The molecular phylogenetics of Trachymyrmex Forel ants and their fungal cultivars provide insights into the origin and coevolutionary history of ‘higher‐attine’ ant agriculture

The fungus‐growing ants and their fungal cultivars constitute a classic example of a mutualism that has led to complex coevolutionary dynamics spanning c. 55–65 Ma. Of the five agricultural systems practised by fungus‐growing ants, higher‐attine agriculture, of which leaf‐cutter agriculture is a derived subset, remains poorly understood despite its relevance to ecosystem function and human agriculture across the Neotropics and parts of North America. Among the ants practising higher‐attine agriculture, the genus Trachymyrmex Forel, as currently defined, shares most‐recent common ancestors with both the leaf‐cutter ants and the higher‐attine genera Sericomyrmex Mayr and Xerolitor Sosa‐Calvo et al. Although previous molecular‐phylogenetic studies have suggested that Trachymyrmex is a paraphyletic grade, until now insufficient taxon sampling has prevented a full investigation of the evolutionary history of this group and limited the possibility of resolving its taxonomy. Here we describe the results of phylogenetic analyses of 38 Trachymyrmex species, including 27 of the 49 described species and at least 11 new species, using four nuclear markers, as well as phylogenetic analyses of the fungi cultivated by 23 species of Trachymyrmex using two markers. We generated new genetic data for 112 ants (402 new gene sequences) and 95 fungi (153 new gene sequences). Our results corroborate previous findings that Trachymyrmex, as currently defined, is paraphyletic. We propose recognizing two new genera, Mycetomoellerius gen.n. and Paratrachymyrmex gen.n. , and restricting the continued use of Trachymyrmex to the clade of nine largely North American species that contains the type species [Trachymyrmex septentrionalis (McCook)] and that is the sister group of the leaf‐cutting ants. Our fungal cultivar phylogeny generally corroborates previously observed broad patterns of ant–fungus association, but it also reveals further violations of those patterns. Higher‐attine fungi are divided into two groups: (i) the single species Leucoagaricus gongylophorus (Möller); and (ii) its sister clade, consisting of multiple species, recently referred to as Leucoagaricus Singer ‘clade B’. Our phylogeny indicates that, although most non‐leaf‐cutting higher‐attine ants typically cultivate species in clade B, some species cultivate L. gongylophorus, whereas still others cultivate fungi typically associated with lower‐attine agriculture. This indicates that the attine agricultural systems, which are currently defined by associations between ants and fungi, are not entirely congruent with ant and fungal phylogenies. They may, however, be correlated with as yet poorly understood biological traits of the ants and/or of their microbiomes.     

Alarm pheromones in the genus Manica derived from the mandibular gland

The alarm pheromones present in the mandibular glands of Manica mutica and M. bradleyi are dominated by a novel C₁₀ ketone, 4,6-dimethyl-4-octene-3-one (manicone). Two other new insect pheromones, 4-methyl-3-hexanone and 3-decanone, are also present. In addition, two characteristic myrmicine alarm pheromones, 3-octanone and 4-methyl-3-heptanone, have been identified as mandibular gland constituents. While manicone functions as a powerful releaser of alarm behaviour for Manica workers a much weaker response was obtained to the other identified 3-alkanones. The significance of the occurrence of 3-ketones in members of the genus Manica and species in other genera of the Myrmicinae is analysed in terms of the accepted phylogeny of this subfamily.     

Phylogenetic patterns of ant–fungus associations indicate that farming strategies,not only a superior fungal cultivar,explain the ecological success of leafcutter ants

