Molecular Systematics of the Filmy Ferns (Hymenophyllaceae) of South India P

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 phylogenetic analysis was conducted by using Distance, Maximum likelihood and Bayesian inference approaches. As indels and gaps were not present in the rbcL gene, all the 1205 positions were considered for the analysis. Before the analysis, evolutionary model test was done in MEGA version 5.0 (Tamura et al. 2011). Rooting of the tree was done with the three Hymenophyllum species (specifically using H. polyanthos (Sw.) Sw.). Bayesian analysis was performed in Mr.Bayes version 3.1 (Huelsenbeck & Ronquist 2001, Ronquist & Huelsenbeck 2003) with two searches run simultaneously for at least two million generations. Flat Dirichlet as the prior and General Time Reversible model with a proportion of invariable sites and a gamma-shaped distribution of rates across sites as the model of nucleotide substitution were opted. Three heated chains (temperature 0.2) and one cold chain were used in each search and trees were sampled every 1000 generations. The parameter was then fixed for a bootstrap analysis with 1000 replicates. Tracer version 1.5 (Rambaut & Drummond 2007) was used to evaluate mixing and convergence, and to estimate appropriate burn-in period. For distance methods and maximum likelihood approaches the software MEGA

TABLE 3 : Percent Identity Score generated by ClustalW2 between the 19 taxa using rbcL sequences (1205 bp)

1. A. obscurum, 2. C. bilabiatum, 3. C. bipunctatum, 4. C. christii, 5. C. indicum, 6. C. insigne, 7. C. intramarginale, 8. C. kurzii, 9. C. lunulatum,

10. C. malabaricum, 11. C. latealatum, 12. C. proliferum var. proliferum, 13. C. saxifragoides, 14. C. schmidianum, 15. D. bimarginatum, 16. D. exiguum,

i 17. H. acanthoides, 18. H. denticulatum, 19. H. polyanthos d

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 ar

1 100 91 91 91 91 91 91 91 91 91 91 90 90 90 89 91 91 91 92

2 91 100 98 99 98 99 99 98 99 99 99 94 94 98 90 90 89 89 89 V

Madusoodana 3 91 98 100 99 98 98 98 98 98 98 98 95 94 98 89 90 89 89 89

4 91 99 99 100 98 99 98 98 98 98 98 95 94 98 90 90 89 89 89

5 91 98 98 98 100 98 98 98 98 98 98 94 94 98 90 90 90 89 89

6 91 99 98 99 98 100 99 98 99 99 99 94 94 98 90 90 89 89 89 M

olecula

7 91 99 98 98 98 99 100 98 100 100 98 94 94 98 90 91 89 89 89

8 91 98 98 98 98 98 98 100 98 98 98 94 93 98 89 90 89 89 89 S

ystem 9 91 99 98 98 98 99 100 98 100 100 98 94 94 98 90 91 89 89 89

tic 10 91 99 98 98 98 99 100 98 100 100 98 94 94 98 90 91 89 89 89

11 91 99 98 98 98 99 98 98 98 98 100 95 94 98 90 90 89 89 89 th

12 90 94 95 95 94 94 94 94 94 94 95 100 96 94 88 89 89 89 89 il

13 90 94 94 94 94 94 94 93 94 94 94 96 100 94 88 89 89 89 89 F

14 90 98 98 98 98 98 98 98 98 98 98 94 94 100 89 90 89 89 89

(

15 89 90 89 90 90 90 90 89 90 90 90 88 88 89 100 95 89 89 89 ymenop

16 91 90 90 90 90 90 91 90 91 91 90 89 89 90 95 100 89 89 89

h 17 91 89 89 89 90 89 89 89 89 89 89 89 89 89 89 89 100 99 97 yllaceae)

18 91 89 89 89 89 89 89 89 89 89 89 89 89 89 89 89 99 100 97

19 92 89 89 89 89 89 89 89 89 89 89 89 89 89 89 89 89 97 100

70 TABLE 4 : Estimation of evolutionary divergence between 19 taxa (as distances) using the rbcL sequences (1205 bp). Standard error estimate were shown above the diagonal

oli

nulatum

bimarginatum exiguum polyanthos

acanthoides denticulatum

p bipunctatum schmidianum malabaricum

indicum

saxifragoides bilabiatum intramarginale

latealatum kurzii christii insigne

.obscurum

. . . . .

