Selection and genomic differentiation during ecological speciation: isolating the contributions of host association via a comparative genome scan of Neochlamisus bebbianae leaf beetles

This study uses a comparative genome scan to evaluate the contributions of host plant related divergent selection to genetic differentiation and ecological speciation in maple- and willow-associated populations of Neochlamisus bebbianae leaf beetles. For each of 15 pairwise population comparisons, we identified "outlier loci" whose strong differentiation putatively reflects divergent selection. Of 447 AFLP loci, 15% were outliers across multiple population comparisons, and low linkage disequilibrium indicated that these outliers derived from multiple regions of the genome. Outliers were further classified as "host-specific" if repeatedly observed in "different-host" population comparisons but never in "same-host" comparisons. Outliers exhibiting the opposite pattern were analogously classified as "host-independent." Host-specific outliers represented 5% of all loci and were more frequent than host-independent outliers, thus revealing a large role for host-adaptation in population genomic differentiation. Evidence that host-related selection can promote divergence despite gene flow was provided by population trees. These were structured by host-association when datasets included host-specific outliers, but not when based on neutral loci, which united sympatric populations. Lastly, three host-specific outliers were highly differentiated in all nine different-host comparisons. Because host-adaptation promotes reproductive isolation in these beetles, these loci provide promising candidate gene regions for future molecular studies of ecological speciation.     

Extensive variation in chromosome number and genome size in sexual and parthenogenetic species of the jumping‐bristletail genus Machilis (Archaeognatha)

Parthenogenesis in animals is often associated with polyploidy and restriction to extreme habitats or recently deglaciated areas. It has been hypothesized that benefits conferred by asexual reproduction and polyploidy are essential for colonizing these habitats. However, while evolutionary routes to parthenogenesis are manifold, study systems including polyploids are scarce in arthropods. The jumping‐bristletail genus Machilis (Insecta: Archaeognatha) includes both sexual and parthenogenetic species, and recently, the occurrence of polyploidy has been postulated. Here, we applied flow cytometry, karyotyping, and mitochondrial DNA sequencing to three sexual and five putatively parthenogenetic Eastern‐Alpine Machilis species to investigate whether (1) parthenogenesis originated once or multiply and (2) whether parthenogenesis is strictly associated with polyploidy. The mitochondrial phylogeny revealed that parthenogenesis evolved at least five times independently among Eastern‐Alpine representatives of this genus. One parthenogenetic species was exclusively triploid, while a second consisted of both diploid and triploid populations. The three other parthenogenetic species and all sexual species were diploid. Our results thus indicate that polyploidy can co‐occur with parthenogenesis, but that it was not mandatory for the emergence of parthenogenesis in Machilis. Overall, we found a weak negative correlation of monoploid genome size (Cx) and chromosome base number (x), and this connection is stronger among parthenogenetic species alone. Likewise, monoploid genome size decreased with elevation, and we therefore hypothesize that genome downsizing could have been crucial for the persistence of alpine Machilis species. Finally, we discuss the evolutionary consequences of intraspecific chromosomal rearrangements and the presence of B chromosomes. In doing so, we highlight the potential of Alpine Machilis species for research on chromosomal and genome‐size alterations during speciation.     

Phylogeographic assessment of mtDNA paraphyly and the evolution of unisexuality in Calligrapha (Coleoptera: Chrysomelidae)

