Do ambush predators prefer rewarding or non‐rewarding orchid inflorescences?

Previous research has shown that the presence of a reward on average doubles reproductive success in orchids by correspondingly increasing the frequency of pollinator visitations. We examined whether such reward-induced increases have a downstream effect on the behaviour of ambush predators concealed in orchid inflorescences, extending observations begun at Downe by Charles Darwin. Specifically, we studied three orchid-rich sites in southern England, in order to compare the occurrence of crab spiders on three coexisting species of terrestrial orchids: the nectariferous Gymnadenia conopsea versus the nectar-less Dactylorhiza fuchsii and Anacamptis pyramidalis . No significant difference was observed between rewarding and non-rewarding inflorescences at Risborough, whereas at Aston Clinton the nectar-less species supported significantly more crab spiders, albeit mainly in relatively short grass. Comparison of the two non-rewarding species present at Downe approximated a significant preference by the spiders for Anacamptis , which more closely resembles the rewarding Gymnadenia . The presence of a floral reward does not result in a higher frequency of crab spiders. We speculate that concealment quality of the inflorescence, the nature of the dominant pollinator(s), and/or ease of movement of spiders between inflorescences may be more important than frequency of visits by potential prey, particularly when no other form of concealment is readily available.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 763–771.     

Genetic differentiation and habitat preference of flowering-time variants within Gymnadenia conopsea

Using fast-evolving microsatellites, more slowly evolving ITS markers and performing habitat analyses, we demonstrated a drastic genetic divergence and significant habitat differentiation between early- and late-flowering variants of plants morphologically belonging to Gymnadenia conopsea ssp conopsea. The two phenological variants can either be found in separate or in mixed populations. Information from microsatellite markers and ITS sequences indicated the occurrence of an early historical split between the two flowering-time variants, a split that has been maintained until the present time even within sympatric populations. Early-flowering variants were also far more genetically diverse, had more alleles per microsatellite locus and a wider habitat amplitude than late-flowering variants. As a comparison, we included G. odoratissima in the sequencing study. We found G. odoratissima to be most closely related to the early-flowering type. This indicates a more ancient divergence event between the two flowering-time variants within G. conopsea ssp conopsea than between the two different species G. odoratissima and the early-flowering variant of G. conopsea. Possible explanations to the results arrived at and possible mechanisms maintaining the genetic separation are discussed.     

Phylogeography and subspecies taxonomy of dunlins (Calidris alpina) in western Palearctic analysed by DNA microsatellites and amplified fragment length polymorphism markers

In a circumpolar wader, the dunlin ( Calidris alpina ), there are 11 named subspecies, but only five mitochondrial DNA (mtDNA) lineages have been found. In the present study, we investigated the genetic structure of dunlins in western Palearctic (from East Greenland to Taimyr peninsula) using DNA microsatellites and amplified fragment length polymorphism (AFLP) markers that may detect more recent differentiation than mtDNA. In this region, we consider four described subspecies; alpina , schinzii , arctica and centralis , together comprising two mtDNA lineages. We analyse seven polymorphic microsatellite loci and 91 AFLP markers in 287 and 152 unrelated individuals, respectively, originating from 17 populations. Neither microsatellites nor AFLPs reveal distinct groups that correspond to currently recognized subspecies. There is a clear pattern of isolation by distance in microsatellites. Our results do not contradict the former mtDNA results that there are two phylogenetic lineages (approximately corresponding to schinzii and centralis ) that have met and formed a cline ( alpina ). We find no difference between schinzii and arctica (East Greenland). We conclude that, given the lack of distinct groups and the gradual changes in microsatellite allele frequencies, these markers provide little genetic support for the dunlin subspecies taxonomy in the western Palearctic.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 713–726.     