......

. .

C C proliferum C C C C C C C C C C C D D H H H A

C. saxifragoides 0.007 0.009 0.009 0.009 0.010 0.009 0.009 0.009 0.010 0.009 0.009 0.010 0.018 0.017 0.017 0.017 0.017 0.015

India C. proliferum 0.040 0.009 0.009 0.009 0.009 0.008 0.009 0.009 0.009 0.009 0.009 0.009 0.018 0.017 0.017 0.017 0.017 0.015

n var. proliferum

C. bilabiatum 0.059 0.053 0.001 0.003 0.003 0.003 0.003 0.002 0.003 0.003 0.003 0.003 0.016 0.015 0.017 0.017 0.016 0.013 n

J C. plicatum 0.058 0.052 0.002 0.003 0.003 0.003 0.003 0.002 0.004 0.003 0.003 0.004 0.016 0.015 0.017 0.017 0.016 0.014 ou

C. intramarginale 0.054 0.055 0.010 0.011 0.003 0.004 0.003 0.003 0.004 0.000 0.000 0.004 0.015 0.015 0.016 0.016 0.016 0.014 l

C. kurzii 0.065 0.057 0.012 0.013 0.013 0.004 0.004 0.003 0.004 0.003 0.003 0.004 0.016 0.015 0.017 0.017 0.016 0.014 olum C. bipunctatum 0.058 0.050 0.012 0.013 0.013 0.015 0.002 0.003 0.004 0.004 0.004 0.004 0.016 0.015 0.016 0.016 0.016 0.014

C. christii 0.058 0.052 0.010 0.011 0.012 0.013 0.003 0.003 0.004 0.003 0.003 0.004 0.016 0.015 0.016 0.016 0.016 0.014 XXX

C. insigne 0.058 0.054 0.003 0.004 0.010 0.012 0.012 0.010 0.003 0.003 0.003 0.003 0.016 0.015 0.016 0.016 0.016 0.014 I

(2014) C. schmidianum 0.061 0.058 0.012 0.013 0.017 0.019 0.019 0.017 0.012 0.004 0.004 0.005 0.016 0.016 0.017 0.017 0.017 0.014 C. malabaricum 0.054 0.055 0.010 0.011 0.000 0.013 0.013 0.012 0.010 0.017 0.000 0.004 0.015 0.015 0.016 0.016 0.016 0.014

C. lunulatum 0.054 0.055 0.010 0.011 0.000 0.013 0.013 0.012 0.010 0.017 0.000 0.004 0.015 0.015 0.016 0.016 0.016 0.014 C. indicum 0.062 0.056 0.012 0.013 0.015 0.017 0.017 0.015 0.012 0.019 0.015 0.015 0.016 0.016 0.016 0.016 0.016 0.014 D. bimarginatum 0.125 0.123 0.110 0.110 0.107 0.113 0.111 0.109 0.109 0.114 0.107 0.107 0.110 0.008 0.016 0.016 0.017 0.016 D. exiguum 0.115 0.115 0.101 0.102 0.098 0.103 0.103 0.101 0.101 0.105 0.098 0.098 0.103 0.047 0.017 0.017 0.017 0.014 H. acanthoides 0.120 0.120 0.116 0.117 0.112 0.118 0.115 0.115 0.116 0.120 0.112 0.112 0.115 0.114 0.119 0.001 0.005 0.014 H. denticulatum 0.121 0.121 0.117 0.118 0.113 0.119 0.116 0.116 0.117 0.121 0.113 0.113 0.116 0.114 0.119 0.002 0.005 0.014 H. polyanthos 0.121 0.119 0.112 0.113 0.111 0.115 0.112 0.112 0.112 0.117 0.111 0.111 0.113 0.121 0.120 0.024 0.024 0.013 A. obscurum 0.107 0.105 0.092 0.093 0.098 0.096 0.092 0.092 0.094 0.099 0.098 0.098 0.095 0.114 0.094 0.088 0.089 0.085

