Natural hybridization and introgression of Abies firma and Abies homolepis along the altitudinal gradient and genetic insights into the origin of Abies umbellata

Two Japanese fir species (Pinaceae), Abies firma and A. homolepis, comprise the majority of the temperate forest vegetation zones of Japan. Their vertical distributions are exclusively segregated: A. firma occurs in low elevations and A. homolepis occurs in elevated regions; they contact each other at an altitude of around 1,000–1,100 m. Around the contact zone, A. umbellata is described as a tree with shoots of A. homolepis. It has the greenish cones of A. firma instead of the violet A. homolepis cones. An albinism hypothesis and a hybrid hypothesis were proposed to explain the origin of A. umbellata, provoking long debates. Therefore, we examined a total of 78 A. firma and A. homolepis trees (including an A. umbellata tree) along the altitudinal gradient in two study sites using 13 nuclear microsatellites and a species-specific chloroplast DNA (cpDNA) marker and examined morphologies of shoots and cone scales. Finally, we tested the two hypotheses. The genetic analyses revealed that shoot morphologies of A. homolepis and A. firma correspond mostly to the genetic identity of the species, whereas nine trees with intermediate shoot morphologies and two trees (including the A. umbellata tree) with A. homolepis shoots were genetically intermediate, indicating natural hybrids of A. firma and A. homolepis. These 11 hybrids occur in the narrow altitude ranges of their contact zones. The genetic analyses also suggested that hybrid backcrosses with parental species occur. These results support the hybrid hypothesis and suggest that the natural hybrids should taxonomically be classified as A. × umbellata.     

Allelic sequence polymorphisms in the intron region of the nuclear-encoded GapC gene preceded the speciation of three closely related Abies species (Pinaceae)

The DNA sequences of GapC intron 8 were determined for three closely related Abies species, Abies firma, Abies homolepis and Abies veitchii, and ten alleles were identified. Although, in most cases, an allele was specifically detected in one species, some rare alleles were found in two species. The phylogenetic analysis of those alleles showed that they trace back to different ancestral sequences, and that a species possessed the different originated alleles. The polymorphism of the GapC gene observed in the three Abies species seemed to have preceded their evolutionary divergence. Received: 1 February 2000 / Accepted: 10 May 2000     

Spatiotemporal distribution of fruitbodies of ectomycorrhizal fungi in an Abies firma forest

 Spatial associations between ectomycorrhizal (ECM) fungi and their presumed host trees, and spatiotemporal associations among ECM fungi were surveyed for 3 years in an Abies firma-dominated forest in central Japan. A total of 39 species in 13 genera of ECM fungi were recorded, with more species in the Russulaceae than any other family. Russula ochroleuca, Russula sp.1 and Strobilomyces confusus tended to produce their fruitbodies on the forest floor directly under the crown of A. firma, whereas those of Inocybe cincinnata, Gomphus floccosus and G. fujisanensis were aggregated in limited areas outside the A. firma crown. Interspecific spatial associations were analysed for Russula sp.1, which was the most dominant species, and three other frequent species, I. cincinnata, S. confusus and R. ochroleuca. Pairwise, Russula sp.1 with I. cincinnata, with S. confusus or with R. ochroleuca showed an association which was exclusive, overlapping or independent, respectively. Fruiting phenologies differed in that S. confusus showed a peak density in the summer, whereas the other three species peaked in the autumn. These results suggest that the formation of ECM fruitbodies can be partitioned among the species both spatially and temporally. Accepted: 7 July 1998     

In vitro ectomycorrhiza formation between Abies firma and Pisolithus tinctorius

 The first in vitro aseptic synthesis of Abies firma Sieb. et Zucc. with Pisolithus tinctorius (Pers.) Coker & Couch is reported. Techniques were improved for the aseptic synthesis of ectomycorrhizas of A. firma, a slow-growing species in vitro, and Pisolithus tinctorius using a novel culture medium and both sterilized and re-rooted seedlings. After 2–3 months incubation, ectomycorrhizas were formed by both methods. The mycorrhizas possessed a mantle and a highly branched nonseptate Hartig net mycelium colonizing the intercellular spaces within the host cortex, features characteristic of ectomycorrhizas. These techniques will prove useful for addressing physiological and biochemical questions on the interactions of microbes with roots of whole plants. Accepted: 29 June 1999     

Identification and characterization of nuclear microsatellite loci in Abies alba Mill.

