Incipient allochronic speciation in the pine processionary moth (Thaumetopoea pityocampa, Lepidoptera, Notodontidae)

A plausible case of allochronic differentiation, where barrier to gene flow is primarily attributable to a phenological shift, was recently discovered in Portugal for the pine processionary moth Thaumetopoea pityocampa. Previous results suggested that the observed 'summer population' (SP) originated from the sympatric winter population (WP). Our objectives were to finely analyse these patterns and test their stability in time, through field monitoring and genetic analyses of larvae and adults across different years. Reproductive activity never overlapped between SP and WP. Microsatellites showed a clear differentiation of the SP, consistent with a strong reduction in gene flow owing to the phenological shift. Assignment tests suggested that some individuals shift from the SP to the WP phenology, causing some hybridization. We discuss these patterns and their maintenance over time. This could be a first stage of allochronic speciation, and SP should be considered as a distinct phenological race.     

Deciphering the demographic history of allochronic differentiation in the pine processionary moth Thaumetopoea pityocampa

Understanding the processes of adaptive divergence, which may ultimately lead to speciation, is a major question in evolutionary biology. Allochronic differentiation refers to a particular situation where gene flow is primarily impeded by temporal isolation between early and late reproducers. This process has been suggested to occur in a large array of organisms, even though it is still overlooked in the literature. We here focused on a well‐documented case of incipient allochronic speciation in the winter pine processionary moth Thaumetopoea pityocampa. This species typically reproduces in summer and larval development occurs throughout autumn and winter. A unique, phenologically shifted population (SP) was discovered in 1997 in Portugal. It was proved to be strongly differentiated from the sympatric “winter population” (WP), but its evolutionary history could only now be explored. We took advantage of the recent assembly of a draft genome and of the development of pan‐genomic RAD‐seq markers to decipher the demographic history of the differentiating populations and develop genome scans of adaptive differentiation. We showed that the SP diverged relatively recently, that is, few hundred years ago, and went through two successive bottlenecks followed by population size expansions, while the sympatric WP is currently experiencing a population decline. We identified outlier SNPs that were mapped onto the genome, but none were associated with the phenological shift or with subsequent adaptations. The strong genetic drift that occurred along the SP lineage certainly challenged our capacity to reveal functionally important loci.     

Emergence of novel fungal pathogens by ecological speciation: importance of the reduced viability of immigrants

Expanding global trade and the domestication of ecosystems have greatly accelerated the rate of emerging infectious fungal diseases, and host-shift speciation appears to be a major route for disease emergence. There is therefore an increased interest in identifying the factors that drive the evolution of reproductive isolation between populations adapting to different hosts. Here, we used genetic markers and cross-inoculations to assess the level of gene flow and investigate barriers responsible for reproductive isolation between two sympatric populations of Venturia inaequalis, the fungal pathogen causing apple scab disease, one of the fungal populations causing a recent emerging disease on resistant varieties. Our results showed the maintenance over several years of strong and stable differentiation between the two populations in the same orchards, suggesting ongoing ecological divergence following a host shift. We identified strong selection against immigrants (i.e. host specificity) from different host varieties as the strongest and likely most efficient barrier to gene flow between local and emerging populations. Cross-variety disease transmission events were indeed rare in the field and cross-inoculation tests confirmed high host specificity. Because the fungus mates within its host after successful infection and because pathogenicity-related loci prevent infection of nonhost trees, adaptation to specific hosts may alone maintain both genetic differentiation between and adaptive allelic combinations within sympatric populations parasitizing different apple varieties, thus acting as a 'magic trait'. Additional intrinsic and extrinsic postzygotic barriers might complete reproductive isolation and explain why the rare migrants and F1 hybrids detected do not lead to pervasive gene flow across years.     

Genetic isolation through time: allochronic differentiation of a phenologically atypical population of the pine processionary moth

Allochronic speciation refers to a mode of sympatric speciation in which the differentiation of populations is primarily due to a phenological shift without habitat or host change. However, it has been so far rarely documented. The present paper reports on a plausible case of allochronic differentiation between sympatric populations of the pine processionary moth (PPM), Thaumetopoea pityocampa. The PPM is a Mediterranean insect with winter larval development. A phenologically atypical population with early adult activity and summer larval development was detected 10 years ago in Portugal. Mitochondrial and nuclear sequences strongly suggest that the 'summer' individuals are closely related to the sympatric winter population, while microsatellite data show a reduction in allelic richness, a distortion of allelic frequencies and significant genetic differentiation. Moreover, monitoring of adult flights suggests that reproductive activity does not overlap between the summer and winter populations. We postulate that the summer population appeared after a sudden phenological shift of some individuals of the sympatric winter population, leading to a founder effect and complete reproductive isolation. Given that the individuals showing this new phenology are subject to different selection pressures, the observed allochronic differentiation may rapidly lead to deeper divergence.     

