Phylogenomic inference and demographic model selection suggest peripatric separation of the cryptic steppe ant species Plagiolepis pyrenaica stat. rev.

The ant Plagiolepis taurica Santschi, 1920 (Hymenoptera, Formicidae) is a typical species of the Eurasian steppes, a large grassland dominated biome that stretches continuously from Central Asia to Eastern Europe and is represented by disjunct outposts also in Central and Western Europe. The extent of this biome has been influenced by the Pleistocene climate, and steppes expanded recurrently during cold stages and contracted in warm stages. Consequently, stenotopic steppe species such as P. taurica repeatedly went through periods of demographic expansion and severe isolation. Here, we explore the impact of these dynamics on the genetic diversification within P. taurica. Delimitation of P. taurica from other Plagiolepis species has been unclear since its initial description, which raised questions on both its classification and its spatiotemporal diversification early on. We re-evaluate species limits and explore underlying mechanisms driving speciation by using an integrative approach based on genomic and morphometric data. We found large intraspecific divergence within P. taurica and resolved geographically coherent western and eastern genetic groups, which likewise differed morphologically. A morphometric survey of type material showed that Plagiolepis from the western group were more similar to P. barbara pyrenaica Emery, 1921 than to P. taurica; we thus lift the former from synonymy and establish it as separate species, P. pyrenaica stat. rev. Explicit evolutionary model testing based on genomic data supported a peripatric speciation for the species pair, probably as a consequence of steppe contraction and isolation during the mid-Pleistocene. We speculate that this scenario could be exemplary for many stenotopic steppe species, given the emphasized dynamics of Eurasian steppes.

Whole-genome analysis of multiple wood ant population pairs supports similar speciation histories, but different degrees of gene flow, across their European ranges.

The application of demographic history modelling and inference to the study of divergence between species has become a cornerstone of speciation genomics. Speciation histories are usually reconstructed by analysing single populations from each species, assuming that the inferred population history represents the actual speciation history. However, this assumption may not be met when species diverge with gene flow, for example, when secondary contact may be confined to specific geographic regions. Here, we tested whether divergence histories inferred from heterospecific populations may vary depending on their geographic locations, using the two wood ant species Formica polyctena and F. aquilonia. We performed whole-genome resequencing of 20 individuals sampled in multiple locations across the European ranges of both species. Then, we reconstructed the histories of distinct heterospecific population pairs using a coalescent-based approach. Our analyses always supported a scenario of divergence with gene flow, suggesting that divergence started in the Pleistocene (c. 500 kya) and occurred with continuous asymmetrical gene flow from F. aquilonia to F. polyctena until a recent time, when migration became negligible (2-19 kya). However, we found support for contemporary gene flow in a sympatric pair from Finland, where the species hybridise, but no signature of recent bidirectional gene flow elsewhere. Overall, our results suggest that divergence histories reconstructed from a few individuals may be applicable at the species level. Nonetheless, the geographical context of populations chosen to represent their species should be taken into account, as it may affect estimates of migration rates between species when gene flow is spatially heterogeneous.

The Gene and Gene Expression (GAGE) Species Concept: An Universal Approach for All Eukaryotic Organisms.

The Gene and Gene Expression (GAGE) species concept, a new version of the Pragmatic Species Concept of Seifert (2014), is proposed as a concept applicable to any described recent or fossil eukaryotic organism independent from its mode of reproduction or evolutionary history. In addition to presenting the concept as such, the article also provides practical recommendations for taxonomists when delimiting species and describing taxa. The wording of the new concept contains a heading core sentence plus five attached sentences addressing essential conditions for its translation into a sound taxonomic practice: "Species are separable clusters that have passed a threshold of evolutionary divergence and are exclusively defined by nuclear DNA sequences and/or their expression products. Nuclear DNA sequences and their expression products are different character systems but have a highly correlated indicative function. Character systems with the least risk of epigenetic or ontogenetic modification have superior indicative value when conflicts between character systems of integrative studies arise. All character systems have to be described by an adequate numerics allowing cluster formation and determination of thresholds. Thresholds for each character system should be fixed by consensus among the experts under the principle of avoiding oversplitting or lumping. Clusters must not be the expression of intraspecific polymorphism." Recognizing the distortions and conflicts caused to taxonomy through barcoding or through assessment on the basis of association with other organisms, the GAGE species concept strongly downgrades the use of cytoplasmic DNA of endosymbiotic origin (mtDNA, cpDNA) or DNA of closely associated microbes (e.g., Wolbachia bacteria) for final taxonomic decision-making. Recognizing the distortion of phylogenies by the high frequency of reticulate evolution, it is argued that delimiting and naming species has to be separated from constructing bifurcating phylogenetic trees. [Cytoplasmic DNA; lumping; nuclear DNA; numeric taxonomy; oversplitting; reticulate evolution.].