P K Vidya Varma & P V Madusoodanan : Molecular Systematics of the Filmy Ferns (Hymenophyllaceae) 71

version 5.0 (Tamura et al. 2011) was used. Treatment by distance was performed with Neighbor Joining method (Saitou & Nei 1987). The evolutionary distances were computed using the Maximum Composite Likelihood method (MCL method-Tamura et al. 2004) and were in the units of the number of base substitutions per site.The analysis using Maximum Likelihood method was based on General Time Reversible model with discrete gamma distribution of rate differences among sites and proportion of invariable sites. In both NJ and ML, bootstrap analysis (Felsenstein 1985) with 10000 replicates was considered for the assessing robustness. Initial tree for the heuristic search was obtained by the option of BIONJ with MCL distance matrix for ML. A majority rule consensus of the bootstrap replicates was calculated in Consensus in the PHYLIP 3.69 package (Felsenstein 1989).

RESULTS

The rbcL sequence data included 1205bp for each of 19 taxa and no positional homology ambiguities were observed. rbcL sequence data of eight species under the genus Crepidomanes (C. Presl) C. Presl, which were generated in this study, were not reported previously. ClustalW2 used for multiple sequence alignment calculated the best match for the selected sequences and lined up the sequences accordingly. The Percent identity score thus generated by ClustalW2 between the 19 rbcL sequences (1205bp length) were shown in the Table 3.The Conserved region analysis of the sequences with DnaSP v5 software showed that there were three highly conserved regions in the 1205bp sequence. The Polymorphic site analysis showed that there were 945 invariable sites (monomorphic sites), 260 variable (polymorphic sites), 188 parsimony informative sites and 72 singleton variable sites in the 1205bp length sequence. There were 312 mutations in the 1205bp region and all were of the silent type. The haplotype distribution analysis showed that there were only 17 haplotypes out of the 19 sequences analysed. The sequences of Crepidomanes intramarginale (Hook. & Grev.) Copel., Crepidomanes lunulatum Madhus. & C. A. Hameed and Crepidomanes malabaricum C. A. Hameed & Madhus. were found to be of the same haplotypes. The results of the estimation of evolutionary divergence between taxa were given in the Table 4. The model test done in the MEGA version 5.0 (Tamura et al. 2011) with Akaike Information Criterion (AIC) as the model selection criteria, showed that the General Time Reversible model with discrete gamma distribution of rate differences among sites and proportion of invariable sites was most suitable for the sequence given. The optimal tree (the sum of branch length = 0.32995444) showing the relationships of the 19 taxa inferred from Neighbor Joining method was given in Figure 5. In Maximum likelihood analysis, the tree with the highest log likelihood (-3936.2865) was shown (Fig. 6) and the tree resulted from the Bayesian analyses was also given (Fig.7). Regardless of the phylogenetic method used for the analysis, strong support (as given by the Bootstrap values (BS>95) and Bayesian posterior probabilities as percentage (PP>95)) was observed for the four different groups. The clustering pattern of different

72 Indian Fern Journal Volume XXXI (2014)

taxa showed no difference in the three trees derived. Group-A comprised of the species viz., Hymenophyllum polyanthos, H. acanthoides and H. denticulatum. In this, H. polyanthos was considered as outgroup for rooting the tree. Group–B consists of only one species Abrodictyum obscurum and Group-C with two species viz., Didymoglossum bimarginatum and D. exiguum. Group-D was the largest group in the tree with thirteen species. Group-D consisted of a smaller cluster (Group-D1) with two taxa (Crepidomanes proliferum var. proliferum and C. saxifragoides) and a larger cluster (Group-D2) with eleven species. Both sub groups of Group-D were with strong support (both BS and PP above 97). The first split in the Group-D2 led to the cluster consisting of Crepidomanes bipunctatum and C. christii (PP=100, BS>90). The second split gave comparatively a larger cluster with very low support (BS<50) both in NJ and ML trees but with PP=63 in the Bayesian tree. This cluster consisted of C. insigne cluster and the C. intramarginale cluster and three other species. Most remarkable result was that newly described endemic species, namely C. lunulatum and C. malabaricum, were nested along with C. intramarginale without any segregation and with extremely strong support (BS≥99, PP=100). The C. insigne cluster was found to be consisting of three species in which C. bilabiatum and C. latealatum formed a clade with weak support (BS≤70) in ML and NJ tree but with good support in Bayesian tree (PP=94) and C. insigne appeared sister to this clade.