To elucidate fungicultural specializations contributing to ecological dominance of leafcutter ants, we estimate the phylogeny of fungi cultivated by fungus‐growing (attine) ants, including fungal cultivars from (i) the entire leafcutter range from southern South America to southern North America, (ii) all higher‐attine ant lineages (leafcutting genera Atta, Acromyrmex; nonleafcutting genera Trachymyrmex, Sericomyrmex) and (iii) all lower‐attine lineages. Higher‐attine fungi form two clades, Clade‐A fungi (Leucocoprinus gongylophorus, formerly Attamyces) previously thought to be cultivated only by leafcutter ants, and a sister clade, Clade‐B fungi, previously thought to be cultivated only by Trachymyrmex and Sericomyrmex ants. Contradicting this traditional view, we find that (i) leafcutter ants are not specialized to cultivate only Clade‐A fungi because some leafcutter species ranging across South America cultivate Clade‐B fungi; (ii) Trachymyrmex ants are not specialized to cultivate only Clade‐B fungi because some Trachymyrmex species cultivate Clade‐A fungi and other Trachymyrmex species cultivate fungi known so far only from lower‐attine ants; (iii) in some locations, single higher‐attine ant species or closely related cryptic species cultivate both Clade‐A and Clade‐B fungi; and (iv) ant–fungus co‐evolution among higher‐attine mutualisms is therefore less specialized than previously thought. Sympatric leafcutter ants can be ecologically dominant when cultivating either Clade‐A or Clade‐B fungi, sustaining with either cultivar‐type huge nests that command large foraging territories; conversely, sympatric Trachymyrmex ants cultivating either Clade‐A or Clade‐B fungi can be locally abundant without achieving the ecological dominance of leafcutter ants. Ecological dominance of leafcutter ants therefore does not depend primarily on specialized fungiculture of L. gongylophorus (Clade‐A), but must derive from ant–fungus synergisms and unique ant adaptations.     

Evolution of Substrate Preparation Behaviors for Cultivation of Symbiotic Fungus in Attine Ants (Hymenoptera: Formicidae)

Despite their importance for the evolution of the symbiosis between Attine ants and their fungal cultivar, substrate preparation behaviors have been the focus of few studies. This study aimed to comparatively examining these behaviors in Acromyrmex disciger, Apterostigma pilosum, Mycetarotes parallelus, Myrmicocrypta sp., Trachymyrmex fuscus and Trachymyrmex sp. Nov. to describe the patterns of their evolution. Behavioral observations were carried out with a set of micro cameras and the behavioral frequencies were analyzed by principal components. Our findings revealed that the process can be divided into three parts: physical treatment, chemical treatment, and incorporation. Two behavioral patterns were revealed. The first is exhibited by basal species (Myrmicocrypta sp, A. pilosum and M. parallelus) and is characterized by the absence or low frequency of chemical treatment behaviors, while the second pattern is exhibited by derived species (Trachymyrmex sp. Nov., T. fuscus and A. disciger) and is characterized by great fragmentation of the substrate and deposit of fecal fluid. This suggests that the evolution of the process is marked by an increase in the importance of the chemical treatment, leading to the adaptations observed in leaf-cutting ants.     

A chemotaxonomic survey of the tribe psoraleeae in africa

A study of the leaf flavonoids, furanocoumarins and essential oils of 51 species (three genera) of the tribe Psoraleeae (Fabaceae) has provided chemical data to support the recent subdivision of the large worldwide genus Psoralea into a number of segregate genera. The flavonoid patterns in most species were very similar, consisting largely of proanthocyanidins and complex mixtures of flavone O- and C-glucosides. Psoralea repens, the only maritime species to be examined, is unique in containing only rutin. Three isoflavones (daidzein, formononetin and genistein) were of widespread occurrence. The genera Psoralea and Otholobium, which could not be separated on flavonoid data, were clearly demarcated by their essential oils. Furanocoumarin patterns were useful for the characterization of species.     

Reduced biological control and enhanced chemical pest management in the evolution of fungus farming in ants

To combat disease, most fungus-growing ants (Attini) use antibiotics from mutualistic bacteria (Pseudonocardia) that are cultured on the ants'' exoskeletons and chemical cocktails from exocrine glands, especially the metapleural glands (MG). Previous work has hypothesized that (i) Pseudonocardia antibiotics are narrow-spectrum and control a fungus (Escovopsis) that parasitizes the ants'' fungal symbiont, and (ii) MG secretions have broad-spectrum activity and protect ants and brood. We assessed the relative importance of these lines of defence, and their activity spectra, by scoring abundance of visible Pseudonocardia for nine species from five genera and measuring rates of MG grooming after challenging ants with disease agents of differing virulence. Atta and Sericomyrmex have lost or greatly reduced the abundance of visible bacteria. When challenged with diverse disease agents, including Escovopsis, they significantly increased MG grooming rates and expanded the range of targets. By contrast, species of Acromyrmex and Trachymyrmex maintain abundant Pseudonocardia. When challenged, these species had lower MG grooming rates, targeted primarily to brood. More elaborate MG defences and reduced reliance on mutualistic Pseudonocardia are correlated with larger colony size among attine genera, raising questions about the efficacy of managing disease in large societies with chemical cocktails versus bacterial antimicrobial metabolites.     