Segregation distortion causes large-scale differences between male and female genomes in hybrid ants.

Hybridization in isolated populations can lead either to hybrid breakdown and extinction or in some cases to speciation. The basis of hybrid breakdown lies in genetic incompatibilities between diverged genomes. In social Hymenoptera, the consequences of hybridization can differ from those in other animals because of haplodiploidy and sociality. Selection pressures differ between sexes because males are haploid and females are diploid. Furthermore, sociality and group living may allow survival of hybrid genotypes. We show that hybridization in Formica ants has resulted in a stable situation in which the males form two highly divergent gene pools whereas all the females are hybrids. This causes an exceptional situation with large-scale differences between male and female genomes. The genotype differences indicate strong transmission ratio distortion depending on offspring sex, whereby the mother transmits some alleles exclusively to her daughters and other alleles exclusively to her sons. The genetic differences between the sexes and the apparent lack of multilocus hybrid genotypes in males can be explained by recessive incompatibilities which cause the elimination of hybrid males because of their haploid genome. Alternatively, differentiation between sexes could be created by prezygotic segregation into male-forming and female-forming gametes in diploid females. Differentiation between sexes is stable and maintained throughout generations. The present study shows a unique outcome of hybridization and demonstrates that hybridization has the potential of generating evolutionary novelties in animals.

A revision of the Palaearctic species of the ant genus Cardiocondyla Emery 1869 (Hymenoptera: Formicidae).

A synopsis of the Palaearctic species of the ant genus Cardiocondyla Emery 1869 is provided. The four species groups which are of Palaearctic origin or which are restricted in their distribution to this faunal zone, the C. elegans, C. ulianini, C. batesii and C. stambuloffii group-consisting of 25 recognized species-are taxonomically revised. Further nine species belonging to species groups of Ethiopic and Oriental origin which may penetrate into and build up true outdoor populations in the southern Palaearctic are only treated in the determination key. The main working rationale of this revision is Numeric Morphology-Based Alpha-Taxonomy (NUMOBAT) with formation of species hypotheses largely based on exploratory data analyses and checking these hypotheses by discriminant analysis. NUMOBAT data of the species considered comprise 727 worker samples with 1555 individuals and 23,300 primary data. Including high-resolution photos of surface microstructures, all species are depicted by z-stack imaging in four standard visual positions. Numeric data on 19 phenotypical characters are presented in comparative tables and supplementary verbal descriptions are given. In contrast to species groups with Ethiopian, Oriental and Australasian origin, no member of the four Palaearctic species groups has developed a tramp species potential to spread globally. Four cryptic species are described as new: Cardiocondyla dalmaticoides n. sp., C. caspiense n. sp., C. verdensis n. sp. and C. rolandi n. sp. Confirmed were the synonymies of Cardiocondyla elegans santschii Forel 1905, C. provincialis Bernard 1956 and Xenometra gallica Bernard 1957 with C. elegans Emery 1869, that of C. elegans eleonorae Forel 1911 with C. bulgarica Forel 1892, that of C. elegans torretassoi Finzi 1936 with C. nigra Forel 1905, and that of C. bogdanovi Ruzsky 1905, C. montandoni Santschi 1912 and C. stambuloffii taurica Karavajev 1927 with C. stambuloffii Forel 1892. Cardiocondyla bicoronata Seifert 2003 was newly synonymized with C. nigra Forel 1905.

Broadly sympatric occurrence of two thief ant species Solenopsisfugax (Latreille, 1798) and S.juliae (Arakelian, 1991) in the East European Pontic-Caspian region (Hymenoptera, Formicidae) is disclosed.

