Conservation genetics of the endangered Iberian steppe plant Ferula loscosii (Apiaceae)

Ferula loscosii (Lange) Willk (Apiaceae) is a threatened endemic species native to the Iberian Peninsula. The plant has a narrow and disjunct distribution in three regions, NE, C and SE Spain. Genetic variability within and among 11 populations from its natural distribution was assessed using allozymes. Intermediate levels of genetic diversity were detected in F. loscosii (P(99%) = 36.83; H(E) = 0.125; H(T) = 0.152). However, the highest genetic diversity (58%) corresponded to the threatened populations from SE and C Spain (H(T) = 0.169) rather than the more abundant and larger populations from NE Spain (Ebro valley) (H(T) = 0.122). Low to moderate levels of genetic structure were found among regional ranges (G(ST) = 0.134), and several statistical spatial correlation analyses corroborated substantial genetic differentiation among the three main regional ranges. However, no significant genetic differentiation was found among the NE Spain populations, except for a northernmost population that is geographically isolated. Outcrossing mating and other biological traits of the species could account for the maintenance of the present values of genetic diversity within populations. The existence of an ancestral late Tertiary wider distribution of the species in SE and C Spain, followed by the maintenance of different Quaternary refugia in these warmer areas, together with a more recent and rapid post-glacial expansion towards NE Spain, are arguments that could explain the low genetic variability and structure found in the Ebro valley and the higher levels of diversity in the southern Iberian populations.     

Population genetic structure in the Iberian cyprinid fish Iberochondrostoma lemmingii (Steindachner, 1866): disentangling species fragmentation and colonization processes

Interplay between the complex geography, hydrogeomorphological history, past climatic changes, and anthropogenic pressures is likely responsible for the current diversity and species' distribution of freshwater fishes in the Iberian Peninsula. To further disentangle the evolutionary processes promoting the diversification of endemic Iberian Cyprinids through time and space, we explored the patterns of genetic diversity of the Iberian arched‐mouth nase, Iberochondrostoma lemmingii (Steindachner, 1866), using molecular markers rendering at different timescales: the mitochondrial gene cytochrome b and seven microsatellite loci. Both markers showed significant differentiation of populations though the relative genetic distances among populations were different between markers. Mitochondrial DNA results indicate the isolation of hydrographic basins as the main driver of population differentiation, with Tejo as the centre of diversification. The results also support connections between Tejo, Guadiana, and Guadalquivir, with levels of divergence suggesting an earlier severance of Guadalquivir, whereas Guadiana and Tejo maintained connections until a more recent past. Establishment of more peripherial populations in small southern basins (Quarteira and Almargem) could have been ruled by founder events. However, the analysis of present‐day genetic configuration suggested by microsatellite data implies, for the first time, the involvement of other factors in the evolution of arched‐mouth Iberian nase populations. Relative low genetic distances between inter‐basin populations (Tejo and Guadiana) and the lack of concordance between differentiation and geography suggest a possible influence of human‐mediated translocations in the population genetic patterns of I. lemmingii. High intra‐basin differentiation levels were found within Tejo and Guadiana and may be associated with factors intrinsic to the species (e.g. low dispersal capability) or natural and/or artificial barriers to gene flow. The low vagility of the species appears to be an important factor influencing the evolutionary processes shaping the phylogeographical patterns of I. lemmingii, which could be relevant for the conservation of this threatened species. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 559–572.     

Ecological and historical determinants of population genetic structure and diversity in the Mediterranean shrub Rosmarinus officinalis (Lamiaceae)

