Indian Fern J. 31 : 65-83 (2014) ISSN 0970-2741 MOLECULAR SYSTEMATICS OF THE FILMY FERNS (HYMENOPHYLLACEAE) OF SOUTH INDIA P. K. VIDYA VARMA1 AND P. V. MADUSOODANAN2* 1Govt. College, Madappally, Vatakara-673102, Kerala 2Malabar Botanical Garden, Calicut-673014, Kerala (Received April 22, 2014; Revised Accepted May 16, 2014) ABSTRACT Molecular systematics of the filmy ferns (Hymenophyllaceae) of South India is done based on rbcL nucleotide sequences. Live materials were collected from Western Ghats of South India. Fresh and silica dried fronds were used for the extraction of DNA. The rbcL gene was amplified and sequenced. For phylogenetic analysis 19 sequences (eight newly sequenced and 11 sequences from GenBank) were considered. Multiple sequence alignment was done with ClustalW2 software. The phylogenetic analysis was done with Neighbor Joining method, Maximum Likelihood method and Bayesian inference. As a result a conspicuous clustering pattern was obtained showing the Trichomanoid lineage of South India comprising of three strongly supported clades which correspond to the three genera recognized by Ebihara et al. (2006) viz., Abrodictyum, Crepidomanes and Didymoglossum. Key Words : Molecular systematics, Trichomanoid lineage, South India, rbcL gene. I NTRODUCTION The filmy ferns are unique group of leptosporangiate monilophytes belonging to the family of Hymenophyllaceae Link comprising around 600 species (Iwatsuki, 1990). They grow in the interior of dense humid forests either as epipetric forms or as epiphytes (except certain terrestrial species such as Abrodictyum obscurum) and are easily mistaken for thalloid bryophytes. The molecular phylogenetic study of the family done by Pryer et al. (2001) showed that there exist two lineages in the family viz., Trichomanoid lineage and Hymenophylloid lineage. In this, the Trichomanoid ferns (about 325 species-Dubuisson 1997) show maximum diversity in their morphology and ecological preferences. The Trichomanoid ferns are well adapted to the humid rain forests of Western Ghats region of South India. A detailed study of Hymenophyllaceae of South India done by Hameed et al. (2003) reported twenty two species of Trichomanoid ferns (including Vandenboschia radicans (Sw.) Copel.), which also include five new species under the same lineage (Hameed & Madhusoodanan 1998, 1999, 2003, Madhusoodanan & Hameed 1998, 1999). But the later studies (Fraser-Jenkins 2008a,b, Varma et al. 2014) on the filmy ferns of South India raised doubts about species status and relationships of the newly described taxa of this region. The present study attempts to determine the relationships exist between the Trichomanoid taxa of South India by employing the rbcL gene sequences and also to * E-mail : [email protected] 66 Indian Fern Journal Volume XXXI (2014) determine the affinities and status of various newly described endemic species prevailing in the region. MATERIALS AND METHODS In this study, Trichomanoid ferns (Hymenophyllaceae) distributed all over South India were selected to deduce the systematic relationship existing between them. The prime aim of the study was to deduce the relationship between these taxa by employing the molecular systematic techniques. The specimens for the present study were collected from Western Ghats forests of South Indian region. A portion of the collected specimen was used for deposition as voucher specimen in the Calicut University Herbarium (CALI). The other portion of the collected specimen was utilized for molecular study. As most of the filmy ferns were epiphytic, they were often found to grow intermingled with habitually similar bryophytes, to avoid the possible contamination by bryophytic species, the specimens were examined under Nikon SMZ 800 (Type – 104) stereomicroscope and bryophytes were removed carefully from the fronds. The photographs were taken using Nikon D 100 digital camera (Figs.1 & 2).The clean fronds were separated from the rhizome and were used fresh or after silica drying for the extraction of DNA. DNA extraction was carried out by a modified CTAB method (Doyle & Doyle 1987). Details of the species used for DNA extraction and rbcL amplification was given in TABLE 1 : Details of taxa used for the DNA e xtraction and rbcL g ene amplification Sl. Name of the Species Herbarium Nature of No. accession number the material 1. Crepidomanes bilabiatum (Nees & Blume) Copel. CU.119929 Silica dried material 2. Crepidomanes indicum C. A. Hameed & Madhus. CU.119958 Silica dried material 3. Crepidomanes insigne (Bosch) S.H.Fu CU.119903 Silica dried material 4. Crepidomanes intramarginale (Hook. &G rev.) Copel. CU.119907 Silica dried material 5. Crepidomanes lunulatum Madhus. & C.A. Hameed CU.119980 Silica dried material 6. Crepidomanes malabaricum