Game of clones: Is Wolbachia inducing speciation in a weevil with a mixed reproductive mode?

Parthenogenesis is widely distributed in Metazoa but it is especially frequent in weevils (Coleoptera, Curculionidae) with one fifth of all known cases. Previous studies have shown that in the tribe Naupactini parthenogenetic reproduction most likely originated with an infection of the endoparasitic bacterium Wolbachia pipientis. In particular, Pantomorus postfasciatus possess a mixed reproductive mode: some populations have males while in others they are absent, and females produce clones by thelytoky. To better understand this scenario, we studied the population structure and infection status in 64 individuals of P. postfasciatus from Argentina and Brazil. We sequenced two mitochondrial (COI and COII) and one nuclear (ITS-1) fragments and obtained two very divergent haplogroups, one corresponding to the sexual populations uninfected with Wolbachia, and another conforming a monophyletic parthenogenetic (or presumptively parthenogenetic) and infected clade. Each of these haplogroups was identified as an independently evolutionary unit by all species delimitation analyses accomplished: multilocus *BEAST and BP&P, and single locus GMYC and K/θ rule. Additionally, present evidence suggests that Wolbachia infection occurred at least twice in all-female populations of P. postfasciatus with two different bacterial strains. Speciation mediated by Wolbachia is a recently described phenomenon and the case of P. postfasciatus is the first known case in a diplo-diploid insect. A model that describes how thelytoky-inducing phenotypes of Wolbachia could generate new lineages is discussed.

Introduction and Establishment of Pissodes castaneus (Coleoptera: Curculionidae) in the Andean Patagonia of Argentina.

The pine weevils that occur in plantations of Pinus spp. in Andean Patagonia of Argentina belong to the species Pissodes castaneus (De Geer), a Eurasian endemic species, according to the identification based on molecular and morphological characters. Sequences of the mitochondrial Cytochrome oxidase subunit I and nuclear genes (28 S rDNA and ITS2) were obtained for individuals of 13 afforestations, covering the entire distribution area of the established populations in the Andean Patagonia of Argentina. Sequence comparison with representative species of the genus (European, North American, and Chinese species) shows that Patagonian specimens are conspecific to those of P. castaneus sequenced from Europe. Phylogenetic analyses indicate that all terminals from Patagonia form a monophyletic unit without evident subclades, eliminating the possibility of existence of more than one species of Pissodes Germar in this area, including cryptic ones. Moreover, the very low genetic divergence between the Patagonian populations suggests that it is plausible that P. castaneus was introduced into Patagonia from just one location. Mitochondrial DNA analysis shows that Patagonian terminals group together with a French haplotype and are clearly separated from other P. castaneus individuals represented in our sample, and reveal that established populations in Andean Patagonia originated via a limited introduction.

On the presence of five species of naupactini (Coleoptera: Curculionidae) damaging soybean in Brazil.

We report five broad-nosed weevils of the tribe Naupactini, Naupactus leucoloma Boheman, Naupactus minor (Buchanan), Naupactus peregrinus (Buchanan), Naupactus tremolerasi Hustache and Pantomorus viridisquamosus (Boheman) (Coleoptera: Curculionidae) damaging soybean plants in the state of Rio Grande do Sul, Brazil. Some of these species are recorded for the first time in Brazil, and they all belong to species groups naturally distributed in the prairies of southern Brazil, Uruguay, Central Argentina and Paraguay. Three of them have been introduced, established and expanded in other countries outside South America. The five species in conjunction with other native species of Naupactini could cause severe damages to soybean crops if the root-feeding larvae attack young growing plants.

Potential geographic distributions and successful invasions of parthenogenetic broad-nosed weevils (Coleoptera: Curculionidae) native to South America.

Ten species of parthenogenetic broad-nosed weevils (Coleoptera: Curculionidae: Entiminae) native to Argentina, southern Brazil, and Uruguay were selected for niche modeling analysis based on climatic data and altitude, to evaluate their potential range expansion inside and outside South America. The selected species belong to five genera of the tribe Naupactini affecting economically important crops. Until present, five of the 10 species analyzed here have invaded prairies and steppes of countries outside South America (Australia, New Zealand, Mexico, United States, and South Africa): Aramigus tessellatus (Say), Atrichonotus sordidus (Hustache), Atrichonotus taeniatulus (Berg), Naupactus leucoloma Boheman, and Naupactus peregrinus (Buchanan). Our niche modeling analyses performed with MAXENT demonstrated that these areas would be also suitable for Aramigus conirostris (Hustache), Eurymetopus fallax (Boheman), Pantomorus auripes Hustache, Pantomorus ruizi (Brèthes), and Pantomorus viridisquamosus (Boheman), consequently, they also have the potential to invade areas outside their native ranges, mainly in southeastern United States, some European countries (e.g., Portugal, France, and southern England), South Africa, New Zealand, and southeastern Australia. All the studied species share similar environmental requirements, the most important variables being the Mean Temperature of Driest Quarter, the Annual Mean Temperature and Isothermality. Long distance dispersal through commercial trade, and parthenogenetic reproduction would increase the threat of these weevils to crop production worldwide.

