Genome and cuticular hydrocarbon-based species delimitation shed light on potential drivers of speciation in a Neotropical ant species complex.

Geographic separation that leads to the evolution of reproductive isolation between populations generally is considered the most common form of speciation. However, speciation may also occur in the absence of geographic barriers due to phenotypic and genotypic factors such as chemical cue divergence, mating signal divergence, and mitonuclear conflict. Here, we performed an integrative study based on two genome-wide techniques (3RAD and ultraconserved elements) coupled with cuticular hydrocarbon (CHC) and mitochondrial (mt) DNA sequence data, to assess the species limits within the Ectatomma ruidum species complex, a widespread and conspicuous group of Neotropical ants for which heteroplasmy (i.e., presence of multiple mtDNA variants in an individual) has been recently discovered in some populations from southeast Mexico. Our analyses indicate the existence of at least five distinct species in this complex: two widely distributed across the Neotropics, and three that are restricted to southeast Mexico and that apparently have high levels of heteroplasmy. We found that species boundaries in the complex did not coincide with geographic barriers. We therefore consider possible roles of alternative drivers that may have promoted the observed patterns of speciation, including mitonuclear incompatibility, CHC differentiation, and colony structure. Our study highlights the importance of simultaneously assessing different sources of evidence to disentangle the species limits of taxa with complicated evolutionary histories.

Within-litter covariance of allele-specific MHC heterozygosity, coccidian endoparasite load and growth is modulated by sibling differences in starting mass.

Although littermates in altricial mammals usually experience highly similar environmental conditions during early life, considerable differences in growth and health can emerge among them. In a study on subadults of a European rabbit (Oryctolagus cuniculus) population with low MHC polymorphism, we tested whether litter-sibling differences in endoparasitic coccidia load and body mass at the end of the vegetation period were associated with within-litter differences in starting body mass (measured around 2 weeks prior to weaning) and in immune-genetic (MHC class II DRB) constitution. We hypothesized that siblings with a lighter starting mass might be more susceptible to endoparasite infections and thus, negative effects of a more unfavourable MHC constitution might be particularly pronounced in such individuals. Within-litter comparisons revealed that animals with a lighter starting mass reached a relatively lower body mass in autumn. Furthermore, there were indications for an allele-specific heterozygote advantage, as animals with heterozygous combinations of the allele Orcu-DRB*4 had relatively lower hepatic coccidia loads than their littermates with certain homozygous allele combinations. Consistent with our hypothesis, significantly higher hepatic coccidia loads and tendentially lower autumn body masses in homozygous compared to heterozygous individuals for the allele Orcu-DRB*4 were evident in initially lighter but not in heavier siblings, suggesting synergistic effects between an unfavourable MHC constitution and a light starting mass. Taken together, these effects might lead to notable differences in fitness among litter siblings, as a low body mass and a high endoparasite burden are key factors limiting young rabbits' survival during winter.

Context dependent life-history shift in Macrodinychus sellnicki mites attacking a native ant host in Colombia.

Ant parasitoidism has been reported in seven of the 26 recognized species of the mite genus Macrodinychus (Machrodynichidae). Macrodynichus sellnicki, previously reported as a parasitoid of the invasive ant Nylanderia fulva in Colombia, is now reported, in the same region, as attacking a native host, Ectatomma sp. 2 (E. ruidum complex). The mite develops within the protective silk cocoon of an Ectatomma pupa and waits for the emergence of the young ant before leaving the cocoon, unmolested. Overall nest prevalence was relatively high (34.6% of the 52 nests containing cocoons) but pupae prevalence was low (4.0%, n = 1401 cocoons). Mite life-history (parasite or parasitoid) was context dependent, shifting according to the intensity of the attack on a same host. Contrary to the strictly parasitoidic association of M. sellnicki with N. fulva, single mite attacks against E. ruidum did not result in host killing and solitary M. sellnicki (78.6% of the cases) behaved as parasites. However, in 21.4% of the attacks (0.9% of all available host pupae) more than one mite was involved and behaved as parasitoids, draining the host of its internal fluids and killing it. This is the first association of a macrodinychid mite with a species of the subfamily Ectatomminae, and the first ant associated mite for which such a context dependent life-style shift is described.

