Testing hypotheses of speciation in the Plethodon jordani species complex with allozymes and mitochondrial DNA sequences

Salamander populations of the Plethodon jordani species complex form a challenging system for applying the general lineage concept of species to diagnose population-level lineages. The present study reports and analyses mitochondrial-DNA haplotypes (∼1200 nucleotide bases from the genes encoding ND2, tRNA^Trp, and tRNA^Ala from 438 salamanders) from 100 populations representing six species of the P. jordani complex ( Plethodon amplus , Plethodon cheoah , Plethodon jordani , Plethodon meridianus , Plethodon metcalfi , and Plethodon montanus ) with comparative analyses of previously published allozymic data to reconstruct the evolutionary history of this group and to diagnose species lineages. Analyses of mitochondrial haplotypic data include nested-cladistic analysis of phylogeography, analysis of molecular variance, hierarchical analysis of nucleotide-diversity measures, and likelihood-based estimates of recent temporal changes in population size. New analyses of allozymic data include multidimensional scaling and principal component analyses, and both data sets are analysed and compared for congruent genetic structure using Mantel correlation tests. These analyses in combination identify the six named species as distinct evolutionary lineages despite sporadic genetic exchanges among them and some discordance between mitochondrial DNA and allozymic markers. Sexual isolation is not complete for any pair of these six species, but they replace each other geographically and appear to block the geographical spreading of their neighbours. The P. jordani complex is a strong study system for investigating the genetic and ecological processes responsible for vicariant speciation.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 89 , 25–51.     

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     

Fine‐scale genetic structure overrides macro‐scale structure in a marine snail: nonrandom recruitment,demographic events or selection?

The planktotrophic littorinid species Littoraria flava occupies a continuous habitat on rocky shores close to brackish and freshwater sources. Previous studies of this species have shown a moderate genetic structure over a broad geographical scale, with high deviations from Hardy–Weinberg expectations in many allozymic loci. Local-scale subdivision in marine species with a long dispersal phase is unexpected, but occasionally found. Using a horizontal transect at three locations, we examined whether microscale and short-term subdivision also occurred in L. flava populations and, if so, whether this could explain the Hardy–Weinberg deviations. Littoraria flava showed even more structuring on a microgeographical scale (4–300 m) than on a large-scale (> 200 km). The Ewens–Watterson neutrality test showed that 18% of the tests deviated significantly from the neutrality model. A homogeneity test for each locus across samples within transects showed homogeneous and high F _IS values in many loci. These results and the apparent genetic patchiness within transects suggest that asynchronous spawning associated with recurrent colonizations in L. flava can explain the local differentiation without a recognizable pattern. In addition, there could be a balance between these factors and diversifying selection acting on different loci at different times and localities. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 23–36.     

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.     

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.     

Genetic variation in Irish pygmy shrews Sorex minutus (Soricomorpha: Soricidae): implications for colonization history

The status of the pygmy shrew ( Sorex minutus L.) as a native or an introduced species in Ireland has been subject to much debate. To examine this and other aspects of the colonization history of the Irish pygmy shrew, genetic variation was determined in 247 pygmy shrews collected throughout Ireland, using mitochondrial control region sequences and five polymorphic microsatellite loci. Genetic diversity was low for both types of marker. The median-joining network for control region sequences was star-like, suggesting that the colonization of Ireland involved a small number of founders and rapid population expansion thereafter; this was supported by other statistics. Molecular dating with both mitochondrial DNA and microsatellite data is consistent with a human introduction. This would have been several thousand years ago; a recent colonization within historical times can be ruled out. This is the first detailed population genetic study of the pygmy shrew anywhere in its range.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 918–927.     

Spatio-temporal genetic variation in sympatric and allopatric Mediterranean Cicada species (Hemiptera, Cicadidae)

Cicada barbara (Stål) and Cicada orni L. are two Mediterranean cicadas, very similar in morphology, that produce distinct acoustic mating signals and that have partially overlapping distribution ranges in the Iberian Peninsula, occurring in sympatry in several locations. In the present study, six microsatellite loci were analysed in C. barbara , four of which were also analysed in C. orni . Geographical and temporal genetic variation in these species was studied. No evidence of hybridization was found, enabling us to infer that the isolating barriers between these species are efficient. Partitioning of geographic variation in each species, revealed the following patterns: Iberian Peninsula and Northwestern African populations of C. barbara showed higher differentiation between than within each region, supporting C. barbara subspecific taxonomy ( C. barbara lusitanica in the Iberian Peninsula and C. barbara barbara in Northwestern Africa) and highlighting isolation coincident with the presence of physical barriers to gene-flow; differentiation between populations of C. orni from both sides of the Pyrenees was very low, and this mountain range may not constitute a significant barrier for the dispersal of this species; Greek populations of C. orni were found to be highly differentiated from Western European populations; and no pattern of isolation-by-distance was found in either species within the Iberian Peninsula.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 249–265.     

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.     