Speciation in the asexual realm: is the parthenogenetic weevil Naupactus cervinus a complex of species in statu nascendi?

Population genetic theory shows that asexual organisms may evolve into species, which behave as independent evolutionary units. As a result, they form genotypic clusters separated by deep gaps due to geographic isolation and/or divergent selection. Identification of several genetically divergent groups of weevils embodied in the nominal species Naupactus cervinus deserves further study, in order to test if these lineages are evolving independently. In the present paper we tested if the parthenogenetic weevil N. cervinus, native to South America and broadly distributed throughout the world, contains more than one evolutionary unit. For this purpose, we applied three different approaches, a multilocus phylogenetic analysis, the GMYC approach and the K/θ method. We accomplished these analyses through a survey of mitochondrial (COI and COII genes) and nuclear (ITS1 sequence) genetic variation and morphometric analysis in a sample which included individuals from different locations within the native geographic range of N. cervinus. In addition, we compared the divergence accumulated in this species with that in another weevil of the same tribe (Naupactini) showing identical reproductive mode to see if similar levels of morphological variation matches similar levels of genetic divergence. We report the presence of two independent evolutionary units living in sympatry in forest areas. The incongruence between mitochondrial and nuclear datasets analyzed herein reflects incomplete lineage sorting of the nuclear marker and different evolutionary rates between genomes. Ecological divergence driven by natural selection (sympatry) or secondary contact after geographic isolation (allopatry) might explain the deep gaps in mitochondrial phylogenies. Instead, Wolbachia infection was ruled out as a causal factor for such differentiation. We conclude that N. cervinus is probably a species complex with at least two well differentiated lineages that would represent a cluster of species in statu nascendi.

Wolbachia infection in the tribe Naupactini (Coleoptera, Curculionidae): association between thelytokous parthenogenesis and infection status.

Several parthenogenetic species of broad-nosed weevils exist, some of them of economic importance because of their pest status. Screening of the maternally inherited Wolbachia bacterium in 29 weevils of the tribe Naupactini, using multilocus sequence typing allowed us to assess a significant correlation between asexuality and infection, and suggests an involvement of Wolbachia in the origin of this reproductive mode. The nine Wolbachia strains retrieved from the Naupactini belong to the B supergroup. Phylogenetic analysis of these strains, along with other 23 strains obtained from arthropods and nematodes, supports previous hypotheses that horizontal transfer of Wolbachia amongst species from unrelated taxa has been pervasive.

Nuclear and mitochondrial sequences confirm complex colonization patterns and clear species boundaries for flightless weevils in the Galápagos archipelago.

Nuclear sequence data were collected from endemic Galápagos species and an introduced close relative, and contrasted with mitochondrial DNA sequences, continuing investigation into the colonization history and modes of diversification in the weevil genus Galapaganus. The current combined phylogeny together with previously published penalized likelihood age estimates builds a complex picture of the archipelago's colonization history. The present reconstruction relies on submerged platforms to explain the early divergence of the young southern Isabela endemics or the Española or San Cristobal populations. Diversity is later built through inter-island divergence starting on older islands and continuing on two simultaneous tracks towards younger islands. The amount of diversity generated through intra-island processes is skewed towards older islands, suggesting that island age significantly influences diversity. Phylogenetic concordance between nuclear and mitochondrial datasets and well-supported monophyletic species in mitochondrial derived topologies appear to reject the possibility of inter-species hybridization. These clear species boundaries might be related to the tight host associations of adult weevils in discrete ecological zones. If shared hosts facilitate hybridization, then host- or habitat-promoted divergences could prevent it, even in the case of species that share islands, since the altitudinal partitioning of habitats minimizes range overlap.

Colonization history, ecological shifts and diversification in the evolution of endemic Galápagos weevils.

Mitochondrial DNA sequence data were obtained for eight species of flightless Galapaganus endemic weevils and one winged close relative in order to study their colonization history and modes of diversification in the Galápagos Archipelago. Contrary to most other insular radiations, the phylogeny estimates we recovered for Galapaganus do not follow the progression rule of island biogeography. The penalized likelihood age estimates of colonization of the archipelago exceed the age of the emerged islands and underscore the potential role of now sunken seamounts for the early evolution of Galapaganus. The phylogeny proposes one intra-island origin for Galapaganus endemics, but monophyly tests suggest a larger contribution of in-situ speciation on older islands. Generalist habitat preferences were reconstructed as ancestral while shifts to highland habitats were reconstructed as having evolved independently on different islands. Magnitudes and patterns of diversification rate were found to differ between older and younger islands. Our analyses reveal that the colonization sequence of islands and timing of colonization of Galapaganus could be linked with the geological and volcanic history of the islands in a rather complex scenario. Even though most islands appear to have been colonized soon after their emergence, there are notable deviations from the pattern of sequential colonization expected under the progression rule when considering only the extant emerged islands. Patterns of diversification rate variation on older and younger islands correspond to the volcanic activity or remnants of such activity, while the pattern of independent evolution of restricted habitat preferences in different islands suggests that habitat shifts could also have contributed to species diversity in Galapaganus.