Eleven polymorphic nuclear microsatellite markers for Abies alba Mill. were developed from an enriched genomic library. An average of 5.2 alleles per locus and a mean expected heterozygosity of 0.532 were found in a sample of 24 Abies alba individuals from different populations within Europe. These loci can be used in studies of genetic diversity for parentage analysis and for estimation of gene flow in silver fir populations. Moreover, successful amplifications were obtained for eight other Mediterranean Abies species, suggesting that these loci may be useful for similar applications in other fir species.     

Terpenoids from seeds of Abies firma

Nine monoterpenoids, 16 sesquiterpenoids including two novel compounds, 12 diterpenoids and sitosterol were identified in seeds of Abies firma. Two new selinane-type compounds were isolated and their structures were determined as 4α-methoxy-selina-11-ene and 11-hydroxy-4α-methoxy-selinane on the basis of chemical and spectroscopic evidence.     

Ceratobasidiaceae mycorrhizal fungi isolated from nonphotosynthetic orchid <Emphasis Type="Italic">Chamaegastrodia sikokiana</Emphasis>

Mycorrhizal fungi were isolated from the nonphotosynthetic orchid Chamaegastrodia sikokiana and identified as members of Ceratobasidiaceae by phylogenetic analysis of the internal transcribed spacer (ITS) region of ribosomal deoxyribonucleic acid. The ITS sequences were similar among geographically separated samples obtained from Mt. Kiyosumi in Chiba Prefecture and Mt. Yokokura in Kochi Prefecture. One of the isolated fungi, KI1-2, formed ectomycorrhiza on seedlings of Abies firma in pot culture, suggesting that tripartite symbiosis exists among C. sikokiana, mycorrhizal fungi, and A. firma in nature, and carbon compounds are supplied from A. firma to C. sikokiana through the hyphae of the mycorrhizal fungi. To our knowledge, this is the second study to suggest the involvement of Ceratobasidiaceae fungi in tripartite symbiosis with achlorophyllous orchids and photosynthetic host plants.     

Genetic variation at chloroplast microsatellites (cpSSRs) in Abies nebrodensis (Lojac.) Mattei and three neighboring Abies species

Abies nebrodensis (Lojac.) Mattei (Sicilian fir) is an endangered species represented by only one population of 29 adult individuals occurring in a limited area of the Madonie Range in northern Sicily (Italy). Taxonomic boundaries between this taxon and the neighboring Abies species are not clear. In this study, we used six chloroplast simple-sequence repeats (cpSSRs) to investigate the population genetic structure and the distribution of chloroplast haplotypic variation in A. nebrodensis and three of the neighboring Abies species: Abies alba (Mill.), Abies numidica (De Lann) and Abies cepha-lonica (Loud.). Our aims were to quantify the level of cpDNA differentiation within the Abies populations and to shed light on the history of A. nebrodensis. Diversity levels based on the haplotype frequency at six cpSSRs were high, especially in A. alba and A. cephalonica. In all, we found 122 haplotypes among the 169 individuals analyzed, and the four species were distinguished from each other by their haplotype composition. The majority of the haplotypes (76%) were detected only once, but in A. nebrodensis seven individuals (41% of the sample population) shared the same haplotype. Moreover, the seven A. nebrodensis individuals with an identical haplotype showed a tendency to be geographically grouped within the population"s limited range. The analysis of molecular variance (AMOVA) showed a significant difference in the level of apportionment of gene diversity between the species A. alba and A. cephalonica (F_ST=0.191 and 0.012, respectively). AMOVA analysis conducted over all populations from the four species showed that 19% of the total cpSSR variation was attributable to differences among species, 6% was due to differences among populations within species, and 74% to differences within populations. The high percentage of unique haplotypes identified confirms the power of cpSSR haplotype analysis for identifying individual trees in individual Abies populations. Our results indicate that A. nebrodensis differs from the other three Abies species investigated and support its classification as an independent taxon. The results also showed a decreased level of variation in A. nebrodensis and suggested that the species has experienced a genetic bottleneck during the last two centuries. Received: 10 April 2000 / Accepted: 13 July 2000     