Population genetic studies of widespread Mediterranean shrubs are scarce compared with those of trees and narrow endemics or studies from phylogeographical perspectives, despite the key role these species may play in Mediterranean ecosystems. Knowledge on the effect of ecological factors in shaping their genetic patterns is also limited. In this study we investigate genetic diversity and population structure across 18 populations of Rosmarinus officinalis, a Mediterranean shrubland plant. Populations were sampled along two elevational gradients, one each on calcareous and siliceous soils in a mountain system in the eastern Iberian Peninsula, to decipher the effect of ecological factors on the genetic diversity and structure based on 11 microsatellite loci. We found overall high levels of genetic diversity and weak population structure. Genetic diversity increased with elevation, whereas population differentiation was stronger among populations growing on siliceous soils. The nested analysis of elevational gradients within soil types revealed that these general patterns were mostly driven by siliceous populations, whereas calcareous populations were more homogeneous along elevational belts. Bayesian analysis of population structure revealed genetic membership of lowland and high‐elevation populations to different genetic clusters and a higher admixture of intermediate‐elevation populations to both clusters. High‐elevation populations were less differentiated from a hypothetical ancestral cluster, suggesting the persistence of their gene pool during the Pleistocene glaciations. In contrast, lowland populations resulted from more recent divergence. We propose that life‐history and reproductive traits mostly contribute to explain the high levels of genetic diversity and weak population structure, whereas ecological and historical factors mostly contribute to the stronger differentiation of siliceous populations and a rapid expansion of R. officinalis on calcareous soils possibly mediated by human landscape transformations, © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180 , 50–63.     

Molecular phylogeography of the microturbellarian Monocelis lineata (Platyhelminthes: Proseriata) in the North‐East Atlantic

Monocelis lineata is a complex of cryptic species (three in the Mediterranean and one in the Atlantic) widespread in midlittoral habitats. Throughout the range, populations with or without an ocular pigmented shield are found. We investigated the genetic structure of the North‐East Atlantic populations with the aim of shedding light on their phylogeography and reconstructing possible patterns of recolonization after the Würmian glaciation. Fourteen samples were investigated using cytochrome c oxidase subunit I (COI) and 13 by inter‐simple sequence repeats (ISSRs). COI did not exhibit a clear pattern of decreased genetic diversity along a latitudinal gradient. Populations from Ferrol (Spain), Doolin (Ireland), and Helsingør (Denmark) showed a higher genetic variability, whereas a reduction in the number of haplotypes was found at the northernmost edge of the distribution and in northern Ireland and Scotland. Two genetically differentiated areas (southern Europe and south‐western Ireland versus northern Atlantic) were revealed by ISSR data. The results obtained provided evidence of three refugia (Iberian Peninsula, south‐western Ireland, and North Sea), and the occurrence of secondary contacts that shaped the genetic variability of some of the populations examined. Two different recolonization pathways of north‐western Europe during the post‐Würmian glaciations have been detected. Furthermore, ISSR analysis provided evidence of genetic divergence among populations with and without pigmented eyespot, suggesting the action of ecological differentiation. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 117–135.     

Conservation genetics of the rare Iberian endemic Cheirolophus uliginosus (Asteraceae)

Cheirolophus uliginosus is a rare species, endemic to the south‐western Iberian Peninsula, and listed as a characteristic taxon from the temperate Atlantic wet heaths, a priority habitat for conservation by the European Union. The conservation status of this species in most of its distribution area is poorly known, but, in recent times, some populations have disappeared and there has been a reduction in the number of individuals in others. In this context, we analysed the effects of population size on genetic diversity, revealing that genetic erosion and inbreeding depression could be having a significant impact on smaller populations. Furthermore, we studied the patterns of genetic structure and variability at the species level, finding a strikingly low within‐population diversity and high among‐population genetic differentiation. Finally, the genetic structure analyses suggested a long and complex phylogeographical history of C. uliginosus in the region, in agreement with the climate relict status proposed for this species. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 157–171.     

Population structure,diversity, and phylogeography in the near‐threatened Eurasian black vultures Aegypius monachus (Falconiformes; Accipitridae) in Europe: insights from microsatellite and mitochondrial DNA variation

The Eurasian black vulture (Aegypius monachus) has experienced a severe decline during the last two centuries and is globally classified as near‐threatened. This has led to the extinction of many traditional breeding areas in Europe and resulted in the present patchy distribution (Iberian and Balkan peninsulas) in the Western Palearctic. In the present study, we describe the current genetic status of the European populations using both mitochondrial cytochrome b sequences and nuclear microsatellite markers, comparing with those found in Asia (Mongolia and Caucasus region). Although, mitochondrial (mt)DNA revealed a relatively low genetic variability (haplotype diversity), no evidence of genome‐wide genetic erosion exists because nuclear diversity exhibits normal levels and strong differentiation. A highly philopatric dispersal behaviour must be invoked to explain the existence of a clear pattern that revealed by the phylogeographic analysis, which indicates a sharp East–West clinal distribution and an allopatric differentiation. The distribution of mtDNA haplotypes one in the Iberian population and two in Balkan population and the significance divergence at nuclear loci fulfill the definitions of those populations as evolutionary significant units. We discuss how management strategies should aim at the maintenance (or increase) of current genetic variability levels, suggesting that independent conservation plans are urgently required to protect these two breeding European populations from extinction. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 859–872.     