Hybrids and fruit set in a mixed flowering-time population of Gymnadenia conopsea (Orchidaceae)

We have recently found that the morphologically determined subspecies Gymnadenia conopsea ssp conopsea in Sweden includes early and late flowering individuals. We were interested in the interactions between the flowering time groups; if there were gene flow between them and if so this was detrimental or advantageous. A spatially mixed population of early and late flowering individuals was studied using three microsatellite loci. We measured patterns in genetic differentiation and inferred occurrence of hybridisation and introgression. Variation in flowering time, fertility and relative and absolute fruit set was measured. The pattern of introgression between flowering-time groups differed between loci. In two of the three investigated loci, allele separation was distinct between early and late flowering plants and one genetically obvious hybrid was infertile. In the third locus, several alleles were shared between the two flowering time variants. The degree of introgression was associated to fruit set failure, which was higher in the late flowering plants and lower in early flowering plants. A small group of early flowering individuals with somewhat delayed flowering compared to the main group was genetically distinct and had lower relative and absolute fruit set. This group was not genetically intermediate, but rather constituting an independent group, with lower fruit set possibly caused by absence of pollinators. There seem to be a strong barrier against introgression into the late flowering group which is kept genetically distinct and less diverse. The early flowering group is diverse, includes two subgroups and seems to benefit from gene flow.     

Patterns of genetic variation in Gymnadenia conopsea, the fragrant orchid

Gymnadenia conopsea (L.) R. Br., or the fragrant orchid, is one of many plant species negatively influenced by new practices in agriculture and forestry during the last decades. This study describes the level of microsatellite variation within and among 10 Swedish populations of this species. It was not possible to detect strong effects of small population size or fragmentation. In general, the species had high genetic variation within and low genetic divergence among populations, although the correlation between population size and number of alleles was close to significance at the 95% level. Also, a significant isolation by distance effect was observed, indicating the presence of modest restrictions in gene dispersal between the investigated populations.     

Genetic differentiation and gene flow of two sparidae subspecies, Diplodus sargus sargus and Diplodus sargus cadenati in Atlantic and south-west Mediterranean populations

A total of nine enzymes coded by 14 loci were assayed for each of six populations (from the north-eastern Atlantic and the Mediterranean) of two sea bream subspecies ( Diplodus sargus sargus and Diplodus sargus cadenati ). Diagnostic alleles were observed for each subspecies, although there were several common alleles. Estimates of variance in allele frequencies among samples ( F _ST) revealed significant differences ( P  < 0.05) among both subspecies. Genetic divergence was found between Atlantic and Mediterranean samples: values for genetic distances were higher than 0.163. Furthermore, D. sargus cadenati populations displayed a higher mean weight and length than D. sargus sargus populations and significant differences in growth were found among subspecies and populations. These results are discussed in terms of levels of gene flow and its respective relationships with water circulation in the Strait of Gibraltar and geological events.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 89 , 705–717.     

Population differentiation in the marginal populations of the great tit (Paridae: Parus major)

The major subspecies group of the great tit, Parus major , has experienced demographic and spatial expansions during the last century in several sites at the edges of its distribution range. These expansions, although temporarily very even, have resulted in dissimilar patterns of molecular diversity. Populations locating at regions of contact to other subspecies groups (in Amur, Kirghizia–Kazakhstan, and Iran) show divergence from central population by nuclear and mitochondrial markers. In Amur, gene flow from minor group could be detected based on the existence of private minor alleles in the major population. In Kirghizia and Kazakhstan, the bokharensis and major groups share almost all the microsatellite alleles detected though frequencies differ. In Iran, three geographically close populations are distinct according to the mitochondrial sequences but also indications of present or recent admixture is detected. Populations, which have expanded to regions previously unoccupied by the species (northern UK and Finland), show divergence only by one of the markers. The variability in molecular differentiation may be due to dissimilar expansions, depending on whether the colonized regions have previously been occupied by another subspecies or not, on the amount of colonizing birds, and on the amount of past and present gene flow.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 201–210.     

