Comparative genomics and transcriptomics in ants provide new insights into the evolution and function of odorant binding and chemosensory proteins

Background

The complex societies of ants and other social insects rely on sophisticated chemical communication. Two families of small soluble proteins, the odorant binding and chemosensory proteins (OBPs and CSPs), are believed to be important in insect chemosensation. To better understand the role of these proteins in ant olfaction, we examined their evolution and expression across the ants using phylogenetics and sex- and tissue-specific RNA-seq.

Results

We find that subsets of both OBPs and CSPs are expressed in the antennae, contradicting the previous hypothesis that CSPs have replaced OBPs in ant olfaction. Both protein families have several highly conserved clades with a single ortholog in all eusocial hymenopterans, as well as clades with more dynamic evolution and many taxon-specific radiations. The dynamically evolving OBPs and CSPs have been hypothesized to function in chemical communication. Intriguingly, we find that seven members of the conserved clades are expressed specifically in the antennae of the clonal raider ant Cerapachys biroi, whereas only one dynamically evolving CSP is antenna specific. The orthologs of the conserved, antenna-specific C. biroi genes are also expressed in antennae of the ants Camponotus floridanus and Harpegnathos saltator, indicating that antenna-specific expression of these OBPs and CSPs is conserved across ants. Most members of the dynamically evolving clades in both protein families are expressed primarily in non-chemosensory tissues and thus likely do not fulfill chemosensory functions.

Conclusions

Our results identify candidate OBPs and CSPs that are likely involved in conserved aspects of ant olfaction, and suggest that OBPs and CSPs may not rapidly evolve to recognize species-specific signals.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-718) contains supplementary material, which is available to authorized users.     

Plant protein peptidase inhibitors: an evolutionary overview based on comparative genomics

Background

Peptidases are key proteins involved in essential plant physiological processes. Although protein peptidase inhibitors are essential molecules that modulate peptidase activity, their global presence in different plant species remains still unknown. Comparative genomic analyses are powerful tools to get advanced knowledge into the presence and evolution of both, peptidases and their inhibitors across the Viridiplantae kingdom.

Results

A genomic comparative analysis of peptidase inhibitors and several groups of peptidases in representative species of different plant taxonomic groups has been performed. The results point out: i) clade-specific presence is common to many families of peptidase inhibitors, being some families present in most land plants; ii) variability is a widespread feature for peptidase inhibitory families, with abundant species-specific (or clade-specific) gene family proliferations; iii) peptidases are more conserved in different plant clades, being C1A papain and S8 subtilisin families present in all species analyzed; and iv) a moderate correlation among peptidases and their inhibitors suggests that inhibitors proliferated to control both endogenous and exogenous peptidases.

Conclusions

Comparative genomics has provided valuable insights on plant peptidase inhibitor families and could explain the evolutionary reasons that lead to the current variable repertoire of peptidase inhibitors in specific plant clades.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-812) contains supplementary material, which is available to authorized users.     

Fly pollination in Ceropegia (Apocynaceae: Asclepiadoideae): biogeographic and phylogenetic perspectives

Background and Aims

Ceropegia (Apocynaceae subfamily Asclepiadoideae) is a large, Old World genus of >180 species, all of which possess distinctive flask-shaped flowers that temporarily trap pollinators. The taxonomic diversity of pollinators, biogeographic and phylogenetic patterns of pollinator exploitation, and the level of specificity of interactions were assessed in order to begin to understand the role of pollinators in promoting diversification within the genus.

Methods

Flower visitor and pollinator data for approx. 60 Ceropegia taxa were analysed with reference to the main centres of diversity of the genus and to a cpDNA–nrDNA molecular phylogeny of the genus.

Key Results

Ceropegia spp. interact with flower-visiting Diptera from at least 26 genera in 20 families, of which 11 genera and 11 families are pollinators. Size range of flies was 0·5–4·0 mm and approx. 94 % were females. Ceropegia from particular regions do not use specific fly genera or families, though Arabian Peninsula species are pollinated by a wider range of Diptera families than those in other regions. The basal-most clade interacts with the highest diversity of Diptera families and genera, largely due to one hyper-generalist taxon, C. aristolochioides subsp. deflersiana. Species in the more-derived clades interact with a smaller diversity of Diptera. Approximately 60 % of taxa are so far recorded as interacting with only a single genus of pollinators, the remaining 40 % being less conservative in their interactions. Ceropegia spp. can therefore be ecological specialists or generalists.

