Did Polyommatus icarus (Lepidoptera: Lycaenidae) have distinct glacial refugia in southern Europe? Evidence from population genetics

Pleistocene climatic oscillations strongly influenced the genetic composition of many species which are often divided into several genetic lineages. In this context, we studied the allozymes of a common and widely distributed butterfly, the common blue Polyommatus icarus, over a large part of Europe. The species had a rather high genetic diversity within populations with a strikingly high mean number of alleles per locus (2.98). In contrast, differentiation between populations was very low ( F _ST: 0.0187). Only a marginal trend of decline in genetic diversity from the south to the north was observed. Isolation-by-distance existed on a European scale ( r =  0.826), but not at a regional level. Regional differentiation between populations in western Germany was extremely low ( F _ST: 0.0041). It is probable that P. icarus was widely distributed in the Mediterranean region during the last ice age and expanded into central Europe in the postglacial period without major genetic erosion. Moderate present and past gene flow in an intact metapopulation structure may have occurred on local, regional and perhaps even continental scales.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 80 , 529–538.     

Genetic structure of fragmented November moth (Lepidoptera: Geometridae) populations in farmland

Habitats are now becoming increasingly fragmented throughout the world due to intense cultivation. As a consequence, populations of some animals with low mobility have become isolated, thus increasing the risk of inbreeding and local extinction. In Britain, weakly flying geometric moths of the genus Epirrita are a good model species with which to test the genetic effects of habitat fragmentation on insect populations. Genetic variation within and between populations of two Epirrita species captured using a network of light traps at two spatial scales (local and national) was assessed using allozyme electrophoresis, with particular reference to the local scale (the 330-ha arable farm estate at Rothamsted, Hertfordshire, in southern Britain). Populations sampled widely in England and Wales displayed low (but statistically significant) levels of genetic differentiation for both species ( F _st  = 0.0051–0.0114 and 0.0226 for E. dilutata and E. christyi , respectively). However, analysis of large samples of E. dilutata from four small woods at Rothamsted revealed low ( F _st  = 0.0046) but significant differentiation, indicating that gene flow was restricted, even at this very small scale. It was concluded that small intervening patches of farmland (often a few fields width) were enough to prevent genetic homogeneity. The close similarity between more distant Epirrita populations was considered to be a result of historical, rather than recurrent gene flow, as genetic equilibrium between drift and gene flow is unlikely over such scales.  © 2003 The Linnean Society of London . Biological Journal of the Linnean Society , 2003, 78 , 467–477.     

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.     

Conservation genetics of Hebe speciosa (Plantaginaceae) an endangered New Zealand shrub

All extant populations of Hebe speciosa (Plantaginaceae), a threatened endemic New Zealand shrub, were investigated using the amplified fragment length polymorphism technique (AFLP). Genetic diversity indices varied significantly among geographical regions and were positively correlated with population size. Among-population genetic differentiation was high (mean pairwise Φ_ST = 0.47), implying complex historical relationships between disjunct populations and negligible contemporary gene flow. Southern populations exhibited extremely low genetic diversity relative to those found in Northland, suggesting that these populations may be more recent in origin. Patterns of genetic relationship among some populations indicate pre-European Māori dispersal and cultivation. The three northernmost populations were found to contain the majority of the species' remaining genetic diversity and thus, should be a focus for future conservation management. Some southern sites may also be culturally significant as evidence of Māori trade and cultivation of Hebe speciosa .  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 149 , 229–239.     

Genetic variation within and between Iliamna corei and I. remota (Malvaceae): implications for species delimitation

Iliamna corei and I. remota are classified as endangered species; however, their designation as separate species has been questioned. In order to address this problem, intersimple sequence repeat (ISSR) data were generated to examine patterns of genetic differentiation within and between these two taxa. ISSRs were used to screen individuals for genetic diversity of I. corei from the single known natural population and two garden populations, and individuals of I. remota from the six natural populations and four garden populations. Using ten primers, 140 informative markers were generated. Ninety-four percent of loci detected revealed polymorphisms. Cluster analysis (neighbour-joining, NJ) revealed that the two species are genetically distinct and that the Illinois populations of I. remota were genetically distinct from the Virginia populations of I. remota . Ordination by Principal Coordinate Analysis (PCoA) supported the findings of the NJ, with a separation of I. corei and I. remota . Analysis of Molecular Variance (AMOVA) revealed that the majority of variation detected was within populations, which is consistent with other self-incompatible plants. The results indicate a correlation between the geographical distributions of the species and gene flow.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 151 , 345–354.     