The phylogeographical history of the Iberian steppe plant Ferula loscosii (Apiaceae): a test of the abundant-centre hypothesis

The geology and climate of the western Mediterranean area were strongly modified during the Late Tertiary and the Quaternary. These geological and climatic events are thought to have induced changes in the population histories of plants in the Iberian Peninsula. However, fine-scale genetic spatial architecture across western Mediterranean steppe plant refugia has rarely been investigated. A population genetic analysis of amplified fragment length polymorphism variation was conducted on present-day, relict populations of Ferula loscosii (Apiaceae). This species exhibits high individual/population numbers in the middle Ebro river valley and, according to the hypothesis of an abundant-centre distribution, these northern populations might represent a long-standing/ancestral distribution centre. However, our results suggest that the decimated southern and central Iberian populations are more variable and structured than the northeastern ones, representing the likely vestiges of an ancestral distribution centre of the species. Phylogeographical analysis suggests that F. loscosii likely originated in southern Spain and then migrated towards the central and northeastern ranges, further supporting a Late Miocene southern-bound Mediterranean migratory way for its oriental steppe ancestors. In addition, different glacial-induced conditions affected the southern and northern steppe Iberian refugia during the Quaternary. The contrasting genetic homogeneity of the Ebro valley range populations compared to the southern Iberian ones possibly reflects more severe bottlenecks and subsequent genetic drift experienced by populations of the northern Iberia refugium during the Pleistocene, followed by successful postglacial expansion from only a few founder plants.     

High genetic diversity of the endangered Iberian three‐spined stickleback (Gasterosteus aculeatus) at the Mediterranean edge of its range

1. The three‐spined stickleback (Gasterosteus aculeatus) on the Iberian Peninsula is only distributed in freshwater habitats and has completely disappeared from most of its range, mainly as a consequence of habitat degradation and invasive fish introductions. Genetic investigations have shown that Mediterranean‐Adriatic sticklebacks constitute an evolutionarily significant unit. Here, we present the first genetic data for Iberian populations living on the southern edge of the stickleback’s range. We used microsatellite markers to study gene diversity, population structure and genetic demography of stickleback populations. 2. High genetic differentiation among collections yielded a model of four genetically homogeneous units related to geography. The observed pattern of isolation by distance resulted mainly from the hydrographical pattern and limited gene flow among rivers. Moreover, low levels of gene diversity, high isolation and recent bottleneck events, which have led to small or even critical effective population sizes in several locations, could be explained by additional recent anthropogenic fragmentation. 3. We defined at least four evolutionarily significant units threatened by habitat fragmentation in north‐eastern Iberian sticklebacks. Because they retain long evolutionary histories, these populations should be considered of high conservation priority, and urgent management measures should be implemented.     

Patterns of nuclear and mitochondrial DNA variation in Iberian populations of <Emphasis Type="Italic">Emys orbicularis</Emphasis> (Emydidae): conservation implications

The European pond turtle (Emys orbicularis) is threatened and in decline in several regions of its natural range, due to habitat loss combined with population fragmentation. In this work, we have focused our efforts on studying the genetic diversity and structure of Iberian populations with a fine-scale sampling (254 turtles in 10 populations) and a representation from North Africa and Balearic island populations. Using both nuclear and mitochondrial markers (seven microsatellites, ∼1048 bp nDNA and ∼1500 bp mtDNA) we have carried out phylogenetic and demographic analyses. Our results show low values of genetic diversity at the mitochondrial level although our microsatellite dataset revealed relatively high levels of genetic variability with a latitudinal genetic trend decreasing from southern to northern populations. A moderate degree of genetic differentiation was estimated for Iberian populations (genetic distances, F _ST values and clusters in the Bayesian analysis). The results in this study combining mtDNA and nDNA, provide the most comprehensive population genetic data for E. orbicularis in the Iberian Peninsula. Our results suggest that Iberian populations within the Iberian–Moroccan lineage should be considered as a single subspecies with five management units, and emphasize the importance of habitat management rather than population reinforcement (i.e. captive breeding and reintroduction) in this long-lived species.     