Conclusions

The genus Ceropegia has largely radiated without evolutionary shifts in pollinator functional specialization, maintaining its interactions with small Diptera. Intriguing biogeographic and phylogenetic patterns may reflect processes of regional dispersal, diversification and subsequent specialization onto a narrower range of pollinators, though some of the findings may be caused by inconsistent sampling. Comparisons are made with other plant genera in the Aristolochiaceae and Araceae that have evolved flask-shaped flowers that trap female flies seeking oviposition sites.Key words: Apocynaceae, Asclepiadoideae, Brachystelma, Ceropegia, Diptera, flower evolution, generalization, mutualism, pollination, Riocreuxia, specialization, Stapeliinae     

Dual African Origins of Global Aedes aegypti s.l. Populations Revealed by Mitochondrial DNA

Background

Aedes aegypti is the primary global vector to humans of yellow fever and dengue flaviviruses. Over the past 50 years, many population genetic studies have documented large genetic differences among global populations of this species. These studies initially used morphological polymorphisms, followed later by allozymes, and most recently various molecular genetic markers including microsatellites and mitochondrial markers. In particular, since 2000, fourteen publications and four unpublished datasets have used sequence data from the NADH dehydrogenase subunit 4 mitochondrial gene to compare Ae. aegypti collections and collectively 95 unique mtDNA haplotypes have been found. Phylogenetic analyses in these many studies consistently resolved two clades but no comprehensive study of mtDNA haplotypes have been made in Africa, the continent in which the species originated.

Methods and Findings

ND4 haplotypes were sequenced in 426 Ae. aegypti s.l. from Senegal, West Africa and Kenya, East Africa. In Senegal 15 and in Kenya 7 new haplotypes were discovered. When added to the 95 published haplotypes and including 6 African Aedes species as outgroups, phylogenetic analyses showed that all but one Senegal haplotype occurred in a basal clade while most East African haplotypes occurred in a second clade arising from the basal clade. Globally distributed haplotypes occurred in both clades demonstrating that populations outside Africa consist of mixtures of mosquitoes from both clades.

Conclusions

Populations of Ae. aegypti outside Africa consist of mosquitoes arising from one of two ancestral clades. One clade is basal and primarily associated with West Africa while the second arises from the first and contains primarily mosquitoes from East Africa     

Sarcophaga (Liosarcophaga) dux (Diptera: Sarcophagidae): A flesh fly species of medical importance

Background

Although tropical climate of Thailand is suitably endowed with biodiversity of insects, flies of medical importance is not well investigated. Using information from literature search, fly survey approach and specialist’s experience, we review database of Sarcophaga (Liosarcophaga) dux Thomson (Diptera: Sarcophagidae), one of the priorities flesh fly species of medical importance in Thailand.

Results

This review deals with morphology, bionomics and medical involvement. Important morphological characteristics of egg, larva, puparia and adult were highlighted with illustration and/or micrographs. Search pertaining to molecular analysis used for fly identification and developmental rate of larvae were included. Medical involvement of larvae was not only myiasis-producing agent in humans and animals, but associated with human death investigations.

Conclusions

This information will enable us to accurate identify this species and to emphasis the increase medically important scene in Thailand.     

Stress resistance and longevity are not directly linked to levels of enzymatic antioxidants in the ponerine ant Harpegnathos saltator

Background

The molecular mechanisms of variations in individual longevity are not well understood, even though longevity can be increased substantially by means of diverse experimental manipulations. One of the factors supposed to be involved in the increase of longevity is a higher stress resistance. To test this hypothesis in a natural system, eusocial insects such as bees or ants are ideally suited. In contrast to most other eusocial insects, ponerine ants show a peculiar life history that comprises the possibility to switch during adult life from a normal worker to a reproductive gamergate, therewith increasing their life expectancy significantly.

Results

We show that increased resistance against major stressors, such as reactive oxygen species and infection accompanies the switch from a life-history trait with normal lifespan to one with a longer life expectancy. A short period of social isolation was sufficient to enhance stress resistance of workers from the ponerine ant species Harpegnathos saltator significantly. All ant groups with increased stress resistances (reproducing gamergates and socially isolated workers) have lower catalase activities and glutathione levels than normal workers. Therewith, these ants resemble the characteristics of the youngest ants in the colony.