Incipient allochronic speciation by climatic disruption of the reproductive period

Disruptive selection of life-cycle timing may cause temporal isolation directly and, ultimately, allochronic speciation. Despite the fact that segregation of the reproductive period among related species has been broadly observed across taxa, it remains controversial whether temporal isolation can function as the primary process of speciation. In the Japanese winter geometrid moth Inurois punctigera, allochronic divergence has resulted from climatic disruption of the reproductive period. In habitats with severe midwinter, two sympatric groups of moth reproduce allochronically in early and late winter. These groups are genetically diverging sister lineages and now co-occur allochronically throughout Japan. By contrast, in habitats with milder midwinter these lineages form a continuous adult period and gene flow has been facilitated between the lineages. These results, together with the fact that there is no difference in larval host use, indicate that temporal isolation has been the sole mechanism for allochronic isolation in colder habitats and that allochrony is not a by-product of other adaptations. Thus, the allochronic divergence of sympatric I. punctigera populations represents an incipient speciation process driven by midwinter disruption of the reproductive period.     

Temperature niche shift observed in a Lepidoptera population under allochronic divergence

A process of adaptive divergence for tolerance to high temperatures was identified using a rare model system, consisting of two sympatric populations of a Lepidoptera (Thaumetopoea pityocampa) with different life cycle timings, a 'mutant' population with summer larval development, Leiria SP, and the founder natural population, having winter larval development, Leiria WP. A third, allopatric population (Bordeaux WP) was also studied. First and second instar larvae were experimentally exposed to daily-cycles of heat treatment reaching maximum values of 36, 38, 40 and 42 °C; control groups placed at 25 °C. A lethal temperature effect was only significant at 42 °C, for Leiria SP, whereas all temperatures tested had a significant negative effect upon Leiria WP, thus indicating an upper threshold of survival c.a. 6 °C above that of the WP. Cox regression model, for pooled heat treatments, predicted mortality hazard to increase for Leiria WP (+108%) and Bordeaux WP (+78%) in contrast to Leiria SP; to increase by 24% for each additional °C; and to decrease by 53% from first to second instar larvae. High variability among individuals was observed, a population characteristic that may favour selection and consequent adaptation. Present findings provide an example of ecological differentiation, following a process of allochronic divergence. Results further contribute to a better understanding of the implications of climate change for ecological genetics.     

Experimental evidence for heritable reproductive time in 2 allochronic populations of pine processionary moth

Phenology allows organisms to overcome seasonally variable conditions through life‐cycle adjustment. Changes in phenology can drastically modify the evolutionary trajectory of a population, while a shift in the reproductive time may cause allochronic differentiation. The hypothesis of heritable reproductive time was experimentally tested, by studying a unique population of the pine processionary moth Thaumetopoea pityocampa (Den. & Schiff.) which has a shifted phenology, and however co‐occurs with the typical population following the classical life cycle. When populations of both types were reared under controlled conditions, the reproductive time was maintained asynchronous, as observed in the field. The shifted population was manipulated in the laboratory to reproduce later than usual, yet the offspring emerged in the next year at the expected dates thus “coming back” to the usual cycle. Hybrids from crosses performed between the 2 populations showed an intermediate phenology. From the emergence times of parents and offspring, a high heritability of the reproductive time (h = 0.76) was observed. The offspring obtained from each type of cross was genetically characterized using microsatellite markers. Bayesian clustering analysis confirmed that hybrids can be successfully identified and separated from the parental genetic classes by genotyping. Findings support the hypothesis that, for this particular population, incipient allochronic speciation is due to a heritable shift in the reproductive time that further causes assortative mating and might eventually cause ecological adaptation/maladaptation in response to environmental changes.     