Comparative analysis of genetic diversity and population genetic structure in Abies chensiensis and Abies fargesii inferred from microsatellite markers

Abies chensiensis Tieghem and Abies fargesii Franchet are two closely related tree species of Pinaceae endemic to China. A. chensiensis is usually found scattered in small forest fragments, whereas A. fargesii is a dominant member of coniferous forest. To evaluate the genetic effect of fragmentation on A. chensiensis, a total of 24 populations were sampled from the whole distribution of the two species. Seven nuclear microsatellite loci were employed to analyze comparatively the genetic diversity and population genetic differentiation. Both A. chensiensis and A. fargesii have high level within-population genetic diversity and low inter-population genetic differentiation. Low microsatellite differentiation (2.1%) between A. fargesii and A. chensiensis was observed. But microsatellite marker was able to discriminate most populations of these two species. Compared to A. fargesii, A. chensiensi has lower allelic diversity and higher genetic differentiation among populations. It suggested the existence of negative genetic impacts of habitat fragmentation on A. chensiensis.     

Fungal content of ectomycorrhizal tips: comparison among 13 tree species

To better understand soil carbon cycling in forest ecosystems, we studied the proportion of fungal sheath area (FSA) in the cross-sectional ectomycorrhizal area in 13 tree species. Ectomycorrhizal samples were collected from subalpine and temperate forests in Japan. The FSA values were in the range of 12% to 56% across all tree species, tree ages, and fungal species. In Abies firma and Quercus serrata, the FSA values were larger in mature trees than in seedlings, whereas no such differences were found in Pinus densiflora and Fagus crenata. In broad-leaved trees, because the plant tissue radii lay within a narrow range, the FSA was affected mainly by the fungal sheath thickness. In conifers, however, the plant tissue radii varied widely among genera, so the FSA was affected by both the plant tissue radius and the fungal sheath thickness. Our findings suggest that the fungal content of ectomycorrhizal tips differs among tree species and fungal species, so that both parameters must be considered in studies of forest carbon cycling. The estimates revealed that data gathering in each type of forest leads to more accurate estimates of the biomass of fungi in ectomycorrhizal tips.     

Development of microsatellite markers in Abies nordmanniana (Stev.) Spach and cross‐species amplification in the Abies genus

Abies nordmanniana (Stev.) Spach is a widely grown Christmas tree in Denmark, from where it is exported to most of Europe. An ongoing breeding programme is taking place, and as a tool for that, we report the development of five polymorphic nuclear microsatellite markers. To investigate the potential for transferring the markers to other species in the Abies genus, polymerase chain reaction amplification was tested in 19 other species. In general, amplification occurred in a very high proportion of the tested species.     

Age structure and regeneration of old growthCryptomeria japonica forests on Yakushima Island

The regeneration process ofCryptomeria japonica forests was studied from surveys of age and initial growth of the coniferous stumps in plots of 2.66 ha in total on Yakushima Island, south Japan. The conifers germinated during particular regeneration periods each of which was shorter than 100 years. The periods repeated themselves several times in each plot, and conifers of each period formed patches. Both tree cutting and natural gap formation of the canopies initiated the regenerations. The saplings that were thought to have germinated before the initiation of the regeneration grew slowly. After the initiation, the saplings that germinated earlier grew more quickly. For the three codominant conifers:Cryptomeria japonica, Tsuga sieboldii, andAbies firma, the shade tolerance was inversely correlated with maximum age. Even the most shade-tolerantA. firma was a gap-dependent species in regeneration periods, and there were no species differences in the colonizing times during the regeneration period. Shade-intolerant long-lived species and shade-tolerant short-lived species coexisted as climax species, not as alternate species in the sere of succession.     

Host Associations Between Fungal Root Endophytes and Boreal Trees

Fungal root endophytes colonize root tissue concomitantly with mycorrhizal fungi, but their identities and host preferences are largely unknown. We cultured fungal endophytes from surface-sterilized Cenococcum geophilum ectomycorrhizae of Betula papyrifera, Abies balsamea, and Picea glauca from two boreal sites in eastern Canada. Isolates were initially grouped on the basis of cultural morphology and then identified by internal transcribed spacer ribosomal DNA sequencing or by PCR restriction fragment length polymorphism. Phylogenetic analysis of the sequence data revealed 31 distinct phylotypes among the isolates, comprising mainly members of the ascomycete families Helotiaceae, Dermateaceae, Myxotrichaceae, and Hyaloscyphaceae, although other fungi were also isolated. Multivariate analyses indicate a clear separation among the endophyte communities colonizing each host tree species. Some phylotypes were evenly distributed across the roots of all three host species, some were found preferentially on particular hosts, and others were isolated from single hosts only. The results indicate that fungal root endophytes of boreal trees are not randomly distributed, but instead form relatively distinct assemblages on different host tree species.     