Conclusions

Social insects with their specific life history including a switch from normal workers to reproducing gamergates during adult life are well suited for ageing research. The regulation of stress resistance in gamergates seemed to be modified compared to foraging workers in an economic way. Interestingly, a switch towards more stress resistant animals can also be induced by a brief period of social isolation, which may already be associated with a shift to a reproductive trajectory. In Harpegnathos saltator, stress resistances are differently and potentially more economically regulated in reproductive individuals, highlighting the significance of reproduction for an increase in longevity in social insects. As already shown for other organisms with a long lifespan, this trait is not directly coupled to higher levels of enzymatic and non-enzymatic antioxidants.     

On the extent and origins of genic novelty in the phylum Nematoda

Background

The phylum Nematoda is biologically diverse, including parasites of plants and animals as well as free-living taxa. Underpinning this diversity will be commensurate diversity in expressed genes, including gene sets associated specifically with evolution of parasitism.

Methods and Findings

Here we have analyzed the extensive expressed sequence tag data (available for 37 nematode species, most of which are parasites) and define over 120,000 distinct putative genes from which we have derived robust protein translations. Combined with the complete proteomes of Caenorhabditis elegans and Caenorhabditis briggsae, these proteins have been grouped into 65,000 protein families that in turn contain 40,000 distinct protein domains. We have mapped the occurrence of domains and families across the Nematoda and compared the nematode data to that available for other phyla. Gene loss is common, and in particular we identify nearly 5,000 genes that may have been lost from the lineage leading to the model nematode C. elegans. We find a preponderance of novelty, including 56,000 nematode-restricted protein families and 26,000 nematode-restricted domains. Mapping of the latest time-of-origin of these new families and domains across the nematode phylogeny revealed ongoing evolution of novelty. A number of genes from parasitic species had signatures of horizontal transfer from their host organisms, and parasitic species had a greater proportion of novel, secreted proteins than did free-living ones.

Conclusions

These classes of genes may underpin parasitic phenotypes, and thus may be targets for development of effective control measures.     

Non-additive benefit or cost? Disentangling the indirect effects that occur when plants bearing extrafloral nectaries and honeydew-producing insects share exotic ant mutualists

Background and Aims

In complex communities, organisms often form mutualisms with multiple different partners simultaneously. Non-additive effects may emerge among species linked by these positive interactions. Ants commonly participate in mutualisms with both honeydew-producing insects (HPI) and their extrafloral nectary (EFN)-bearing host plants. Consequently, HPI and EFN-bearing plants may experience non-additive benefits or costs when these groups co-occur. The outcomes of these interactions are likely to be influenced by variation in preferences among ants for honeydew vs. nectar. In this study, a test was made for non-additive effects on HPI and EFN-bearing plants resulting from sharing exotic ant guards. Preferences of the dominant exotic ant species for nectar vs. honeydew resources were also examined.

Methods

Ant access, HPI and nectar availability were manipulated on the EFN-bearing shrub, Morinda citrifolia, and ant and HPI abundances, herbivory and plant growth were assessed. Ant-tending behaviours toward HPI across an experimental gradient of nectar availability were also tracked in order to investigate mechanisms underlying ant responses.

Key Results

The dominant ant species, Anoplolepis gracilipes, differed from less invasive ants in response to multiple mutualists, with reductions in plot-wide abundances when nectar was reduced, but no response to HPI reduction. Conversely, at sites where A. gracilipes was absent or rare, abundances of less invasive ants increased when nectar was reduced, but declined when HPI were reduced. Non-additive benefits were found at sites dominated by A. gracilipes, but only for M. citrifolia plants. Responses of HPI at these sites supported predictions of the non-additive cost model. Interestingly, the opposite non-additive patterns emerged at sites dominated by other ants.

Conclusions

It was demonstrated that strong non-additive benefits and costs can both occur when a plant and herbivore share mutualist partners. These findings suggest that broadening the community context of mutualism studies can reveal important non-additive effects and increase understanding of the dynamics of species interactions.     

