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CHAPTER 8 MOLECULAR SYSTEMATIC OF DIPLAZIUM FROM WEST MALESIA
8.1. Introduction
The lack of informative morphological characters in ferns led to the search for new sources of characters in molecular data, including restriction site and
nucleotide sequence data, to infer phylogenetic relationships Eastwood et al 2004. Phylogenetic analysis on Diplazium based on morphological characters
Chapter 6 revealed that Diplazium has only a little informative morphological characters and a hight homoplasy HI=0.76. The lack of the informative
morphological characters and the high of homoplasy suggests that additonal caharcters are required to asses the relationship within this genus.
Advances in molecular biology have provided systematists with a valuable source of characters. Correlating in inferring phylogeny of the vascular plants,
plant sistematists are depending upon chloroplast genome. Most phylogenetic reconstructions in plant systematics conducted so far is based on molecular data
from cpDNA genes. The chloroplast genome is well suited for evolutionary and phylogenetic studies, because: 1 the chloroplast genome is small typically
between 120 and 200 kb, making it relatively easy to examine the entire genome via ristriction site analysis; 2 it contains primarily single copy genes; 3 has a
conservative rate of nucleotide subtitution; and 4 extensive background for molecular information on the chloroplast genome is available Soltis Soltis
1998. The most common gene used to provide sequence data for plant
phylogenetic analysis is the plastid-encoded rbcL gene Chase et al 1993. It is located in the large single-copy region of the chloroplast genome and encodes the
large subunit or ribulose 1,5-bisphosphate carboxylaseoxygenase RUBISCO. This single copy gene is aproximately 1430 base pairs in length; insertions or
deletions indels are extremely rare.
147 The use of gene rbcL sequences for phylogenetic analysis has been
reviewed in Palmer et al 1988. On family level and above, rbcL is preferred for inferring phylogeny. rbcL are not only widely used in the analyzing the extant
taxa, but also found in the leaves fossil samples from Miocene, ca. 17-20 millions ago Golenberg et al. 1990, Soltis et al, 1992; Manen et al, 1995. This gene have
proved useful in elucidating higher-order phylogenetic relationships in angiosperms Chase et al, 1993; Duvall et. al. 1993.
Recent use of rbcL sequence data in constructing pteridophyte phylogeny showed that interspecific as well as higher order relationship could be resolved
Hasebe et al. 1994; Wolf et al. 1994; Hennequin et al 2006. rbcL also could circumscribe the genera of the fern family Crane 1997; Smith et.al. 2006.
Phylogenetic trees based on rbcL sequences support the current classification that the Matoniaceae and its genera, Matonia and Phanerosorus, each are treated as
monophyletic groups Kato Setoguchi 1999. Moreover a large number of rbcL sequence variation has been reported in various species such as Asplenium nidus
Yatabe et al 2001, Hymenasplenium obliquissimum Murakami et al 1998, Stegnogramma pozoi
Thelypteridaceae Yatabe et al 1998, Osmunda cinnamomea
, O. claytonia, and O. regalis Osmundaceae Yatabe et al 1999 and Cheiropleuria bicupsis
Dipteridaceae Kato et al 2001. The substitutions rates of rbcL in Osmundaceae were estimated to be one nucleotide substitution
between two rbcL sequences occurs only one in 5 million years on average Yatabe et al 1999. Considering this slow evolutionary rate for rbcL, the large
amount of rbcL sequence variation within a single morphological species may suggest that these species contain several cryptic species that are reproductively
isolated from each other. rbcL
gene sequence has been used as a tool in unraveling the taxonomic problems in ferns. Based on rbcL gene sequence which are also supported by
morphological and cytological characters, Sano et al 2000a moved D.
subsinuatum and D. tomitaroanum into Deparia lancea and Di. tomitaroana,
respectively. The phylogenetic analysis on nucleotide sequences of the chloroplast-encoded rbcL gene that included Loxoscaphe thecifera Aspleniaceae
and Actiniopteris radiata Pteridaceae results robust clades and consequently the
148 two species should be included into Asplenium and Onychium, respectively
Gastony Johnson 2001. Moreover characters of rbcL sequences information are useful in the discovery of cryptic species in ferns, e.g. Asplenium nidus
Yatabe et al 2001. rbcL
gene sequence has been applied in studying the phylogeny of the Lady fern group, tribe Physematieae Dryopteridaceae Sano et al 2000a. Based
on the chloroplast rbcL gene sequence included 42 species of tribe Physematieae, including Diplazium, Sano et al 2000a showed that: 1 the monophyletic
Diplazium clade included Monomelangium; 2 Athyrium, Cornopteris,
Pseudocystopteris , and Anisochampium form a monophyletic clade and Athyrium
is polyphyletic. However the result of Sano et al 2000a is preliminary because they examined only few 10 species of the ca. 400 species of Diplazium. In their
preliminary inferred phylogeny also showed that Diplazium wichurae from eastern Asia and D. lonchophyllum from central America form a clade in the rbcL
trees. They suggested that more detailed studies including species from wider areas should be useful for biogeographic studies of Diplazium.
In this present study, gene rbcL sequences from 54 collections number representing 29 species of West Malesian Diplazium and 9 collections number of
9 references species outside Malesia were analyzed. The objectives of this were five-fold: 1 to recognize genetic diversity within species; 2 to obtain supporting
evidences in species delimitation; 3 to obtain more informative characters for inferring phylogenetic hypothesis of Diplazium; 4 to evaluate the monophyly of
Diplazium , and 5 to investigate species-level relationships within the genus.
8.2. Materials and Methods
Kategori