The leaf beetle genus Calligrapha is one of the few examples of animals with several obligate unisexual, female‐only species. Previous work showed that each one arose independently from interspecific hybridization events involving different species. However, all of them clustered in a single mtDNA clade together with some individuals of the parental bisexual species, which appeared as deeply polyphyletic in the mtDNA genealogy of the genus. The dating of these splits using a molecular clock placed them in the Quaternary and it was hypothesized that climatic change during this period may have favored range expansions and secondary contacts required for hybridization. In this work, we test this hypothesis and the origins of unisexuality in Calligrapha examining the diversity of mitochondrial (cox1) and nuclear (wingless, Wg) genes and the Bayesian continuous mtDNA phylogeography of a sample of more than 500 specimens of two bisexual species of Calligrapha at a continental scale and two unisexual species derived from them. Besides a major topological difference, whereby each bisexual species is monophyletic for Wg but paraphyletic for cox1, both gene datasets are consistent with a minimum of seven evolutionary lineages, coherent with geography and consistent with an ordered expansion to occupy their current ranges. The results also imply their survival in well‐established glacial refuges during the Last Glacial Maximum (LGM). Thus, for bisexual C. multipunctata there are two main, southern and northern lineages. The southern lineage expanded its range in two evolutionary branches, to the Rocky Mountains and to the northern Mississippi and Ohio River basins, respectively. The northern lineage has one branch in the Upper Mississippi and one that expanded west to the Pacific Northwest and east to the northeastern North Atlantic, finding refuge in both areas. These major lineages are parapatric in the Northern Great Plains, an area consistent with them having found refuge in the so‐called Driftless region during the LGM. For bisexual C. philadelphica, one northern lineage expanded west from the northern Appalachians and one east and southwest along the axis of the Appalachians, and the timing of events is consistent with their persistence in glacial refugia at both sides of the main Great Lakes lobe of the Laurentide Ice Sheet. There is evidence that the northeastern North Atlantic lineages of both species hybridized at the edge of their ranges after the LGM. The additional, divergent mtDNA lineage of these species shows evidence of range expansions of two lineages, one for each species, in an area south of the Laurentide Ice Sheet, and giving origin to the unisexual species by way of hybridization with other species in the Alleghanian region after the LGM. Interestingly, the individuals of supposedly bisexual species in this clade are all females. This suggests that unisexuality actually predates the origin of unisexual taxa in this system and that some bisexual species in Calligrapha may be species complexes instead, with cryptic species differing in their reproductive mode.     

Assessment of the role of Wolbachia in mtDNA paraphyly and the evolution of unisexuality in Calligrapha (Coleoptera: Chrysomelidae)

Calligrapha is a New World leaf beetle genus that includes several unisexual species in northeastern North America. Each unisexual species had an independent hybrid origin involving different combinations of bisexual species. However, surprisingly, they all cluster in a single mtDNA clade and with some individuals of their parental species, which are in turn deeply polyphyletic for mtDNA. This pattern is suggestive of a selective sweep which, together with mtDNA taxonomic incongruence and occurrence of unisexuality in Calligrapha, led to hypothesize that Wolbachia might be responsible. I tested this hypothesis studying the correlation between diversity of Wolbachia and well‐established mtDNA lineages in >500 specimens of two bisexual species of Calligrapha and their derived unisexual species. Wolbachia appears highly prevalent (83.4%), and fifteen new supergroup‐A strains of the bacteria are characterized, belonging to three main classes: wCallA, occupying the whole species ranges, and wCallB and wCallC, narrowly parapatric, infecting beetles with highly divergent mtDNAs where they coexist. Most beetles (71.6%) carried double infections of wCallA with another sequence class. Bayesian inference of ancestral character states and association tests between bacterial diversity and the mtDNA genealogy show that each mtDNA lineage of Calligrapha has specific types of infection. Moreover, shifts can be explained by horizontal or vertical transfer from local populations to an expanding lineage and cytoplasmic incompatibility between wCallB and wCallC types, suggesting that the symbionts hitchhike with the host and are not responsible for selective mtDNA sweeps. Lack of evidence for sweeps and the fact that individuals in the unisexual clade are uninfected or infected by the widespread wCallA type indicate that Wolbachia does not induce unisexuality in Calligrapha, although they may manipulate host reproduction through cytoplasmic incompatibility.     

AFLP analyses demonstrate genetic divergence, hybridization, and multiple polyploidization in the evolution of Achillea (Asteraceae-Anthemideae)

