| Pinus tabulaeformis f. sekannesis, a Pinus taxa, has a similar morphology with P.tabulaeformis, only distributes in Fuxian county of Shaanxi province with a total area about337.3hectares. Compare with P. taulaeformis, besides high stress-tolerance and adaptabilityto the environment, P. tabulaeformis f. sekannesis has many advantages, such as fast-growth,straight stem, well natural pruning, etc. It is praised as a precious genetic resource in regionsof Loess Plateau of northwest China and worthy to be protected and exploited reasonably andefficiently. Currently, the disputation on the taxonomic status of P. tabulaeformis f. sekannesisin academic fields resulted in restriction of systemic research and efficient protection andutilization on such a superior genetic resource. In order to study the taxonomic status of P.tabulaeformis f. shekannesis, we selected other five Pinus taxa (P. tabulaeformis, P.tabulaeformis var. umbraculifera, P. tabulaeformis var. mukdensis, P. henryi and P.massoniana) as taxonomic criteria. We discussed the taxonomic status of P. tabulaeformis f.shekannesis based on three molecular makers (RAPD, ISSR and SRAP) andmicromorphological traits (anatomical features of needle and palynostratigraphiccharacteristics). The main results are included as follows:1. The genetic diversity and genetic differentiation of six Pinus taxa were revealed byRAPD, ISSR and SRAP. A high level of genetic diversity was founded on the overall level,the percentage of polymorphic loci (PPB) from93.93%(SRAP) to97.71%(RAPD+ISSR+SRAP), an average of96.74%. Compare the level of genetic diversity in termsof each texon, very low level of genetic diversity (PPB average of24.90%) was founded in P.tabulaeformis var. umbraculifera, due to its small number of individuals. The remaining fivetaxa mean PPB descending order was P. tabulaeformis f. sekannesis (PPB=76.85%)> P.henryi (PPB=71.56%)> P. tabulaeformis (PPB=67.24%)> P. tabulaeformis var. mukdensis(PPB=67.16%)> P. massoniana (PPB=57.88%). The same gene differentiation coefficientand gene flow were calculated by RAPD, ISSR and SRAP. Gene differentiation coefficient(Gst) ranged from0.2644(RAPD) to0.3332(SRAP), which indicated26.44%to33.32%ofthe variation existed among taxa, while gene flow (Nm) from1.0004(SRAP) to1.6916(ISSR)greater than1, indicated that gene flow can prevent taxa differentiation caused by genetic drift. Different methods of molecular variance (AMOVA) analysis revealed that there were26%(RAPD) to34%(SRAP) genetic variation among taxa,66%(SRAP) to74%(RAPD) geneticvariation within texon.2. Pollen characteristics of six pines were described by light microscope (LM) andscanning electron microscope (SEM). The results showed that all pollen grains had bisaccate,as same as other Pinus L and their outline represented elliptical from both proximal side viewand distal side view. The pollen size of six Pinus taxa obtained by LM was consistent withSEM, that was P. tabulaeformis var. mukdensis> P. tabulaeformis var. umbraculifera> P.tabulaeformis> P. massoniana> P. henryi> P. tabulaeformis f. sekannesis. The size of pollenobtained by LM (67.84～80.59μm) was greater than by SEM (60.44～69.38μm). Surfaceveins of pollen cappa of the six Pinus taxa were typical of the Pinus L., belonging togranulate-lines like texture by SEM. The surface veins of bladder and aperture were smoothand there was not distinct variation among taxa. Structure and texture of pollen exine in sixtaxa were described by transmission electron microscopy (TEM). Pollen exine consisted ofsexine and nexine and sexine consisted of tectum, columellac and foot layer. The type oftectum belonged to tectum-no perforation and there was not distinct variation of texture ofcolumellac among taxa.3. Cluster analysis and principal component analysis (PCA) were produced based on theneedle anatomy and pollen measurements calculated data in six taxa. The results showed thattwo kinds of contradictory conclusions. The first conclusion, via taxa clustering obtained bythe mean of each index, indicated that the relationship between P. tabulaeformis f. sekannesisand P. massoniana and P. henryi was close to P. tabulaeformis, P. tabulaeformis var.mukdensis and P. tabulaeformis var. umbraculifera. The second conclusion, according to103individuals cluster analysis and PCA mapping by the raw data for each index, showed thatindividuals of the six taxa disorganized mingled together and cannot be distinguished fromeach other. This phenomenon indicated that there was no specificity of needle anatomy andpollen morphologic characteristics among the six taxa. Therefore, it was not reliable thatclustering was obtained by the mean of each index when analyzing the genetic relationshipamong the six taxa of Pinus L. Application of the raw data by103individuals cluster analysisand PCA mapping was more accurate, but individuals of six taxa cannot be separated fromeach other.4. The study on the molecular classification of P. tabulaeformis f. shekannesis wasconducted based on the RAPD, ISSR and SRAP markers data. The results of structureclustering analysis, neighbor-joining cluster analysis and principal coordinate analysisclustering showed that P. henryi and P. massoniana were two distinct species, while P. tabulaeformis f. sekannesis, P. tabulaeformis, P. tabulaeformis var. umbraculifera and P.tabulaeformis var. mukdensis were conspecific (P. tabulaeformis).5. This study find nine highly significant or significant (stable) differences between P.tabulaeformis f. sekannesis and P. tabulaeformis, depending on single-factor analysis ofvariance (SFANOVA) in morphological characteristics, which were needle size and shape,vascular bundle size, endothelial layer size and proportion of needle cross section, the numberand size of resin canals, the number of fat cells, pollen size, corpus size, airsacs size, aperturewidth. Meanwhile, depending on the relevant previous research, we identified eleven stabledifferences between P. tabulaeformis f. sekannesis and P. tabulaeformis, which were needlecolour, bark cracks and shape, the angle between branch and trunk, natural pruning condition,stem straightness, cone shape and size, wood stiffness, the number and size of resin canals inthe inner stem, the number and size of resin canals in the outer stem, the number of resinducts and medullary rays. Therefore, the evidence of molecular classification, twenty stabledifferences between P. tabulaeformis f. sekannesis and P. tabulaeformis and the concept ofsubspecies, varieties and forms, indicated that P. tabulaeformis f. shekanensis should beconsidered a variety of P. tabuliformis. |
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