Colonization patterns and genetic structure of peripheral populations of the trumpeter finch (Bucanetes githagineus) from Northwest Africa, the Canary Islands and the Iberian Peninsula

Aim This paper has three aims: (1) to reconstruct the colonization history of two peripheral populations of the trumpeter finch (Bucanetes githagineus) presumably originating from the same source, one the result of an ancient expansion process and the other recently established and still expanding; (2) to estimate the importance of key events, such as past and current gene flow and bottlenecks, in both expansion processes and their contribution to the present population structure and genetic diversity; and (3) to find out whether two peripheral populations that established at widely differing times also differ in terms of genetic diversity. Location Northwest Africa (assumed source population), Canary Islands (long‐established peripheral) and south‐eastern Iberian Peninsula (recently established peripheral). Methods Bayesian analysis of population structure, individual assignment tests, F‐statistics, maximum likelihood migration estimates, genetic diversity indices and bottleneck tests were calculated with microsatellite data from 194 trumpeter finches from five breeding and two seasonal non‐breeding sites. Results Our data support the existence of two subpopulations (Canary Island and Ibero‐African) as the most likely population structure. Seasonal sites in the Iberian Peninsula had the highest percentage of birds assigned to other, mainly Iberian, sites. Pairwise F_ST values showed that the Canary Island localities were very similar to each other, but differed from the rest. Gene flow estimates within subpopulations were only slightly higher in the Canary Island population than in the Ibero‐African one. Gene diversity indices were similar at all localities. Canary Island sites show evidence of bottlenecks, whereas the Ibero‐African sites do not. Main conclusions Our data show that, at present, birds from the Canary Islands are genetically differentiated from those in North Africa and continental Spain. We could not unequivocally confirm the African origin of Canary populations because the contrary is also plausible. The Iberian Peninsula seems to have repeatedly received individuals from North Africa, which would have led to the relatively high genetic diversity found in these recently established localities and prevented bottlenecks. Movements of individuals towards sites outside their current range during the non‐breeding season are likely to precede the establishment of new breeding sites at the periphery of the distribution range.     

Genetic diversity,genetic structure and phylogeography of the Iberian endemic Gypsophila struthium (Caryophyllaceae) as revealed by AFLP and plastid DNA sequences: connecting habitat fragmentation and diversification

Iberian gypsum outcrops are highly fragmented and ecologically challenging environments for plant colonization. As gypsophytes occur exclusively in such habitats, they are ideal models for the study of both the effects of habitat fragmentation and selection on population genetic diversity and structure. In this study, we used amplified fragment length polymorphism (AFLP) and plastid DNA sequences to investigate the phylogeographical history of the Iberian plant Gypsophila struthium (Caryophyllaceae), a widespread endemic restricted to Iberian gypsum outcrops. Gypsophila struthium consists of two subspecies that differ in the architecture of their inflorescence and have mostly allopatric ranges. Gypsophila struthium subsp. struthium occurs in central, eastern and south‐eastern Iberia, whereas G. struthium subsp. hispanica occurs in northern and eastern areas. AFLPs revealed low but significant genetic differentiation between the subspecies, probably as a result of a recent diversification during the Pliocene–Pleistocene. In the geographical contact zone between the taxa, the Bayesian analyses revealed populations with mixed ancestries and genetic clusters predominantly of one or the other subspecies, indicating incomplete reproductive barriers between them. Plastid DNA haplotypes revealed strong geographical structure and testified to processes of isolation by distance and continuous range expansion for some haplotype clades. The Bayesian analyses of the population structure of AFLP data and nested clade phylogeographical analysis (NCPA) of plastid haplotypes revealed that the putative ancestral range corresponded to central and eastern populations of G. struthium subsp. struthium, with those lineages contributing through more recent expansion to increased genetic diversity and structure of the south‐eastern and eastern ranges of this subspecies and to the diversification of G. struthium subsp. hispanica in northern and eastern gypsum outcrops. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 654–675.     