Parnassia palustris: a genetically diverse species in Scandinavia

Patterns of variation at nine enzyme loci were examined in 528 plants representing diploid and tetraploid populations of Parnassia palustris s. l. in Europe to assess genetic variation patterns and migration history. Half of the plants showed a unique multilocus phenotype and 75% of all phenotypes occurred only in Scandinavia. Diploid populations showed similar levels of genetic diversity as other widespread outbreeding species with animal-mediated pollination and F -statistics indicated excessive heterozygosity and low rates of gene flow among them. In spite of dramatic population histories caused by the ice ages, diploid populations have maintained the same genetic diversity in Scandinavia as in central and southern Europe. Northern populations have apparently been established through the gradual advance of genetically variable populations and patterns of variation at individual loci indicate different migration routes, from the south-south-west and the east-north-east, respectively. The data strongly support a repeated autoploid origin of the tetraploid cytotype which has been much more successful than the diploid progenitors in colonizing new land since the last ice age. High genetic diversity in Scandinavia has apparently been obtained by a combination of immigration of plants from different source areas and recurrent formation of autotetraploids from diploid progenitors.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 142 , 347−372.     

Systematics of the black rat in Madagascar: consequences for the transmission and distribution of plague

The plague has been present for a century in Madagascar; the only known reservoir is the black rat, which is also the main victim of this disease. Whereas the two plague foci are restricted to an altitude above 800 m, the black rat is distributed over the whole island, the only morphological variation being a within-site variability in belly colour. To resolve these apparent contradictions, an analysis of the different belly colour morphs and of populations living in different habitats and altitudes was undertaken. An allozymic study demonstrated that sympatric white- and grey-bellied rats ( N  = 26) living within the plague foci, all belonged to the same species: Rattus rattus . This specific assignment was confirmed by a chromosomal analysis which showed that all 90 individuals studied presented the same diploid number, 2 n  = 38, whatever the habitat and altitude. This study indicates that all specimens investigated in Madagascar could be referred to the same black rat species. Survival of rat populations, despite a century of coexistence with the plague, is most likely related to their high reproductive and recolonization potential, as well as selection of plague resistant genotypes. As the altitudinal limit of the plague foci does not seem to be related to characteristics of the rodent reservoir, investigations concerning the vectors are now essential.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 78 , 335–341.     

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.     

Possible cryptic speciationwithin the sea anemone Actinia equina complex detected byAFLP markers

The population genetic profiles of 60 specimens of thecommon intertidal sea anemone Actinia equina taken from foursampling sites (Peroj, Barbariga and the north and south shoresof the Limski Canal), along a 25 km strip of the coastof the Istra Peninsula, Croatia, were evaluated by AFLP markers(144 loci). All populations were characterized by aggregates ofspecimens confined within a few square meters. One of the populations(on the north shore of the canal) contained both large and small-sizedindividuals. Results revealed high within-aggregate genetic heterogeneity(36.1–55.6%). With respect to interpopulationprofiles, geographically remote populations from Peroj, Barbarigaand the south shore of the canal were more closely related geneticallythan those on the north and south shores of the canal. The small-sizedsubpopulation from the north shore exhibits a distinct AFLP pattern,suggesting that it warrants recognition as a cryptic species.  © 2002The Linnean Society of London, Zoological Journal of the LinneanSociety , 2002, 136 , 315−320     

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.     

Genetic differentiation amongst fragrant orchids (Gymnadenia conopsea s.l.) in the British Isles

Genetic variation was examined in five microsatellite loci to seek evidence of genetic differentiation and restricted gene flow that would support the taxonomic division of Gymnadenia into three species ( G. borealis , G. conopsea , and G. densiflora ). A total of 107 alleles was detected in 17 populations from England, Scotland, and Ireland. The mean expected heterozygosities ranged from 0.48 to 0.81. The differentiation in allele frequencies amongst populations that had been assigned to each taxon on the basis of morphology was sufficiently large to support the taxa as distinct species. Phylogenetic trees based on microsatellite allele frequencies, as well as assignment tests, supported the existence of three distinct groups with at least partial restriction of gene flow between them. There was substantial homozygote excess, leading to high F _IS estimates, for most loci in most populations. This is unlikely to have been a result of widespread null alleles, and more probably reflects a high level of inbreeding in G. conopsea . This inference requires further investigation. The implications of the results of this and other taxonomic studies for the conservation of Gymnadenia in Britain are discussed.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 155 , 349–360.     

Rapid radiation of the Mediterranean Luciobarbus species (Cyprinidae) after the Messinian salinity crisis of the Mediterranean Sea, inferred from mitochondrial phylogenetic analysis

Phylogenetic relationships of 64 freshwater Barbus s.s. species distributed in basins around the Mediterranean Sea were assessed using cytochrome b sequences. Our results are in concordance with previous morphological and genetic studies, which proposed that these species belong to two major lineages (or subgenera): Barbus and Luciobarbus . We were particularly interested in phylogenetic relationships among species of the Luciobarbus lineage that are primarily found in the southern Mediterranean region from the Iberian Peninsula to the Middle East. In the Luciobarbus lineage, species that were previously attributed to the Capoeta genus were clustered. In this study, we observed short internodes between monophyletic groups having a geographical agreement around the Mediterranean. However, groups from the opposite sides of the Mediterranean Sea (Iberian Peninsula– Capoeta , north-western Africa–Middle East) seem to be phylogenetically close. We therefore infer that rapid radiation of Luciobarbus species in the Late Miocene better fits our data rather than gradual founder events in the southern Mediterranean. We propose that the biogeographical event along an east–west route, responsible for the present distribution of Luciobarbus species, was the 'Lago Mare' phase of the Mediterranean Sea that provided a rapid dispersal route over extensive distances. This provides new insights into the speciation pattern of this group, and may be of general use in the study of freshwater species in these regions.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 80 , 207–222.     