This paper presents numeric morphology-based evidence on the broadly overlapping distribution of two thief ant species Solenopsisfugax (Latreille, 1798) and S.juliae (Arakelian, 1991) in the East European Pontic-Caspian region. The paper integrates two autonomous data collections and independent analyses performed by different researchers, using different equipment, considering different character combinations, and evaluating partially different samples. Five type series, the neotype series of Solenopsisfugax (Latreille 1798) and the type series of S.flavidula (Nylander, 1849), S. (Diplorhoptrum) fugax var. furtiva Santschi, 1934, S. (Diplorhoptrum) fugax var. pontica Santschi, 1934, S. (Diplorhoptrum) fugax var. scytica Santschi, 1934 were nested in one cluster and we propose the junior synonymy of the latter four taxa names with S.fugax. The other cluster contained only one type specimen of Solenopsisnitida (Dlussky & Radchenko, 1994) measured from AntWeb images. The naming of this cluster was based on both verbal statements and measurements of gynes given in the original description of Solenopsisjuliae (Arakelian, 1991), which represents the oldest available name for this cluster. Hence, S.nitida is proposed as junior synonym of S.juliae. Solenopsiscypridis Santschi, 1934 is raised to species rank based on investigation of worker and gyne type specimens.

Specificity and transmission mosaic of ant nest-wall fungi.

Mutualism, whereby species interact to their mutual benefit, is extraordinary in a competitive world. To recognize general patterns of origin and maintenance from the plethora of mutualistic associations proves a persisting challenge. The simplest situation is believed to be that of a single mutualist specific to a single host, vertically transmitted from one host generation to the next. We characterized ascomycete fungal associates cultured for nest architecture by the ant subgenera Dendrolasius and Chthonolasius. The ants probably manage their fungal mutualists by protecting them against fungal competitors. The ant subgenera display different ant-to-fungus specificity patterns, one-to-two and many-to-one, and we infer vertical transmission, in the latter case overlaid by horizontal transmission. Possible evolutionary trajectories include a reversal from fungiculture by other Lasius subgenera and inheritance of fungi through life cycle interactions of the ant subgenera. The mosaic indicates how specificity patterns can be shaped by an interplay between host life-cycles and transmission adaptations.

Integrative taxonomy: a multisource approach to exploring biodiversity.

Good alpha taxonomy is central to biology. On the basis of a survey of arthropod studies that used multiple disciplines for species delimitation, we evaluated the performance of single disciplines. All included disciplines had a considerable failure rate. Rigor in species delimitation can thus be increased when several disciplines chosen for complementarity are used. We present a flexible procedure and stopping rule for integrative taxonomy that uses the information from different disciplines separately. Disagreement among disciplines over the number and demarcation of species is resolved by elucidating and invoking evolutionary explanations for disagreement. With the identification of further promising study organisms and of new questions for in-depth analysis, evolutionary biology should profit from integrative taxonomy. An important rationale is clarity in researcher bias in the decision-making process. The success of integrative taxonomy will further increase through methodological progress, taxonomic training of evolutionary biologists, and balanced resource allocation.

Courtship with two spoons-Anatomy and presumed function of the bizarre antennae of Cardiocondyla zoserka ant males.

Mating in ants often occurs on the wing during nuptial flights or on the ground when scattered female sexuals attract males by pheromones. In both scenarios, there is little opportunity for males to engage in prolonged aggressive competition or elaborate courtship displays. Male morphology is therefore adapted to locating female sexuals and mating, and it lacks specific weapons or other traits associated with courtship. In contrast, sexuals of the ant genus Cardiocondyla typically mate in their natal nests. As a consequence, in many species winged males have been replaced by wingless fighter or territorial males, which kill or expel rival males with their strong mandibles and show complex mating behavior. However, no wingless males are known from Cardiocondyla zoserka from West Africa, and instead, winged males have evolved a bizarre secondary sexual trait: uniquely shaped antennae with spoon-like tips that show heavily sculptured ventral surfaces with numerous invaginations. We here report on the courtship behavior of C. zoserka males and describe antennal glands with class 3 gland cells, which presumably secrete a close range sex pheromone. Antennal glands have not yet been found in males of other ant species, including a close relative of C. zoserka, suggesting that in ants with intranidal mating sexual selection can rapidly lead to highly divergent adaptations and the evolution of novel structures.

Cytoplasmic incompatibility between Old and New World populations of a tramp ant.

