Lack of mitochondrial genetic structure in hamlets (Hypoplectrus spp.): recent speciation or ongoing hybridization?

Species in the genus Hypoplectrus (hamlet fish) have been recognized primarily on the basis of colour morphology, which varies substantially. Limited differentiation in other morphological characters, however, has led to ongoing debate about their taxonomy. Our analysis of mtDNA sequences demonstrates neither reciprocal monophyly nor significant genetic differentiation among hamlet colour morphs. These data are potentially consistent with a model of recent speciation due to sexual selection on colour morphology. The presence within hamlets of two divergent mtDNA clades, however, suggests a longer history during which hybridization and gene flow have prevented the differentiation of hamlet colour morphs, at least in neutral genetic markers.     

Does intraspecific hybridization contribute to the evolution of invasiveness?: an experimental test

One of the major objectives of research on invasive species is to determine the relative importance of different evolutionary and ecological forces in the invasion process. It was recently suggested that post-introduction intraspecific hybridization between previously isolated genotypes could produce novel and/or heterotic progeny that might express enhanced invasiveness. We tested this hypothesis with Silene latifolia, a European native that has successfully invaded North America and has previously been shown to have undergone genetic change since its introduction. In a common garden experiment we compared the performance of plants derived from within and between population crosses from eight European and 18 North American populations. Results showed that there was no significant effect of crossing distance on progeny phenotype. Furthermore, progeny from within or between population crosses did not differ in size, reproductive output or survival. Collectively, these results suggest that the invasive phenotype of S. latifolia is likely the result of natural selection and/or genetic drift rather than intraspecific hybridization.     

Multiple generalist morphs of Lake Trout: Avoiding constraints on the evolution of intraspecific divergence?

A generalist strategy, as an adaptation to environmental heterogeneity, is common in Arctic freshwater systems, often accompanied, however, by intraspecific divergence that promotes specialization in niche use. To better understand how resources may be partitioned in a northern system that supports intraspecific diversity of Lake Trout, trophic niches were compared among four shallow‐water morphotypes in Great Bear Lake (N65^° 56′ 39″, W120^° 50′ 59″). Bayesian mixing model analyses of stable isotopes of carbon and nitrogen were conducted on adult Lake Trout. Major niche overlap in resource use among four Lake Trout morphotypes was found within littoral and pelagic zones, which raises the question of how such polymorphism can be sustained among opportunistic generalist morphotypes. Covariances of our morphological datasets were tested against δ¹³C and δ¹⁵N values. Patterns among morphotypes were mainly observed for δ¹⁵N. This link between ecological and morphological differentiation suggested that selection pressure(s) operate at the trophic level (δ¹⁵N), independent of habitat, rather than along the habitat‐foraging opportunity axis (δ¹³C). The spatial and temporal variability of resources in Arctic lakes, such as Great Bear Lake, may have favored the presence of multiple generalists showing different degrees of omnivory along a weak benthic–pelagic gradient. Morphs 1–3 had more generalist feeding habits using both benthic and pelagic habitats than Morph 4, which was a top‐predator specialist in the pelagic habitat. Evidence for frequent cannibalism in Great Bear Lake was found across all four morphotypes and may also contribute to polymorphism. We suggest that the multiple generalist morphs described here from Great Bear Lake are a unique expression of diversity due to the presumed constraints on the evolution of generalists and contrast with the development of multiple specialists, the standard response to intraspecific divergence.     

Critical swimming speed of sterlet (Acipenser ruthenus): Does intraspecific hybridization affect swimming performance?

We studied the effect of intraspecific hybridization on swimming performance in sterlet, hypothesizing that such hybridization increases the performance by inducing the hybrid vigor. A total of 12 purebred and hybrid crosses were reproduced from Danube (D) and Volga (V) populations of Acipenser ruthenus. Within each cross, one group of fish was exposed to temperature challenges mimicking the temperature variation in the natural environment during summer. Temperature challenges comprised a constant increase from 19°C to 24°C and then return to 19°C within 12 hr (dT<1°C/hr), and were carried out every third day over the experimental period of 20 days. As a control, fish from each cross were kept at a constant temperature of 19°C. Critical swimming speed (U_crit) was assessed on day 0 (29 days post hatch, dph), 10 (39 dph) and 20 (49 dph). The critical swimming speeds ranged from 5.12 cm/s (1.63 TL/s) to 16.44 cm/s (2.4 TL/s) during the experimental period (29–49 dph). There were no significant differences observed in U_crit between repeatedly temperature challenged and control groups, indicating that the temperature challenge did not alter the swimming performance. The critical swimming speed showed positive relationship with total body length. Comparing intraspecific hybrid crosses with purebred crosses, no significant difference in swimming performance was observed. It is thus concluded that swimming performance is a family specific trait. There is no indication that intraspecific hybridization affects swimming performance nor that close‐to‐natural temperature regimes increase swimming performance.     

