Population genetic analysis of Phialocephala fortinii s.l. and Acephala applanata in two undisturbed forests in Switzerland and evidence for new cryptic species

The genetic structure of the root endophytes Phialocephala fortinii s.l. and Acephala applanata was analyzed in two undisturbed forests. A total of 606 strains isolated from surface-sterilized, fine roots of Picea abies and Vaccinium myrtillus were examined. Two new cryptic species of P. fortinii were recognized and host specialization of A. applanata was confirmed. This species was almost exclusively isolated from roots of P. abies. The index of association did not deviate significantly from zero within any population, suggesting that recombination occurs or had occurred. Significant gene but no genotype flow was detected among study sites for P. fortinii s.l. In contrast, several isolates of A. applanata with both identical multi-locus haplotype and identical ISSR fingerprint were found in both study sites indicating genotype flow or a recent common history.     

Suitability of methods for species recognition in the Phialocephala fortinii-Acephala applanata species complex using DNA analysis

Sequence data of two coding and three non-coding loci were used to study the taxonomic identity within and relatedness among seven previously defined cryptic species (CSP) of Phialocephala fortinii and Acephala applanata using two approaches of species recognition. Identification of taxonomic groups corresponding to CSP was ambiguous in some cases when applying solely the genealogical concordance phylogenetic species recognition (GCPSR) concept. The definition of groups corresponding to CSP using GCPSR was complicated due to shared sequence haplotypes between CSP, unresolved CSP for several loci, and possible introgression. GCPSR in conjunction with a population genetic approach improved resolution significantly and the CSP status could be confirmed for all seven CSP of P. fortinii s.l. The most critical step in both analyses was the definition of groups. The combination of several classes of markers differing in resolution helped to define species boundaries.     

Assignment of species rank to six reproductively isolated cryptic species of the Phialocephala fortinii s.1.-Acephala applanata species complex

Phialocephala fortinii s.1. and Acephala applanata are the dominant dark septate endophytes (DSE) in roots of many trees and shrubs. Population genetic analysis led to the discovery of morphologically indistinguishable but reproductively isolated cryptic species (CSP) within Phialocephala fortinii s.1. In the present study we show that sequence data of two coding (beta-tubulin and translation elongation factor [EF-lalpha]) and three noncoding DNA loci confirm subdivision of P. fortinii s.1. and allow to differentiate seven CSP of P. fortinii. In addition we show that strains collected throughout Europe can be classified correctly based on these sequence markers. Statistically significant differences in growth response on different media were observed among CSP of P. fortinii and A. applanata. Growth inhibition on MEA amended with 100 mgl(-1) cycloheximide had the strongest differential effect of all physiological traits examined. In contrast exoenzyme production (laccase, proteinase, pectinase, phenol-oxidase, amylase, cytochrome oxidase and tyrosinase) rarely helped to differentiate CSP of P. fortinii. However A. applanata was a strong producer of amylases, laccases and proteinases. Based on these data we propose to assign species rank to six CSP of P. fortinii: P. turiciensis, P. letzii, P. europaea, P. helvetica, P. uotolensis, P. subalpina spp. nov. and P. fortinii s.s.     

Community structure of Phialocephala fortinii s. lat. in European tree nurseries, and assessment of the potential of the seedlings as dissemination vehicles

Patterns of colonization of conifer roots by dark septate endopyhtes of the Phialocephala fortinii s. lat. species complex in nurseries in Switzerland and Lithuania were studied. The potential for man-mediated genotype flow was estimated for two Swiss nurseries based on customers' addresses and the number of delivered plants. Two hundred and forty-nine strains from three Swiss and five Lithuanian nurseries and an afforestation site were characterized using a combination of inter-simple sequence repeat-anchored PCR (ISSR-PCR), single-copy RFLP analysis, and sequence analysis. P. fortinii s. lat. was abundant in nursery seedlings, but the frequency of seedlings colonized varied considerably among and within nurseries. Ten cryptic species (CSP) of P. fortinii s. lat. were identified, including four hitherto undiscovered CSP. P. helvetica was the dominant species in Swiss nurseries, whereas P. fortinii s. str. was the most abundant species in Lithuanian nurseries and the afforestation site. Swiss nurseries deliver plants over distances of more than 200 km indicating the high potential for man-mediated genotype flow in P. fortinii s. lat.     