Phylogeography of five Polytrichum species within Europe

Using allozymes and microsatellites we have analysed the genetic structure among European populations for several Polytrichum species to infer relevant factors, such as historical events or gene flow, that have shaped their genetic structure. As we observed low levels of genetic differentiation among populations, and no decreasing levels of genetic variation with increasing latitude within most of the examined species, no genetic evidence was obtained for a step-wise recolonization of Europe from southern refugia after the latest glacial period for P. commune , P. uliginosum , P. formosum and P. piliferum . The near absence of population substructuring within these species does indicate that extensive spore dispersal is the most important factor determining the genetic structure among European Polytrichum populations. Gene flow levels have apparently been sufficient to prevent genetic differentiation among populations caused by genetic drift, and to wipe out any genetic structure caused by the postglacial recolonization process. On the other hand, increased genetic differentiation of alpine P. formosum populations suggests that mountain ranges might restrict gene flow significantly among Polytrichum populations. In contrast to most examined Polytrichum species, P. juniperinum showed high levels of genetic differentiation and a profound genetic structure. Assuming that gene flow is not more restricted in P. juniperinum , these findings suggest that this species has recolonized Europe after the latest glacial period from two different refugia, one possibly being the British Isles.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society 2003, 78, 203–213.     

The genetic structure of the steppe plant Iris aphylla L. at the northern limit of its geographical range

Genetic diversity of Iris aphylla was examined in seven Polish populations located at the northern limit of the geographical range of the species. Using RAPD analysis, 491 DNA fragments were amplified, all of which were polymorphic. The peripheral populations were characterized by a low number of unique bands (one to three per population) and moderate values of genetic diversity parameters (percent of polymorphic loci = 30.6%, Shannon's index = 0.149 and Nei's gene diversity = 0.097, on average), which can be explained as being due to the recent expansion of the European biota and the extremely low frequency or absence of seed set and of juveniles, as well as limited pollen and seed dispersal. There was also no significant correlation between ecological properties (population size; number of flowering ramets and fruits) and genetic diversity parameters. Despite this fact, more fixed loci were noted in the smallest populations than were in the largest ones. All populations were significantly differentiated and geographically structured. Analysis of molecular variance between regions showed a low and nonsignificant value, but relatively high molecular variation was partitioned among and within populations. Only one group of south-eastern populations was structured according to isolation by distance pattern and neighbour-joining cluster analysis. © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 152 , 245–255.     

Phylogeography of a host-specific insect: genetic structure of Ips typographus in Europe does not reflect past fragmentation of its host

The phylogeography of the bark beetle Ips typographus was assessed using five microsatellite markers. Twenty-eight populations were sampled throughout Europe on the host tree Picea abies . I. typographus showed very low levels of genetic diversity, and the study revealed a lack of genetic structure across Europe. No significant barrier to gene flow was found, even though P. abies has a fragmented distribution. A weak but significant effect of isolation by distance was found. These results suggest a high dispersal capacity of I. typographus , which leads to low genetic differentiation between populations. Its high dispersal capacity is likely to have prevented I. typographus from developing important local adaptations to its host, which would have influenced its genetic structure. The nuclear data was compared to previously published mitochondrial data that showed strong differentiation between Central–Northern European populations and Russian–Baltic populations, and a founder effect in Scandinavia, probably reflecting the postglacial history of I. typographus . Discrepancies between nuclear and mitochondrial markers could be due to the maternal inheritance of mitochondrial DNA, and to sex-biased dispersal in I. typographus . The overall low genetic diversity observed on both markers on a large geographical scale is discussed. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 239–246.     

Population genetics of Dioon edule Lindl. (Zamiaceae, Cycadales): biogeographical and evolutionary implications

Dioon edule Lindl. (Zamiaceae) is a cycad endemic to Mexico, that occurs as one species D. edule and the geographical variety D. edule var. angustifolium (Miq.) Miq. Dioon edule has a north to south distribution in eastern Mexico. In this study, we analysed 14 allozymic loci in eight populations of D. edule from its total distribution range by sampling all known populations. Patterns of diversity and genetic variability, within and among populations, were obtained. The mean number of alleles per locus ( A ) was 1.44 and the percentage of polymorphic loci was relatively high ( P  = 54.78). The mean observed ( H _O ) and expected heterozygosity ( H _E ) were 0.27 and 0.24, respectively. F -statistics revealed an excess of heterozygous genotypes, locally and globally ( F  = −0.17 and f  = −0.27, respectively). The genetic variation explained by differences among populations was only 7.5%. We also detected a negative relationship between genetic diversity and latitude. On average, the gene flow between population pairs was relatively high ( Nm  = 2.98); furthermore, gene flow between population pairs was significantly correlated with geographical distances ( r  = −0.38, P  = 0.025). Therefore, patterns of genetic diversity in D. edule appear to be associated with the post-Pleistocene spread of the species, from its southerly (origin) to its northerly range (derived populations, including its central distribution). The biogeographical and evolutionary aspects of the results of this study are discussed. We recognize Dioon angustifolium Miq. for the northernmost disjunct populations.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 80 , 457–467     

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.     