Late Pleistocene genetic diversification and demographic expansion in the widely distributed neotropical ant Neoponera villosa (Ponerinae).

Phylogeographic studies of continent-wide distributed species are key to understand population dynamics processes that occurred at large geographical scales. Here, we examined two mitochondrial (mt) DNA sequence (COI, Cyt b) and eight nuclear microsatellites markers to investigate the cohesiveness, genetic diversity and demographic history of Neoponera villosa (Fabricius), a ponerine ant species widely distributed along most part of the Neotropics and southern Nearctic. The reconstructed phylogeny and mt variation supported the cohesiveness of the examined populations of N. villosa. The species probably originated in South America during the late Pliocene/middle Pleistocene and subsequently dispersed to Central America and the Transitional Nearctic-Neotropical zone during the late Pleistocene, with an increase in its population size ca. 30 thousand years ago. The limited phylogeographic structure observed in N. villosa supports its late Pleistocene range expansion and gene flow among distant geographic areas in central and southern Mexico and Central America.

Extensive mitochondrial heteroplasmy in the neotropical ants of the Ectatomma ruidum complex (Formicidae: Ectatomminae).

We assembled mitogenomes from 21 ant workers assigned to four morphospecies (E. ruidum spp. 1-4) and putative hybrids of the Ectatomma ruidum complex (E. ruidum spp. 2x3), and to E. tuberculatum using NGS data. Mitogenomes from specimens of E. ruidum spp. 3, 4 and 2 × 3 had a high proportion of polymorphic sites. We investigated whether polymorphisms in mitogenomes are due to nuclear mt paralogues (numts) or due to the presence of more than one mitogenome within an individual (heteroplasmy). We did not find loss of function signals in polymorphic protein-coding genes, and observed strong evidence for purifying selection in two haplotype-phased genes, which indicate the presence of two functional mitochondrial genomes coexisting within individuals instead of numts. Heteroplasmy due to hybrid paternal leakage is not supported by phylogenetic analyses. Our results reveal the presence of a fast-evolving secondary mitochondrial lineage of uncertain origin in the E. ruidum complex.

Fertility Signaling and Partitioning of Reproduction in the Ant Neoponera apicalis.

All individuals in social insect colonies benefit from being informed about the presence and fertility state of reproducers. This allows the established reproductive individuals to maintain their reproductive monopoly without the need for physical control, and the non-reproductive individuals to make appropriate reproductive choices. Here, we studied whether fertility signaling is responsible for the partitioning of reproduction in the ant Neoponera apicalis. This species forms small colonies from one single-mated queen, with workers establishing reproductive hierarchies when hopelessly queenless. Previous studies identified putative fertility signals, particularly the hydrocarbon 13-methylpentacosane (13-MeC25), and have shown that precise status discrimination based on these signals could be involved in the regulation of reproductive activities. Here, we extend these findings and reveal that all individuals, be they queens or workers, differ in their cuticular hydrocarbon profile according to fertility state. Proportions of 13-MeC25 were a strong predictor of an individual's ovarian activity, and could, thus, advertise the established reproducer(s) in both queenright and queenless conditions. Furthermore, this compound might play a key role in the establishment of the reproductive hierarchy, since workers with low fertility at the onset of hierarchy formation already have relatively high amounts of 13-MeC25. Dyadic encounters showed that individuals with experimentally increased amounts of 13-MeC25 triggered less agonistic interactions from top rankers, in accord with them "advertising" higher status. Thus, these bioassays supported the use of 13-MeC25 by competing ants. This simple recognition system potentially allows permanent regulation of partitioning of reproduction in this species.

Asexual queen succession in the higher termite Embiratermes neotenicus.

Asexual queen succession (AQS), in which workers, soldiers and dispersing reproductives are produced sexually while numerous non-dispersing queens arise through thelytokous parthenogenesis, has recently been described in three species of lower termites of the genus Reticulitermes. Here, we show that AQS is not an oddity restricted to a single genus of lower termites, but a more widespread strategy occurring also in the most advanced termite group, the higher termites (Termitidae). We analysed the genetic structure in 10 colonies of the Neotropical higher termite Embiratermes neotenicus (Syntermitinae) using five newly developed polymorphic microsatellite loci. The colonies contained one primary king accompanied either by a single primary queen or by up to almost 200 neotenic queens. While the workers, the soldiers and most future dispersing reproductives were produced sexually, the non-dispersing neotenic queens originated through thelytokous parthenogenesis of the founding primary queen. Surprisingly, the mode of thelytoky observed in E. neotenicus is most probably automixis with central fusion, contrasting with the automixis with terminal fusion documented in Reticulitermes. The occurrence of AQS based on different mechanisms of ploidy restoration raises the hypothesis of an independent evolutionary origin of this unique reproductive strategy in individual lineages of lower and higher termites.