Monitoring the spatial and temporal dynamics of a community of the tree-root endophyte Phialocephala fortinii s.l

Phialocephala fortinii sensu lato was isolated from Picea abies roots that had been collected from the same 3 x 3-m forest plot in 2001 and 2004, to examine the spatial and temporal dynamics of this fungal community. RFLP analysis was used to define the multilocus haplotype (MLH) of each isolate. Pielou's measure of association and chi(2) tests of independence were employed to examine the randomness of patterns of spatial distribution of MLH observed in 2001 and 2004. Population differentiation between the two samplings was tested using the G(ST) statistic. In 2001, 144 strains of 28 MLH were isolated; in 2004, 139 strains of 29 MLH. Abundant MLH in 2001 also prevailed in 2004, and the same two cryptic species were dominant in both collections. The probability of being isolated in both years increased with increasing frequency of an MLH. The patterns of spatial distribution of most MLH did not differ between years. The G(ST) values indicated identity of the two collections. Communities of P. fortinii sensu lato remain spatially and genetically stable for at least 3 yr.     

Molecular and phenotypic description of the widespread root symbiont Acephala applanata gen. et sp. nov., formerly known as dark-septate endophyte type 1

Acephala applanata gen. et sp. nov. is described. A. applanata is a dark-septate endophyte (DSE) of conifer roots and belongs to the Phialocephala fortinii species complex. Several genetic markers, including isozymes, inter-simple-sequence-repeat (ISSR) fingerprints, single-copy restriction fragment length polymorphisms (RFLP) and sequences of the internal transcribed spacers (ITS), let us unambiguously separate isolates of A. applanata from isolates of P. fortinii s.l. and other dark-septate endophytes. Alleles at four RFLP loci and two fixed nucleotides in the ITS region were diagnostic for A. applanata. One of the fixed nucleotides resulted in the addition of an Afa I restriction site. PCR amplification with primers prITS4 and the newly developed primer PF-ITS_F (ACT CTG AAT GTT AGT GAT GTC TGA GT) and restriction digestion with Afa I yielded three fragments (203 bp, 117 bp, 56 bp) in A. applanata but only two (260 bp and 117 bp) in P. fortinii s.l. Population differentiation (GST) between A. applanata and other cryptic species of P fortinii was pronounced, and the index of association (IA) did not deviate significantly from zero, showing that recombination occurs or had occurred in A. applanata. Although isolates of A. applanata never were observed to sporulate, it can be distinguished morphologically from P fortinii s.l. by the scarcity of aerial mycelium, significantly slower growth and denser mycelium on cellophane overlaid on water agar. These phenotypic characteristics, combined with diagnostic RFLP alleles and/or PCR-RFLP of the ITS fragment with the fixed Afa I restriction site, unequivocally allow identification of A. applanata.     

Population structure and species boundary delimitation of cryptic Dioryctria moths: an integrative approach

Accurate delimitation of species boundaries is especially important in cryptic taxa where one or more character sources are uninformative or are in conflict. Rather than relying on a single marker to delimit species, integrative taxonomy uses multiple lines of evidence such as molecular, morphological, behavioural and geographic characters to test species limits. We examine the effectiveness of this approach by testing the delimitation of two cryptic Nearctic species of Dioryctria (Lepidoptera: Pyralidae) using three independent molecular markers [cytochrome c oxidase I (COI), second internal transcribed spacer unit (ITS2), and elongation factor 1alpha (EF1alpha)], forewing variation and larval host plant association. Although mitochondrial DNA (mtDNA) haplotypes do not form reciprocally monophyletic clades, restricted gene flow between COI haplotype groups, and concordance with ITS2 genotypes, forewing variation and host plant associations support delimitation of two Nearctic species: eastern Dioryctria reniculelloides and western Dioryctria pseudotsugella. Conversely, EF1alpha genotype variation was incongruent with the two previous markers. A case of discordance between COI and ITS2 was detected, suggesting either introgression due to hybridization or retained ancestral polymorphism due to incomplete coalescence. This study is consistent with other similar literature where molecular loci in closely related species progress from shared to fixed haplotypes/alleles, and from polyphyletic to reciprocally monophyletic relationships, although loci may vary in these characteristics despite maintenance of genomic integrity between distinct species. In particular, mtDNA in other studies generally showed a lower rate of fixation of differences than did X-linked or autosomal loci, reinforcing the need to use an integrative approach for delimiting species.     

