The coevolution of fungus-ant agriculture.

Fungus-farming ants cultivate multiple lineages of fungi for food, but, because fungal cultivar relationships are largely unresolved, the history of fungus-ant coevolution remains poorly known. We designed probes targeting >2000 gene regions to generate a dated evolutionary tree for 475 fungi and combined it with a similarly generated tree for 276 ants. We found that fungus-ant agriculture originated ~66 million years ago when the end-of-Cretaceous asteroid impact temporarily interrupted photosynthesis, causing global mass extinctions but favoring the proliferation of fungi. Subsequently, ~27 million years ago, one ancestral fungal cultivar population became domesticated, i.e., obligately mutualistic, when seasonally dry habitats expanded in South America, likely isolating the cultivar population from its free-living, wet forest-dwelling conspecifics. By revealing these and other major transitions in fungus-ant coevolution, our results clarify the historical processes that shaped a model system for nonhuman agriculture.

Genomic Signatures of Domestication in a Fungus Obligately Farmed by Leafcutter Ants.

The naturally selected fungal crop (Leucoagaricus gongylophorus) farmed by leafcutter ants shows striking parallels with artificially selected plant crops domesticated by humans (e.g., polyploidy, engorged nutritional rewards, dependence on cultivation). To date, poorly resolved L. gongylophorus genome assemblies based on short-read sequencing have constrained hypotheses about how millions of years under cultivation by ants shaped the fungal crop genome and potentially drove domestication. We use PacBio HiFi sequencing of L. gongylophorus from the leafcutter ant Atta colombica to identify 18 putatively novel biosynthetic gene clusters that likely cemented life as a cultivar (e.g., plant fragment degradation, ant-farmer communication, antimicrobial defense). Comparative analyses with cultivated and free-living fungi showed genomic signatures of stepwise domestication transitions: 1) free-living to ant-cultivated: loss of genes conferring stress response and detoxification, 2) hyphal food to engorged nutritional rewards: expansions of genes governing cellular homeostasis, carbohydrate metabolism, and siderophore biosynthesis, and 3) detrital provisioning to freshly cut plant fragments: gene expansions promoting cell wall biosynthesis, fatty acid metabolism, and DNA repair. Comparisons across L. gongylophorus fungi farmed by three leafcutter ant species highlight genomic signatures of exclusively vertical clonal propagation and widespread transposable element activity. These results show how natural selection can shape domesticated cultivar genomes towards long-term ecological resilience of farming systems that have thrived across millennia.

Distinct and enhanced hygienic responses of a leaf-cutting ant toward repeated fungi exposures.

Leaf-cutting ants and their fungal crops are a textbook example of a long-term obligatory mutualism. Many microbes continuously enter their nest containing the fungal cultivars, destabilizing the symbiosis and, in some cases, outcompeting the mutualistic partners. Preferably, the ant workers should distinguish between different microorganisms to respond according to their threat level and recurrence in the colony. To address these assumptions, we investigated how workers of Atta sexdens sanitize their fungal crop toward five different fungi commonly isolated from the fungus gardens: Escovopsis sp., Fusarium oxysporum, Metarhizium anisopliae, Trichoderma spirale, and Syncephalastrum sp. Also, to investigate the plasticity of these responses toward recurrences of these fungi, we exposed the colonies with each fungus three times fourteen days apart. As expected, intensities in sanitization differed according to the fungal species. Ants significantly groom their fungal crop more toward F. oxysporum, M. anisopliae, and Syncephalastrum sp. than toward Escovopsis sp. and T. spirale. Weeding, self-, and allogrooming were observed in less frequency than fungus grooming in all cases. Moreover, we detected a significant increase in the overall responses after repeated exposures for each fungus, except for Escovopsis sp. Our results indicate that A. sexdens workers are able to distinguish between different fungi and apply distinct responses to remove these from the fungus gardens. Our findings also suggest that successive exposures to the same antagonist increase hygiene, indicating plasticity of ant colonies' defenses to previously encountered pathogens.

Climate Change Influences Basidiome Emergence of Leaf-Cutting Ant Cultivars.

Maintaining symbiosis homeostasis is essential for mutualistic partners. Leaf-cutting ants evolved a long-term symbiotic mutualism with fungal cultivars for nourishment while using vertical asexual transmission across generations. Despite the ants' efforts to suppress fungal sexual reproduction, scattered occurrences of cultivar basidiomes have been reported. Here, we review the literature for basidiome occurrences and associated climate data. We hypothesized that more basidiome events could be expected in scenarios with an increase in temperature and precipitation. Our field observations and climate data analyses indeed suggest that Acromyrmex coronatus colonies are prone to basidiome occurrences in warmer and wetter seasons. Even though our study partly depended on historical records, occurrences have increased, correlating with climate change. A nest architecture with low (or even the lack of) insulation might be the cause of this phenomenon. The nature of basidiome occurrences in the A. coronatus-fungus mutualism can be useful to elucidate how resilient mutualistic symbioses are in light of climate change scenarios.