Formation of a fluvial non-parasitic population of Lethenteron camtschaticum as the first step in petromyzontid speciation

To elucidate the petromyzontid speciation process, the genetic independence of the fluvial non-parasitic populations within the anadromous parasitic Lethenteron camtschaticum was estimated by using polymorphic microsatellite loci. Abundant gene flow was revealed in multitemporal scales between potentially sympatric populations, suggesting ongoing gene flow resulting from imperfect size-assortative mating between them and plastic determination of life histories. On the contrary, landlocked fluvial non-parasitic populations in the upper region of dams were genetically divergent from anadromous parasitic populations. The temporal heterogeneity of gene flow, i.e. contemporary little gene flow but significant gene flow over the long-term between the landlocked fluvial non-parasitic and anadromous parasitic populations was elucidated. In addition, the divergence time of isolation of the landlocked populations from the ancestral anadromous parasitic population was estimated to have occurred 17.9-428.2 years ago, which includes the construction times of an initial dam c. 90 years ago. These instances indicate that the landlocked populations should have very recently been established, and subsequent accumulation of divergence and development of reproductive isolation are predicted. The present landlocked fluvial non-parasitic populations should be analogous to the founder populations in terms of petromyzontid speciation. The data also strongly support the hypothesis of multitemporal and multispatial speciation in the petromyzontid stem-satellite species complex.     

Phenotypic divergence in reproductive traits of a moth population experiencing a phenological shift

Allochrony that is reproductive isolation by time may further lead to divergence of reproductive adaptive traits in response to different environmental pressures over time. A unique “summer” population of the pine processionary moth Thaumetopoea pityocampa, reproductively isolated from the typical winter populations by allochronic differentiation, is here analyzed. This allochronically shifted population reproduces in the spring and develops in the summer, whereas “winter” populations reproduce in the late summer and have winter larval development. Both summer and winter populations coexist in the same pine stands, yet they face different climatic pressures as their active stages are present in different seasons. The occurrence of significant differences between the reproductive traits of the summer population and the typical winter populations (either sympatric or allopatric) is thus hypothesized. Female fecundity, egg size, egg covering, and egg parasitism were analyzed showing that the egg load was lower and that egg size was higher in the summer population than in all the studied winter populations. The scales that cover the egg batches of T. pityocampa differed significantly between populations in shape and color, resulting in a looser and darker covering in the summer population. The single specialist egg parasitoid species of this moth was almost missing in the summer population, and the overall parasitism rates were lower than in the winter population. Results suggest the occurrence of phenotypic differentiation between the summer population and the typical T. pityocampa winter populations for the life‐history traits studied. This work provides an insight into how ecological divergence may follow the process of allochronic reproductive isolation.     

Nuclear DNA recapitulates the cryptic mitochondrial lineages of Lumbricus rubellus and suggests the existence of cryptic species in an ecotoxological soil sentinel

Mitochondrial DNA analysis has revealed two distinct phylogenetic lineages within the ecotoxological sentinel earthworm model Lumbricus rubellus Hoffmeister, 1843. The existence of these lineages could complicate ecotoxicological studies that use the species as a sentinel for soil contamination testing, as they may respond differently to contamination; however, as mitochondrial haplotypes are not always expected to segregate in the same way as chromosomal DNA in natural populations, we further investigated this issue by using nuclear DNA markers (microsatellites) to measure genetic diversity, differentiation, and gene flow in sympatric populations of the two L. rubellus lineages at two sites in South Wales. Our results show that sympatric populations of the two lineages are more genetically differentiated than geographically distant populations of the same lineage, and Bayesian clustering analysis revealed no evidence of gene flow between the lineages at either site. Additionally, DNA sequencing of these microsatellite loci uncovered substantial differentiation between lineages at homologous flanking regions. Overall our findings indicate a high degree of nuclear genetic differentiation between the two lineages of L. rubellus, implying reproductive isolation at the two study sites and therefore the potential existence of cryptic species. The existence of two cryptic taxa has major implications for the application of L. rubellus as an ecotoxicological sentinel. It may therefore be necessary to consider the lineages as separate taxa during future ecotoxicological studies. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 780–795.     