Reproductive manipulation by endosymbiotic Wolbachia can cause unequal inheritance, allowing the manipulator to spread and potentially impacting evolutionary dynamics in infected hosts. Tramp and invasive species are excellent models to study the dynamics of host-Wolbachia associations because introduced populations often diverge in their microbiomes after colonizing new habitats, resulting in infection polymorphisms between native and introduced populations. Ants are the most abundant group of insects on earth, and numerous ant species are classified as highly invasive. However, little is known about the role of Wolbachia in these ecologically dominant insects. Here, we provide the first description of reproductive manipulation by Wolbachia in an ant. We show that Old and New World populations of the cosmotropic tramp ant Cardiocondyla obscurior harbor distinct Wolbachia strains, and that only the Old World strain manipulates host reproduction by causing cytoplasmic incompatibility (CI) in hybrid crosses. By uncovering a symbiont-induced mechanism of reproductive isolation in a social insect, our study provides a novel perspective on the biology of tramp ants and introduces a new system for studying the evolutionary consequences of CI.

Insect morphometry is reproducible under average investigation standards.

Morphometric research is being applied to a growing number and variety of organisms. Discoveries achieved via morphometric approaches are often considered highly transferable, in contrast to the tacit and idiosyncratic interpretation of discrete character states. The reliability of morphometric workflows in insect systematics has never been a subject of focused research, but such studies are sorely needed. In this paper, we assess the reproducibility of morphometric studies of ants where the mode of data collection is a shared routine.We compared datasets generated by eleven independent gaugers, that is, collaborators, who measured 21 continuous morphometric traits on the same pool of individuals according to the same protocol. The gaugers possessed a wide range of morphometric skills, had varying expertise among insect groups, and differed in their facility with measuring equipment. We used intraclass correlation coefficients (ICC) to calculate repeatability and reproducibility values (i.e., intra- and intergauger agreements), and we performed a multivariate permutational multivariate analysis of variance (PERMANOVA) using the Morosita index of dissimilarity with 9,999 iterations.The calculated average measure of intraclass correlation coefficients of different gaugers ranged from R = 0.784 to R = 0.9897 and a significant correlation was found between the repeatability and the morphometric skills of gaugers (p = 0.016). There was no significant association with the magnification of the equipment in the case of these rather small ants. The intergauger agreement, that is the reproducibility, varied between R = 0.872 and R = 0.471 (mean R = 0.690), but all gaugers arrived at the same two-species conclusion. A PERMANOVA test revealed no significant gauger effect on species identity (R 2 = 0.69, p = 0.58).Our findings show that morphometric studies are reproducible when observers follow the standard protocol; hence, morphometric findings are widely transferable and will remain a valuable data source for alpha taxonomy.

Escape to the high canopy-thermal deficiency causes niche expansion in a forest-floor ant.

The small formicoxenine ant Temnothorax saxonicus was known from about 40 localities in Central Europe nesting in anorganic substrates on floor of xerothermous forests whereas investigations of 198 tree canopies in 19 forest sites of the same region provided no indication for arboreal nesting or foraging. We present the first evidence for canopy-nesting populations of T. saxonicus on old Quercus trees in 3 sites having maximum calibrated topsoil temperatures of 17.9 ± 0.3 °C which were significantly (P < 0.007) lower than 22.8 ± 2.0 °C measured in 5 sites with ground-nesting populations. The thermal deficit on forest floor inhibits brood development in ground nests and caused a moving to canopy were maximum calibrated temperatures of the, now wooden, substrates are at least 26.1 °C for the whole canopy and 30.8 °C in more sun-exposed spots. T. saxonicus competed here successfully with the obligatory canopy ants T. affinis and T. corticalis. The distributional data of this rope-climbing study support former results that highest nest densities of small arboreal ants occur in temperate climate over the entire canopy mantle of single trees situated in open land or in park-like environments but occur in the top of the canopy in tree stands with high degree of canopy closure.

The Westpalaearctic Lasius paralienus complex (Hymenoptera: Formicidae) contains three species.

Application of Numeric Morphology-Based Alpha-Taxonomy (NUMOBAT) demonstrated the existence of three cryptic species within the Westpalaearctic Lasius paralienus species complex: L. paralienus Seifert, 1992, having a wider European distribution north to Sweden, L. casevitzi sp. nov., an endemic of Corsica, and Lasius bombycina sp. nov. from southeast Central Europe, the Balkans and Asia Minor. Hierarchical NC-Ward clustering and non-hierarchical NC-k-means clustering of 16 morphological characters resulted in 98.7% identical classifications within 76 examined nest samples of the three species. The classification error in 180 worker individuals was 0% in a linear discriminant analysis (LDA) and 1.3% in a LOOCV-LDA. Differential characters to other species groups and an identification key of the six European members of the Lasius alienus Förster species group are provided.