C. A. Hameed & Madhus. CU.119973 Silica dried material 7. Crepidomanes proliferum (Blume) Bostock var. CU.119985 Fresh material proliferum 8. Crepidomanes saxifragoides (C. Presl) P.S. Green CU.119989 Fresh material P K Vidya Varma & P V Madusoodanan : Molecular Systematics of the Filmy Ferns (Hymenophyllaceae) 67 the Table 1. The quality of the DNA isolated was checked using agarose gel electrophoresis (0.8%) using Rice DNA (30ng) as the control. The gels were visualized in a UV transilluminator (Genei) and the image (Fig.3) was captured under UV light using Gel documentation system (Bio-Rad). PCR amplification of rbcL gene was done by using the four primers (two forward and two reverse). The primer combination aF x JYDS5 and RBCF x 1390R was used (sequences of aF, JYDS5, 1390R from Pryer et al. (2001) and that of RBCF from (Rajiv Gandhi Centre for Biotechnology (RGCB) Thiruvananthapuram, Kerala). The PCR amplifications were carried out in 20 µl reaction volume which contained 1X PCR buffer (100mM TrisHCl, pH-8.3; 500mM KCl), 0.2mM each of dNTPs (dATP, dGTP, dCTP and dTTP), 2.0mM MgCl2, 20ng template DNA, 1 unit of AmpliTaq Gold DNA polymerase enzyme, 0.15 mg/ml BSA and 3% DMSO, 0.5M Betaine, 5pM of forward and reverse primers.The PCR amplification was carried out in a PCR thermal cycler (GeneAmpPCR System 9700, Applied Biosystems) with an initial 95 o C denaturation cycle for 5 min, followed by 40 cycles of 95 o C denaturation for 30 sec, primer annealing at 52o C for 40 sec, elongation at 72o C for 1 min and a final one terminal elongation at 72o C for 7 min. The primer combinations aF x JYDS5 amplified 912bp and the combination RBCF x 1390R amplified about 790bp of the rbcL gene. The PCR products were viewed in 1.2% agarose gel along with 100bp DNA ladder (NEB) as the molecular standard (Fig.4). The complete sequences of the gene were obtained by sequencing each of the amplicon in ABI 3730/3500 Genetic Analyzer (Applied Biosystems). The quality was checked using Sequence Scanner Software v1 (Applied Biosystems). The sequence fragments were edited and assembled into contiguous alignment by using Geneious Pro v5.6 (Drummond et al. 2012). For the phylogenetic analysis of Trichomanoid ferns of South India using rbcL gene sequences, eight newly sequenced taxa, rbcL sequences of eleven species from GenBank (including three sequences under the genus Hymenophyllum) were considered (Table 2). The genus Vandenboschia Copel, which is represented by a single species viz., V. radicans (Sw.) Copel., is not included in this work as its occurrence in South India is doubtful as it is first reported by d‟Almeida (1926) from the region but later works could not collect this species from the region. The sequence of Crepidomanes latealatum (Bosch) Copel. was included in the present study as DNA sequence of C. plicatum (Bosch) R.C. Ching was not available and C. latealatum (Bosch) Copel. was considered to be conspecific with C. plicatum (Bosch) R.C. Ching (Hameed et al. 2003).The details of the sequences from GenBank were given in the Table 2. Multiple sequence alignment of all the 19 species considered for the analysis was done by the ClustalW2 software (Larkin et al. 2007). The software Jalview version 2 (Waterhouse et al. 2009) was used to view the SNPs and conserved regions of the aligned sequences. After the alignment, the sequences of the newly sequenced taxa were trimmed to 1205bp (which was equal to the length of sequences retrieved from GenBank) by using the software BioEdit v7.1.11 (Hall 1999). DnaSP v5 (Librado & Rozas 2009) 68 Indian Fern Journal Volume XXXI (2014) TABLE 2 : Details of the rbcL sequences of taxa accessed from GenBank Sl. Name of the Species GenBank No. accession number 1. Abrodictyum obscurum (Blume) Ebihara & K. Iwats. AB574701 2. Crepidomanes bipunctatum (Poir.) Copel. EU122964 3. Crepidomanes christii (Copel.) Copel. HQ638660 4. Crepidomanes kurzii (Bedd.) Tagawa & K. Iwats. EU122969 5. Crepidomanes latealatum (Bosch) Copel. AB064297 6. Crepidomanes schmidianum (Zenker ex Taschner) K. Iwats. AB378492 7. Didymoglossum exiguum (Bedd.) Copel. AB257488 8. Didymoglossum bimarginatum (Bosch) Ebihara & K. Iwats. AB257494 9. Hymenophyllum acanthoides (Bosch) Rosenst. AB064291 10. Hymenophyllum denticulatum Sw. AB574718 11. Hymenophyllum polyanthos (Sw.) Sw. AB574722 software was used to identify the polymorphic sites, conserved DNA regions, mutations and also for finding the haplotype distribution in the sequences. Estimation of evolutionary divergence between different taxa was done by applying the distance estimation analysis in MEGA version 5.0 (Tamura et al. 2011) using the Maximum Composite Likelihood method (MCL method-Tamura et al. 2004) with gamma distribution for the rate variation among sites. The