Are similarity- or phylogeny-based methods more appropriate for classifying internal transcribed spacer (ITS) metagenomic amplicons?

? The internal transcribed spacer (ITS) of the nuclear ribosomal DNA region is a widely used species marker for plants and fungi. Recent metagenomic studies using next-generation sequencing, however, generate only partial ITS sequences. Here we compare the performance of partial and full-length ITS sequences with several classification methods. ? We compiled a full-length ITS data set and created short fragments to simulate the read lengths commonly recovered from current next-generation sequencing platforms. We compared recovery, erroneous recovery, and coverage for the following methods: best BLAST hit classification, MEGAN classification, and automated phylogenetic assignment using the Statistical Assignment Program (SAP). ? We found that summarizing results with more inclusive taxonomic ranks increased recovery and reduced erroneous recovery. The similarity-based methods BLAST and MEGAN performed consistently across most fragment lengths. Using a phylogeny-based method, SAP runs with queries 400 bp or longer worked best. Overall, BLAST had the highest recovery rates and MEGAN had the lowest erroneous recovery rates. ? A high-throughput ITS classification method should be selected, taking into consideration read length, an acceptable tradeoff between maximizing the total number of classifications and minimizing the number of erroneous classifications, and the computational speed of the assignment method.     

Woodiness within the Spermacoceae–Knoxieae alliance (Rubiaceae): retention of the basal woody condition in Rubiaceae or recent innovation?

Background and Aims

The tribe Spermacoceae is essentially a herbaceous Rubiaceae lineage, except for some species that can be described as ‘woody’ herbs, small shrubs to treelets, or lianas. Its sister tribe Knoxieae contains a large number of herbaceous taxa, but the number of woody taxa is higher compared to Spermacoceae. The occurrence of herbaceous and woody species within the same group raises the question whether the woody taxa are derived from herbaceous taxa (i.e. secondary woodiness), or whether woodiness represents the ancestral state (i.e. primary woodiness). Microscopic observations of wood anatomy are combined with an independent molecular phylogeny to answer this question.

Methods

Observations of wood anatomy of 21 woody Spermacoceae and eight woody Knoxieae species, most of them included in a multi-gene molecular phylogeny, are carried out using light microscopy.

Key Results

Observations of wood anatomy in Spermacoceae support the molecular hypothesis that all the woody species examined are secondary derived. Well-known wood anatomical characters that demonstrate this shift from the herbaceous to the woody habit are the typically flat or decreasing length vs. age curves for vessel elements, the abundance of square and upright ray cells, or even the (near-) absence of rays. These so-called paedomorphic wood features are also present in the Knoxieae genera Otiophora, Otomeria, Pentas, Pentanisia and Phyllopentas. However, the wood structure of the other Knoxieae genera observed (Carphalea, Dirichletia and Triainolepis) is typical of primarily woody taxa.

Conclusions

In Spermacoceae, secondary woodiness has evolved numerous times in strikingly different habitats. In Knoxieae, there is a general trend from primary woodiness towards herbaceousness and back to (secondary) woodiness.Key words: Knoxieae, LM, primary woodiness, Rubiaceae, Rubioideae, secondary woodiness, Spermacoceae, wood anatomy     

Ecological variation within Sellaphora species complexes (Bacillariophyceae): specialists or generalists?