Flowering frequency in a small population of Gymnadenia conopsea-a five year study

This investigation evaluates the usage of genetic markers, microsatellites, to distinguish and re-identify individual plants in a population of the orchid Gymnadenia conopsea. The study also illustrates the problem in estimating the size of a population from single year sampling as individuals can rest underground or occur in vegetative states; information extremely important for the understanding of population dynamics as well as providing information for conservation management. The total population size was, based on information from microsatellite loci, estimated to 84 individuals and vastly larger than the annual number of flowering plants (mean 31.4 individuals/year). Flowering frequency varied from 24–49%, 53 individuals flowered once, five individuals were flowering four years and a single individual was flowering five years. A common pattern was one or two flowering periods followed by a non-flowering period. The observed number of alleles and the observed and expected heterozygosity varied among loci, although allele frequencies and genotype frequencies did not vary significantly among years.     

Genetic diversity and structure of the cycad Zamia loddigesii Miq. (Zamiaceae): implications for evolution and conservation

The genetic diversity and structure of four populations of the cycad Zamia loddigesii were studied throughout its range in Mexico. Allozyme electrophoresis of 15 loci was conducted. The mean number of alleles per locus was 1.80 ± 0.09, the percentage of polymorphic loci was 66.6 ± 5.4, and the expected heterozygosity was 0.266 ± 0.02. The results indicated that the genetic diversity was relatively higher, with respect to tropical tree species and other cycads. The genetic variation explained by differences among populations was 18%. On average, gene flow between paired populations was similar ( Nm  = 1.6) to other tropical forest trees and cycad species. Our results indicated that the geographical isolation among populations of Z. loddigesii generated allele loss, as well as a clinal variation in the frequencies of two loci ( MDH and MNR2 ), in relation to the latitudinal distribution of populations. The populations have become fragmented due to increasingly higher pressure of habitat conversion and disturbance. The importance of the establishment of sanctuaries and protected areas and a reduction in deforestation is highlighted in this research as a way of preserving the high genetic diversity of this and other endemic species.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 152 , 533–544.     

Morphometric study and taxonomy of Genista pulchella Vis. s.l. (Fabaceae), a south European species

A morphometric study of the known populations of Genista pulchella has been undertaken, based on herbarium specimens and field research. This has made it possible to provide a new taxonomic outline as the disjunct populations of G. pulchella (western part of the Balkan peninsula, central Italy and southern France) seem to be distinct. A new taxon is described: G. pulchella ssp. aquilana ssp. nov. (central Italy), a new combination G. pulchella ssp. villarsiana comb. nov. is proposed for the population from southern France, while G. pulchella ssp. pulchella can be considered endemic to the Balkan Peninsula. Genista pulchella Vis., G. villarsii Clementi and G. villarsiana Jord. are lectotypified.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 153 , 245–254.     

Molecular and morphometric data demonstrate the presence of cryptic lineages among freshwater crabs (Decapoda: Potamonautidae: Potamonautes) from the Drakensberg Mountains, South Africa

The evolutionary importance of cryptic taxa is well documented, yet few studies have examined them with a view to conservation. In the present study, the significance of cryptic speciation in freshwater crabs is examined. Allozyme and 16S rRNA sequence data were used to explore the degree of population differentiation between mountain stream populations of two distinct freshwater crab species. Marked patterns of differentiation were evident among populations; those in close geographical proximity were characterized by the fixation of alternate alleles at certain loci, indicating that currently there is no gene flow among populations. Both allozyme and sequence data provide evidence for the recognition of at least five distinct evolutionary lineages with pronounced levels of genetic differentiation. Morphometric data failed to detect any distinct geographically intermediate population groupings. Our findings indicate the presence of five phylogeographical units, all worthy of conservation, three of which are evolutionarily significant.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 78 , 129–147.     