Genome wide AFLP markers support cryptic species in Coniophora (Boletales)

Numerous fungal morphospecies include cryptic species that routinely are detected by sequencing a few unlinked DNA loci. However, whether the patterns observed by multi-locus sequencing are compatible with genome wide data, such as amplified fragment length polymorphisms (AFLPs), is not well known for fungi. In this study we compared the ability of three DNA loci and AFLP data to discern between cryptic fungal lineages in the three morphospecies Coniophora olivacea, Coniophora arida, and Coniophora puteana. The sequences and the AFLP data were highly congruent in delimiting the morphotaxa into multiple cryptic species. However, while the DNA sequences indicated introgression or hybridization between some of the cryptic lineages the AFLP data did not. We conclude that as few as three polymorphic DNA loci was sufficient to recognize cryptic lineages within the studied Coniophora taxa. However, based on analyses of a few (three) sequenced loci the hybridization could not easily be distinguished from incomplete lineage sorting. Hence, great caution should be taken when concluding about hybridization based on data from just a few loci.     

Genetic variation within and among populations of Arabidopsis thaliana.

We investigated levels of nucleotide polymorphism within and among populations of the highly self-fertilizing Brassicaceous species, Arabidopsis thaliana. Four-cutter RFLP data were collected at one mitochondrial and three nuclear loci from 115 isolines representing 11 worldwide population collections, as well as from seven commonly used ecotypes. The collections include multiple populations from North America and Eurasia, as well as two pairs of collections from locally proximate sites, and thus allow a hierarchical geographic analysis of polymorphism. We found no variation at the mitochondrial locus Nad5 and very low levels of intrapopulation nucleotide diversity at Adh, Dhs1, and Gpa1. Interpopulation nucleotide diversity was also consistently low among the loci, averaging 0.0014. gst, a measure of population differentiation, was estimated to be 0.643. Interestingly, we found no association between geographical distance between populations and genetic distance. Most haplotypes have a worldwide distribution, suggesting a recent expansion of the species or long-distance gene flow. The low level of polymorphism found in this study is consistent with theoretical models of neutral mutations and background selection in highly self-fertilizing species.     

Spatial patterns of genetic differentiation in <Emphasis Type="Italic">Brachionus</Emphasis> <Emphasis Type="Italic">calyciflorus</Emphasis> species complex collected from East China in summer

In this study, the mtDNA COI genes of 124 individuals in Brachionus calyciflorus complex were sequenced and analyzed. Overall, 84 mtDNA haplotypes were defined, and were split into three clades by the phylogenetic trees. The divergences of COI gene sequence among the three clades ranged from 11.4 to 22.5%, indicating the occurrence of three cryptic species (cryptic species 1, cryptic species 2, and cryptic species 3). Within cryptic species 3, a remarkable degree of differentiation (F _st = 0.0947) might be attributable to habitat fragmentation, restricted gene flow, and founder effect, most probably together with local adaptation. The more shared haplotypes were observed, and a non-significant correlation existed between geographic and genetic distance, suggesting that some ancestral haplotypes might be involved in a past range expansion, and as founders give a similar distribution pattern among geographic populations. The networks were also in agreement with the pattern of genetic differentiation in B. calyciflorus species complex revealed by molecular phylogeny. The nested clade analysis suggested a slight geographic structure in the network I, network II, and the highest nesting clade levels within the network III. Under the possible effect of the Younger Dryas Event, the three cryptic species might have survived in multiple relict refugia throughout the south of China. The co-occurrence of cryptic species 2 and 3 might be found in Guangzhou and Hainan due to the possible interference of the barrier and contiguous range expansion. After the Younger Dryas Event, cryptic species 3 with high colonization ability might have arrived and colonized whole habitats at first, and reduce effective gene flow among cryptic species, thus effectively increasing persistence of founder events. Cryptic species 2 might, respectively, have expanded their ranges into Wuhu and Nanjing regions only because of the probable barrier of the Yangtze River, its weak colonization abilities and the ‘priority effects’ of cryptic species 3. Cryptic species 1 distributed exclusively in Danzhou probably failed to expand its ranges into mainland China because of small population size, weaker dispersal capacity, and the barrier of the Qiongzhou Strait.     