Achillea, a temperate genus of herbaceous allogamous perennials, is a model for evolutionary radiation through hybridization and polyploidization. AFLP analyses were performed on 300 individuals of 66 populations and 27 taxa/cytotypes, mainly from the polyploid A. millefolium aggregate and its suspected hybrid links with other clades of the genus. The mosaic genetic structure of hybrids and polyploids is revealed by specific AFLP bands shared with their assumed parents. In E Asia, A. alpina-4x and A. wilsoniana-4x are allotetraploids between A. acuminata-2x (sect. Ptarmica) and A. asiatica-2x (sect. Achillea-A. millefolium agg.). A. virescens-4x is a hybrid species linking A. nobilis agg. and A. millefolium agg. in S Europe. The hybrid swarm A. clypeolata-2x yen A. collina-4x recently formed in Bulgaria shows no AFLP bands additive to its parents; by contrast, other more ancient allopolyploids exhibit genetic innovations. Relationships within A. millefolium agg. are complex. Five 2x-taxa, mostly well separated and regressive, are limited to Eurasia; seven 4x- and 6x-taxa are intimately linked by hybridization, are expansive, and through A. asiatica-2x/4x have formed the N American polyploids. All these results from AFLPs correspond well to other evidence, and indicate a long history of reticulate evolution in Achillea.     

The genetic ghost of an invasion past: colonization and extinction revealed by historical hybridization in Senecio

Hybridization is an important evolutionary factor in the diversification of many plant and animal species. Of particular interest is that historical hybridization resulting in the origin of new species or introgressants has occurred between species now geographically separated by great distances. Here, we report that Senecio massaicus, a tetraploid species native to Morocco and the Canary Islands, contains genetic material of two distinct, geographically separated lineages: a Mediterranean lineage and a mainly southern African lineage. A time-calibrated internal transcribed spacer phylogeny indicates that the hybridization event took place up to 6.18 Ma. Because the southern African lineage has never been recorded from Morocco or the Canary Islands, we hypothesize that it reached this area in the distant past, but never became permanently established. Interestingly, the southern African lineage includes S. inaequidens, a highly invasive species that has recently become widespread throughout Europe and was introduced at the end of the 19th century as a 'wool alien'. Our results suggest that this more recent invasion of Europe by S. inaequidens represents the second arrival of this lineage into the region.     

Postmating isolation and genetically variable host use in ecologically divergent host forms of Neochlamisus bebbianae leaf beetles

Ecological speciation studies have more thoroughly addressed premating than postmating reproductive isolation. This study examines multiple postmating barriers between host forms of Neochlamisus bebbianae leaf beetles that specialize on Acer and Salix trees. We demonstrate cryptic isolation and reduced hybrid fitness via controlled matings of these host forms. These findings reveal host-associated postmating isolation, although a nonecological, 'intrinsic' basis for these patterns cannot be ruled out. Host preference and performance results among cross types further suggest sex-linked maternal effects on these traits, whereas family effects indicate their genetic basis and associated variation. Genes of major effect appear to influence these traits. Together with previous findings of premating isolation and adaptive differentiation in sympatry, our results meet many assumptions of 'speciation with gene flow' models. Here, such gene flow is likely asymmetric, with consequences for the dynamics of future ecological divergence and potential ecological speciation of these host forms.     

Phylogeography of Eriotheca species complex: insights into the origin and range expansion of apomictic and polyploid trees in Neotropical Savannas

• Polyploidy and whole genome duplication are major evolutionary drivers in plants. Climate variations during the Pleistocene have influenced distribution and range expansion worldwide. Similar trends have been reported for Cerrado plants, but no attempt has been made to link phylogeography with ploidy and breeding changes. Thus, we aimed to (i) assess ploidy and genome size of Eriotheca estevesiae Carv.-Sobr., and compare it with E. pubescens (Mart.) Schott & Endl. (Both included into the Eriotheca Stellate Trichome Species Complex – ESTSC). (ii) Subsequently, we investigated their phylogeography to see whether genetic structure and range expansion trends were similar to those previously described for the Cerrado biome. Finally (iii), we discuss whether ESTSC phylogeographic patterns could be associated with geographic parthenogenesis processes.
• Common cytogenetic techniques and flow cytometry were used to confirm chromosome number and genome size of E. estevesiae. We used three cpDNA regions to analyse 14 ESTSC Cerrado populations, for which we also obtained ploidy level and breeding information. We investigated haplotype diversity, population structure and tested neutrality, aiming to reconstruct phylogeographic scenarios.
• We found three ploidy levels and eight cpDNA haplotypes in ESTSC, one shared by most populations. Haplotype and ploidy distribution corroborated that E. pubescens, the widely distributed polyploid and apomictic species, may have originated from northern diploid and probably sexual E. estevesiae.
• Matrilinear cpDNA links support the idea that apomixis and polyploidy in ESTSC may have allowed range expansion during the Pleistocene, in a process analogous to the geographic parthenogenesis described elsewhere.