Relationships between resource availability and elevation vary between metrics creating gradients of nutritional complexity.

Plant and animal community composition changes at higher elevations on mountains. Plant and animal species richness generally declines with elevation, but the shape of the relationship differs between taxa. There are several proposed mechanisms, including the productivity hypotheses; that declines in available plant biomass confers fewer resources to consumers, thus supporting fewer species. We investigated resource availability as we ascended three aspects of Helvellyn mountain, UK, measuring several plant nutritive metrics, plant species richness and biomass. We observed a linear decline in plant species richness as we ascended the mountain but there was a unimodal relationship between plant biomass and elevation. Generally, the highest biomass values at mid-elevations were associated with the lowest nutritive values, except mineral contents which declined with elevation. Intra-specific and inter-specific increases in nutritive values nearer the top and bottom of the mountain indicated that physiological, phenological and compositional mechanisms may have played a role. The shape of the relationship between resource availability and elevation was different depending on the metric. Many consumers actively select or avoid plants based on their nutritive values and the abundances of consumer taxa vary in their relationships with elevation. Consideration of multiple nutritive metrics and of the nutritional requirements of the consumer may provide a greater understanding of changes to plant and animal communities at higher elevations. We propose a novel hypothesis for explaining elevational diversity gradients, which warrants further study; the 'nutritional complexity hypothesis', where consumer species coexist due to greater variation in the nutritional chemistry of plants.

Nutritional niches reveal fundamental domestication trade-offs in fungus-farming ants.

During crop domestication, human farmers traded greater productivity for higher crop vulnerability outside specialized cultivation conditions. We found a similar domestication trade-off across the major co-evolutionary transitions in the farming systems of attine ants. First, the fundamental nutritional niches of cultivars narrowed over ~60 million years of naturally selected domestication, and laboratory experiments showed that ant farmers representing subsequent domestication stages strictly regulate protein harvest relative to cultivar fundamental nutritional niches. Second, ants with different farming systems differed in their abilities to harvest the resources that best matched the nutritional needs of their fungal cultivars. This was assessed by quantifying realized nutritional niches from analyses of items collected from the mandibles of laden ant foragers in the field. Third, extensive field collections suggest that among-colony genetic diversity of cultivars in small-scale farms may offer population-wide resilience benefits that species with large-scale farming colonies achieve by more elaborate and demanding practices to cultivate less diverse crops. Our results underscore that naturally selected farming systems have the potential to shed light on nutritional trade-offs that shaped the course of culturally evolved human farming.

Genome Size Versus Genome Assemblies: Are the Genomes Truly Expanded in Polyploid Fungal Symbionts?

Each day, as the amount of genomic data and bioinformatics resources grows, researchers are increasingly challenged with selecting the most appropriate approach to analyze their data. In addition, the opportunity to undertake comparative genomic analyses is growing rapidly. This is especially true for fungi due to their small genome sizes (i.e., mean 1C = 44.2 Mb). Given these opportunities and aiming to gain novel insights into the evolution of mutualisms, we focus on comparing the quality of whole genome assemblies for fungus-growing ants cultivars (Hymenoptera: Formicidae: Attini) and a free-living relative. Our analyses reveal that currently available methodologies and pipelines for analyzing whole-genome sequence data need refining. By using different genome assemblers, we show that the genome assembly size depends on what software is used. This, in turn, impacts gene number predictions, with higher gene numbers correlating positively with genome assembly size. Furthermore, the majority of fungal genome size data currently available are based on estimates derived from whole-genome assemblies generated from short-read genome data, rather than from the more accurate technique of flow cytometry. Here, we estimated the haploid genome sizes of three ant fungal symbionts by flow cytometry using the fungus Pleurotus ostreatus (Jacq.) P. Kumm. (1871) as a calibration standard. We found that published genome sizes based on genome assemblies are 2.5- to 3-fold larger than our estimates based on flow cytometry. We, therefore, recommend that flow cytometry is used to precalibrate genome assembly pipelines, to avoid incorrect estimates of genome sizes and ensure robust assemblies.

Cryptic Diversity in Colombian Edible Leaf-Cutting Ants (Hymenoptera: Formicidae).