The genus Sellaphora has become a model system for studies of the species concept, speciation and automated identification in diatoms. Three species, S. pupula, S. bacillum and S. laevissima, have proved to be complexes containing several or many species, which are difficult to distinguish morphologically but which are genetically differentiated and (where tested) reproductively isolated. Until now, however, there has been little information about the ecology of the species within this complex, except in relation to parasite sensitivity. In order to test whether semi- and pseudo-cryptic Sellaphora species are ecologically differentiated with respect to trophic status, we used tools recently developed in the UK in response to the EU Water Framework Directive (WFD). Diatom samples from three substrata (plants, rocks, sediment) were taken from 22 lakes in Scotland and England, covering a gradient from oligotrophic mountain lakes to eutrophic lowland ponds. The epilithic and epiphytic diatom assemblages were used to evaluate lake trophic status according to the UK WFD assessment system and showed that there was a strong environmental gradient in the dataset. Sellaphora species occurred in the sediment-derived epipelon and their distributions were analysed in relation to the trophic gradient. A total of 28 Sellaphora phenodemes (putative species) were found, and some of them differed in their environmental demands. Two groups were distinguished: (1) a group indicating rather oligotrophic conditions and containing several demes with linear valves and subcapitate or capitate poles (referred to here as S. [pupula] cap-A, cap-B and cap-C and (2) a group occurring in eutrophic lakes and containing the recently described species S. blackfordensis, S. capitata and S. obesa, as well as S. [pupula] Φ ‘small lanceolate’. The data obtained are also discussed with respect to Finlay’s hypothesis on microalgal cosmopolitanism. Handling editor: J. Padisak     

Species divergence in offspring begging intensity: difference in need or manipulation of parents?

Conflicts over the delivery and sharing of food among family members are expected to lead to evolution of exaggerated offspring begging for food. Coevolution between offspring begging intensity and parent response depends on the genetic architecture of the traits involved. Given a genetic correlation between offspring begging intensity and parental response, there may be fast and arbitrary divergence in these behaviours between populations. However, there is limited knowledge about the genetic basis of offspring solicitation and parental response and whether these traits are genetically correlated. In this study, we performed a partial cross-fostering experiment of young between pied and collared flycatchers (Ficedula hypoleuca and Ficedula albicollis) and recorded the behaviour of individual offspring and their (foster)parents. We found that nestling collared flycatchers reached a higher phenotypic quality, estimated both as mass at fledging and as intensity of their T-lymphocyte-mediated immune response when raised by heterospecific foster parents. However, although collared flycatchers begged relatively more intensively, we found no evidence of corresponding higher resistance (i.e. lower feeding rate) of adult collared flycatchers than of adult pied flycatchers. Thus, the difference in offspring begging intensity between the two species seems not to be a result of a difference in escalation of the parent-offspring conflict. Instead, the species' divergence in exaggeration of offspring begging intensity 'honestly' matches a difference between the species in offspring need. This interpretation is strengthened by the fact that the difference in begging intensity between the two species increased as the season progressed, coinciding with the higher sensitivity of nestling collared flycatchers to the seasonal decline in food availability. Thus, the behavioural differentiation appears to be a direct consequence of a life-history differentiation (offspring growth patterns).     

“Forms” of water mites (Acari: Hydrachnidia): intraspecific variation or valid species?

In many groups of organisms, especially in the older literature, it has been common practice to recognize sympatrically occurring phenotypic variants of a species as “forms”. However, what these forms really represent often remains unclear, especially in poorly studied groups. With new algorithms for DNA‐based species delimitation, the status of forms can be explicitly tested with molecular data. In this study, we test a number of what is now recognized as valid species of water mites (Hydrachnidia), but have in the past been treated as forms sympatrically occurring with their nominate species. We also test a form without prior taxonomical status, using DNA and morphometrics. The barcoding fragment of COI, nuclear 28S and quantitative analyses of morphological data were used to test whether these taxa merit species status, as suggested by several taxonomists. Our results confirm valid species. Genetic distances between the form and nominate species (Piona dispersa and Piona variabilis, COI 11%), as well as likelihood ratio tests under the general mixed‐Yule coalescent model, supported that these are separately evolving lineages as defined by the unified species concept. In addition, they can be diagnosed with morphological characters. The study also reveals that some taxa genetically represent more than one species. We propose that P. dispersa are recognized as valid taxa at the species level. Unionicola minor (which may consist of several species), Piona stjordalensis, P. imminuta s. lat., and P. rotundoides are confirmed as species using this model. The results also imply that future studies of other water mite species complexes are likely to reveal many more genetically and morphologically distinct species.     

Population structure of the pumpkin fruit fly Bactrocera depressa (Tephritidae) in Korea and Japan: Pliocene allopatry or recent invasion?