Temnothoraxcrasecundus sp. n. - a cryptic Eurocaucasian ant species (Hymenoptera, Formicidae) discovered by Nest Centroid Clustering.

The paper integrates two independent studies of numeric morphology-based alpha-taxonomy of the cryptic ant species Temnothoraxcrassispinus (Karavajev, 1926) and Temnothoraxcrasecundus sp. n. conducted by different investigators, using different equipment, considering different character combinations and evaluating different samples. Samples investigated included 603 individual workers from 203 nests - thereof 104 nest samples measured by Seifert and 99 by Csösz. The material originated from Europe, Asia Minor and Caucasia. There was a very strong interspecific overlap in any of the 29 shape characters recorded and subjective expert determination failed in many cases. Primary classification hypotheses were formed by the exploratory data analysis Nest Centroid (NC) clustering and corrected to final species hypotheses by an iterative linear discriminant analysis algorithm. The evaluation of Seifert's and Csösz's data sets arrived at fully congruent conclusions. NC-Ward and NC-K-means clustering disagreed from the final species hypothesis in only 1.9 and 1.9% of the samples in Seifert's data set and by 1.1 and 2.1% in Csösz's data set which is a strong argument for heterospecificity. The type series of Temnothoraxcrassispinus and Temnothoraxcrasecundus sp. n. were allocated to different clusters with p = 0.9851 and p = 0.9912 respectively. The type series of the junior synonym Temnothoraxslavonicus (Seifert, 1995) was allocated to the Temnothoraxcrassispinus cluster with p = 0.9927. Temnothoraxcrasecundus sp. n. and Temnothoraxcrassispinus are parapatric species with a long contact zone stretching from the Peloponnisos peninsula across Bulgaria northeast to the southern Ukraine. There is no indication for occurrence of interspecifically mixed nests or intraspecific polymorphism. However, a significant reduction of interspecific morphological distance at sites with syntopic occurrence of both species indicates local hybridization. The results are discussed within the context of the Pragmatic Species Concept of Seifert (2014). The taxonomic description and a differential diagnosis of Temnothoraxcrasecundus sp. n. are given.

Temnothorax pilagens sp. n. - a new slave-making species of the tribe Formicoxenini from North America (Hymenoptera, Formicidae).

A new species of the ant genus Temnothorax Forel, 1890 - Temnothorax pilagens sp. n. is described from eastern North America. T. pilagens sp. n. is an obligate slave-making ant with two known hosts: T. longispinosus (Roger, 1863) and T. ambiguus (Emery, 1895). A differential diagnosis against Temnothorax duloticus (Wesson, 1937), the other dulotic congener from the Nearctic, is presented and a biological characteristics of the new species is given.

Cardiocondyla pirata sp. n. - a new Philippine ant with enigmatic pigmentation pattern (Hymenoptera, Formicidae).

A new species of the ant genus Cardiocondyla Emery, 1869 - Cardiocondyla pirata sp. n. - is described from the Philippines. The species belongs to an Indo-Malayan group of six species that is characterized by workers having a strongly bilobate postpetiolar sternite and a thickset mesosoma with strongly convex dorsal profile as well as wingless, ergatoid males with sickle-shaped mandibles. The female castes show a pigmentation pattern not known from any ant worldwide. If having any adaptive value, a possible function of this structure is supposed to be visual dissolution of body shape in order to irritate predators.

Phylogenetic relationships of Palaearctic Formica species (Hymenoptera, Formicidae) based on mitochondrial cytochrome B sequences.