Population structure of the boll weevil in cotton fields and subtropical forests of South America: a bayesian approach.

The main goal of this contribution is to investigate the genetic structure of boll weevil populations from South America (Argentina and Brazil) and to make further comparisons with a putative source population from USA. Samples were collected in a Paranaense forest under reserve protection, cotton fields and non-cultivated areas. Data from anonymous molecular markers were analysed using both traditional methods of population genetics and Bayesian approaches. Results help to support a previous hypothesis on the presence of two lineages of boll weevil populations in South America: one with characteristics of recent invaders and the other with characteristics of ancient populations. The sample from Urugua-í Provincial Park (Misiones, Argentina) shows the highest percentage of polymorphic loci, the highest values of mean heterozigosity, and the largest number of population-specific alleles, all being typical features of ancient populations. Furthermore, the Urugua-í sample shows two gene pools occurring in sympatry, probably as a consequence of a secondary contact. The remaining samples reveal not only lower percentages of polymorphic loci and heterozygosity values, but also an almost negligible presence of specific alleles. Bayesian methods also suggest the occasional migration of some individuals of ancient lineages from their natural habitats in fragments of the Paranaense forest into cotton fields, and vice versa.

Are flightless Galapaganus weevils older than the Galápagos Islands they inhabit?

The 15 species in the weevil genus Galapaganus Lanteri 1992 (Entiminae: Curculionidae: Coleoptera) are distributed on coastal Perú and Ecuador and include 10 flightless species endemic to the Galápagos islands. These beetles thus provide a promising system through which to investigate the patterns and processes of evolution on Darwin's archipelago. Sequences of the mtDNA locus encoding cytochrome oxidase subunit I (COI) were obtained from samples of seven species occurring in different ecological zones of the oldest south-eastern islands: San Cristóbal, Española and Floreana, and the central island Santa Cruz. The single most parsimonious tree obtained shows two well-supported clades that correspond to the species groups previously defined by morphological characters. Based on a mtDNA clock calibrated for arthropods, the initial speciation separating the oldest species, G. galapagoensis (Linell) on the oldest island, San Cristóbal, from the remaining species in the Galápagos occurred about 7.2 Ma. This estimate exceeds geological ages of the extant emerged islands, although it agrees well with molecular dating of endemic Galápagos iguanas, geckos and lizards. An apparent explanation for the disagreement between geological and molecular time-frames is that about 7 Ma there were emerged islands which subsequently disappeared under ocean waters. This hypothesis has gained support from the recent findings of 11-Myr-old submarine seamounts (sunken islands), south-east of the present location of the archipelago. Some species within the darwini group may have differentiated on the extant islands, 1-5 Ma.

Dispersal of the cotton boll weevil (Coleoptera: Curculionidae) in South America: evidence of RAPD analysis.

RAPD technique provides useful information on the geographic origin and dispersal of the boll weevil Anthonomus grandis in South America. Nine populations from Argentina, Brazil, Paraguay, Mexico and USA were analyzed. Weevils were captured on native plants (Misiones province, Argentina) and on cotton cultures, except the sample from the United States (USDA laboratory-reared colony). A sample of the 'Peruvian square weevil', A. vestitus, from Ecuador, was included in the analysis in order to compare interspecific variation. The four primers used in the analysis revealed 41 'anonymous loci'. The neighbor-joining tree based on Nei's distances and values of Nm (migrants per generation), indicate that genetic similarity between samples from Tecomán (Mexico) and Puerto Iguazú (Argentina), is higher than among remaining South American populations. This result supports an hypothesis of natural occurrence of the boll weevil in South America, prior to extensive cotton cultivation. Population outbreaks of the species would be associated with increase of agricultural lands.

Incongruence between morphological and mitochondrial-DNA characters suggests hybrid origins of parthenogenetic weevil lineages (Genus aramigus).

An expanded matrix of morphological characters for the genus Aramigus (Coleoptera: Curculionidae), which includes numerous polyploid parthenogenetic lineages, was compared and combined with a published matrix of mitochondrial DNA (mtDNA) characters. The matrix of morphological characters provides little resolution of the A. tessellatus and A. uruguayensis species complexes but does resolve previously unresolved relationships among other morphologically defined species (A. globoculus + A. intermedius, A. curtulus + A. planioculus). The morphological and mtDNA characters are significantly incongruent (0.435 < or = IM < or = 0.463; IMF = 0.0735), according to the tests of Farris et al. (P = 0.010) and Templeton (P < 0.005), probably because of hybrid origins of polyploid parthenogenetic lineages. For the few sexual lineages included in both matrices, morphology and mtDNA provide congruent estimates of phylogeny. In spite of recent injunctions against combining data sets that are incongruent because of differing histories, the results of the combined analyses were used to select one of the most-parsimonious mtDNA trees as the best estimate of maternal-lineage genealogy and to reconstruct the evolution of parthenogenesis under the assumption that transitions from sexuality to parthenogenesis are irreversible. Where cytogenetically justified, as in weevils, the irreversibility assumption is useful for producing conservative estimates of the age of parthenogenetic lineages in spite of potential sampling bias against sexuals.