Gene flow and melanism in garter snakes revisited: a comparison of molecular markers and island vs. coalescent models

Within populations, the stochastic effect of genetic drift and deterministic effect of natural selection are potentially weakened or altered by gene flow among populations. The influence of gene flow on Lake Erie populations of the common garter snake has been of particular interest because of a discontinuous colour pattern polymorphism (striped vs. melanistic) that is a target of natural selection. We reassessed the relative contributions of gene flow and genetic drift using genetic data and population size estimates. We compared all combinations of two marker systems and two analytical approaches to the estimation of gene flow rates: allozymes (data previously published), microsatellite DNA (new data), the island model ( F _ST-based approach), and a coalescence-based approach. For the coalescence approach, mutation rates and sampling effects were also investigated. While the two markers produced similar results, gene flow based on F _ST was considerably higher (Nm > 4) than that from the coalescence-based method (Nm < 1). Estimates of gene flow are likely to be inflated by lack of migration-drift equilibrium and changing population size. Potentially low rates of gene flow (Nm < 1), small population size at some sites, and positive correlations of number of microsatellite DNA alleles and island size and between M , mean ratio of number of alleles to range in allele size, and island size suggest that in addition to selection, random genetic drift may influence colour pattern frequencies. © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 79, 389–399.     

Microsatellite DNA analysis of northern pike (Esox lucius L.) populations: insights into the genetic structure and demographic history of a genetically depauperate species

The northern pike Esox lucius L. is a freshwater fish exhibiting pronounced population subdivision and low genetic variability. However, there is limited knowledge on phylogeographical patterns within the species, and it is not known whether the low genetic variability reflects primarily current low effective population sizes or historical bottlenecks. We analysed six microsatellite loci in ten populations from Europe and North America. Genetic variation was low, with the average number of alleles within populations ranging from 2.3 to 4.0 per locus. Genetic differentiation among populations was high (overall θ_ST = 0.51; overall ρ_ST = 0.50). Multidimensional scaling analysis of genetic distances between populations and spatial analysis of molecular variance suggested a single phylogeographical race within the sampled populations from northern Europe, whereas North American and southern European populations were highly distinct. A population from Ireland was monomorphic at all loci, presumably reflecting founder events associated with introduction of the species to the island in the sixteenth century. Bayesian analysis of demographic parameters showed differences in θ (a product of effective population size and mutation rate) among populations from large and small water bodies, but the relative differences in θ were smaller than expected, which could reflect population subdivision within the larger water bodies. Finally, the analyses showed drastic population declines on a time scale of several thousand years within European populations, which we ascribe to either glacial bottlenecks or postglacial founder events.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 91–101.     

Extremely low levels of genetic diversity in the terrestrial orchid Epipactis thunbergii (Orchidaceae) in South Korea: implications for conservation

Levels of allozyme variation, population genetic structure, and fine-scale genetic structure (FSGS) of the rare, both sexually and clonally reproducing terrestrial orchid Epipactis thunbergii were examined for eight ( N  = 734) populations in a 20 × 20-km area in South Korea. Twenty-three putative allozyme loci resolved from 15 enzyme systems were used. Extremely low levels of allozyme variation were found within populations: the mean frequency of polymorphic loci was 3.8% [isocitrate dehydrogenase ( Idh-2 ) with two alleles was polymorphic across populations], the mean number of alleles per locus was 1.04, and the mean expected heterozygosity was 0.013. The overall fixation index was not significantly different from zero ( F _IS = 0.069), although the species is self-compatible. However, a significantly high degree of population differentiation was found between populations at Idh-2 ( F _ST = 0.388) in the studied area. Furthermore, spatial autocorrelation analyses revealed a significant FSGS (up to 3 m) within populations. These observations suggest that the main explanatory factors for the extremely low levels of genetic diversity and the shaping of the population genetic structure of E. thunbergii are genetic drift as a result of a small effective population size, a restricted gene flow, and the isolation of populations. Considering the current genetic structure of E. thunbergii , three guidelines are suggested for the development of conservation strategies for the species in South Korea: (1) protection of habitats of standing populations; (2) prohibition by law of any collection of E. thunbergii ; and (3) protection of nearby pollinator populations, given the fact that fruit set in natural habitats is very low.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 155 , 161–169.     