Leaf-cutting ants are often considered agricultural pests, but they can also benefit local people and serve important roles in ecosystems. Throughout their distribution, winged reproductive queens of leaf-cutting ants in the genus Atta Fabricius, 1804 are consumed as a protein-rich food source and sometimes used for medical purposes. Little is known, however, about the species identity of collected ants and the accuracy of identification when ants are sold, ambiguities that may impact the conservation status of Atta species as well as the nutritional value that they provide to consumers. Here, 21 samples of fried ants bought in San Gil, Colombia, were identified to species level using Cytochrome Oxidase I (COI) barcoding sequences. DNA was extracted from these fried samples using standard Chelex extraction methods, followed by phylogenetic analyses with an additional 52 new sequences from wild ant colonies collected in Panama and 251 publicly available sequences. Most analysed samples corresponded to Atta laevigata (Smith, 1858), even though one sample was identified as Atta colombica Guérin-Méneville, 1844 and another one formed a distinct branch on its own, more closely related to Atta texana (Buckley, 1860) and Atta mexicana (Smith, 1858). Analyses further confirm paraphyly within Atta sexdens (Linnaeus, 1758) and A. laevigata clades. Further research is needed to assess the nutritional value of the different species.

New species of Cortinarius sect. Austroamericani, sect. nov., from South American Nothofagaceae forests.

In this study, we document and describe the new Cortinarius section Austroamericani. Our results reveal high species diversity within this clade, with a total of 12 recognized species. Of these, only C. rufus was previously documented. Seven species are described as new based on basidiomata collections. The four remaining species are only known from environmental sequences. All examined species form ectomycorrhizal associations with species of Nothofagaceae and are currently only known from Argentinean and Chilean Patagonia. The phylogenetic analysis based on the nuc rDNA internal transcriber spacer (ITS1-5.8S-ITS2 = ITS) and partial 28S gene (28S) sequences shows that this section is related to other taxa from the Southern Hemisphere. Species in this group do not belong to subg. Telamonia, where C. rufus was initially placed. Cortinarius rufus and the newly described C. subrufus form a basal clade within sect. Austroamericani that has a weakly supported relationship with the core clade. Because the two species are morphologically similar to species from the core clade and share their distribution and Nothofagaceae associations, we include them here as part of sect. Austroamericani sensu lato (s.l.) until more material is available to refine the delimitation.

Myrteae phylogeny, calibration, biogeography and diversification patterns: Increased understanding in the most species rich tribe of Myrtaceae.

Myrteae (c. 2500 species; 51 genera) is the largest tribe of Myrtaceae and an ecologically important groups of angiosperms in the Neotropics. Systematic relationships in Myrteae are complex, hindering conservation initiatives and jeopardizing evolutionary modelling. A well-supported and robust phylogenetic hypothesis was here targeted towards a comprehensive understanding of the relationships within the tribe. The resultant topology was used as a base for key evolutionary analyses such as age estimation, historical biogeography and diversification rate patterns. One nuclear (ITS) and seven chloroplast (psbA-trnH, matK, ndhF, trnl-trnF, trnQ-rps16, rpl16 and rpl32-trnL) DNA regions for 115 taxa representing 46 out of the 51 genera in the tribe were accessed and analysed using maximum likelihood and Bayesian inference tools for phylogenetic reconstruction. Dates of diversification events were estimated and contrasted using two distinct fossil sets (macro and pollen) in BEAST. The subsequent dated phylogenies were compared and analysed for biogeographical patterns using BioGeoBEARS and diversification rates using BAMM. Myrteae phylogeny presents strong statistical support for three major clades within the tribe: Australasian group, Myrtus group and Main Neotropical Lineage. Dating results from calibration using macrofossil are an average of 20 million years older and show an early Paleocene origin of Myrteae, against a mid-Eocene one from the pollen fossil calibration. Biogeographic analysis shows the origin of Myrteae in Zealandia in both calibration approaches, followed by a widespread distribution throughout the still-linked Gondwana continents and diversification of Neotropical endemic lineages by later vicariance. Best configuration shift indicates three points of acceleration in diversification rates, all of them occurring in the Main Neotropical Lineage. Based on the reconstructed topology, several new taxonomic placements were recovered, including: the relative position of Myrtus communis, the placement of the Blepharocalyx group, the absence of generic endemism in the Caribbean, and the paraphyletism of the former Pimenta group. Distinct calibration approaches affect biogeography interpretation, increasing the number of necessary long distance dispersal events in the topology with older nodes. It is hypothesised that biological intrinsic factors such as modifications of embryo type and polyploidy might have played a role in accelerating shifts of diversification rates in Neotropical lineages. Future perspectives include formal subtribal classification, standardization of fossil calibration approaches and better links between diversification shifts and trait evolution.