Apolipophorin-III mediates antiplasmodial epithelial responses in Anopheles gambiae (G3) mosquitoes

Background

Apolipophorin-III (ApoLp-III) is known to play an important role in lipid transport and innate immunity in lepidopteran insects. However, there is no evidence of involvement of ApoLp-IIIs in the immune responses of dipteran insects such as Drosophila and mosquitoes.

Methodology/Principal Findings

We report the molecular and functional characterization of An. gambiae apolipophorin-III (AgApoLp-III). Mosquito ApoLp-IIIs have diverged extensively from those of lepidopteran insects; however, the predicted tertiary structure of AgApoLp-III is similar to that of Manduca sexta (tobacco hornworm). We found that AgApoLp-III mRNA expression is strongly induced in the midgut of An. gambiae (G3 strain) mosquitoes in response to Plasmodium berghei infection. Furthermore, immunofluorescence stainings revealed that high levels of AgApoLp-III protein accumulate in the cytoplasm of Plasmodium-invaded cells and AgApoLp-III silencing increases the intensity of P. berghei infection by five fold.

Conclusion

There are broad differences in the midgut epithelial responses to Plasmodium invasion between An. gambiae strains. In the G3 strain of An. gambiae AgApoLp-III participates in midgut epithelial defense responses that limit Plasmodium infection.     

Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed,paraffin-embedded samples: insights from liver tissue

Background

The growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun analysis of FFPE samples, but a critical comparison of the different methods is currently lacking.

Experimental design

DT, FASP and ISD workflows were compared by subjecting to the same label-free quantitative approach three independent technical replicates of each method applied to FFPE liver tissue. Data were evaluated in terms of method reproducibility and protein/peptide distribution according to localization, MW, pI and hydrophobicity.

Results

DT showed lower reproducibility, good preservation of high-MW proteins, a general bias towards hydrophilic and acidic proteins, much lower keratin contamination, as well as higher abundance of non-tryptic peptides. Conversely, FASP and ISD proteomes were depleted in high-MW proteins and enriched in hydrophobic and membrane proteins; FASP provided higher identification yields, while ISD exhibited higher reproducibility.

Conclusions

These results highlight that diverse sample preparation strategies provide significantly different proteomic information, and present typical biases that should be taken into account when dealing with FFPE samples. When a sufficient amount of tissue is available, the complementary use of different methods is suggested to increase proteome coverage and depth.     

The Fynbos and sUcculent Karoo biomes do not have exceptional local ant richness

Background

The Fynbos (FB) and Succulent Karoo biomes (SKB) have high regional plant diversity despite relatively low productivity. Local diversity in the region varies but is moderate. For insects, previous work suggests that strict phytophages, but not other taxa, may have high regional richness. However, what has yet to be investigated is whether the local insect species richness of FB and SKB is unusual for a region of this productivity level at this latitude, and whether regional richness is also high. Here we determine whether this is the case for ants.

Methodology/Principal Findings

We use species richness data from pitfall traps in the FB and SKB in the Western Cape Province, South Africa and a global dataset of local ant richness extracted from the literature. We then relate the globally derived values of local richness to two energy-related predictors—productive energy (NDVI) and temperature, and to precipitation, and compare the data from the FB and SKB with these relationships. We further compare our local richness estimates with that of similar habitats worldwide, and regional ant richness with estimates derived from other regions. The local ant species richness of the FB and SKB falls within the general global pattern relating ant richness to energy, and is similar to that in comparable habitats elsewhere. At a regional scale, the richness of ants across all of our sites is not exceptional by comparison with other regional estimates from across the globe.

Conclusions/Significance

Local richness of ants in the FB and SKB is not exceptional by global standards. Initial analyses suggest that regional diversity is also not exceptional for the group. It seems unlikely that the mechanisms which have contributed to the development of extraordinarily high regional plant diversity in these biomes have had a strong influence on the ants.     

Hyaluronidase of bloodsucking insects and its enhancing effect on leishmania infection in mice

Background

Salivary hyaluronidases have been described in a few bloodsucking arthropods. However, very little is known about the presence of this enzyme in various bloodsucking insects and no data are available on its effect on transmitted microorganisms. Here, we studied hyaluronidase activity in thirteen bloodsucking insects belonging to four different orders. In addition, we assessed the effect of hyaluronidase coinoculation on the outcome of Leishmania major infection in BALB/c mice.