Morphological analysis of some cryptic species in the Acanthocyclops vernalis species complex from North America

Patterns of morphological variation and reproductive isolation were examined for several North American populations of copepods in the Acanthocyclops vernalis Fischer A., 1853 (Copepoda, Cyclopinae) species complex. The copepods were collected from six sites in Wisconsin, U.S.A. Morphological analysis of 120 adult females revealed that a character used previously to distinguish species in this group was unreliable because of phenotypic plasticity. Most of the morphological variance was due to environment (Laboratory vs. field) and to field site. Relatively little of the variation was due to measurement error or asymmetry. Multivariate ordination analysis produced poorly-defined clusters of individuals, suggesting that different biological species are difficult or impossible to distinguish using a set of easily-measurable morphological characters. In our study, morphological similarity was independent of geographic distance among sites, between 0.05 and 300 km. Isofemale lines within sites showed little or no reproductive isolation, but nearly complete isolation among sites. Reproductive isolation was also independent of morphology. These results suggest that the Acanthocyclops population at each site could be considered a distinct cryptic biological species. These copepods expressed morphological stasis – persistence of morphological uniformity despite reproductive isolation. Because of the effect of site and environment on morphology, we recommend using much larger collections (many sites), common garden experiments, and a multi-disciplinary approach (morphological, reproductive, chromosomal, and molecular) as the basis for future taxonomic research on putative copepod species.     

Mitochondrial DNA Sequence Variation in a Sea Star (Leptasteriasspp.) Species Complex

A 551-bp region of a PCR product containing the putative mitochondrial control region and flanking sequences was analyzed for sequence variation among 19 sea stars representing 10 previously described PCR–RFLP haplotypes within a cryptic species complex (Leptasteriasspp.). Most (97%) of the sequence variation was interhaplotypic rather than intrahaplotypic, which greatly reduced the utility of sequence polymorphisms in this mtDNA region as markers of intrahaplotypic population structure and gene flow. The estimated number of transition and transversion substitutions per nucleotide site, corrected for multiple hits, was 0.0364 and 0.0158, respectively. Most of the sequence variation occurred in the first half of the putative control region. Phylogenetic analysis (both maximum parsimony and maximum likelihood) revealed three well-supported clades, but the position of two PCR-RFLP haplotypes was not completely resolved. Low intraspecific mtDNA sequence divergence over large geographic distances may be a general pattern for echinoderm species.     

The global genetic structure of the wheat pathogen Mycosphaerella graminicola is characterized by high nuclear diversity,low mitochondrial diversity,regular recombination,and gene flow

A total of 1673 Mycosphaerella graminicola strains were assayed for DNA fingerprints and restriction fragment length polymorphism (RFLP) markers in the nuclear and mitochondrial genomes. The isolates were collected from 17 wheat fields located in 11 countries on five continents over a six year period (1989-1995). Our results indicate that genetic diversity in the nuclear genome of this fungus was high for all but three of the field populations surveyed and that populations sampled from different continents had similar frequencies for the most common RFLP alleles. Hierarchical analysis revealed that more than 90% of global gene diversity was distributed within a wheat field, while approximately 5% of gene diversity was distributed among fields within regions and approximately 3% was distributed among regions on different continents. These findings suggest that gene flow has occurred on a global scale. On average, each leaf was colonized by a different nuclear genotype. In contrast, only seven mtDNA haplotypes were detected among the 1673 isolates and the two most common mtDNA haplotypes represented approximately 93% of the world population, consistent with a selective sweep. Analysis of multilocus associations indicated that all field populations were in gametic equilibrium, suggesting that sexual recombination is a regular occurrence globally.     