     

Spread of violets in polluted pine forests: morphological and molecular evidence for the ecological importance of interspecific hybridization

Hybridization between plant species occurs frequently but hybrids are often restricted to ecotones or disturbed habitats. In this study we show that introgressive hybrids between the tetraploid Viola riviniana and the diploid V. reichenbachiana invaded pine forests of the Dübener Heide (central Germany), an area affected by calcareous pollutants. The spread of these violet populations was correlated with the impact of pollution on habitat conditions. We compared morphology, cytology and random amplified polymorphic DNA (RAPD) bands among six Viola populations from the Dübener Heide and three populations of each pure species. RAPD analysis using 12 primers revealed 141 scorable bands. We considered bands as species specific if they occurred in at least 75% of the plants in one pure violet species but in none of the other. Seven bands were specific to V. riviniana and 11 bands were specific to V. reichenbachiana . Two plants of a V. reichenbachiana population were identified as hybrids. Of the Viola populations from the Dübener Heide, one was diploid and could be classified as V. reichenbachiana by morphology and RAPD markers. However, the majority of the Dübener Heide populations were tetraploid, and of a more variable morphology than V. riviniana and V. reichenbachiana , showing different combinations of intermediate characters, characters of the pure species and extreme characters. Despite their overall genetic similarity to V. riviniana , these plants could be identified as introgressive hybrids between V. riviniana and V. reichenbachiana by species-specific RAPD bands. Therefore, we propose that recurrent hybridization and backcrossing resulted in novel genotypes adapted to the changed environment of polluted pine forests.     

Spread of violets in polluted pine forests: morphological and molecular evidence for the ecological importance of interspecific hybridization

Hybridization between plant species occurs frequently but hybrids are often restricted to ecotones or disturbed habitats. In this study we show that introgressive hybrids between the tetraploid Viola riviniana and the diploid V. reichenbachiana invaded pine forests of the Dübener Heide (central Germany), an area affected by calcareous pollutants. The spread of these violet populations was correlated with the impact of pollution on habitat conditions. We compared morphology, cytology and random amplified polymorphic DNA (RAPD) bands among six Viola populations from the Dübener Heide and three populations of each pure species. RAPD analysis using 12 primers revealed 141 scorable bands. We considered bands as species specific if they occurred in at least 75% of the plants in one pure violet species but in none of the other. Seven bands were specific to V. riviniana and 11 bands were specific to V. reichenbachiana . Two plants of a V. reichenbachiana population were identified as hybrids. Of the Viola populations from the Dübener Heide, one was diploid and could be classified as V. reichenbachiana by morphology and RAPD markers. However, the majority of the Dübener Heide populations were tetraploid, and of a more variable morphology than V. riviniana and V. reichenbachiana , showing different combinations of intermediate characters, characters of the pure species and extreme characters. Despite their overall genetic similarity to V. riviniana , these plants could be identified as introgressive hybrids between V. riviniana and V. reichenbachiana by species-specific RAPD bands. Therefore, we propose that recurrent hybridization and backcrossing resulted in novel genotypes adapted to the changed environment of polluted pine forests.     

The evolution of cranial form and function in theropod dinosaurs: insights from geometric morphometrics

Theropod dinosaurs, an iconic clade of fossil species including Tyrannosaurus and Velociraptor, developed a great diversity of body size, skull form and feeding habits over their 160+ million year evolutionary history. Here, we utilize geometric morphometrics to study broad patterns in theropod skull shape variation and compare the distribution of taxa in cranial morphospace (form) to both phylogeny and quantitative metrics of biting behaviour (function). We find that theropod skulls primarily differ in relative anteroposterior length and snout depth and to a lesser extent in orbit size and depth of the cheek region, and oviraptorosaurs deviate most strongly from the "typical" and ancestral theropod morphologies. Noncarnivorous taxa generally fall out in distinct regions of morphospace and exhibit greater overall disparity than carnivorous taxa, whereas large-bodied carnivores independently converge on the same region of morphospace. The distribution of taxa in morphospace is strongly correlated with phylogeny but only weakly correlated with functional biting behaviour. These results imply that phylogeny, not biting function, was the major determinant of theropod skull shape.     