Sesquiterpenoids as chemosystematic markers in the subtribe Hypochaeridinae (Lactuceae,Asteraceae)

Sesquiterpene lactones are well established as chemosystematic markers in the Asteraceae family. From the Lactuceae tribe of the Asteraceae family a large number of sesquiterpene lactones – mainly of the guaiane type – have been isolated. One of the 11 subtribes of the Lactuceae recognized by Bremer is the subtribe Hypochaeridinae, which encompasses 10 genera with approximately 170 species. The present communication summarizes the sesquiterpene derivatives reported from these 10 genera, points out, which constituents are characteristic for particular groups, and discusses the occurrence of these secondary metabolites in a chemosystematic context. To this end, each of the reported sesquiterpenoids reported for the Hypochaeridinae is classified into one of three main compound classes (MCCs; eudesmane-, germacrane-, guaiane-derivatives) and into one of a number of compound classes (CCs) within these principal groups. The distribution of sesquiterpenoids belonging to these particular classes of sesquiterpenoids generally follows the currently accepted generic limits. However, the genus Helminthoteca, which is included into Picris by many authors, possesses an array of compounds implying a closer relationship to the genus Hypochaeris than to the remainder of the genus Picris. Furthermore, Leontodon subgenus Oporinia shows closer similarity in secondary metabolite patterns to the genus Picris than to Leontodon subgenus Leontodon. On the other hand Leontodon subgenus Leontodon has more chemical characters in common with the genus Hedypnois than with Leontodon subgenus Oporinia. These findings are in-line with recent results of molecular analyses, which imply that current generic limits within the Hypochaeridinae might not reflect the phylogeny of the subtribe.     

The sesquiterpenoids and their chemotaxonomic implications in Senecio L. (Asteraceae)

Sesquiterpenoids are important characteristic compounds in Asteraceae plants. These compounds have been proposed to be potential chemotaxonomic markers, but this application has not been comprehensively investigated. In this paper, sesquiterpenoids from 149 species of the genus Senecio were investigated to assess their taxonomic utility. The presence and absence were encoded as binary taxonomic characteristics and subsequently utilised in a clustering analysis. The previously biosynthesis pathway of sesquiterpenoids was taken into account to explore the relationship among these species contained different sesquiterpenoid types. In addition, the DNA phylogeny was also considered to explore the distribution and evolution of sesquiterpenoids in genus Senecio further. As a result, seven chemical major sections within the Senecio were recognised based on the chemical character. Although with several exceptions, the biosynthesis pathway, molecular phylogeny and the geographical origin of these sesquiterpenoid were found to be related to the chemical sections, which largely confirmed the validity of our chemo-classification for genus Senecio. In conclusion, we suggest that the sesquiterpenoid character in Senecio has some value for the genus taxonomy but should be analysed carefully and critically.     

UV-mediated antibiotic activity of some compositae species

Roots, leaves, and flowers of 80 species of Compositae were tested for phototoxic activity against Candida albicans. Many genera showed activity, especially in the roots. No active genera were found in the tribe Cichorieae. Phototoxic compounds were isolated from Chrysanthemum leucanthemum florets and Cirsium arvense roots. Chemotaxonomic evidence plus preliminary chemical data suggests that the compounds are polyacetylenic in nature. Unlike other phototoxic compounds, these are inactive against human skin.     