The impact of management practices and past demographic history on the genetic diversity of red deer (Cervus elaphus): an assessment of population and individual fitness

The influences of management practices and past demographic history on genetic diversity are of critical relevance to sustainable practices and the conservation of wildlife populations. The red deer (Cervus elaphus) is an interesting model species to address these questions because it has a wide geographical distribution and it has been intensively managed for humans in the last decades. In the present study, we have analyzed the impact of recent management practices on the genetic diversity of Iberian red deer populations and assessed the genetic variation effects on population and individual fitness‐related traits. Four populations subjected to distinct management systems were selected: Cabañeros (CB) and Doñana (DN), not hunted populations; Fraga/Caspe (FG/CP), open hunting area with very low or absent management; and PE, fenced private hunting estate founded 31 years ago through the introduction of deer of different origins. Ten microsatellites were amplified in a total of 172 individuals. Additionally, several fitness‐related traits such as the presence of tuberculosis compatible lesions (TBCL), spleen weight (SW), and body length (BL) were estimated. We found a marked genetic variation and differentiation among populations, suggesting a strong population structure. In the fenced population, the introduction of genetically distinct animals has led to high genetic variability (no evidence of inbreeding) despite intensive management. Lower levels of genetic diversity were observed in two historically isolated natural populations (DN and FG/CP). The past demographic history of Iberian populations appears to be more relevant than the current management policy in shaping the genetic variability of natural populations. Population genetic diversity may correlate with life‐history traits and disease susceptibility, which could compromise the conservation and management of these wildlife populations. Although no significant effects of individual genetic diversity (general and local effect hypotheses) were observed on TBCL, SW and BL, some single‐locus effects had almost significant trends for the TBCL and SW traits. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 209–223.     

Climatic niche and neutral genetic diversity of the six Iberian pine species: a retrospective and prospective view

Quaternary climatic fluctuations have left contrasting historical footprints on the neutral genetic diversity patterns of existing populations of different tree species. We should expect the demography, and consequently the neutral genetic structure, of taxa less tolerant to particular climatic extremes to be more sensitive to long‐term climate fluctuations. We explore this hypothesis here by sampling all six pine species found in the Iberian Peninsula (2464 individuals, 105 populations), using a common set of chloroplast microsatellite markers, and by looking at the association between neutral genetic diversity and species‐specific climatic requirements. We found large variation in neutral genetic diversity and structure among Iberian pines, with cold‐enduring mountain species (Pinus uncinata, P. sylvestris and P. nigra) showing substantially greater diversity than thermophilous taxa (P. pinea and P. halepensis). Within species, we observed a significant positive correlation between population genetic diversity and summer precipitation for some of the mountain pines. The observed pattern is consistent with the hypotheses that: (i) more thermophilous species have been subjected to stronger demographic fluctuations in the past, as a consequence of their maladaptation to recurrent glacial cold stages; and (ii) altitudinal migrations have allowed the maintenance of large effective population sizes and genetic variation in cold‐tolerant species, especially in more humid regions. In the light of these results and hypotheses, we discuss some potential genetic consequences of impending climate change.     

Phylogeography of Hyalomma (Euhyalomma) lusitanicum (Acarina,Parasitiformes, Ixodidae) in Andalusia based on mitochondrial cytochrome oxidase I gene

The genetic population structure relationships of Hyalomma (Euhyalomma) lusitanicum in Andalusia (the south of the Iberian Peninsula) were examined using mtDNA sequence data from 887 bp of cytochrome oxidase subunit I (COI) gene. The sequence for the COI region was determined for 84 individuals collected in several localities of Andalusia, and 10 for other localities (i.e., five from Toledo, central Iberian Peninsula, four from Sicily (Italy) and one from Canary Island). Seventeen haplotypes were detected, including 27 polymorphic sites. The number of amino acid substitutions per site from mean diversity calculations for the entire population was 0.017. AMOVA analysis revealed a low gene flow that characterises the genetic population structure of this species in South Iberian Peninsula, with a haplotype diversity (h) value of 0.815. No geographically induced differentiation was observed, and separate evolutionary units were not detected. Our results indicate low genetic diversity across the geographical range of H. lusitanicum tick in Andalusia. Our data do not show any genetic discontinuity between the tick populations studied, including specimens from Canary Island and Sicily (Italy).