Diversity and natural hybridization in a highly endemic species of Petunia (Solanaceae): a molecular and ecological analysis

Intrinsic reproductive barriers among the species of Petunia are weak and genetic isolation is obtained mainly by geographical separation and ecological diversification. The Serra do Sudeste region in the extreme south of Brazil is one of the centres of diversity of this genus and is characterized by the presence of species with different pollination syndromes. Petunia exserta is known only from four sandstone towers in a restricted area of this region (about 500 km(2)) and is characterized by its differentiated habitat (shelters in the sandstone towers) and by its floral characteristics adapted to ornithophily. In towers where this species is sympatric with the sphingophilous Petunia axillaris, phylogenetically close to P. exserta, we found plants with intermediate floral morphology, suggesting hybridization between them. To test this hypothesis and to better understand its consequences we analysed the sequences of the plastid trnH-psbA, trnS-trnG and psbB-psbH intergenic spacers in 121 individuals sampled all over the P. exserta distribution. The joint analysis of the three markers revealed 13 haplotypes and the network showed two main genetic clades, which probably represent the original gene pool of the two species in the region. In general, individuals of a given population presented the same haplotype, independently of phenotype, corroborating the hybridization hypothesis. Field observations suggest that hummingbirds are responsible for the interspecific gene flow. Analysis of molecular variance revealed high interpopulational diversity among the towers. The low gene flow between populations is possibly related to the autochoric seed dispersion system.     

From kissing to belly stridulation: comparative analysis reveals surprising diversity,rapid evolution,and much homoplasy in the mating behaviour of 27 species of sepsid flies (Diptera: Sepsidae)

Our understanding of how fast mating behaviour evolves in insects is rather poor due to a lack of comparative studies among insect groups for which phylogenetic relationships are known. Here, we present a detailed study of the mating behaviour of 27 species of Sepsidae (Diptera) for which a well‐resolved and supported phylogeny is available. We demonstrate that mating behaviour is extremely diverse in sepsids with each species having its own mating profile. We define 32 behavioural characters and document them with video clips. Based on sister species comparisons, we provide several examples where mating behaviour evolves faster than all sexually dimorphic morphological traits. Mapping the behaviours onto the molecular tree reveals much homoplasy, comparable to that observed for third positions of mitochondrial protein‐encoding genes. A partitioned Bremer support (PBS) analysis reveals conflict between the molecular and behavioural data, but behavioural characters have higher PBS values per parsimony‐informative character than DNA sequence characters.     

Amplified fragment length polymorphisms and sequence data in the phylogenetic analysis of polyploids: multiple origins of Veronica cymbalaria (Plantaginaceae)

The origin of polyploid Veronica cymbalaria (Plantaginaceae) was investigated using DNA sequence data and amplified fragment length polymorphism (AFLP) fingerprints to reveal the parentage of this taxon. The use of AFLP fingerprints in phylogenetic analysis is problematic and various methods have therefore been compared. DNA sequence data (for the internal transcribed spacer (ITS) region and the plastid trnL-F region (trnL intron, 3'exon, and trnL-F spacer)) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the ITS region suggested a reliable hypothesis for the evolution of the V. cymbalaria complex. This hypothesis allowed evaluation of the effect of different distance measures (Jaccard and Nei-Li) in phenetic, character-state weighted parsimony, and Bayesian analyses of AFLP markers. The study establishes that tetraploid V. cymbalaria originated at least twice in the eastern Mediterranean, with one parent differing in the two separate origins. Hexaploid V. cymbalaria originated even more often. The results illustrate that even subtle differences in the analyses of AFLP markers can lead to drastically different conclusions. The study reveals multiple origins of a Mediterranean polyploid species. Furthermore, it demonstrates that the analysis of a complex marker system such as AFLP fingerprints using only one type of analysis can easily be misleading.     