Because of their widespread agricultural impact and rapid range expansions, true fruit flies (Tephritidae) are the subject of quarantine and control efforts worldwide. Among these flies, the pumpkin fruit fly Bactrocera depressa, which infests squash and other cucurbitaceous plants in Korea, Japan and Taiwan, was recently isolated from produce shipments entering Japan and identified as a regulatory target. This species was described in 1933 from collections in Japan and discovered in 1974 in Korea, suggesting that it may have recently invaded mainland Asia. We analysed the genetic structure of Asian populations of B. depressa using sequence variation for mitochondrial gene cytochrome-oxidase I and three nuclear loci: elongation factor 1alpha, tubulinbeta1 and tubulinbeta3, using frequency-based approaches, nested clade analysis and assignment tests. Contrary to the hypothesis of recent invasion, high levels of genetic subdivision were found among five Korean and three Japanese populations. Nested clade analysis suggested a variety of processes operating over different time scales, including ancient isolation between Korea and Japan and more recent range expansions within each country. Contrary to a priori expectations, the results also suggested the recent introduction of a mitochondrial haplotype into Yokohama, Japan that is related closely to a widespread haplotype found throughout Korea. Assignment tests also supported these conclusions. The combination of a genealogical approach and probabilistic assignments of individuals to populations of origin was able to provide statistical support for the identification of cryptic introductions within an otherwise widespread indigenous species.     

Incipient genetic divergence or cryptic speciation? Procamallanus (Nematoda) in freshwater fishes (Astyanax)

Hosts provide the main environmental traits parasites have to deal with, resulting in covariation between both associates at both micro- and macro-evolutionary scales; phylogenetic analyses of highly host-specific parasites have shown that parasite and host phylogeny might be highly congruent, and adaptation of a host species to new environments may lead to concordant changes of their parasites. Procamallanus (Spirocamallanus) neocaballeroi is a highly host-specific parasitic nematode of the Neotropical freshwater fish genus Astyanax in Mexico. One of the host species of the nematode is the emblematic Mexican tetra, A. mexicanus, which exhibits two contrasting phenotypes, a cave-dwelling morph (with troglomorphic features), and the surface-dwelling morph; other congeneric species inhabit rivers and lakes, and some of them occur in sympatry, displaying trophic specializations. Here, we explored the hypothesis that contrasting environments (surface rivers vs cave rivers), and host morphological divergence (sympatric ecomorphs in a lacustrine environment) might result in the divergence of their parasites, even though the hosts maintain a cohesive genetic structure as the same species. To test the hypothesis, several populations of Astyanax spp. were sampled to search for P. (S.) neocaballeroi. The nematode was found in 10 of the 52 sampled sites; two localities corresponded to cave populations. The phylogenetic analysis based on COI sequences yielded three major lineages for P. (S.) neocaballeroi. We found no concordance between the three lineages and the habitat where they occur in Astyanax mexicanus, even considering those living in drastic environmental conditions (caves), or between these lineages and lacustrine ecomorphs of Astyanax aeneus and A. caballeroi occurring in sympatry. Instead, genetic lineages of the nematode exhibit a clearer pattern of host species association and geographical distribution; our results showed that P. (S.) neocaballeroi is experiencing an incipient divergence although the morphological study of lineages shows no conspicuous differences.     

The evolution of early animal embryos: conservation or divergence?

There is a remarkable similarity in the appearance of groups of animal species during periods of their embryonic development. This classic observation has long been viewed as an emphatic realization of the principle of common descent. Despite the importance of embryonic conservation as a unifying concept, models seeking to predict and explain different patterns of conservation have remained in contention. Here, we focus on early embryonic development and discuss several lines of evidence, from recent molecular data, through developmental networks to life-history strategies, that indicate that early animal embryos are not highly conserved. Bringing this evidence together, we argue that the nature of early development often reflects adaptation to diverse ecological niches. Finally, we synthesize old and new ideas to propose a model that accounts for the evolutionary process by which embryos have come to be conserved.     

Geographic variation in Mexican jays (Aphelocoma ultramarina): local differentiation,polyphyly or hybridization?