Principal Findings

High hyaluronidase activity was detected in several Diptera tested, namely deer fly Chrysops viduatus, blackflies Odagmia ornata and Eusimilium latipes, mosquito Culex quinquefasciatus, biting midge Culicoides kibunensis and sand fly Phlebotomus papatasi. Lower activity was detected in cat flea Ctenocephalides felis. No activity was found in kissing bug Rhodnius prolixus, mosquitoes Anopheles stephensi and Aedes aegypti, tse-tse fly Glossina fuscipes, stable fly Stomoxys calcitrans and human louse Pediculus humanus. Hyaluronidases of different insects vary substantially in their molecular weight, the structure of the molecule and the sensitivity to reducing conditions or sodium dodecyl sulphate. Hyaluronidase exacerbates skin lesions caused by Leishmania major; more severe lesions developed in mice where L. major promastigotes were coinjected with hyaluronidase.

Conclusions

High hyaluronidase activities seem to be essential for insects with pool-feeding mode, where they facilitate the enlargement of the feeding lesion and serve as a spreading factor for other pharmacologically active compounds present in saliva. As this enzyme is present in all Phlebotomus and Lutzomyia species studied to date, it seems to be one of the factors responsible for enhancing activity present in sand fly saliva. We propose that salivary hyaluronidase may facilitate the spread of other vector-borne microorganisms, especially those transmitted by insects with high hyaluronidase activity, namely blackflies (Simuliidae), biting midges (Ceratopogonidae) and horse flies (Tabanidae).     

Risk of Buruli ulcer and detection of Mycobacterium ulcerans in mosquitoes in southeastern Australia

Background

Buruli ulcer (BU) is a destructive skin condition caused by infection with the environmental bacterium, Mycobacterium ulcerans. The mode of transmission of M. ulcerans is not completely understood, but several studies have explored the role of biting insects. In this study, we tested for an association between the detection of M. ulcerans in mosquitoes and the risk of BU disease in humans in an endemic area of southeastern Australia.

Methodology/Principal Findings

Adult mosquitoes were trapped in seven towns on the Bellarine Peninsula in Victoria, Australia, from December 2004 to December 2009 and screened for M. ulcerans by real-time PCR. The number of laboratory-confirmed cases of BU in permanent residents of these towns diagnosed during the same period was tallied to determine the average cumulative incidence of BU in each location. Pearson''s correlation coefficient (r) was calculated for the proportion of M. ulcerans-positive mosquitoes per town correlated with the incidence of BU per town. We found a strong dose-response relationship between the detection of M. ulcerans in mosquitoes and the risk of human disease (r, 0.99; 95% CI, 0.92–0.99; p<0.001).

Conclusions/Significance

The results of this study strengthen the hypothesis that mosquitoes are involved in the transmission of M. ulcerans in southeastern Australia. This has implications for the development of intervention strategies to control and prevent BU.     

Does Kin Recognition and Sib-Mating Avoidance Limit the Risk of Genetic Incompatibility in a Parasitic Wasp?

Background

When some combinations of maternal and paternal alleles have a detrimental effect on offspring fitness, females should be able to choose mates on the basis of their genetic compatibility. In numerous Hymenoptera, the sex of an individual depends of the allelic combination at a specific locus (single-locus Complementary Sex Determination), and in most of these species individuals that are homozygous at this sexual locus develop into diploid males with zero fitness.

Methods and Findings

In this paper, we tested the hypothesis of genetic incompatibility avoidance by investigating sib-mating avoidance in the solitary wasp parasitoid, Venturia canescens. In the context of mate choice we show, for the first time in a non-social hymenopteran species, that females can avoid mating with their brothers through kin recognition. In “no-choice” tests, the probability a female will mate with an unrelated male is twice as high as the chance of her mating with her brothers. In contrast, in choice tests in small test arenas, no kin discrimination effect was observed. Further experiments with male extracts demonstrate that chemical cues emanating from related males influence the acceptance rate of unrelated males.

Conclusions

Our results are compatible with the genetic incompatibility hypothesis. They suggest that the female wasps recognize sibs on the basis of a chemical signature carried or emitted by males possibly using a “self-referent phenotype matching” mechanism.     