Morphological diversity and structure of West Mediterranean Abies species

The aim of this study was the biometrical comparison of Abies pinsapo Boiss., Abies maroccana Trab. and Abies tazaotana Côzar ex Villar from Spain and Morocco. The morphological and anatomical analysis of the needle characters of these firs revealed significant taxonomic differences. The results showed considerable distances between regions and very small differences among the Moroccan fir taxa. The examination of the needles and earlier genetic analysis did not provide evidence for distinguishing A. tazaotana at the species level.     

Isolation and characterization of microsatellite markers in Fraser fir (Abies fraseri)

We describe the isolation and characterization of 14 microsatellite loci from Fraser fir (Abies fraseri). These markers originated from cloned inserts enriched for DNA sequences containing tandem di‐ and tri‐nucleotide repeats. In total, 36 clones were selected, sequenced and evaluated. Polymerase chain reaction (PCR) primers for 14 of these sequences consistently produced simple PCR profiles and were found to be polymorphic among 13 Fraser fir samples. In addition, more than half of these loci were found to amplify a wide range of samples from several Abies taxa.     

Predicting tree death for Fagus sylvatica and Abies alba using permanent plot data

Question: How well can mortality probabilities of deciduous trees(Fagus sylvatica) and conifers (Abies alba) be predicted using permanent plot data that describe growth patterns, tree species, tree size and site conditions? Location: Fagus forests in the montane belt of the Jura folds (Switzerland). Method: Permanent plot data were used to develop and validate logistic regression models predicting survival probabilities of individual trees. Backward model selection led to a reduced model containing the growth‐related variable ‘relative basal area increment’ (growth‐dependent mortality) and variables not directly reflecting growth such as species, size and site (growth‐independent mortality). Results: The growth‐mortality relationship was the same for both species (growth‐dependent mortality). However, species, site and tree size also influenced mortality probabilities (growth‐independent mortality). The predicted survival probabilities of the final model were well calibrated, and the model showed an excellent discriminatory power (area under the receiver operating characteristic curve = 0.896). Conclusion: Mortality probabilities of Fagus sylvatica and Abies alba can be predicted with high discriminatory power using a well calibrated logistic regression model. Extending this case study to a larger number of tree species and sites could provide species‐ and site‐specific tree mortality models that allow for more realistic projections of forest succession.     

Post‐glacial history and introgression in Abies (Pinaceae) species of the Russian Far East inferred from both nuclear and cytoplasmic markers

Aim The main aim of the present study is to infer the post‐glacial history of Abies species from north‐east Asia and to test the hypotheses that coastal Abies populations suffered less from climatic fluctuations during Pleistocene glacial periods than their more continental counterparts, and that Sakhalin was a major area of introgression. Location Natural ranges of the fir species Abies nephrolepis, Abies sachalinensis and Abies holophylla in the Russian Far East, and of Abies gracilis, which is endemic to the Kamchatka Peninsula. Methods Nineteen populations were sampled for allozyme analysis. Seventeen of these populations were also screened for variation at two paternally inherited chloroplast DNA microsatellite loci (cpSSR) and variation at one maternally inherited mitochondrial marker (nad4‐3/4). Finally a subset of 11 populations was analysed with amplified fragment length polymorphism (AFLP). Comparisons were made with already available Abies sibirica data. For all sets of markers, we estimated genetic diversity and differentiation using an analysis of molecular variance (AMOVA). Population clustering was assessed with a Bayesian approach implemented in structure v.2.3. Results Among the three major species, A. sibirica, A. nephrolepis and A. sachalinensis, A. sachalinensis demonstrated the highest cytoplasmic and nuclear diversity and the most continental species, A. sibirica, the lowest. Both nuclear and mitochondrial DNA markers revealed the presence of a transitional zone on Sakhalin Island between A. nephrolepis and A. sachalinensis of south Sakhalin. The structure analysis delivered very clear results confirming the admixed origin of A. sachalinensis, with a genetic contribution from A. nephrolepis. No variation in cytoplasmic markers was found in A. gracilis, suggesting the occurrence of a recent bottleneck. Main conclusions There is a clear reduction of genetic diversity in Abies species from the Pacific coast into the continent. The higher diversity in A. sachalinensis could have two causes: a larger effective population size in the islands due to relatively stable climatic conditions and consequently less pronounced demographic fluctuations in population size and/or hybridization with continental and Japanese populations. Sakhalin Island is a major transitional zone for conifer species. Finally, the fir from Kamchatka, A. gracilis, should be regarded as a separate species closely related to the A. nephrolepis–A. sachalinensis complex.     