DISCUSSION

The present analysis has yielded a good clustering pattern that reflects relationships among the taxa. The two lineages in the family Hymenophyllaceae can be seen as two separate clusters in tree (Fig.7) as presented by other molecular studies (Dubuisson 1997, Pryer et al. 2001, Ebihara et al. 2006, 2007, Hennequin et al. 2008). The lineage of Hymenophylloid ferns is represented by three species (Hymenophyllum polyanthos, H. acanthoides and H. denticulatum) which are clustered together and denoted in the trees as Group-A. The Trichomanoid lineage of South India is represented by three well supported groups (Fig.7) which correspond to the three genera of Ebihara et al. (2006). The genera are Abrodictyum C. Presl (Group-B), Didymoglossum Desv. (Group-C) and Crepidomanes (C. Presl) C. Presl (Group-D). The Group-B corresponds to genus Abrodictyum s.s. Ebhihara et al. (2006) and is represented by a single species, viz., A. obscurum (Blume) Ebihara & K. Iwats. This is the only S o u t h Indian species under Trichomanoid lineage that is terrestrial. The genus Abrodictyum C. Presl corresponds to the „Pa‟ clade (Pachychaetum clade) in Ebihara et al. (2007), and also to the TE (Terrestrial) clade of Hennequin et al. (2008). The taxa in these clades ( including A. obscurum) have a basal position in the Trichomanoid lineage in the molecular phylogenetic analysis (Ebihara et al. 2006, 2007, Hennequin et al. 2008). The basal position of this clade is reasonable as evidenced by the studies c o n d u c t e d by Dubuisson (1997), Dubuisson et al. (2003b) and Hennequin et al. (2008). The fossil evidence from the

P K Vidya Varma & P V Madusoodanan : Molecular Systematics of the Filmy Ferns (Hymenophyllaceae) 73

F ig. 1 A. Abr odictyum obscurum; B . Cr e pidomanes bilabia tum, C. C. bipuncta tum; D. C. c hristii; E. C. indicum; F. C. insigne; G. C. intramarginale

74 Indian Fern Journal Volume XXXI (2014)

Fig. 2 A. Crepidomanes kurzii; B. C. lunulatum; C. C. malabaricum; D. C. proliferum var. proliferum; E. C. saxifragoides; F. Didymoglossum bimarginatum; G. C. schmidianum; H. D. exiguum

P K Vidya Varma & P V Madusoodanan : Molecular Systematics of the Filmy Ferns (Hymenophyllaceae) 75

76 Indian Fern Journal Volume XXXI (2014)

Upper Triassic by Axsmith et al. (2001) suggests the terrestrial habitat (with its associated morphological characters) as the ancestral state for Trichomanoids. Furthermore, the divergence of TE clade occurred before the divergence of „HE‟ clade (chronogram in Hennequin et al. 2008) and also before the divergence of genera Didymoglossum and Crepidomanes. This aspect makes it sensible for this terrestrial genus to separate out from other epiphytic genera in the present study (Figs. 5, 6, 7) as in the other previous molecular studies (Ebihara et al. 2006, 2007, Hennequin et al. 2008). Moreover, the phenetic study done by Varma et al. (2014) of the taxa under Trichomanoid lineage of South India also illustrates the basal position of A. obscurum to other Trichomanoid taxa in UPGMA tree. The genera Didymoglossum and Crepidomanes correspond to the „HE‟ (Hemi- epiphytic/Epiphytic) clade of Dubuisson (1997) encompassing taxa that are either epiphytic or epipetric. The two genera have split out from a single node in the NJ and Bayesian analysis done here (Figs. 5, 7) which is in accordance with the previous molecular studies (Dubuisson 1997, Pryer et al. 2001, Dubuisson et al. 2003a, Ebihara et al. 2006, 2007, Hennequin et al. 2008). According to the study related to the evolution of epiphytism and divergence times in the Hymenophyllaceae done by Hennequin et al. (2008), epiphytism have evolved during Cretaceous in the family. The beginning of Cretaceous was the time at which these two genera started their split from their preceding ancestor (chronogram given in