Microscale variation in alpine grasslands: AFLPs reveal a high level of genotypic diversity in Primula minima (Primulaceae)

The microscale variation and spatial genetic structure of the alpine plant species Primula minima L. was analysed using AFLPs. AFLP analysis based on three primer combinations and 123 fragments revealed no identical genotypes among the 86 studied samples from a 300 × 300 cm plot. Variation within the study plot was high: Nei's gene diversity was 0.22, Shannon's information index 0.33 and the percentage of polymorphic bands was 60.9. Cluster analysis revealed four main groups of genetically similar individuals and mapping these individuals resulted in a clear spatial pattern, with samples from the same group often located close together. The observed microscale structure was corroborated using a Mantel test, which revealed significant correlation of genetic and spatial distances, and by the results of a spatial autocorrelation analysis that indicated a high level of similarity between adjacent samples. An analysis of molecular variance revealed clear differentiation (18%) between the spatial groups. Overall gene flow within the plot was 1.11 and ranged from 0.33 between the spatially most distant groups to 2.33 between directly neighbouring groups. The extraordinary level of diversity detected in this study indicates an unexpectedly strong relevance of reproduction by seed for the species P. minima in alpine grasslands. The strong microscale variation suggests, however, that there is limited dispersal of seeds. Clonal reproduction is of subsidiary importance to sexual reproduction and seems to occur only over very small distances.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 155 , 549–556.     

Patterns of genetic diversity in related taxa of Antirrhinum L. assessed using allozymes

Allozyme diversity was studied within and among populations of five related taxa of Antirrhinum L. endemic to the Iberian Peninsula ( A. graniticum Rothm. ssp. graniticum , ssp. brachycalyx Sutton and ssp. ambiguum (Lange) Mateu & Segarra, A. boissieri Rothm. and A. onubensis (Fdez. Casas) Fdez. Casas). All of the studied taxa are obligate outcrossing endemic perennial herbs which form isolated populations. However, the taxa vary in range and population sizes, and are found on different soil types. The level and distribution of allozyme diversity differed widely between taxa: A. graniticum ssp. brachycalyx had the lowest level of allozyme diversity (H_T = 0.09), whilst the highest level was detected in A. boissieri (H_T = 0.25). Total variation was partitioned into within- and among-population variation. The proportion attributable to variation within populations varied from about 67% up to 84.3% and 89.5% in A. graniticum ssp. brachycalyx and A. graniticum ssp. ambiguum , respectively. Both these subspecies also showed little population divergence (G_ST = 0.10 and 0.09, respectively) and had high levels of estimated gene flow (Nm = 2.18 and 2.62, respectively). These results are discussed in relation to geographical proximity of populations and habitat continuity. Isolation by distance was not detected in any of the studied taxa. This result suggests that divergence among populations is due to random genetic drift.  © 2003 The Linnean Society of London . Biological Journal of the Linnean Society , 2003, 79 , 299–307.     

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.     

Little genetic differentiation across Europe between early-flowering and late-flowering populations of the rapidly declining orchid Neotinea ustulata

Amplified fragment length polymorphism (AFLP) was used to characterize genetic diversity of the endangered Burnt Orchid, Neotinea (formerly Orchis ) ustulata . Fingerprinting of Estonian and British populations revealed surprisingly little genetic differentiation between populations but larger amounts of diversity within populations, especially in Britain. The resulting mean F _st value of 0.51 is unusually high for an orchid species. Much of the variation follows a west–east cline across Europe, whereas the much-discussed early- and late-flowering taxa of N. ustulata are considered insufficiently distinct to be viewed as separate subspecies. The later flowering N. ustulata var. aestivalis probably evolved independently on two or three occasions, each time diverging from the earlier flowering nominate race. The identity of the genes underpinning phenology in the species, and the potential selective advantages of phenological divergence, merit further study. Overall genetic diversity within populations is sufficiently high to render impoverishment an unlikely cause of their recent, precipitous decline.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 87 , 13–25.     