南川绣线菊和细枝绣线菊分子水平研究

对6个野外居群(南川绣线菊和细枝绣线各3个)36个个体进行叶绿体(chloroplast,cp)DNA trnL-trnF片断测序分析.在南川绣线菊中发现了3个单倍型(Ros1-Ros3),在细枝绣线菊中发现了2个单倍型(Myr1-Myr2).两个种的序列联合分析对位排列后得到850 bp,共有9个变异位点,其中一个为碱基插入或缺失,另外8个为碱基置换,变异位点的百分率为0.11.对单倍型的遗传多样性分析表明同一区域亲缘关系相近的单倍型发生于同一居群中,并且存在着明显的分子系统地理学关系.以蔷薇科另两个外属植物Rosa californica 和 Sorbaria sorbifolia为外类群构建这两个种的最大简约(MP)树、最大似然(ML)树及贝叶斯树,结果获得了分辨良好的种间关系树.这表明在分子水平上两个种之间存在明显的差异,这与形态学上的表现是相一致的.遗传多样性分析结果表明了cpDNA trnL-trnF 片段对于绣线菊属的分子地理学研究还是比较有效的,可以通过进一步的大面积采样和分析来揭示植物的遗传结构、冰期避难所等问题.     

Mitochondrial diversity in European and Chinese pigs is consistent with population expansions that occurred prior to domestication

Mitochondrial DNA (mtDNA) diversity in European and Asian pigs was assessed using 1536 samples representing 45 European and 21 Chinese breeds. Diagnostic nucleotide differences in the cytochrome b (Cytb) gene between the European and Asian mtDNA variants were determined by pyrosequencing as a rapid screening method. Subsequently, 637bp of the hypervariable control region was sequenced to further characterize mtDNA diversity. All sequences belonged to the D1 and D2 clusters of pig mtDNA originating from ancestral wild boar populations in Europe and Asia, respectively. The average frequency of Asian mtDNA haplotypes was 29% across European breeds, but varied from 0 to 100% within individual breeds. A neighbour-joining (NJ) tree of control region sequences showed that European and Asian haplotypes form distinct clusters consistent with the independent domestication of pigs in Asia and Europe. The Asian haplotypes found in the European pigs were identical or closely related to those found in domestic pigs from Southeast China. The star-like pattern detected by network analysis for both the European and Asian haplotypes was consistent with a previous demographic expansion. Mismatch analysis supported this notion and suggested that the expansion was initiated before domestication.     

DNA barcoding reveals 24 distinct lineages as cryptic bird species candidates in and around the Japanese Archipelago

DNA barcoding using a partial region (648 bp) of the cytochrome c oxidase I (COI) gene is a powerful tool for species identification and has revealed many cryptic species in various animal taxa. In birds, cryptic species are likely to occur in insular regions like the Japanese Archipelago due to the prevention of gene flow by sea barriers. Using COI sequences of 234 of the 251 Japanese‐breeding bird species, we established a DNA barcoding library for species identification and estimated the number of cryptic species candidates. A total of 226 species (96.6%) had unique COI sequences with large genetic divergence among the closest species based on neighbour‐joining clusters, genetic distance criterion and diagnostic substitutions. Eleven cryptic species candidates were detected, with distinct intraspecific deep genetic divergences, nine lineages of which were geographically separated by islands and straits within the Japanese Archipelago. To identify Japan‐specific cryptic species from trans‐Paleartic birds, we investigated the genetic structure of 142 shared species over an extended region covering Japan and Eurasia; 19 of these species formed two or more clades with high bootstrap values. Excluding six duplicated species from the total of 11 species within the Japanese Archipelago and 19 trans‐Paleartic species, we identified 24 species that were cryptic species candidates within and surrounding the Japanese Archipelago. Repeated sea level changes during the glacial and interglacial periods may be responsible for the deep genetic divergences of Japanese birds in this insular region, which has led to inconsistencies in traditional taxonomies based on morphology.     