P K Vidya Varma & P V Madusoodanan : Molecular Systematics of the Filmy Ferns (Hymenophyllaceae) 77

Hennequin et al. 2008). This makes sense w i t h their clubbing up in the phylogenetic analysis. The Group-C consists of the two species viz., Didymoglossum bimarginatum (Bosch) Ebihara & K. Iwats. and D. exiguum (Bedd.) Copel. They represent the genus Didymoglossum s.s. of Ebihara et al. (2006) and the „Di‟ clade (Didymoglossum clade) of Ebihara et al. (2007). In this, D. exiguum represents subclade „Le‟ (Lecanium) and D. bimarginatum corresponds to subclade „Mg‟ (Microgonium) of Ebihara et al. (2007). Group-D corresponds to the genus Crepidomanes s.s. of Ebihara et al. (2006) and clade „PT‟ (Pan-tropical) and subclade „Cr‟ (Crepidomanes) of Ebihara et al. (2007). The two prominent clusters (Group-D1 and D2) in this group represent the two sections described by Ebihara et al. (2006). Group-D1 consists of the taxa under the sect. Gonocormus s.s. of Ebihara et al. (2006) and Group-D2 consists of the taxa under the sect. Crepodimanes s.s. of Ebihara et al. (2006). The two taxa clustered together in the Group-D1 are Crepidomanes proliferum (Blume) Bostock var. proliferum and C. saxifragoides (C. Presl) P. S. Green. Recent revision of Hymenophyllaceae done by Ebihara et al. (2006) places all the taxa and several other obscure names under sect. Gonocormus into one polymorphic species complex viz., Crepidomanes minutum (Genus Crepidomanes subg. Crepidomanes sect. Gonocormus) and advocates further studies on these taxa. The study done by Nitta et al. (2011) reveals that C. minutum complex is a reticulate network including multiple diploid lineages with stabilized hybrid crosses. They also advocate further study in the complex for defining the natural taxa. From the analysis of rbcL sequences done here, the taxa, Crepidomanes proliferum var. proliferum and C. saxifragoides appear distinct and can be treated as separate species. The percent identity score (Table 3) between Crepidomanes proliferum var. proliferum and C. saxifragoides is 96. The percent identity score for C. bipunctatum and C. christii is 99 and these taxa are considered as separate species by the former workers (Ebihara et al. 2007, Hennequin et al. 2008, Nitta et al. 2011). The estimation of evolutionary distance between Crepidomanes proliferum var. proliferum and C. saxifragoides shows that the evolutionary distance between them is 0.040 (Table 4) which is almost similar to the distance between Didymoglossum bimarginatum and D. exiguum (0.047). Moreover, Sledge (1968) have definitively shown that Trichomanes saxifragoides C. Presl (= Crepidomanes saxifragoides (C. Presl) P.S. Green) is distinct from T. proliferum Blume (=Crepidomanes proliferum (Blume) Bostock). The latter has a more pinnate and less radiating lamina and frequently bears adventive fronds from the stipes (proliferous) whereas T. saxifragoides has flabellate lamina and non-proliferous stipes. Haplotype distribution analysis also shows the distinctiveness of the two taxa viz., Crepidomanes proliferum var. proliferum and C. saxifragoides. In addition to this, the status of the C. proilferum (Blume) Bostock var. minutum (Blume) C. A. Hameed, the variety described by Hameed et al. (2003), has to be clarified.