Flies in the ointment: a morphological and molecular comparison of Lucilia cuprina and Lucilia sericata (Diptera: Calliphoridae) in South Africa

Abstract.  Complementary nuclear (28S rRNA) and mitochondrial (COI) genes were sequenced from blowflies that phenotypically resembled Lucilia cuprina (W.), Lucilia sericata (Meigen) or exhibited characters of both species. The aim was to test a long-held hypothesis that these species hybridize under natural conditions in South Africa ( Ullyett, 1945 ). Blowflies were obtained predominantly from the Cape Town metropolitan area, but reference samples were acquired for L. sericata from Pretoria. Several L. cuprina -like flies were shown to possess a conflicting combination of nuclear and mitochondrial genes that has also been seen in Hawaiian specimens. Homoplasy, sampling of pseudogenes, hybridization and incomplete lineage sorting are discussed as possible hypotheses for the pattern and the latter is concluded to represent the most likely explanation.     

Chemical defences in leaf beetles and their larvae: The ecological, evolutionary and taxonomic significance

The chemical defences of the Chrysomelinae are reviewed. Defensive glandular secretions have evolved independently in larvae and adults, and faster than the morphology of the glands. Both characters are used in a phylogenetic study of the Chrysomelini, disclosing suprageneric affinities. First, a close relationship between the Chrysomelina and Phratora is proposed. Secondly, Leptinotarsa and Gonioctena are probably more closely related to the Chrysolinina than to the Chrysomelina and Phratora. The qualitative and quantitative composition of larval and adult secretions sometimes differs between geographically isolated populations of the same species, which are therefore probably genetically distinct. The host plant shows an influence which explains discrepancies between the current classification and the chemical nature of the secretions. Some beetles sequester secondary plant metabolites or use them as precursors for their own defences. The original biosynthetic pathway has been lost in these species. Other factors which could affect the chemical diversity of the secretions are discussed: random events such as neutral mutations and genetic drift, conditioning or adaptation of predators to specific defensive mixtures, and the diversity of potential predators. The latter could help to explain the divergence in the chemical nature of the larval and adult secretions. The volatile irritants produced by the larvae are well suited to repel small arthropods, such as ants, whereas poisons associated with aposematic coloration in the adults could be directed against small vertebrates, such as birds.     

Environmental and geographical determinants of beta diversity of leaf beetles (Coleoptera: Chrysomelidae) in the Iberian Peninsula

Abstract.  1. The effect and relative importance of both environmental and geographic factors on species turnover of Chrysomelidae assemblages in the Iberian Peninsula was explored.
2. Ordination and classification analysis of species lists from well-sampled local areas identified two main biogegraphic groups of inventories, discriminating between Eurosiberian and Mediterranean faunas.
3. After describing this pattern, the underlying determinants were explored, separating pure from combined effects of environment and geography by means of partial canonical correspondence analysis.
4. Results indicate that pure effects of environment and geographic position are of similar magnitude (about 20% of the variation from each), and quite important in comparison with combined effects.
5. The power of the pure effect of geographical variables, together with beta-diversity independence of species richness gradients, point to the inadequacy of environmental measurement explanation of beta-diversity patterns, and to the existence of a major component of geographical variation independent of environment.     

The evolution of jumping performance in anurans: morphological correlates and ecological implications

We investigated the evolution of anuran locomotor performance and its morphological correlates as a function of habitat use and lifestyles. We reanalysed a subset of the data reported by Zug (Smithson. Contrib. Zool. 1978; 276: 1–31) employing phylogenetically explicit statistical methods (n = 56 species), and assembled morphological data on the ratio between hind-limb length and snout-vent length (SVL) from the literature and museum specimens for a large subgroup of the species from the original paper (n = 43 species). Analyses using independent contrasts revealed that classifying anurans into terrestrial, semi-aquatic, and arboreal categories cannot distinguish between the effects of phylogeny and ecological diversification in anuran locomotor performance. However, a more refined classification subdividing terrestrial species into 'fossorials' and 'non-fossorials', and arboreal species into 'open canopy', 'low canopy' and 'high canopy', suggests that part of the variation in locomotor performance and in hind-limb morphology can be attributed to ecological diversification. In particular, fossorial species had significantly lower jumping performances and shorter hind limbs than other species after controlling for SVL, illustrating how the trade-off between burrowing efficiency and jumping performance has resulted in morphological specialization in this group.