Cryptic species, native populations and biological invasions by a eucalypt forest pathogen

Human‐associated introduction of pathogens and consequent invasions is very evident in areas where no related organisms existed before. In areas where related but distinct populations or closely related cryptic species already exist, the invasion process is much harder to unravel. In this study, the population structure of the Eucalyptus leaf pathogen Teratosphaeria nubilosa was studied within its native range in Australia, including both commercial plantations and native forests. A collection of 521 isolates from across its distribution was characterized using eight microsatellite loci, resulting in 112 multilocus haplotypes (MLHs). Multivariate and Bayesian analyses of the population conducted in structure revealed three genetically isolated groups (A, B and C), with no evidence for recombination or hybridization among groups, even when they co‐occur in the same plantation. DNA sequence data of the ITS (n = 32), β‐tubulin (n = 32) and 27 anonymous loci (n = 16) were consistent with microsatellite data in suggesting that T. nubilosa should be considered as a species complex. Patterns of genetic diversity provided evidence of biological invasions by the pathogen within Australia in the states of Western Australia and New South Wales and helped unravel the pattern of invasion beyond Australia into New Zealand, Brazil and Uruguay. No significant genetic differences in pathogen populations collected in native forests and commercial plantations were observed. This emphasizes the importance of sanitation in the acquisition of nursery stock for the establishment of commercial plantations.     

Unexpected cryptic species diversity in the widespread coral Seriatopora hystrix masks spatial‐genetic patterns of connectivity

Mounting evidence of cryptic species in a wide range of taxa highlights the need for careful analyses of population genetic data sets to unravel within‐species diversity from potential interspecies relationships. Here, we use microsatellite loci and hierarchical clustering analysis to investigate cryptic diversity in sympatric and allopatric (separated by 450 km) populations of the widespread coral Seriatopora hystrix on the Great Barrier Reef. Structure analyses delimited unique genetic clusters that were confirmed by phylogenetic and extensive population‐level analyses. Each of four sympatric yet distinct genetic clusters detected within S. hystrix demonstrated greater genetic cohesion across regional scales than between genetic clusters within regions (<10 km). Moreover, the magnitude of genetic differentiation between different clusters (>0.620 G”_ST) was similar to the difference between S. hystrix clusters and the congener S. caliendrum (mean G”_ST 0.720). Multiple lines of evidence, including differences in habitat specificity, mitochondrial identity, Symbiodinium associations and morphology, corroborate the nuclear genetic evidence that these distinct clusters constitute different species. Hierarchical clustering analysis combined with more traditional population genetic methods provides a powerful approach for delimiting species and should be regularly applied to ensure that ecological and evolutionary patterns interpreted for single species are not confounded by the presence of cryptic species.     

Reproductive isolation among cryptic species in the ectomycorrhizal genus Strobilomyces: population-level CAPS marker-based genetic analysis

Among the higher fungi, reproductively isolated cryptic species exist that are morphologically difficult to distinguish owing to a lack of taxonomically useful morphological characters. Mating tests are helpful for detecting reproductive isolation between cryptic species, but are often difficult to perform for higher fungi, especially ectomycorrhizal fungi. In order to identify cryptic species of the ectomycorrhizal genus Strobilomyces more efficiently, lineages were defined based on the nucleotide sequence of two mitochondrial genes. Then the gene flow among lineages was measured using cleaved amplified polymorphic sequences (CAPS) markers designed for single copy nuclear genes. No heterozygosity was observed between different lineages, but within the same lineage heterozygosity was present at the ratio expected given Hardy Weinberg equilibrium. These results show that the mtDNA lineages are separate Mendelian populations, possibly cryptic species that are reproductively isolated from each other.     

Genetic identification of three species of the genus Clarias using allozyme and mitochondrial DNA markers

Genetic variability in three clariid species, Clarias batrachus, C. gariepinus and C. macrocephalus, were investigated by allozyme electrophoresis and mitochondrial DNA (ND 5/6) RFLP markers. The catfish species C. batrachus and C. macrocephalus are native to the Indian subcontinent and Southeast Asia, respectively, while the African catfish C. gariepinus was introduced to Asian countries for culture. Sixteen gene loci from 12 enzyme systems were analyzed. Fixed allelic differences were evident between pairs of species at least at four loci. In mtDNA RFLP analysis, eight composite haplotypes were observed and each species was characterized by a set of haplotypes. The UPGMA phylogenetic tree revealed three distinct clusters: C. batrachus; C. gariepinus (India and Thailand); and C. macrocephalus.