEBA, ref. ANR-10-LABX-25-01). A.P.K. and D.B. were supported by grants from the Alberta Mennega Foundation. N.J.M. was supported by the National Institute for Mathematical and Biological Synthesis, an Institute sponsored by the National Science Foundation (NSF) through NSF Award no. EFJ0832858, with additional support from The University of Tennessee, Knoxville, and is currently supported by a Discovery Early Career Researcher Award DE150101773, funded by the Australian Research Council, and by The Australian National University. D.S. is funded by the Swedish Research Council (2015-04748). A.A. is supported by grants from the Swedish Research Council, the European Research Council under the European Union’s Seventh Framework Program (FP/2007-2013, ERC Grant Agreement no. 331024), the Swedish Foundation for Strategic Research and a Wallenberg Academy Fellowship

The origin and speciation of orchids

SummaryOrchids constitute one of the most spectacular radiations of flowering plants. However, their origin, spread across the globe, and hotspots of speciation remain uncertain due to the lack of an up-to-date phylogeographic analysis.We present a new Orchidaceae phylogeny based on combined high-throughput and Sanger sequencing data, covering all five subfamilies, 17/22 tribes, 40/49 subtribes, 285/736 genera, and c. 7% (1921) of the 29 524 accepted species, and use it to infer geographic range evolution, diversity, and speciation patterns by adding curated geographical distributions from the World Checklist of Vascular Plants.The orchids' most recent common ancestor is inferred to have lived in Late Cretaceous Laurasia. The modern range of Apostasioideae, which comprises two genera with 16 species from India to northern Australia, is interpreted as relictual, similar to that of numerous other groups that went extinct at higher latitudes following the global climate cooling during the Oligocene. Despite their ancient origin, modern orchid species diversity mainly originated over the last 5 Ma, with the highest speciation rates in Panama and Costa Rica.These results alter our understanding of the geographic origin of orchids, previously proposed as Australian, and pinpoint Central America as a region of recent, explosive speciation