High genetic variability in Neotropical myophilous orchids

The patterns of genetic variability of seven Bulbophyllum Thouars species were investigated using 14 enzymatic loci, and compared with those of other Neotropical myophilous orchid species. The genetic variability estimated was very high ( H _e = 0.39–0.61, P  = 86–100%, A  = 2.6–3.8), despite the small population sizes, which may be explained by the vegetative reproduction. Of the three species with multiple populations, only B. epiphytum showed moderate values of genetic structuring. Environmental characteristics and seed dispersal mechanisms are important in understanding the differences in the population structure observed. Almost all pairs of species showed low genetic similarity, indicating a long period of divergence. Our results suggest that B. adiamantinum and B. insectiferum , species currently classified in different sections of the genus, are more closely related than previously thought.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 153 , 33–40.     

Genetic variation and its evolutionary implications in a Mediterranean island endemic lizard

The peculiar bioclimatic and geographic features of Corso–Sardinian islands may provide an ideal scenario for investigating microevolutionary processes, given their large heterogeneity of environments, which could affect dispersal and gene flow among populations, as well as processes of local adaptation. The genetic variation and differentiation among populations of the endemic lizard Archaeolacerta bedriagae were studied by allozyme electrophoresis at 20 presumptive loci. The genetic structure of this species is characterized by relatively high levels of polymorphism and low differentiation among populations. The pattern of genetic differentiation cannot be explained by genetic drift as a function of geographic distance. Genetic distance data show that genetic variation is distributed into three geographically coherent population groups and suggest a recent (Late Pleistocene) origin for the observed geographic fragmentation. The analysis of environmental correlates of allozymic variation indicates a strong correlation of the Idh-1 locus with climatic variables. The frequency of the Idh-1¹⁰⁶ allele is negatively correlated with annual temperature, and positively correlated with annual precipitation. In addition, the observed heterozygosity at this locus decreases towards more arid climatic regimes. The results obtained support the assumption of differential selection acting on Idh-1 allozymes under diverse climates. An association between Idh-1 allozymes and local bioclimatic regimes was also observed for the sympatric lizard Podarcis tiliguerta , suggesting a key role for such selective agents on Idh-1 polymorphism in these two Corso–Sardinian lacertids.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 661–676.     

Two new species of Galium (Rubiaceae) from the Iberian Peninsula

Two new species of the genus Galium are described from the Iberian Peninsula: G. moralesianum Ortega-Olivencia & Devesa, and G. talaveranum Ortega-Olivencia & Devesa. The first grows on limestone or dolomitic-limestone outcrops in south-east Spain (the Segura and Gádor mountain ranges) and shows morphological similarities with G. boissieranum Ehrend. & Krendl, which is endemic to southern Spain (mountains of Málaga). The second inhabits the margins of water courses, alder stands and cork-oak dehesas (parkland-type systems) of the west of the Iberian Peninsula, and is morphologically reminiscent of G. mollugo L. The chromosome numbers of these two species and of G. boissieranum are given.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 143 , 177−187.     

Genetic differentiation among the Maculinea species (Lepidoptera: Lycaenidae) in eastern Central Europe

The present study aimed to analyse the level of genetic variation in the eastern Central European (Slovenia, Hungary, and Romania/Transylvania) populations of the Large Blues ( Maculinea ) to analyse the pattern of differentiation both between and within the species. One objective was to compare the level of differentiation between the two disputed species ( Maculinea alcon and Maculinea rebeli ) with that among the other species. Imagos were collected from 23 localities in eastern Central Europe in 2002. Enzyme polymorphism was analysed using polyacrylamide gel electrophoresis. Fourteen enzyme loci were studied in all samples. In the analysis of the data, F -statistics and Nei's genetic distances were calculated and a dendrogram (unweighted pair group method with arithmetic mean) was constructed on the basis of the distance matrix. A multivariate analysis of variance was performed to study the pattern of genetic differentiation among the samples. Principal component analysis analysis was also carried out using the allele frequency data of the samples. Our results indicated that the large blues are generally less polymorphic than other European lycaenid butterflies studied. At the same time, the level of genetic differentiation was high, even among local populations within the species. A low level of genetic variation within the populations coupled with strong differentiation among them implies the effect of genetic drift. Strong genetic differentiation of four Maculinea species ( M. alcon , Maculinea teleius, Maculinea nausithous , and Maculinea arion ) was confirmed. Significant differentiation was not found between M. alcon and M. rebeli .  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 11–21.