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T he taxa described by Hameed et al . (2003) as C. proliferum var. prolifer um is T. proliferum Blume of Manickam and Irudayaraj (1992) whereas, C. proliferum var. minutum is T. proliferum forma minutum of Manickam and Irudayaraj (1992) which seems to be T. minutum of Blume (1828). It seems that the above mentioned workers followed Sledge‟s (1968) observation that “Trichomanes minutum Blume and T. proliferum Blume cannot be regarded as more than individual states of same variable species” and thus treated T. minutum Blume (=Crepidomanes minutum (Blume) K. Iwats.) as a forma/variety of T. proliferum Blume (=Crepidomanes proliferum (Blume) Bostock). But the affinity of C. proliferum var. minutum has been questioned by Varma et al. (2014) as this taxon shows more affinity towards C. saxifragoides in the phenetic analysis and Hameed et al. (2003) too agrees with the similarity shown by C. proliferum var. minutum to C. saxifragoides. The rbcL data of C. proliferum var. minutum taxa is not available and the confusion related its status has remained unsolved. Further studies (probably cytological, anatomical and data from other molecular markers) are needed to clarify the relationships between the three taxa of section Gonocormus of South India. Group-D2 comprises of eleven species which corresponds to the sect. Crepidomanes s.s. Ebihara et al. (2006). In this, the cluster of Crepidomanes intramarginale consists of three taxa viz., Crepidomanes intramarginale (Hook. & Grev.) Copel. C. lunulatum Madhus. & C. A. Hameed and C. malabaricum C. A. Hameed & Madhus. This clade has good support (PP=100, BS=99) in the three dendrograms obtained in the analysis. In both Bayesian and ML tree this group appears sister to other six species whereas in the NJ tree it is sister to C. kurzii with a very low support (BS=39). In this group, C. lunulatum and C. malabaricum are newly described species from South India and are endemic to the region (actually endemic to Kerala and are reported only from the type of locality i.e., from the Athirapally waterfalls, Thrissur Dt.). C. intramarginale is the type species of the genus Crepidomanes and is confined to South India and Srilanka (Iwatsuki 1985, Hameed et al. 2003). The clustering pattern obtained from the analysis of rbcL gene pose doubts on the species identity of the two newly described taxa which are endemic to South India. The estimation of evolutionary divergence shows that the distance between Crepidomanes intramarginale, C. lunulatum and C. malabaricum is 0.00 (Table 4) i.e., there is no divergence between the taxa when rbcL sequences is considered. The percent identity score (Table 3) of the three taxa are 100 which also supports the clustering pattern. The haplotype distribution analysis also shows that Crepidomanes intramarginale, C. lunulatum and C. malabaricum consist of one and the same haplotype and are not distinct. Varma et al. (2014) has reported 75% morphological similarity between C. intramarginale and C. malabaricum. Hameed et al. (2003) have also commented on the resemblances of C. malabaricum with C. intramarginale. According to them, C. malabaricum is larger, clearly pinnatifid or pinnatisect, unistratose fronds with close and slightly overlapping segments, with wavy margin, campanulate indusia with abruptly dilated

P K Vidya Varma & P V Madusoodanan : Molecular Systematics of the Filmy Ferns (Hymenophyllaceae) 79