Disentangling structural genomic and behavioural barriers in a sea of connectivity

Genetic divergence among populations arises through natural selection or drift and is counteracted by connectivity and gene flow. In sympatric populations, isolating mechanisms are thus needed to limit the homogenizing effects of gene flow to allow for adaptation and speciation. Chromosomal inversions act as an important mechanism maintaining isolating barriers, yet their role in sympatric populations and divergence with gene flow is not entirely understood. Here, we revisit the question of whether inversions play a role in the divergence of connected populations of the marine fish Atlantic cod (Gadus morhua), by exploring a unique data set combining whole‐genome sequencing data and behavioural data obtained with acoustic telemetry. Within a confined fjord environment, we find three genetically differentiated Atlantic cod types belonging to the oceanic North Sea population, the western Baltic population and a local fjord‐type cod. Continuous behavioural tracking over 4 year revealed temporally stable sympatry of these types within the fjord. Despite overall weak genetic differentiation consistent with high levels of gene flow, we detected significant frequency shifts of three previously identified inversions, indicating an adaptive barrier to gene flow. In addition, behavioural data indicated that North Sea cod and individuals homozygous for the LG12 inversion had lower fitness in the fjord environment. However, North Sea and fjord‐type cod also occupy different depths, possibly contributing to prezygotic reproductive isolation and representing a behavioural barrier to gene flow. Our results provide the first insights into a complex interplay of genomic and behavioural isolating barriers in Atlantic cod and establish a new model system towards an understanding of the role of genomic structural variants in adaptation and diversification.     

Phylogenetic and molecular evidence for allochronic speciation in gall-forming aphids (Pemphigus)

Sympatric populations can diverge when variation in phenology or life cycle causes them to mate at distinctly different times. We report patterns consistent with this process (allochronic speciation) in North American gall-forming aphids, in the absence of a host or habitat shift. Pemphigus populi-transversus Riley and P. obesinymphae Aoki form a monophyletic clade within the North American Pemphigus group. They are sympatric on the eastern cottonwood, Populus deltoides (Salicaceae), but have distinctly different life cycles, with sexual stages offset by approximately six months. Field evidence indicates that intermediate phenotypes do not commonly occur, and mitochondrial and bacterial endosymbiont DNA sequences show no maternal gene flow between the two species. Because a genetically distinct population of P. obesinymphae occurs in the southwestern United States on Populus fremontii, we consider the possibility of an initial allopatric phase in the divergence. We discuss the likely origins of the host use patterns in P. obesinymphae, and the larger sequence of evolutionary changes that likely led to the sympatric divergence of P. populi-transversus and P. obesinymphae. A plausible interpretation at this stage of investigation is that a shift in timing of the life cycle in an ancestral population, correlated with an underlying phenological complexity in its host plant, spurred divergence between the incipient species.     

Low reproductive isolation and highly variable levels of gene flow reveal limited progress towards speciation between European river and brook lampreys

Ecologically based divergent selection is a factor that could drive reproductive isolation even in the presence of gene flow. Population pairs arrayed along a continuum of divergence provide a good opportunity to address this issue. Here, we used a combination of mating trials, experimental crosses and population genetic analyses to investigate the evolution of reproductive isolation between two closely related species of lampreys with distinct life histories. We used microsatellite markers to genotype over 1000 individuals of the migratory parasitic river lamprey (Lampetra fluviatilis) and freshwater‐resident nonparasitic brook lamprey (Lampetra planeri) distributed in 10 sympatric and parapatric population pairs in France. Mating trials, parentage analyses and artificial fertilizations demonstrated a low level of reproductive isolation between species even though size‐assortative mating may contribute to isolation. Most parapatric population pairs were strongly differentiated due to the joint effects of geographic distance and barriers to migration. In contrast, we found variable levels of gene flow between sympatric populations ranging from panmixia to moderate differentiation, which indicates a gradient of divergence with some population pairs that may correspond to alternative morphs or ecotypes of a single species and others that remain partially isolated. Ecologically based divergent selection may explain these variable levels of divergence among sympatric population pairs, but incomplete genome swamping following secondary contact could have also played a role. Overall, this study illustrates how highly differentiated phenotypes can be maintained despite high levels of gene flow that limit the progress towards speciation.     