Comparative study of the mandibular gland secretion of four species of Myrmica ants

The mandibular glands of the two species of ant, Myrmica ruginodis and Myrmica sabuleti contain a similar mixture of compounds, but the proportions are different. M. sabuleti produces ethanol, propanone, methylpropanal, 3-hexanone, 3-hexanol, 3-heptanone, 3-heptanol, 3-octanone, 3-octanol, 6-methyl-3-octanone, 6-methyl-3-octanol and 3-decanone. With the exception of 3-decanone these compounds were also found in M. ruginodis. These compounds were also found in M. rubra and M. scabrinodis.In both species now studied, the mandibular gland contents attract the workers and cause a large increase in their linear speed. In M. sabuleti these behavioural activities are due to 3-octanone and 3-octanol: the attraction of these two compounds in a synthetic mixture is exactly like that of an isolated mandibular gland; the compounds act in synergy to cause an increase in the ants' linear speed. Workers of M. ruginodis specifically respond to a mixture of ethanol, 3-octanone and 3-octanol: the alcohol only moderates the ethological action of the ketone, which is a true attractant and causes a very large increase in the ants' velocity; ethanol also attracts workers, acting in this respect in synergy with 3-octanone.These chemical and behavioural results are combined with those previously reported (Cammaerts-Tricot, 1973; Morganet al., 1978) to explain the responses of M. rubra, M. ruginodis, M. sabuleti and M. scabrinodis workers to isolated mandibular glands from each of these four species.     

Heavy metal resistant strains are widespread along Streptomyces phylogeny

The genus Streptomyces comprises a group of bacteria species with high economic importance. Several of these species are employed at industrial scale for the production of useful compounds. Other characteristic found in different strains within this genus is their capability to tolerate high level of substances toxic for humans, heavy metals among them. Although several studies have been conducted in different species of the genus in order to disentangle the mechanisms associated to heavy metal resistance, little is known about how they have evolved along Streptomyces phylogeny. In this study we built the largest Streptomyces phylogeny generated up to date comprising six genes, 113 species of Streptomyces and 27 outgroups. The parsimony-based phylogenetic analysis indicated that (i) Streptomyces is monophyletic and (ii) it appears as sister clade of a group formed by Kitasatospora and Streptacidiphilus species, both genera also monophyletic. Streptomyces strains resistant to heavy metals are not confined to a single lineage but widespread along Streptomyces phylogeny. Our result in combination with genomic, physiological and biochemical data suggest that the resistance to heavy metals originated several times and by different mechanisms in Streptomyces history.     

Floral and vegetative cues in oil-secreting and non-oil-secreting Lysimachia species

Background and Aims

Unrelated plants pollinated by the same group or guild of animals typically evolve similar floral cues due to pollinator-mediated selection. Related plant species, however, may possess similar cues either as a result of pollinator-mediated selection or as a result of sharing a common ancestor that possessed the same cues or traits. In this study, visual and olfactory floral cues in Lysimachia species exhibiting different pollination strategies were analysed and compared, and the importance of pollinators and phylogeny on the evolution of these floral cues was determined. For comparison, cues of vegetative material were examined where pollinator selection would not be expected.

Methods

Floral and vegetative scents and colours in floral oil- and non-floral oil-secreting Lysimachia species were studied by chemical and spectrophotometric analyses, respectively, compared between oil- and non-oil-secreting species, and analysed by phylogenetically controlled methods.

Key Results

Vegetative and floral scent was species specific, and variability in floral but not vegetative scent was lower in oil compared with non-oil species. Overall, oil species did not differ in their floral or vegetative scent from non-oil species. However, a correlation was found between oil secretion and six floral scent constituents specific to oil species, whereas the presence of four other floral compounds can be explained by phylogeny. Four of the five analysed oil species had bee-green flowers and the pattern of occurrence of this colour correlated with oil secretion. Non-oil species had different floral colours. The colour of leaves was similar among all species studied.

Conclusions

Evidence was found for correlated evolution between secretion of floral oils and floral but not vegetative visual and olfactory cues. The cues correlating with oil secretion were probably selected by Macropis bees, the specialized pollinators of oil-secreting Lysimachia species, and may have evolved in order to attract these bees.     