Studies of genetic variation within highly variable taxa can provide valuable insight into the factors influencing biological diversification. We examined six microsatellite loci, a nuclear intron and the mitochondrial control region to determine if the Mexican jay subspecies Aphelocoma ultramarina couchii and A. u. potosina have hybridized with western scrub-jays (A. californica). We suspected hybridization because these Mexican Jay populations resemble scrub-jays in several traits. We sampled six Mexican jay (N = 105) and four scrub-jay (N = 78) populations. Suspected hybrid Mexican Jay populations did not share any mitochondrial types or intron alleles with scrub-jays. All microsatellite alleles found in the suspected hybrid Mexican jay populations are also found in the control Mexican jay populations. Genetic distance-based trees from microsatellites supported reciprocal monophyly of Mexican jays and scrub-jays with bootstrap support > 80%. We randomized genotypes among populations to test scenarios consistent with hybridization. In some areas where Mexican jays and scrub-jays occur in sympatry or geographical proximity, randomization tests yield results expected under hybridization (100% support for seven of nine scenarios). However, these populations were not the primary candidates for hybridization based on phenotype. Even if low-level hybridization did occur, hybridization does not appear to be the main reason some Mexican jay populations resemble scrub-jays more than others. The scrub-jay-like traits in these populations may be due to drift, adaptation or plasticity. Alternatively, ancient hybridization, followed by selection for scrub-jay like traits in some Mexican jay populations, might have given rise to the observed variation.     

Genetic divergence of the seminal signal-receptor system in houseflies: the footprints of sexually antagonistic coevolution?

To understand fully the significance of cryptic female choice, we need to focus on each of those postmating processes in females which create variance in fitness among males. Earlier studies have focused almost exclusively on the proportion of a female's eggs fertilized by different males (sperm precedence). Yet, variance in male postmating reproductive success may also arise from differences in ability to stimulate female oviposition and to delay female remating. Here, we present a series of reciprocal mating experiments among genetically differentiated wild-type strains of the housefly Musca domestica. We compared the effects of male and female genotype on oviposition and remating by females. The genotype of each sex affected both female oviposition and remating rates, demonstrating that the signal-receptor system involved has indeed diverged among these strains. Further, there was a significant interaction between the effects of male and female genotype on oviposition rate. We discuss ways in which the pattern of such interactions provides insights into the coevolutionary mechanism involved. Females in our experiments generally exhibited the weakest, rather than the strongest, response to males with which they are coevolved. These results support the hypothesis that coevolution of male seminal signals and female receptors is sexually antagonistic.     

Russian wheat aphids (Diuraphis noxia) in China: native range expansion or recent introduction?

In this study, we explore the population genetics of the Russian wheat aphid (RWA) (Diuraphis noxia), one of the world's most invasive agricultural pests, in north-western China. We have analysed the data of 10 microsatellite loci and mitochondrial sequences from 27 populations sampled over 2 years in China. The results confirm that the RWAs are holocyclic in China with high genetic diversity indicating widespread sexual reproduction. Distinct differences in microsatellite genetic diversity and distribution revealed clear geographic isolation between RWA populations in northern and southern Xinjiang, China, with gene flow interrupted across extensive desert regions. Despite frequent grain transportation from north to south in this region, little evidence for RWA translocation as a result of human agricultural activities was found. Consequently, frequent gene flow among northern populations most likely resulted from natural dispersal, potentially facilitated by wind currents. We also found evidence for the long-term existence and expansion of RWAs in China, despite local opinion that it is an exotic species only present in China since 1975. Our estimated date of RWA expansion throughout China coincides with the debut of wheat domestication and cultivation practices in western Asia in the Holocene. We conclude that western China represents the limit of the far eastern native range of this species. This study is the most comprehensive molecular genetic investigation of the RWA in its native range undertaken to date and provides valuable insights into the history of the association of this aphid with domesticated cereals and wild grasses.     

Extensive color polymorphism in the southeast Asian oriental dwarf kingfisher Ceyx erithaca: a result of gene flow during population divergence?

Extensive plumage color polymorphism in the oriental dwarf kingfisher Ceyx erithaca has long intrigued ornithologists of the Indo–Malayan region. A large proportion of birds in Sumatra, Peninsular Malaysia and Borneo have plumages intermediate between the northerly black form and the southerly rufous form. We used molecular genetic techniques to investigate whether the pattern is likely caused by selection, contemporary hybridization or past introgression of genes. These data consist of DNA sequences of a mitochondrial gene (ND2) and five nuclear intronic loci. Phylogenetic analyses indicated that birds from continental southeast Asia (excluding the Thai–Malay Peninsula) are well differentiated from those from insular southeast Asia. This genetic distinction correlates well with a fixed difference in mantle coloration. Northern birds have black mantles and are consistently dark; southern birds, though highly variable in parts of the plumage, consistently possess a rusty brown mantle. Multivariate analyses of morphometric data also support clustering birds in the two groups defined by mantle coloration. Coalescent analyses suggest that gene flow occurred after initial population splitting. We hypothesized that the dynamic geography of the Indo–Malayan Archipelago, as driven by eustatic sea‐level changes related to glacial cycles, is responsible for extensive secondary contact, and gene flow, between the two color forms. Such periodic contacts, after initial separation of the populations, may be equally common in other geographically dynamic regions of the world.     