Inferences of selection and migration in the Danish house mouse hybrid zone

We analysed the patterns of allele frequency change for ten diagnostic autosomal allozyme loci in the hybrid zone between the house mouse subspecies Mus musculus domesticus and M. m. musculus in central Jutland. After determining the general orientation of the clines of allele frequencies, we analysed the cline shapes along the direction of maximum gradient. Eight of the ten clines are best described by steep central steps with coincident positions and an average width of 8.9 km (support limits 7.6–12.4) flanked by tails of introgression, indicating the existence of a barrier to gene flow and only weak selection on the loci studied. We derived estimates of migration from linkage disequilibrium in the centre of the zone, and by applying isolation by distance methods to microsatellite data from some of these populations. These give concordant estimates of σ =  0.5–0.8 km generation     . The barrier to gene flow is of the order of 20 km (support limits 14–28), and could be explained by selection of a few per cent at 43–120 underdominant loci that reduces the mean fitness in the central populations to 0.45. Some of the clines appear symmetrical, whereas others are strongly asymmetrical, and two loci appear to have escaped the central barrier to gene flow, reflecting the differential action of selection on different parts of the genome. Asymmetry is always in the direction of more introgression into musculus , indicating either a general progression of domesticus into the musculus territory, possibly mediated by differential behaviour, or past movement of the hybrid zone in the opposite direction, impeded by potential geographical barriers to migration in domesticus territory.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 593–616.     

Cryptic species within the freshwater isopod Mesamphisopus capensis (Phreatoicidea: Amphisopodidae) in the Western Cape, South Africa: allozyme and 12S rRNA sequence data and morphometric evidence

The freshwater phreatoicidean isopod Mesamphisopus capensis has been regarded as the most widespread of the four Mesamphisopus species occurring in the Western Cape, South Africa. To determine whether this species was monotypic across its distribution over two mountainous regions, separated by a low-lying coastal plain remnant, genetic differentiation among populations from 11 localities was studied through allozyme electrophoresis of 12 loci and sequencing of a 338-bp 12S rRNA mtDNA fragment from representative individuals. Populations of the two regions were separated by a mean identity value of 0.477. Fixed allele differences at two loci distinguished these regions. Estimates of θ indicated substantial differentiation among populations across the entire sample, as well as within each of the regions. Topologies derived through parsimony and neighbour joining supported the monophyly of the two regions. On the basis of these topologies, allele frequencies and an allozyme dendrogram, five groups were identified. Discriminant function analyses, performed on body and pereopod variables independently, revealed these groups to be well differentiated with a high rate of correct a posteriori reclassification. Using genetic distance criteria these five distinct forms may be considered to be putative species. From a conservation perspective, the two regions can be seen to represent two evolutionarily significant units, while the five groups should be regarded as management units.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 81 , 235–253.     

Contrasting temporal dynamics and spatial patterns of population genetic structure correlate with differences in demography and habitat between two closely-related African freshwater snails

The relationship between habitat stability, demography, and population genetic structure was explored by comparing temporal microsatellite variability spanning a decade in two closely-related hermaphroditic freshwater snails from Cameroon, Bulinus forskalii and Bulinus camerunensis . Although both species show similar levels of preferential selfing, microsatellite analysis revealed significantly greater allelic richness and gene diversity in populations of the highly endemic B. camerunensis compared to those of the geographically-widespread B. forskalii . Additionally, B. camerunensis populations showed significantly lower spatial genetic differentiation, higher dispersal rates, and greater temporal stability compared to B. forskalii populations over a similar spatial scale. This suggests that a more stable demography and greater gene flow account for the elevated genetic diversity observed in this geographically-restricted snail. This contrasts sharply with a metapopulation model (which includes extinction/contraction, recolonization/expansion, and passive dispersal) invoked to account for population structuring in B. forskalii . As intermediate hosts for medically important schistosome parasites, these findings have ramifications for determining the scale at which local adaptation may occur in the coevolution of these snails and their parasites.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 747–760.     