Evolutionary relationships among species of Puccinia and Uromyces (Pucciniaceae,Uredinales) inferred from partial protein coding gene phylogenies

The two rust genera with the largest number of species are Puccinia Pers. ex Pers. and Uromyces (Link) Unger in the family Pucciniaceae (Uredinales). The hosts of these pathogens include representatives from almost all major angiosperm orders. Despite their ecological and economic importance, the status of Puccinia and Uromyces as distinct genera has been disputed, and little is known about relationships within and among these groups. Here we present phylogenetic analyses based on sequence data from the translation elongation factor 1α gene for over 60 species in the family Pucciniaceae. In particular, we investigate evolutionary relationships between Puccinia and Uromyces. A relatively smaller phylogeny using the beta-tubulin 1 gene was generated to test support for this phylogeny. Two main phylogenetic clades were identified and indicate at least two radiations within the Pucciniaceae. As expected neither Puccinia s. lat. nor Uromyces s. lat. are supported as monophyletic groups by either of the protein coding genes. However, both Puccinia sensu stricto (type P. graminis), and Uromyces sensu stricto (type U. appendiculatus) constitute distinct clades. In general, members of Uromyces spp. occurred scattered throughout the phylogeny suggesting that they represent more recent radiations. Several host families are found in both of the two main clades while two families, Poaceae and Cyperaceae, are separated, with one in each of the two main clades.     

Woodiness within the Spermacoceae–Knoxieae alliance (Rubiaceae): retention of the basal woody condition in Rubiaceae or recent innovation?

Background and Aims

The tribe Spermacoceae is essentially a herbaceous Rubiaceae lineage, except for some species that can be described as ‘woody’ herbs, small shrubs to treelets, or lianas. Its sister tribe Knoxieae contains a large number of herbaceous taxa, but the number of woody taxa is higher compared to Spermacoceae. The occurrence of herbaceous and woody species within the same group raises the question whether the woody taxa are derived from herbaceous taxa (i.e. secondary woodiness), or whether woodiness represents the ancestral state (i.e. primary woodiness). Microscopic observations of wood anatomy are combined with an independent molecular phylogeny to answer this question.

Methods

Observations of wood anatomy of 21 woody Spermacoceae and eight woody Knoxieae species, most of them included in a multi-gene molecular phylogeny, are carried out using light microscopy.

Key Results

Observations of wood anatomy in Spermacoceae support the molecular hypothesis that all the woody species examined are secondary derived. Well-known wood anatomical characters that demonstrate this shift from the herbaceous to the woody habit are the typically flat or decreasing length vs. age curves for vessel elements, the abundance of square and upright ray cells, or even the (near-) absence of rays. These so-called paedomorphic wood features are also present in the Knoxieae genera Otiophora, Otomeria, Pentas, Pentanisia and Phyllopentas. However, the wood structure of the other Knoxieae genera observed (Carphalea, Dirichletia and Triainolepis) is typical of primarily woody taxa.

Conclusions

In Spermacoceae, secondary woodiness has evolved numerous times in strikingly different habitats. In Knoxieae, there is a general trend from primary woodiness towards herbaceousness and back to (secondary) woodiness.Key words: Knoxieae, LM, primary woodiness, Rubiaceae, Rubioideae, secondary woodiness, Spermacoceae, wood anatomy     

Major Clades of Australasian Rutoideae (Rutaceae) Based on rbcL and atpB Sequences

Background

Rutaceae subfamily Rutoideae (46 genera, c. 660 species) is diverse in both rainforests and sclerophyll vegetation of Australasia. Australia and New Caledonia are centres of endemism with a number of genera and species distributed disjunctly between the two regions. Our aim was to generate a high-level molecular phylogeny for the Australasian Rutoideae and identify major clades as a framework for assessing morphological and biogeographic patterns and taxonomy.

Methodology/Principal Findings

Phylogenetic analyses were based on chloroplast genes, rbcL and atpB, for 108 samples (78 new here), including 38 of 46 Australasian genera. Results were integrated with those from other molecular studies to produce a supertree for Rutaceae worldwide, including 115 of 154 genera. Australasian clades are poorly matched with existing tribal classifications, and genera Philotheca and Boronia are not monophyletic. Major sclerophyll lineages in Australia belong to two separate clades, each with an early divergence between rainforest and sclerophyll taxa. Dehiscent fruits with seeds ejected at maturity (often associated with myrmecochory) are inferred as ancestral; derived states include woody capsules with winged seeds, samaras, fleshy drupes, and retention and display of seeds in dehisced fruits (the last two states adaptations to bird dispersal, with multiple origins among rainforest genera). Patterns of relationship and levels of sequence divergence in some taxa, mostly species, with bird-dispersed (Acronychia, Sarcomelicope, Halfordia and Melicope) or winged (Flindersia) seeds are consistent with recent long-distance dispersal between Australia and New Caledonia. Other deeper Australian/New Caledonian divergences, some involving ant-dispersed taxa (e.g., Neoschmidia), suggest older vicariance.