Chemical signature and reproductive status in the facultatively polygynous ant Pachycondyla verenae.

In insects, cuticular hydrocarbons (CHCs) generally are used as cues and signals for within colony processes, such as signaling reproductive status, and between colony processes, such as colony membership. We examined CHC profiles of the facultatively polygynous ant Pachycondyla verenae in order to identify chemical signals of reproductive queens within colonies containing many gynes. Colonies of P. verenae, belonging to two different members of a complex of morphospecies, were collected from three geographic localities within South America. We also tested whether CHC profiles differed between geographic localities and morphospecies. We found three alkenes, two isomers of pentacosene and heptacosene, which were more abundant in CHC profiles of reproductive queens of this morphospecies complex. When we tested whether these differences were consistent across geographic localities, we found the abundance of these alkenes differed according to morphospecies, with the isomers of pentacosene being more abundant in queens from morph one, and heptacosene being more abundant in queens from morph two. Our study has given further insight into the mechanisms behind maintenance of reproductive dominance, and has demonstrated that chemical signatures associated with reproductive status in Pachycondyla verenae are not conserved within this species complex.

The role of microgynes in the reproductive strategy of the neotropical ant Ectatomma ruidum.

Miniaturized queens, microgynes, are regarded as an alternative reproductive strategy sparsely present through the ant world. The described roles of miniaturized queens include alternative short-distance dispersal morphs, an adaptation to polygyny and inquiline parasites. Some of these inquiline parasite microgynes have been described as a separate species from their host. In the poneromorph group, miniaturized queens are only reported in two Mexican populations of two Ectatomminae: Ectatomma tuberculatum, in which small queens represent an inquiline species (Ectatomma parasiticum) and Ectatomma ruidum. E. ruidum presents apparently facultative polygyny with microgynes. We used mitochondrial DNA markers and newly developed microsatellite loci to investigate the status as well as the role of microgynes in E. ruidum. We confirmed that microgynes and macrogynes are from the same species. This species is almost exclusively monogynous and monoandrous, supernumerary dealate queens of both types being actually daughters of the mother queen. An apparently polygynous nest was more often headed by a macrogyne than a microgyne. We didn't find any inbreeding or isolation by distance in the studied population, indicating that new gynes are inseminated by unrelated males and can establish a new nest far from their natal nest. However, re-adoption of daughter queens seems to be the rule and rate of microgyny appears to be linked to nest density and environmental factors.

Stridulations reveal cryptic speciation in neotropical sympatric ants.

The taxonomic challenge posed by cryptic species underlines the importance of using multiple criteria in species delimitation. In the current paper we tested the use of acoustic analysis as a tool to assess the real diversity in a cryptic species complex of Neotropical ants. In order to understand the potential of acoustics and to improve consistency in the conclusions by comparing different approaches, phylogenetic relationships of all the morphs considered were assessed by the analysis of a fragment of the mitochondrial DNA cytochrome b. We observed that each of the cryptic morph studied presents a morphologically distinct stridulatory organ and that all sympatric morphs produce distinctive stridulations. This is the first evidence of such a degree of specialization in the acoustic organ and signals in ants, which suggests that stridulations may be among the cues used by these ants during inter-specific interactions. Mitochondrial DNA variation corroborated the acoustic differences observed, confirming acoustics as a helpful tool to determine cryptic species in this group of ants, and possibly in stridulating ants in general. Congruent morphological, acoustic and genetic results constitute sufficient evidence to propose each morph studied here as a valid new species, suggesting that P. apicalis is a complex of at least 6 to 9 species, even if they present different levels of divergence. Finally, our results highlight that ant stridulations may be much more informative than hitherto thought, as much for ant communication as for integrative taxonomists.