Study On Systematic Biology Of Sect. Enantiophyllum In Dioscorea L. In China

Sect. Enantiophyllum belongs to Dioscorea L. Dioscoreaceae. There are about 120species in the section in the world, but only 14 species, 5 varieties in China. They widelydistribute in many provinces south of Yangtse Rive. Stem twining to right. Maleinflorescences spikes, solitary or clustered, or grouped into axillary or terminal panicles.Flowers solitary, sessile; Female spike sometimes branched. Seeds inserted near middle ofcapsule, winged all round. This section is an evolutive natural group in Dioscorea L.Researches on Sect. Enantiophyllum were conducted by micromorphological, anatomical andmolecular biological means. The results were stated as follows:1 The ultrastructure of leaf epidermis cells, epidermis hair and stomatal of 10 species and1 variety in Sect. Enantiophyllum were observed by SEM (Scanning Electron Microscopy)There are epidermis hairs both on nervure and mesophyll. And there exsit 2 types ofepidermis hair: single cell nonglandular hairs and multicell glandular hairs. Leaf epidermiscells of most species are irregular. Appurtenances of leaf epidermis are grain, bits or pieces,which are sparseness or denseness. Most stomatal are ambiquity. The inner margin of stomatalis smooth or lumpy. The appurtenances of periclinal wall is grain, grain-stripe, stripe or cluster.The style of anticlinal wall is arciform. The decorations around stomatal are curving stripe,diffusing stripe after circle or stripe diffusing in 4 directions. This research proved thatultrastructure of leaf epidermis cells, epidermis hair and stomatal are of significance inphylogeny of Sect. Enantiophyllum.2 Using LM (Light microscopy), leaf venation was studied. The vein type of Sect.Enantiophyllum is actinodromous; the number of primary vein is 5-9; the grade of primaryvein is 5-6; the secondary veins' angle is more than 65°; the intersecondaries is complex;areola develops unconsummate, which is square or polygon; bland vein is not ramified; mostmarginal vein ateliosis. Venation characters should not be neglected in the phylogeny of Sect.Enantiophyllum. 3 Based on the former results, the conditions of staminodes of 5 species wereobserved. There are 6 staminodes in the female flowers except for D.aspersa and D.decipiens.Generally, the staminodes against chapiters are bigger. The colour of anthers is darker thanthat of the chapiters. Staminodes have no pollen except that of D.alata. Staminodes in femaleflowers may be one of the evolutive characters in Sect. Enantiophyllum.4 The tuber of Sect. Enantiophyllum is built up by periderm, ground tissue and vascularbundle. Outside the periderm, there is brown defence tissue, which is differentiation fromground weak-wall tissue under epidermis, including phellem, phellem cambium andendophellem weak-wall cells. The outer ground tissue is made up with ground weak-wallcells, thinner, which contains mucous cells (raphides), resiniferous cells. The inner groundtissue is also made up of ground weak-wall cells, vascilar bundle scattering, without anycambium, tracheid or vessel in the center of metaxylem; sieve tube in the center of phloem.There are mucous cells and resiniferous cells in inner ground tissue in some species.5 The micromorphology of starch granules of Sect. Enantiophyllum were described withthe help of LM. There have two forms of starch granules:single and compound. Most singlegranules are similar to round, and a few of them are like shells, triangle or stip. The hilumsseem like dot, flying bird or slit. There are 2 types of compound granules. Type A is consistof 2-3 single granules, which mainly are ovum and trangle and the hilums are like dot whichcan't be seen clearly, but only annular striation of a few species are clear. Type B is consist ofmore than 10 granules, which are round and have illegible hilum and no annular striation.According to the size, starch granules can be devided into 3 types:tiny (long diameterrange, LR＜14μm), Middle (40μm＞LR＞14μm),and Big (LR＞41μm).The hilum & annularstriation and size relate to one another. Specifically, tiny granule has illegible hilum & annularstriation while Big grain has clear hilum & annular striation. Some of the middle sizedgranules have clear hilum & annular striation and some do not.6 Total DNA was isolated by CTAB technique and the PCR products were applied tosequence directly. We analyzed cpDNA trnL-F, rbcL and matK of 11 species of Sect. Enantiophyllum. The results were as follows.: The length of trnL-F is 689-834bp. When thegaps were always treated as missing, there were 67 variable sites, of which 11 wereporsim-info, account for 1.52% of the total length. The (G+C) content was 32.5%. Thepairwise distance between species was 2.2%, of which 0.8% was transitions and 1.4% wastransversions. The total length of rbrL was 1096-1160bp. When the gaps were always treatedas missing, there wa 42 variable sites, of which 10 were parsim-info ones, and account for0.93% of the total length. The pairwise distance between species was 0.9%, of which 0.6%was the transitions and 0.3% was the transversions. The (G+C) content was 44.1%. The totallength of matK was 1032-1165bp. When the gaps were always treated as missing, there were67 variable sites, of which 8 were parsim-info ones, and account for 0.78% of the total length.The pairwise distance between species was 1.3%, of which 0.8% was transitions and 0.5%was the transversions. The (G+C) content was 32.4%. Based on the 3 sequences, thephylogeny trees were constructed by bootstrap test. The molecular trees held the same pointwith the classification of the morphological and anatomical characters. Besides, it is provedthat the cpDNA sequecing is the rapid, efficient, credible way to distinguish RhizomaDioscoreae and its related species.7 Genetic character from 10 species in Sect. Enantiophyllum of Dioscorea L. was studied byISSR markers.92 loci were identified with 11 primers, out of which 88 were polymorphic andaccount for 95.67% of total genetic diversity above species level, shannons indices of diversity(I) was 0.5236 at the section among species level, and nei's gene diversity was 0.3516 and theeffective number of alleles was 1.6068. The genetic distance between D.persimilis andD.cirrhosa is farthest to 0.7051, while the genetic distance between D.polystachya andD.glabra or D.decipiens is nesearest to 0.2733.Cluster analysis grouped all the 10 speciesinto 3 groups. Besides, the results proved that the ISSR marker can be successfully used todistinguish the plants in Sect. Enantiophyllum that have similar exterior.8 Based on 46 morphological characters, using SPSS13.0, phenetic tree was constructedby UPGMA. The tree grouped 10 species and 1 variety into 3 groups. Throwing awaycharacters without plesiomorphy or apomorphy (e.g., the characer of starch and venation), using PAUP, the cladistic tree was constructed by MP. 6 trees were obtained. According to thephylogeny, D.aspersa and D.deipiens are original species, while D.cirrhosa and D.cirrhosavar.cylindrica are more evolutive ones. Phenetic cluster held the same points on the relative ofSect. Enantiophyllumwith cladistic analysis. But they didn't agree with the tree base oncoefficients of ISSR.9 Genetic diversity from 8 populations of D. polystachya Turcz. was studied by RAPDmarkers. 82 loci were identified with 11 primers, out of which 65 were polymorphic andaccounted for 79.27% of total genetic diversity among species level. Shannons indices ofdiversity (I) was 0.4626 among species level, while nei's gene diversity was 0.3159 and theeffective number of alleles was 1.5552. Cluster analysis grouped all the 8 population into 2groups. The results of genetic diversity analysis showed that the gene library of wildpopulation of D.polystachya Turcz. is shrunk and the genetic diversity on molecular leveal isdropping. It is necessary to quick the breeding programe, and this will be helpful to use theimportant medical resource reasonably, sufficiently and efficitively.