Conclusions/Significance

This comprehensive molecular phylogeny of the Australasian Rutoideae gives a broad overview of the group’s evolutionary and biogeographic history. Deficiencies of infrafamilial classifications of Rutoideae have long been recognised, and our results provide a basis for taxonomic revision and a necessary framework for more focused studies of genera and species.     

Species richness of herbivorous insects on Nothofagus trees in South America and New Zealand: The importance of chemical attributes of the host

Several studies have evaluated the relative contribution of various host-plant attributes to the species richness of the associated insect herbivores, with and without the inclusion of the phylogeny of the host species for Northern hemisphere trees. In general these studies reached the same conclusion: tree availability (range and abundance) was a good predictor of insect species richness, although chemical attributes of the trees were not tested. The present study evaluates the relative contribution of ecological attributes of host-plant species within the Southern hemisphere genus Nothofagus, to the species richness of their associated insect fauna in South America and New Zealand. The variables included were: area of distribution (a), including longitudinal (rln) and latitudinal ranges (rlt), architectural complexity (c) and phytochemical attributes of the species, including chemical diversity (d) and chemical uniqueness (u). The analysis by independent contrasts revealed that the latitudinal range is an important factor, which explained the insect richness associated with Nothofagus for all guilds and taxonomic orders, except for the sap feeders. Compared to the non-phylogenetic analyses, including host-plant phylogeny led to the inclusion of additional variables in the regression equations. Phytochemical uniqueness of the host plants was an important factor to explain insect species richness. For example, Nothofagus alessandrii, the species with the most unique chemistry, had very few and mostly specialised herbivores, whereas Nothofagus dombeyi, with the least unique chemistry, had the highest number of related insect species. We conclude that, in addition to geographic range and phylogenetic relatedness, studies of insect herbivore diversity must also examine plant chemical composition.     

Volatile components from the mandibular glands of the turtle ants, Cephalotes alfaroi and Cephalotes cristatus

GC–MS analysis of whole head extracts from the turtle ants, Cephalotes alfaroi and Cephalotes cristatus, showed that 4-heptanone and 4-heptanol were the major volatile components in the mandibular glands. 4-Heptanone and 4-heptanol have rarely been identified in mandibular gland secretions from other ant genera. Thus, these compounds may be chemotaxonomic markers for the genus Cephalotes, since they have been identified in the mandibular glands from all members of this genus that have been investigated to date. Minor components identified in the whole head extracts of these ants were 3-methyl-1-butanol, 3-heptanone, 3-hexanol, 2- and 3-methylbutanoic acids, 2-methyl-4-heptanone, 2-phenylethanol and phenol. To our knowledge, this is the first time that 2-methyl-4-heptanone and phenol have been reported in the mandibular gland secretion from any Formicid.     

Systematic implications of the exocrine chemistry of some Hypoclinea species

The exocrine compounds produced by several species of Hypoclinea were analysed and compared to those identified in species in other genera of the ant subfamily Dolichoderinae. Two new natural products, 2-hydroxy-6-methylaceto-phenone and 2-acetoxy-6-methylacetophenone, were identified as anal gland products of three Hypoclinea species. The significance of two “forms” of H. bidens possessing completely different glandular secretions is discussed, and the relationship of the genera Hypoclinea and Dolichoderus is explored in terms of the exocrine chemistry of species in these two dolichoderine taxa.     

New furanosesquiterpenes from Eumorphia species

Eumorphia sericea and E. prostata contained several new furanosesquiterpenes and a new triterpene aldehyde. Structures were elucidated mainly by spectroscopic methods and by some chemical reactions. Structures of three compounds however could not be established with certainty and are partially based on biogenetic considerations. Gymnopentzia bifurcata also contained known furanosesquiterpenes. The number of South African genera from the tribe Anthemideae, where these furanosesquiterpenes are characteristic, has now increased to eight. The importance of these observations for the botanical position of these genera is still not clear.