Does damming of the Colorado River affect the nursery area of blue shrimp Litopenaeus stylirostris (Decapoda: Penaeidae) in the Upper Gulf of California?

After damming the Colorado River the freshwater flow was reduced to 1% of its virgin flow to the Upper Gulf of California (UGC). The ecological effects need to be properly documented. The UGC is the nursery area for Litopenaeus stylirostris, the most profitable fishery in the zone. In order to know the relative abundance of L. stylirostris postlarval stage we conducted a sampled survey every 14 days in 1993, 1994 and 1997, plus an intensive sampling during a complete tide cycle in July 1995 and 1996. We did 10 min trawls each hour during the flood tide. Relative abundance of postlarvae was higher (p < 0.05) in those years when freshwater flow reached the UGC.     

A willow drawing from 1786: the earliest depiction of intraspecific trait variation in plants?

Background and AimsThe study of intraspecific trait variation (ITV) in plants has a long history, dating back to the fourth century BC. Its existence was widely acknowledged by the end of the 18th century, although systematic and experimental studies commenced only a century later. However, the historiography of ITV has many gaps, especially with regard to early observations and visual documents. This note identifies an early depiction of plant ITV.MethodsThe botanical works of Johann Wolfgang von Goethe (1749–1832), a German writer and naturalist, were subjected to close reading. This included all publications and unpublished sources related to botany between 1785 and 1832 (e.g. notes, drafts, diaries, letters, drawings). This material is accessible in the multi-volume historical-critical edition of Goethe’s studies in natural science (Leopoldina-Ausgabe).Key ResultsA diary entry from 9 September 1786 described changes in leaf morphology along an elevation gradient in the Bavarian Alps. The leaves of an unidentified species of willow (Spix sp.) and gentian (Gentiana sp.) were said to become narrower with increasing elevation; leaves also stood further apart on twigs, and the latter became thinner. A crude drawing of two willow twigs illustrated the differences. Goethe conjectured that the differences were due to environmental conditions.ConclusionsGoethe’s notes were anecdotal, and it is unclear whether the observed plant individuals actually belonged to the same species. Nevertheless, the notes represent an early and clear articulation of the hypothesis that changes in environmental conditions can cause ITV in a natural plant population. The drawing may be the earliest visual record of environmentally caused plant ITV in the wild.     

Genetic Differentiation in Tetranychus Urticae (Acari: Tetranychidae): polymorphism,host races or sibling species?

Based on allozyme electrophoresis at the Pgm locus and nuclear ribosomal DNA (ITS2) sequences, we studied the genetic variation of the two-spotted spider mite Tetranychus urticae Koch collected on rose bay, Nerium oleander L. (Apocynaceae), from several localities around the Mediterranean basin. In addition, we compared these results with those of Navajas et al. (1998) and Tsagkarakou (1997) who collected from several other host plants from the Mediterranean. In the western part of this area (Spain, France, Tunisia), we found the individuals collected from rose bay to be clearly genetically differentiated from other samples. No evidence of such host-associated differentiation was detected in the eastern Mediterranean (Italy and Greece). The genetic differentiation of mites collected on rose bay was investigated further by studying the reproductive incompatibilities between populations in Greece and in France and a laboratory strain reared on bean, Phaseolus vulgaris, in France. Reciprocal crosses performed between these strains revealed variable levels of incompatibility, spanning from partial to complete reproductive isolation. In all cases incompatibility was asymmetric. We designed a test based on double-mating to establish the fertilization status of females in fully incompatible crosses. These crosses showed that the females had been inseminated, which suggests that the barrier to reproduction is not of a prezygotic behavioral nature. The data raises the question of the relative role of ecological factors (host plant) and geographical distance, in the ongoing differentiation process potentially leading to speciation.