Reduced genetic variation and strong genetic population structure in the freshwater killifish Valencia letourneuxi (Valenciidae) based on nuclear and mitochondrial markers

The genetic variation of the critically endangered Corfu killifish (Valencia letourneuxi), an endemic freshwater fish species of the western Balkans, was assessed for nine populations sampled in eight water systems in western continental Greece, the Peloponnese and the Ionian Island of Corfu, using mitochondrial and microsatellite markers. The analyses were based on data from three mtDNA regions (D‐loop, COI and 16S rRNA sequences) and 14 microsatellite loci. Samples from the congeneric species Valencia hispanica and the phylogenetically closely related species Aphanius fasciatus were also used in the study as outgroups. Both the mitochondrial and the microsatellite analyses revealed three distinct population groupings associated with the geographical distribution of the populations: one southern group occupying rivers draining to the Patraikos Gulf, the second one including the populations flowing into the Amvrakikos Gulf and the third, more northern group, including the other populations from rivers in Corfu Island and Epirus flowing into the Ionian Sea. Within these groupings there is limited genetic differentiation between populations; in addition, there is reduced intrapopulation genetic variation, evidenced by low heterozygosity values, number of alleles and haplotype diversity. In terms of taxonomic implications and appropriate management actions for conservation, our data suggest that the major population groups should be regarded at least as three distinct conservation units (CUs), with translocation and restocking actions to take place only within the geographical range of the CU concerned. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 334–349.     

Genetic variation and clonal diversity in island Cephalanthera rubra populations from the Biebrza National Park, Poland

Three populations of the orchid species Cephalanthera rubra localized on neighbouring mineral islands in the Biebrza National Park (north-east Poland) were examined using 16 allozyme loci. The percentage of polymorphic loci in these C. rubra populations ranged from 25 to 31.3% and the mean number of alleles per polymorphic locus from 1.25 to 1.31. The levels of observed heterozygosity at polymorphic loci were higher than expected for all populations. The overabundance of heterozygotes was noted in every population. The values of fixation indices were highly negative. The genetic differentiation among the three populations was small ( F _ST = 0.0173), albeit statistically significant ( P  < 0.001). The level of gene flow varied from 13 to 16. Fourteen multilocus genotypes were found among the 401 ramets sampled from the populations. Only four of them were common for all populations. The frequency of genotypes was different in these three populations of C. rubra . Some of the multilocus genotypes dominated in the given populations, others were found sporadically. Genetic variation within C. rubra populations is mainly the result of small population sizes and reflects the influences of breeding system and type of reproduction. On the other hand, these properties are also connected with habitat conditions.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 143 , 99–108.     

Genetic differentiation between populations of the European rose hip fly Rhagoletis alternata

We tested for genetic differentiation between populations of Rhagoletis alternata Fall. (Diptera: Tephritidae) on three different host species. We collected larvae from three rose species of the section Caninae ( Rosa canina L., Rosa corymbifera Borkh . , and Rosa rubiginosa L.) from 15 sites across Germany, where the three roses occurred together. Additionally, we sampled three sites in Switzerland. Roses differ in morphology (e.g. leaf glands) as well as phenology. We were able to score nine allozyme loci (five polymorphic). Populations from the three hosts did not differ in genetic variability. We found significant genetic differentiation between populations from different host species. However, the differentiation was very low (0.9%). Hence, we found no indication for host races. Furthermore, surprisingly little geographical structure of genetic differentiation was found between populations of this fruit fly across central Europe. We offer three mutually non-exclusive explanations for these findings. First, gene flow between populations of Rh. alternata is high. Second, the pattern of genetic differentiation is based on a recent expansion of the distributional range . Third, the ongoing gene flow between roses of the section Caninae acts as a hybrid bridge.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 619–625.