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
These results revealed that rbcL nucleotides variations in Diplazium are large enough, but between the two pair of the closely related species D.
sorzogonense TNgP 1744 – D. batuayauense and D. xiphophyllum TNgP 1791 -
D. hottae TNgP 1911. Among species are differentiated from each other by 4-37
nucleotides between pair number 1– 721 Table 8.4.. The more closely related species the lower the differences number of nucleotides. Morphological character
of D. cordifoliuman is very similar to D. angustipinna. The two species are sharing in: imparipinnate fronds with copiously anastomosing veins; scales light
brown, lanceolate, margin entire. The pinnate fronds plant of D. cordifolium differs from D. angustipinna by 11 nucleotides between pair number 103-363.
While the simple fronds plant of D. cordifolium differs from D. angustipinna by 4 nucleotides between pair number 63-717. The distantly related West Malesian
species of D. megasegmentum and D. porphyrorachis are difference each other in 19 nucleotides. The first species is a huge plant with tripinnate fronds whereas the
second species is a small plant with pinnatifid fronds. These results revealed that gene rbcL sequences are well supporting in delimiting species in Diplazium.
Recently comparison of rbcL sequences among species on ferns showed that five or more nucleotides differences are enough to differentiate species. In
genus Diplazium Sano et al. 2000a, 2000b also showed that rbcL nucleotide variation are great, more than four nucleotides, between different species, while
there are no or only two sequence changes within species. The intraspecific variation study at the molecular level using rbcL sequencing in the Japanese
Asplenium of sect. Thamnopteris showed that there are as many as 14-23
differences in the rbcL sequences between Asplenium australasicum J. Sm. and A. setoi
N. Murak. et Seriz. Murakami et al. 1999b.
164 Tabel 8.4. Interspecific Genetic Variaton of Diplazium based on Gene rbcL
Sequences
Species Number of
Nucleotides Difference
Notes D. donianum – D. bantamense
18 D. bantamense – D. lobbianum
TNgP 1031
13 D. cordifolium
TNgP 1737 – D. riparium TML4
16 D.esculentum – D. cavalerianum
37 D. esculentum – D. chinense
16 D. wichurae – D. okudairaekami
5 D. wichurae – D.lonchophyllum
17 D. squamigerum – D. sibiricum
6 D. mesososrum – D. rhachidosorus
12 D. cavalerianum – D. okudairaekami
36 D. pallidum
= D. procumbens TNgP 1163 D. cordifolium
‘simple’ – D. angustipinna TNgP 1905
4 D. cordifolium
TNgP 1238 – D. angustipinna
TNgP 1905 11
D. xiphophyllum TNgP 1791 – D. hottae
TNgP 1911 1
D. speciosum TNgP 1243– D. batuayauense
TNgP 1909 15
D. xiphophyllum TNgP 1791 – D. riparium
TML4 7
D. dilatatum TNgP 1011 – D.
simplicivenium TNgP 1025
16 Missing
data in
13 sites
D. dilatatum TNgP 1011 – D. procumbens TNgP 1015
12 D. bantamense
TNgP 1160 – D. lobbianum TNgP 1181
11 D. sorzogonense
TNgP 1720- 1725 -1731 – 1744 – D. batuayauense
D. simplicivenium TNgP 1179 – D.
dilatatum TNgP 1011
18 Missing
data on
many place, mainly in nucleotide
number 711-721 D.megasegmentum
TNgP 1450 – D. prophyrorachis
19 TNgP
1450 nucleotide
number 63-76 is in N D. porphyrorachis
missing data on nucleotides number 699 –
721
D. poiense – D. porphyrorachis 14
D. poiense missing data
on nucleotide number 649- 721
165 The lack or low of nucleotides difference between D. sorzogonense TNgP
1744 – D. batuayauense and D. xiphophyllum TNgP 1791 - D. hottae TNgP 1911 should be re-checked again in the future studies as the length of the gene rbcL
compared in this study are only 721-bp region from the pair number 1 to 721. The nucleotides differences between the two pair of these species may be existed
between the pair number 722 – 1445. Because morphological studies See Chapter 9 showed D. batuayauense and D. sorzogonense are having many
differences. D. batuayauense differs from D. sorzogonense in the following characters: Scales sharply toothed with thickening dark brown stand irregularly,
fronds are much smaller, never reaching 1 m, lacks the fibrillose scales on stipe and rachis, pinnae lobed only to ½ way to costa, sori not impressed. Therefore D.
batuayauense is propsed as new species. The morphogical differences between D.
hottae and D. xiphophyllum can be seen in the Chapter 9.
8.3.3. Informative Characters of Gene rbcL Sequences for Inferring Phylogenetic Hypothesis of Diplazium
In the molecular phylogenetic relationships studies on ferns, generally rbcL
shows a high parsimony informative characters. Wolf et al. 1994 reported 465 phylogenetic informative characters of the 1320 nucleotide sites of
Dennstaedtioid ferns. In the molecular phylogenetic relationships study in the
heterosporous fern genus Marsilea Nagalingum et al 2007 obtained 58 parsimony informative characters from 1233-pb. Sano et al. 2000a reported 342
parsimony informative characters in the phylogenetic analysis of 68 taxa of ferns in inferring phylogenetic relationship of the Lady fern group, tribe Physematieae,
including Diplazium, by using 1206 bp region between 73-1278. Lu et al. 2007 showed 144 parsimony informative characters from 1320-necleotide segment of
the rbcL gene in studying the phylogeny of the Polystichoid ferns in Asia. Hauk et al
2003, in the phylogenetic studies of Ophioglossaceae, the adder’s tongue ferns that has limited the number of characters available for understanding
relationships, got 341 potentially parsimony-informative characters of the 1321- bp.
The DNA sequence of West Malesian Diplazium that only included 721 base pair bp contained a high parsimony informative characters, 544 75 of
166 parsimony-informative characters. This result showed that gene rbcL sequence is
a good enough marker for inferring phylogenetic hiphothesis of Diplazium.
8.3.4. Phylogenetic Analysis