mouth whereas C. intramarginale has bistratose, subpinnatifid fronds, and winged rachis with bivalvate indusia. In this, more striking character is the unistratose lamina of C. malabaricum which can be distinguishable from the bistratose lamina of C. intramarginale. Fraser-Jenkins (2008a, b) considers C. malabaricum as “probable synonym of C. kurzii”, whereas the study done by Varma et al. (2014) shows 70% similarity between C. kurzii and C. malabaricum. The clustering of C. lunulatum with C. malabaricum and C. intramarginale is a surprising one. Earlier workers have different opinions about the affinity of C. lunulatum. Hameed et al. (2003) have commented that C. lunulatum shows resemblance with C. agasthianum Madhus. & C. A. Hameed, another newly described endemic species of South India. But, Chandra et al. (2008) and Fraser-Jenkins (2008a, b) consider C. lunulatum as the “probable synonym of C. agasthianum”. The study done by Varma et al. (2014), C. lunulatum has 75% morphological similarity with C. malabaricum and C. kurzii whereas it shows only 60% morphological similarity with C. agasthianum. The estimate of evolutionary divergence by rbcL sequences shows that distance between C. kurzii and C. lunulatum is 0.013 (Table 4) and the percent identity score between them is 98 (Table 3) and both values are similar to that of C. kurzii and C. malabaricum. As the rbcL sequence of C. agasthianum is not included in the current analysis the affinity of C. lunulatum with this species cannot be commented here. To deduce the exact identity of the C. lunulatum and C. malabaricum further studies using other molecular markers that show relatively more variation than rbcL is needed (e.g.: matk). In addition to this, evidences from cytology and anatomy may also be useful. A combined phylogenetic analysis using molecular data and non-molecular data is advisable along with additional sequences from other taxa such as C. agasthianum in order to clarify the doubts regarding the status of these two newly described taxa. In the Group-D2, another important cluster is the C. insigne cluster. This cluster consists of three species which are considered to be closely related (Iwatsuki 1985, Hameed et al. 2003). Here, Crepidomanes insigne appears as sister to a cluster with C. bilabiatum and C. latealatum. Iwatsuki (1985) considers C. insigne as conspecific to C. latealatum along with C. plicatum. Manickam and Irudayaraj (1992) and Fraser-Jenkins (2008a) consider C. insigne as synonym of C. latealatum. But in the description, Fraser-Jenkins (2008a) states that Trichomanes (Crepidomanes) insigne are plants with a single submarginal false veinlet. Iwatsuki (1985) clearly states that the taxa under Crepidomanes latealatum complex (including Crepidomanes latealatum, C. insigne and C. plicatum) are without submarginal false veinlet, but with oblique striae and the plants with submarginal veinlet are those under C. bipunctatum. So it seems that specimens studied by Fraser-Jenkins (2008a) do not belong to Trichomanes (Crepidomanes) latealatum complex. As reported by Hameed et al. (2003) and I wa t s uk i (1985), C. insigne have no submarginal false veinlet but with oblique striae. In the present study, C . insigne is not clustered together with C. latealatum (Figs. 5, 6, 7) but placed as sister to C. bilabiatum-

80 Indian Fern Journal Volume XXXI (2014)

C. latealatum cluster. The percent identity score (Table 3), estimates of evolutionary divergence (Table 4) and the haplotype distribution analysis support the distinctiveness of C. insigne from other related taxa considered in this study. Crepidomanes indicum C. A. Hameed & Madhus. is a newly described species from South India which is endemic to the region. The status and affinity of this endemic species is a matter of dispute. Hameed et al. (2003) has reported that C . indicum shows resemblances with C. kurzii a n d C. agasthianum. The study done by Varma et al. (2014) has showed that C. indicum is more closely allied to C. agasthianum than C. kurzii. Fraser-Jenkins (2008a, b) considers C. indicum as “probable synonym of C. kurzii”. In the Bayesian tree (Fig.7), C. indicum clusters with C. insigne g r o u p along with C. schmidianum and this cluster h a s good support too (PP=94). But in the ML tree (Fig.6) C. indicum is placed above to C. kurzii. But the estimate of evolutionary divergence (Table 4) shows that C. indicum appears to be more close to C. bilabiatum and C. insigne than C. kurzii. In the present study, the C. indicum appears as a distinct species as revealed by the haplotype distribution analysis and also by its positioning in the three trees obtained. It seems that additional rbcL data from taxa such as C. agasthianum will be necessary to clarify the status and affinities of C. indicum. Further studies on South Indian Trichomanoid lineage are needed to clarify ambiguities observed in the present study. Additional rbcL sequences data from species such as Crepidomanes agasthianum Madhus. & C. A. Hameed, which has not been included in the present study, is needed to obtain more clarity in the relationships of species such as C. indicum C. A. Hameed & Madhus. and C. lunulatum Madhus. & C. A. Hameed. A combined phylogenetic analysis using both molecular and non-molecular data (morphology, cytology, anatomy etc.) will be advisable to get clearer picture about the relationships and status of the above mentioned problematic taxa. Preferably, a rbcL sequence based phylogenetic study of Trichomanoid lineage with a pantropical range will be more useful to discuss the taxonomical position of every species and to propose historical bio-geographical hypotheses for Indian taxa. Taxonomy is a „never ending synthesis‟. Being bereft of own data, Taxonomy has to depend on other disciplines of biology; as and when new disciplines arise Taxonomy has to undergo vicissitudes redefining the phylogenetic aspects and lineages restructuring the g roups and their affinities. Earlier, taxonomists used to depend onl y the visible morphological characters for classification and elucidation of evolutionary relationships. Later, palynological, anatomical, cytological, phytochemical characters, etc. were utilized. The emergence of Molecular Systematics in which the molecular sequences are considered to be the most dependable character in the determination of species circumscription and phylogenetic relationships has overtopped the present evidences used in taxonomy. There are several advantages in using rbcL for systematic analysis. It is situated in the single copy region of the chloroplast genome which is 1.4bp in length and has fairly conservative rate of evolution. The gene is universal among all photosynthetic plants which