Chemical Composition and Content of Lipophilic Seed Extractives of Some Abies and Picea Species

The chemical content and composition of the lipophilic extracts from seeds of some fir species: Abies alba, A. cephalonica, A. concolor, and A. koreana, as well as of a few spruce species: Picea abies, P. orientalis, and P. pungens, were examined. The amount of lipophilic extractives is diverse among the tree species and it varies from 9.8% to 41% of seeds. The chemical characterization showed significant differences, not only in the content, but also in the composition of extractives. However, most of the identified compounds like resin alcohols, ‐aldehydes, and ‐acids, as well as fatty acids, were detected in the seed extracts of all the examined tree species. The dominating identified compound group was esterified fatty acids (2.5 – 55.4% w/w of dry extract), occurring mainly as tri‐ and diglycerides, as well as free acids. The main representatives of this group were linoleic and oleic acids. The resin acids, among which the main were abietic, neoabietic, dehydroabietic, and palustric acids, were also detected at high levels, from 1.8% to 16.9% of the dry seed extracts. Phytosterols, tocopherols, resin hydrocarbons, and resin esters, as well as fatty alcohols were also identified. The coniferous tree seeds, as a renewable natural material, could represent a prospective raw material for producing valuable chemicals.     

Isolation and characterization of 10 new compound microsatellite markers for a mangrove tree species,Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

Avicennia marina is an ecologically important mangrove tree species. We isolated 10 polymorphic microsatellite loci from this species using an improved technique. Our isolated loci provided compound microsatellite markers with polymorphism of two to six alleles per locus. The observed and expected heterozygosities ranged from 0.025 to 0.625 and from 0.096 to 0.767, respectively. These markers would be the useful tools for researching on the genetic diversity and population genetic structure of A. marina.     

Allozyme,chloroplast DNA and RAPD markers for determining genetic relationships between Abies alba and the relic population of Abies nebrodensis

Allozyme, chloroplast (cpDNA) and random amplified polymorphic DNA (RAPD) markers have been used to estimate genetic and taxonomic relationships among different populations of Abies alba and the relic population of A. nebrodensis. Twelve isozyme gene loci, as well as restriction fragment length polymorphism (RFLP) at cpDNA spacer regions between t-RNA genes were analysed. Moreover, a set of 60 random sequence 10-mer primers were tested. Over all isozyme loci, evident differences in allele frequencies among A. nebrodensis and A. alba populations were found, particularly at 2 loci, phosphoglucose isomerase (Pgi-a) and shikimate dehydrogenase (Skd-a). More than 10% of the total genetic diversity was due to differences among populations. High values of genetic distances among populations were also found. Out of the 60 primers tested, 12 resulted in a polymorphic banding pattern both within and among populations. A total of 84 RAPD fragments were produced by the 12 selected primers. A phenogram of relationships among populations was constructed based on RAPD band sharing: the differentiation of the A. nebrodensis population was evident. The analysis of molecular variance (AMOVA) was used to apportion the variation among individuals within populations and among populations. There was considerable variation within each population: even so, genetic divergence was found among populations. This pattern of genetic variation was very different from that reported for inbred species. Identical cpDNA amplification and restriction patterns were observed among all the individuals sampled from the populations. Taken together, the results of allozyme and RAPDs show a clear differentiation among A. nebrodensis and A. alba populations and provide support for their classification into two different taxonomic groups.