Tropical Mosquito Assemblages Demonstrate ‘Textbook’ Annual Cycles

Background

Annual biological rhythms are often depicted as predictably cyclic, but quantitative evaluations are few and rarely both cyclic and constant among years. In the monsoon tropics, the intense seasonality of rainfall frequently drives fluctuations in the populations of short-lived aquatic organisms. However, it is unclear how predictably assemblage composition will fluctuate because the intensity, onset and cessation of the wet season varies greatly among years.

Methodology/Principal Findings

Adult mosquitoes were sampled using EVS suction traps baited with carbon dioxide around swamplands adjacent to the city of Darwin in northern Australia. Eleven sites were sampled weekly for five years, and one site weekly for 24 years, the sample of c. 1.4 million mosquitoes yielding 63 species. Mosquito abundance, species richness and diversity fluctuated seasonally, species richness being highly predictable. Ordination of assemblage composition demonstrated striking annual cycles that varied little from year to year. The mosquito assemblage was temporally structured by a succession of species peaks in abundance.

Conclusion/Significance

Ordination provided strong visual representation of annual rhythms in assemblage composition and the means to evaluate variability among years. Because most mosquitoes breed in shallow freshwater which fluctuates with rainfall, we did not anticipate such repeatability; we conclude that mosquito assemblage composition appears adapted to predictable elements of the rainfall.     

Effects of light on direct and indirect defences against herbivores of young plants of Mallotus japonicus demonstrate a trade-off between two indirect defence traits

Background and Aims

Although most studies on plant defence strategies have focused on a particular defence trait, some plant species develop multiple defence traits. To clarify the effects of light on the development of multiple defence traits, the production of direct and indirect defence traits of young plants of Mallotus japonicus were examined experimentally under different light conditions.

Methods

The young plants were cultivated under three light conditions in the experimental field for 3 months from May to July. Numbers of ants and pearl bodies on leaves in July were examined. After cultivation, the plants were collected and the developments of trichomes and pellucid dots, and extrafloral nectaries (EFNs) on the leaves were examined. On plants without nectar-collecting insects, the size of EFNs and the volume of extrafloral nectar secreted from the EFNs were examined.

Key results

Densities of trichomes and pellucid dots did not differ significantly among the plants under the different light conditions, suggesting that the chemical and physical defences function under both high and low light availability. The number of EFNs on the leaves did not differ significantly among the plants under the different light conditions, but there appeared to be a trade-off between the size of EFNs and the number of pearl bodies; the largest EFNs and the smallest number of pearl bodies were found under high light availability. EFN size was significantly correlated with the volume of extrafloral nectar secreted for 24 h. The number of ants on the plants was smaller under low light availability than under high and moderate light availability.

Conclusions

These results suggest that direct defence traits function regardless of light conditions, but light conditions affected the development of indirect defence traits.     

Ecological Niche Models and Coalescent Analysis of Gene Flow Support Recent Allopatric Isolation of Parasitoid Wasp Populations in the Mediterranean

Background

The integration of multiple complementary approaches is a powerful way to understand the processes of diversification and speciation. The parasitoid wasp Aphidius transcaspicus Telenga (Hymenoptera: Braconidae) is a parasitoid of Hyalopterus aphids across a wide geographic range. This species shows a remarkable degree of genetic structure among western, central, and eastern Mediterranean population clusters. In this paper we attempt to better characterize this genetic structure.

Methodology/Principal Findings

We use a Bayesian coalescent analysis of gene flow under the Isolation with Migration model using mitochondrial and microsatellite markers together with climate-based ecological niche models to better understand the genetic structure of A. transcaspicus in the Mediterranean. The coalescent analysis revealed low levels of migration among western and eastern Mediterranean populations (Nm<1) that were not statistically distinguishable from zero. Niche models showed that localities within population clusters each occupy areas of continuously high environmental suitability, but are separated from each other by large regions of completely unsuitable habitat that could limit dispersal. Overall, environmental characteristics were similar among the population clusters, though significant differences did emerge.

Conclusions/Significance

These results support contemporary allopatric isolation of Mediterranean populations of A. transcaspicus, which together with previous analyses indicating partial behaviorally mediated reproductive isolation, suggest that the early stages of cryptic speciation may be in progress.