Prominent intraspecific genetic divergence within Anopheles gambiae sibling species triggered by habitat discontinuities across a riverine landscape

The Anopheles gambiae complex of mosquitoes includes malaria vectors at different stages of speciation, whose study enables a better understanding of how adaptation to divergent environmental conditions leads to evolution of reproductive isolation. We investigated the population genetic structure of closely related sympatric taxa that have recently been proposed as separate species (An. coluzzii and An. gambiae), sampled from diverse habitats along the Gambia river in West Africa. We characterized putatively neutral microsatellite loci as well as chromosomal inversion polymorphisms known to be associated with ecological adaptation. The results revealed strong ecologically associated population subdivisions within both species. Microsatellite loci on chromosome‐3L revealed clear differentiation between coastal and inland populations, which in An. coluzzii is reinforced by a unusual inversion polymorphism pattern, supporting the hypothesis of genetic divergence driven by adaptation to the coastal habitat. A strong reduction of gene flow was observed between An. gambiae populations west and east of an extensively rice‐cultivated region apparently colonized exclusively by An. coluzzii. Notably, this ‘intraspecific’ differentiation is higher than that observed between the two species and involves also the centromeric region of chromosome‐X which has previously been considered a marker of speciation within this complex, possibly suggesting that the two populations may be at an advanced stage of differentiation triggered by human‐made habitat fragmentation. These results confirm ongoing ecological speciation within these most important Afro‐tropical malaria vectors and raise new questions on the possible effect of this process in malaria transmission.     

Genetic variation and structuring in the threatened koala populations of Southeast Queensland

Habitat fragmentation can act to cause reproductive isolation between conspecifics and undermine species’ persistence, though most studies have reported the genetic condition of populations that have already declined to a very small size. We examined genetic diversity within the vulnerable, declining koala (Phascolarctos cinereus) population in Southeast Queensland, Australia to determine the genetic impact of ongoing threatening processes. Five hundred and twelve koalas from ten Southeast Queensland Local Government Areas on the mainland and one island were genotyped at six polymorphic microsatellite loci. Based on Bayesian cluster analysis incorporating spatial data, the regional koala population was subdivided into six clusters, with location of major roads and rivers appearing to be consistent with being barriers to gene flow. The distribution of mtDNA control region haplotypes identified distinct coastal and inland clades suggesting that historically there was gene flow between koalas along the coast (though little interchange between coastal and inland animals). In contrast, koalas from the Koala Coast (Brisbane City, Logan City and Redland Shire) were shown by microsatellite analysis to be genetically distinct from adjacent areas. It is likely, therefore, that more recent reductions in population size and restricted gene flow through urbanisation have contributed to the genetic differentiation of koalas in the Koala Coast region.     

GENETIC EVIDENCE FOR REPRODUCTIVE ISOLATION AND MULTIPLE ORIGINS OF SYMPATRIC TROPHIC ECOTYPES OF WHITEFISH (COREGONUS)

We assessed variation in mitochondrial DNA (mtDNA) by restriction fragment length polymorphism (RFLP) analysis and in nuclear genes by allozyme analysis among sympatric pairs of limnetic and benthic ecotypes of whitefish (Coregonus) coexisting in three lakes of southern Yukon to address three evolutionary questions regarding their origins. Are sympatric low and high gill-raker count ecotypes genetically differentiated? Are they issued from monophyletic or polyphyletic evolutionary events? If they are polyphyletic in origins, did they originate from multiple allopatric speciation events or intralacustrine radiation? Our results corroborated previous genetic and ecological studies of these ecotypes, indicating that they represent genetically distinct reproductive units, and therefore refuting the hypothesis of phenotypic polymorphism within a single population. However, the amount of gene flow between ecotypes varied among lakes, correlating with the extent of morphological differentiation and the potential for premating reproductive isolation. The results indicated a polyphyletic origin of ecotypes whereby each of them have been expressed independently more than once. In the two lakes of Squanga Creek drainage, the existence of sympatric pairs was best explained by the secondary contact of two monophyletic whitefish groups that evolved in allopatry during the last glaciation events. In Dezadeash L. of Alsek R. drainage, our results could not verify either sympatric or allopatric (or microallopatric) origin of ecotypes. Regardless of the mode of speciation involved in their origins, these sympatric whitefish populations provided further evidence that Pleistocene glaciation events created conditions favoring rapid divergence and phenotypic differentiation among northern freshwater fishes.     