Ants of genus Formica demonstrate variation in social organization and represent model species for ecological, behavioral, evolutionary studies and testing theoretical implications of the kin selection theory. Subgeneric division of the Formica ants based on morphology has been questioned and remained unclear after an allozyme study on genetic differentiation between 13 species representing all subgenera was conducted. In the present study, the phylogenetic relationships within the genus were examined using mitochondrial DNA sequences of the cytochrome b and a part of the NADH dehydrogenase subunit 6. All 23 Formica species sampled in the Palaearctic clustered according to the subgeneric affiliation except F. uralensis that formed a separate phylogenetic group. Unlike Coptoformica and Formica s. str., the subgenus Serviformica did not form a tight cluster but more likely consisted of a few small clades. The genetic distances between the subgenera were around 10%, implying approximate divergence time of 5 Myr if we used the conventional insect divergence rate of 2% per Myr. Within-subgenus divergence estimates were 6.69% in Serviformica, 3.61% in Coptoformica, 1.18% in Formica s. str., which supported our previous results on relatively rapid speciation in the latter subgenus. The phylogeny inferred from DNA sequences provides a necessary framework against which the evolution of social traits can be compared. We discuss implications of inferred phylogeny for the evolution of social traits.

The evolution of invasiveness in garden ants.

It is unclear why some species become successful invaders whilst others fail, and whether invasive success depends on pre-adaptations already present in the native range or on characters evolving de-novo after introduction. Ants are among the worst invasive pests, with Lasius neglectus and its rapid spread through Europe and Asia as the most recent example of a pest ant that may become a global problem. Here, we present the first integrated study on behavior, morphology, population genetics, chemical recognition and parasite load of L. neglectus and its non-invasive sister species L. turcicus. We find that L. neglectus expresses the same supercolonial syndrome as other invasive ants, a social system that is characterized by mating without dispersal and large networks of cooperating nests rather than smaller mutually hostile colonies. We conclude that the invasive success of L. neglectus relies on a combination of parasite-release following introduction and pre-adaptations in mating system, body-size, queen number and recognition efficiency that evolved long before introduction. Our results challenge the notion that supercolonial organization is an inevitable consequence of low genetic variation for chemical recognition cues in small invasive founder populations. We infer that low variation and limited volatility in cuticular hydrocarbon profiles already existed in the native range in combination with low dispersal and a highly viscous population structure. Human transport to relatively disturbed urban areas thus became the decisive factor to induce parasite release, a well established general promoter of invasiveness in non-social animals and plants, but understudied in invasive social insects.

A multidisciplinary approach reveals cryptic diversity in Western Palearctic Tetramorium ants (Hymenoptera: Formicidae).

Diversity of ants of the Tetramorium caespitum/impurum complex was investigated in a multidisciplinary study. Focusing on morphologically hardly distinguishable Western Palearctic samples, we demonstrate the genetic and phenotypic diversity, demarcate phylogenetic entities, and discuss the clades in terms of biogeography. Sequences of 1113bp of the mitochondrial COI gene revealed 13 lineages. COII data, worker morphometry and male genitalia morphology corroborated the COI results for seven lineages; the remaining six were disregarded because of small sample size. A comparison with published data on cuticular hydrocarbons showed correspondence. The seven entities show different distribution patterns, though some ranges overlap in Central Europe. Since no major discrepancy between the results of the different disciplines became apparent, we conclude that the seven entities within the T. caespitum/impurum complex represent seven species. Geographical evidence allows the identification of T. caespitum and T. impurum, and we therefore designate neotypes and redescribe the two species in terms of morphology and mtDNA. As the revision of about 50 taxon names would go beyond the scope of this study, we refer to the remaining five species under code names. We discuss our findings in terms of plesiomorphy and convergent evolution by visualizing the mtDNA phylogeny in morphological space.

Evolution of male morphology in the ant genus Cardiocondyla.

The ant genus Cardiocondyla is characterized by a striking male polymorphism, with wingless, local fighter males (ergatoid males) with life-long spermatogenesis, and winged, peaceful disperser males with limited sperm supply. We examined the evolution of male morphology by reconstructing the phylogeny of Cardiocondyla from sequences of the mitochondrial COI/COII and 16S RNA genes from 13 of the 15 species, of which males are known. Data suggest that male polymorphism is ancestral and that winged males were lost convergently in several taxa, such as C. elegans, C. batesii, and C. mauritanica. Saber-shaped mandibles and lethal fighting among adult ergatoid males might probably have been the original condition, from which strong, shear-shaped mandibles and attacks directed predominantly against freshly eclosed, not yet sclerotized males might have evolved once. The evolution of queen number from ancestral polygyny to derived monogyny appears to be associated with a switch in the behavior of ergatoid males from fighting to mutual tolerance.