     

Phylogeography and species delineation of the genus Phoxinus Rafinesque, 1820 (Actinopterygii: Leuciscidae) in the Iberian Peninsula

The genus Phoxinus is comprised of at least 15 currently recognized species inhabiting Eurasia. Morphological traits have been traditionally used to delineate species in Phoxinus; however, the high level of phenotypic plasticity observed in the genus has confounded this process. Molecular genetic analyses have revealed a higher than expected genetic structure within Phoxinus. Here, we analyzed both nuclear and mitochondrial molecular genetic markers to infer the phylogeography and divergence times of Phoxinus in the Iberian Peninsula. Our results show that the Iberian lineages of Phoxinus were polyphyletic. They also support the co‐existence of three species in the Iberian Peninsula, two corresponding to two previously recognized species (Phoxinus bigerri and Phoxinus septimaniae) and a third undescribed species (Phoxinus sp.). Phoxinus bigerri is structured into western Cantabrian, eastern Cantabrian, and Artibai basins. We hypothesize that this structure is a consequence of glaciation–deglaciation cycles during the Pleistocene. While the presence of P. septimaniae in the Iberian Peninsula is possibly the result of human translocation, that of Phoxinus sp. in lower Ebro rivers may be attributed to past fluvial captures. Our study represents the first report to show a relationship among Phoxinus populations from central Pyrenean rivers of Spain and France. Furthermore, we found genetic hybridization between Phoxinus sp. and P. septimaniae in the shared localities, a likely consequence of anthropogenic activities. Overall, our findings provide insight into the genetic structure of Iberian Phoxinus populations, including the presence of an undescribed species and the putative introduction of some species that may have implications for conservation.     

Narrow endemics on coastal plains: Miocene divergence of the critically endangered genus Avellara (Compositae)

Critically endangered species representing ancient, evolutionarily isolated lineages must be given priority when allocating resources for conservation projects. Sound phylogenetic analyses and divergence time estimations are required to detect them, and studies on their population genetics, ecological requirements and breeding system are needed to understand their evolutionary history and to design efficient conservation strategies. Here we present the paradigmatic case of Avellara, a critically endangered monotypic genus of Compositae inhabiting a few swamps in the west–southwest Iberian coastal plains. Our phylogenetic and dating analyses based on nuclear (ITS) and plastid (matK) DNA sequences support a Miocene (>8.6 Ma) divergence between Avellara and closely related genera, resulting in marked morphological and ecological differentiation. We found alarmingly low levels of genetic diversity, based on AFLPs and plastid DNA sequences, and confirmed the prevalence of clonal reproduction. Species distribution modelling suggested a large macroclimatically suitable area for Avellara in the western Iberian Peninsula, but its apparently narrow microecological requirements restrict its distribution to peatlands with low‐mineralised waters. Although five populations have been recorded from Spain and Portugal in the past, its current distribution may be reduced to only one population, recurrently found in the last decade but threatened by herbivory and habitat degradation. All this confirms the consideration of Avellara as a threatened species with high phylogenetic singularity, and makes it a flagship species for plant conservation in both Spain and Portugal that should be given priority in the design of in situ and ex situ conservation programmes.     

Genetic diversity and structure in fragmented populations of the endangered species Ranunculus cabrerensis (Ranunculaceae): implications for conservation

Ranunculus cabrerensis is an endemic and endangered species of the Northwestern Iberian Peninsula. The molecular markers AFLP and ISSR were used to investigate the genetic diversity and population structure of four populations across its known distribution. Fifteen selective primer combinations of AFLP and seventeen ISSR primer combinations produced a total of 2830 and 103 unambiguously repeatable fragments respectively, of which 97.57 and 81.38% were polymorphic for both markers. The genetic diversity of R. cabrerensis at species level was high (H _E = 0.294 by ISSR and H _E = 0.191 by AFLP) and differentiation between sampled locations was also relatively high (G _ST = 0.316 and 0.158 by ISSR and AFLP analysis respectively) compared to other studies of endangered and rare species using the same techniques. The analysis of molecular variance (AMOVA) indicated that the main genetic variation was within sampled locations (73% by AFLP; 52% by ISSR), even though the variation among locations was also significant. Principal Coordinates, NeighborNet and Bayesian analyses revealed a weak but significant relationship between the genetic structures of different populations in R. cabrerensis, with gene flow acting as a homogenizing force that prevents stronger differentiation of populations. Finally, suggestions for conservation strategies to preserve the genetic resources of this species are outlined.     