Divergent selection maintains adaptive differentiation despite high gene flow between sympatric rainbow smelt ecotypes (Osmerus mordax Mitchill)

In this study, we investigate the relative role of historical factors and evolutionary forces in promoting population differentiation in a new case of sympatric dwarf and normal ecotypes of the rainbow smelt (Osmerus mordax Mitchill) in Lac Saint-Jean (Québec, Canada). Our first objective was to test the hypothesis that the evolution of sympatric smelt ecotypes in Lac Saint-Jean has been contingent upon the secondary contact between two evolutionary lineages in postglacial times. Secondly, the QST method was applied to test the null hypothesis that the extent of phenotypic differences relative to that of neutral marker variation would be similar in comparisons involving populations within and among ecotypes. Thirdly, we applied a quantitative-genetic method as an exploratory assessment as to whether the amount of gene flow observed between populations could affect divergence in adaptive traits under specific conditions. This study revealed a unique situation of dwarf and normal smelt ecotypes that are, respectively, characterized by selmiparous and iteroparous life histories and the occurrence in each of two genetically distinct populations that synchronously use the same spawning habitat in two tributaries. Historical contingency has apparently played little role in the origin of these populations. In contrast, an important role of divergent natural selection in driving their phenotypic divergence was suggested. While divergent selection has apparently been strong enough to maintain phenotypic differentiation in the face of migration, this study suggests that gene flow has been sufficiently important to modulate the extent of adaptive differentiation being achieved between ecotypes, unless the extent of stabilizing selection acting on smelt ecotypes is much more pronounced than usually reported in natural populations.     

Restricted gene flow within and between rapidly diverging Neotropical plant species

Speciation involves the evolution of traits and genetic differences that contribute to reproductive isolation and the cessation of gene flow, and studying closely related species and divergent populations gives insight into how these phenomena proceed. Here, we document patterns of gene flow within and between two members of a rapid Neotropical species radiation, Costus pulverulentus and Costus scaber (Costaceae). These species co‐occur in the tropical rainforest and share pollinators, but are reproductively isolated by a series of prezygotic barriers, some of which show evidence of reinforcement at sympatric sites. Here, we genotype microsatellite markers in plants from eight sites that span the geographical range of both species, including four sympatric sites. We also genotype putative hybrids found at two sympatric sites. We find high levels of genetic isolation among populations within each species and low but detectable levels of introgression between species at sympatric sites. Putative hybrids identified by morphology are consistent with F1 or more advanced hybrids. Our results highlight the effectiveness of prezygotic isolating mechanisms at maintaining species boundaries in young radiations and provide empirical data on levels of gene flow consistent with reinforcement.     

Population differentiation in plethodontid salamanders: divergence of allozymes and sexual compatibility among populations of Desmognathus imitator and D. ochrophaeus (Gaudata: Plethodontidae)

Plethodontid salamanders of the genus Desmognathus exhibit varying levels of genetic differentiation among and within both allopatric and sympatric taxa. This provides excellent opportunities to study population differentiation and speciation. Two morphologically similar species in this genus, D. imitator and D. ochrophaeus, are genetically well-differentiated from one another and occur in sympatry with no evidence of hybridization and introgression. We report that the degree of sexual isolation between these two species is very high, regardless of whether the populations under comparison are allopatric or sympatric with one another. Neither reinforcement nor reproductive character displacement are required to explain the evolution of sexual incompatibility in sympatry. Sexual behaviour apparently diverges while populations are allopatric with one another. Preliminary study indicates that D. imitator consists of populations among which there may be significant sexual isolation in the absence of detectable genetic differentiation.     

Spatial genetic structure and reproductive success in fragmented and continuous populations of<Emphasis Type="Italic">Primula vulgaris</Emphasis>

In Flanders (northern Belgium),Primula vulgaris, a self-incompatible long-lived perennial herb, is rare and consists of a network of fragmented populations in the intensively used agricultural landscape. We investigated genetic variation and structure using 27 allozyme loci in 41 populations, and reproductive success to assess the effect of fragmentation on gene flow and the influence of the nearest neighbouring (large and/or highly genetically diverse) population on within-population genetic variation and reproductive success. Isolation by distance was found among and within populations. Smaller and more isolated populations showed a slight loss of allelic variation, but maintained high levels of observed heterozygosity. They were not more differentiated from each other than large populations. No significant difference in the regression slopes of the spatial autocorrelation analysis was found between two continuous populations and two groups of fragmented populations with similar distance classes. Multiple regression showed that population allelic richness and reproductive success were higher when the nearest neighbouring population was genetically more diverse. These results suggest moderate current gene flow within and among populations rather than historical gene flow. We conclude that small and isolated populations ofP. vulgaris should be considered not only as remnants of previously larger populations, but also as potential stepping stones insuring gene flow processes. For conservation, all highly variable and flowering populations should be considered, irrespective of their size or their isolation from large and continuous populations.