P K Vidya Varma & P V Madusoodanan : Molecular Systematics of the Filmy Ferns (Hymenophyllaceae) 81

makes it a good phylogenetic tool. Introns are absent and positional coding of information permits the unambiguous alignment of rbcL which is advantageous for among phylogenetic analysis. The CBOL (Consortium for the Barcode of Life) has approved the two plastid loci rbcL and matK as the official DNA barcode for all land plants including ferns (Li et al., 2011) thus highlighting the utility of rbcL phylogenetic interpretations. The protein coding genes in cpDNA are conserved ones and most exploited in molecular studies. The genes that code for subunit ATP Synthase (atpB), enzyme maturase (matK) subunit F of NADP Hydrogenase (ndhF) and ribosomal proteins (rpS2 and rpS4) are often employed in phylogenetic studies. The atpB genes are without any introns and found to have a rate of evolution as that of rbcL gene (Hoot et al. 1995) whereas matK evolves relatively as much at rapid rate than rbcL (Soltis & Soltis, 1998). Now matK along with rbcL are considered to be the potential barcoding markers in the land plants (CBOL Plant Working Group, 2009). The present data on nucleotide sequences evidently help to draw the phylogenetic relationships of Trichomanoid species of South India. This is partly in agreement with the results of the phenetic analysis done with morphological features of trichomanoid ferns as three distinct groups (Varma et al., 2014). While morphological features show distinction of the newly reported species viz., Crepidomanes lunulatum and Crepidomanes malabaricum, molecular data categorically deny the validity of these species. Even though the rbcL nucleotide sequences are almost reliable in determining the identity at species and subspecific level, the process is cumbersome and time consuming deterring field taxonomists from its applications and to think that Molecular Systematics is useful only in the understanding of the phylogenetic relationships and not for primary taxonomic procedures (description, nomenclature, identification and classification) or it can be applied only to demystify the „taxonomically problematic taxa‟

ACKNOWLEDGEMENTS

The first author is thankful to University Grants Commission, Govt. of India for sanctioning Minor Research Project which provided the financial help for this study. The authors acknowledge the technical support given by DBT, Distributed Information Sub Centre (IISR) for the computational analysis of data. Many thanks are also addressed to Mr. Suresh Kumar U., DNA Examiner, RGCB, Thiruvananthapuram for his helps and suggestions during the molecular study and also for providing the sequence of primer, RBCF. The authors also thank Dr. Santhosh J. Eapen, Principal Scientist & Co-ordinator (Bioinformatics), and Ms. Rosana O.B., Senior Research Fellow, DISC, IISR, Calicut for their help and suggestions during phylogenetic analysis. We are also thankful to Mr. Anoop K.P. for his help in photoshop.

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