Genetic variation in Femeniasia (Compositae, Cardueae), an endemic and endangered monotypic genus from the Balearic Islands (Spain)

Femeniasia balearica is the only representative of its genus and is endemic to a small area on the northern coast of Minorca (Balearic Islands, Spain). The entire range of the species covers only 18 km of coastline. It is extremely rare and classified as endangered in the National Catalogue of Threatened Species and as a priority species in the EU Habitats Directive. We carried out a census of current population size and estimated genetic diversity based on AFLP markers to facilitate conservation of this unique species. In the 66 individuals analysed (∼10% of population), 225 bands were scored and the level of diversity was relatively high. Three divergent population groups corresponding to geographical areas (Western, Central and Eastern) were identified. In AMOVA and Bayesian analyses, most of the diversity was found within populations but there was strong differentiation between the three population groups. Genetic and geographical distances between the populations were strongly correlated. Our results show that the populations of F. balearica are not genetically depauperate, in spite of their small sizes. Unexpectedly, our results suggest that as many as three MUs (management units) should be recognized in this very small area, because the low levels of gene flow among them indicate contemporary demographic independence.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 153 , 97–107.     

Population genetic structure of New Zealand's endemic corophiid amphipods: evidence for allopatric speciation

Allozyme electrophoresis was used to examine population genetic structure at inter- and intraspecific levels for the New Zealand endemic corophiid amphipods, Paracorophium lucasi and P. excavatum. Individuals were collected from estuarine and freshwater habitats from North, South and Chatham Islands. Analyses of genetic structure among interspecific populations indicated clear allelic differentiation between the two Paracorophium species (Nei's genetic distance, D  = 1.62), as well as considerable intraspecific substructuring ( D  = 0.15–0.65). These levels of divergence are similar to interspecific levels for other amphipods and it is proposed that at least two groups from the P. lucasi complex and three from the P. excavatum complex correspond to sibling species. In most cases allopatry can account for the differentiation among the putative sibling species. For populations that share a common coastline we found low levels of differentiation and little or no correlation with geographical distance, suggesting that gene flow is adequate to maintain homogeneous population genetic structure. By contrast, populations on separate coastlines (i.e. isolated by land) showed moderate levels of geographical differentiation indicating restricted gene flow. The juxtaposition of population genetic and biogeographical data for Paracorophium in conjunction with the geological record infers past histories of glacial extirpation, and possible isolating effects of sea-level and landmass changes that have occurred throughout the Plio-Pleistocene.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 81 , 119–133.     

Low population differentiation and high genetic diversity in the invasive species Carduus acanthoides L. (Asteraceae) within its native range in the Czech Republic

Colonizing species are predicted to suffer from reductions in genetic diversity during founding events. Although there is no unique mode of reproduction that is characteristic of successful plant colonizers, many of them are predominantly self-fertilizing or apomictic species, and almost all outcrossing colonizers are self-compatible. Carduus acanthoides comprises a species of disturbed habitats with wind-dispersed seeds that colonizes open spaces of various sizes. Population genetic diversity was expressed by assessing patterns of variation at nine putatively neutral allozyme loci within and among 20 natural populations in its native distribution range in the Czech Republic. Overall, C. acanthoides displayed high levels of genetic diversity compared to other herbaceous plants. The percentage of polymorphic loci was 84.5, with values of 2.37, 0.330, and 0.364 for the mean number of alleles per polymorphic locus ( A ), observed heterozygosity ( H _o), and expected heterozygosity ( H _e), respectively. There was only weak evidence of inbreeding within populations ( f  = 0.097) and very low genetic differentiation among populations ( θ  = 0.085). Analyses of the data provide strong evidence for isolation-by-distance for the whole study area. Even the colonizing species, C. acanthoides , currently supports a substantial amount of allozyme variation at both the species and population levels.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 596–607.     

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.     

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.     

Evolution in the slow lane: molecular rates of evolution in sexual and asexual ostracods (Crustacea: Ostracoda)

Parthenogenetic lineages within non-marine ostracods can occur either in mixed (with sexual and asexual females) or exclusively asexual taxa. The former mode of reproduction is associated with a high intraspecific diversity at all levels (genetic, morphological, ecological) and, at least in the Cypridoidea, with geographical parthenogenesis. Obligate asexuality is restricted to the Darwinuloidea, the strongest candidate for an ancient asexual animal group after the bdelloid rotifers, and is characterized by low diversity. We have compared rates of molecular evolution for the nuclear ITS1 region and the mitochondrial COI gene amongst the three major lineages of non-marine ostracods with sexual, mixed and asexual reproduction. Absolute rates of molecular evolution are low for both regions in the darwinulids. The slow-down of evolution in ITS1 that has been observed for Darwinula stevensoni (Brady & Robertson) apparently does not occur in other darwinulid species. ITS1 evolves more slowly than COI within non-marine ostracod families, including the darwinulids, but not between superfamilies. The ancient asexuals might have a higher relative substitution rate in ITS1, as would be expected from hypotheses that predict the accumulation of mutations in asexuals. However, the speed-up of ITS could also be ancient, for example through the stochastic loss of most lineages within the superfamily after the Permian–Triassic mass extinction. In this case, the difference in rate would have occurred independently from any effects of asexual reproduction.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 79 , 93–100.