161
8.2.2. Phylogenetic analysis
DNA sequences were edited and aligned by using Clustal W. All characters were treated as equally weighted and unordered. Maximum
Parsimony MP analysis was performed with PAUP 4.0b10 Swofford 1998 using the heuristic search option with 100 random replicates of stepwise data
addition with TBR swapping and Multrees set to 200 trees limit. Boostrap analysis Felsenstein 1985 was performed with 200 replicates to evaluate internal support.
Athyrium filix-femina, A. distentifolium, A. niponicum, A. sheareri. A.vidalii and
A. yokoscence were selected as outgroup taxa.
8.3. Results and Discussion
Chloroplast rbcL gene sequences of 25 species of Diplazium from West Malesia were newly generated Table 1. Length of the sequences of the 29
species are varying, from 640 to 1445 base pair bp. There are many missing data on gene between pair number 722 – 1445. Therefore 721-bp region from the
pair number 1 to 721 were used to make comparison between two cytotypes in a species or among the closely related species and used for phylogenetic analysis.
However the 721-pb region possess many informative characters, viz. 544 75.
8.3.1. Infraspecific Genetic Diversity in Diplazium
Intraspecific nucleotides differences in Diplazium are showed in Table 8.3. The differences among individuals in the same species are 0 – 5 nucleotides,
but in D. dilatatum, D. procumbens and D. subvirescens. There are not any differences on gene rbcL squences in D. esculentum, D. polypodioides, D.
subserratum, D.simplicivenium, D. silvaticum, and D. xiphophyllum
TNgP1717 - 1719
. As revealed in Chapter 5, the two first species are diploid. D. subserratum showed diploid, triploid and tetraploid. D. simplicivenium and D. silvaticum are
triploid and tetraploid, respectively. Whereas the voucher specimen of the last species was not know its chromosomes number. These results indicate that these
species are autoploid.
162 The diploid and tetraploid of D. pallidum differ from each other by five
nucleotides between pair number 74 – 721. There are missing data on the diploid TNgP 1406 on pair number 1-73. Takamiya et al 2001 reported genetic
comparison between triploid and tetraploid of Diplazium doederleinii. The two cytotypes differ from each other by only two nucleotides in the rbcL sequences,
and there are no variations in cytotype except for a single plant of the triploid. Tabel 8.3. Infraspecific Genetic Variaton of Diplazium based on Gene rbcL
Sequences
Species Number of
Nucleotides Difference
Notes D. esculentum
TNgP 730 – TNgP 1227 D. esculentum
Kagoshima-Japan – Java TNgP 1227
3 D. accedens
TNgP 1001 – TNg 1161 4
In the
same locality,
G. Gede-G.
Pangrango National Park D. accedens
TNgP 1161 G. Gede-Pangrango – TNgP 1211 G. Salak
1 On
nucleotide number
649, in
which TNgP 1161 has Thymine and TNgP 1211 has Cytosine
D. cordifolium ‘simple’ TNgP 1202 – D.
cordifolium ‘pinnate’ TNgP 1237
1 On
nucleotide number
340, in
TNgP 1202 Cytosine pyrimidine base and TNgP 1237 has Adenin
purine base D. cordifolium
TNgP1238 - 1237 10
D. cordifolium TNgP 1912 berbulu – D.
cordifolium TNgP 1237
8 D. xiphophyllum
TD902 – TNgP1717 1
D. xiphophyllum TNgP1717 - 1719
D. polypodioides TNgP 677, TNgP 1184, 1185
D. procumbens 1015 - 1047
5 D. procumbens
1015-1216 7
D. procumbens 1047-1216
6 Missing
data on
nucleotide number 1-4 and 649 to 721 in
TNgP 1216 D. pallidum tetraploid 1172– diploid TNgP
1406 5
Missing data
on TNgP
1406 in
1-73 D. subserratum
TNgP 1070 - 1072 D. lobbianum
TNgP 1031-1181 5
Missing data
on TNgP
1181 in
nucleotide number 1-3 D. lobbianum
TNgP 1181 - 1008 4
D. lobbianum TNgP 1031 - 1008
4 D. silvaticum TNgP 1300 – 1302
D.simplicivenium TNgP 1025, 1073, 1141,
1171, 1175, 1179 D. dilatatum
TNgP 1011 G. Gede Java – ABO21719 Japan
16 D. subvirescens
TNgP 1012 – D. subvirescens TNgP 1178
22
163 The high number of nucleotides differences of D. dilatatum, D.
procumbens and D. subvirescens are presumed to due they are hybrid. In Java,
the three species mentioned above are triploid apogamous. However their putative parents have not been found. Further study are needed to prove this suggestion.
8.3.2. Species Delimitation in Diplazium based on Gene rbcL Sequence
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