Genetic diversity in the endangered dysploid larkspur Delphinium bolosii and its close diploid relatives in the series Fissa of the Western Mediterranean area

Allozyme diversity was evaluated in four closely-related taxa of the Delphinium series Fissa distributed throughout the Western Mediterranean area. All are considered threatened plants. Delphinium bolosii and Delphinium mansanetianum are narrowly endemic to the Eastern Iberian Peninsula, whereas Delphinium fissum ssp. sordidum is found in a few populations across the Peninsula. Delphinium fissum ssp. fissum is more widely distributed but often in small and isolated populations. In this group, Delphinium bolosii is dysploid (2 n  = 18) whereas the other taxa are diploid (2 n  = 16). A total of 12 populations were surveyed, including all known locations for D. bolosii , D. mansanetianum , and D. fissum ssp. sordidum . Eleven enzyme systems were assayed and 15 loci were resolved. Markedly depauperate values for genetic diversity were obtained for D. mansanetianum ( H _e = 0.013) and D. fissum ssp. sordidum ( H _e = 0.044). The estimates for D. fissum ssp. fissum ( H _e = 0.071) were below the values expected for widespread species. Small population size and marginal distribution have probably contributed to the low variability observed in this group. By contrast, D. bolosii exhibited comparatively larger populations and greater genetic diversity ( H _e = 0.138). We suggest that, apart from population size and local adaptation, genetic diversity during speciation may have been promoted by dysploidy through genomic recombination.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 773–784.     

Contrasting levels of genetic diversity between the historically rare orchid Cypripedium japonicum and the historically common orchid Cypripedium macranthos in South Korea

Many terrestrial orchids are historically rare and occur in small, spatially isolated populations. Theory predicts that such species will harbour low levels of genetic variation within populations and will exhibit a high degree of population genetic divergence, primarily as a result of genetic drift. If the origin of the present‐day populations is relatively recent from the same genetically depauperate source population, a complete lack of genetic differentiation between conspecific populations is expected. If a terrestrial orchid was historically common with moderate or high levels of genetic diversity, but has experienced more recent anthropogenic disturbance as a result of over‐collection, it would still exhibit initial levels of genetic variation within populations and a low degree of genetic divergence between populations. To test these predictions, we examined the genetic diversity in six populations (N = 131) of the historically and currently rare Cypripedium japonicum and in four populations (N = 94) of the historically common but now rare C. macranthos from South Korea. Fourteen putative allozyme loci resolved from eight enzyme systems revealed no variation either within or among populations of C. japonicum, which supports the first prediction. In contrast, populations of C. macranthos harboured high levels of genetic variation (mean percentage of polymorphic loci %P = 46.7; mean expected heterozygosity H_e = 0.185) and exhibited a low degree of population genetic divergence (G_ST = 0.059), supporting the second prediction. The lack of genetic variation both within and among conspecific populations of C. japonicum may suggest that populations originated from the same genetically depauperate ancestral population. The high levels of genetic diversity maintained in populations of C. macranthos suggest that the collection‐mediated decrease in the number of individuals is still too recent for long‐term effects on genetic variation. Based on current demographic and genetic data, in situ and ex situ conservation strategies should be provided to preserve genetic variation and to ensure the long‐term survival of the two species in the Korean Peninsula. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160 , 119–129.     

Population history of the terrestrial orchid Cremastra appendiculata var. variabilis from Korea,inferred from levels and distribution of genetic diversity

Cremastra appendiculata var. variabilis is a self‐compatible, insect‐pollinated, terrestrial orchid that is a typical member of the warm‐temperate vegetation in the Korean Peninsula. Here we examine levels and partitioning of allozyme diversity (22 loci) in 12 populations of this orchid to gain insight into its genetic structure and post‐glacial colonization history in Korea. It harboured considerably higher levels of genetic variation within populations (%P = 48.1, A = 1.70 and H_e = 0.217) and lower degree of differentiation among populations (F_ST = 0.068) than those typical of allozyme‐based studies in other terrestrial orchid species. These patterns suggest that extant populations were derived from multiple source populations (i.e. from multiple glacial refugia), although further studies are needed to confirm this scenario. In addition to population history, traits such as high potential of seed dispersal, a mixed mating system and its occurrence in large and continuous populations would have contributed to the current levels and distribution of genetic diversity in Korean populations of C. appendiculata var. variabilis. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 721–732.