PHENETIC RELATIONSHIP BETWEEN LEPISORUS ( J . SM . ) CHING ( PTERIDOPHYTA : POLYPODIACEAE ) AND ITS RELATED GENERA

Multivariate analyses based on morphological and anatomical characters have been performed to investigate the phenetic relationship and to clarify the circumscriptions of the genus Lepisorus (J.Sm.) Ching and its related genera, namely Neocheiropteris Christ, Paragramma T. Moore and Platygyria Ching & S.K. Wu. The dendrogram of cluster analysis separated the plants into three groups at Gower similarity coefficient 0.75. Group 1 and Group 2 consisted of Neocheiropteris palmatopedata (Baker) H. Christ and four species of Platygyria, respectively. Group 3 was Neocheiropteris ensata Ching and two species of Paragramma deeply embedded in the Lepisorus s.s. Canonical discriminant analysis supported the classification inferred from the clustering results. Based on these results, Platygyria and N. palmatopedata should be recognized as distinct genera. On the other hand, N. ensata and the genus Paragramma should be merged to the genus Lepisorus. Introduction Lepisorus (J.Sm.) Ching s.l. (including Paragramma T. Moore) is the fern genus of Polypodiaceae, which comprised approximately 30 species (Verdcourt, 2001) or 70 species (Lin, 2000) naturally occurring in the tropical and subtropical Old World and extending northwards to the Far East of Russia with one species in Hawaii (Verdcourt, 2001). Lepisorus s.s. (excluding the Paragramma), however, was first treated by J. Smith in 1846 (in Zink, 1993) as a section of a highly heterogeneous Drynaria, and Ching (1933) raised the section Lepisorus to generic rank. The common features of Lepisorus s.l. are epiphytic, epilithic or terrestrial ferns with shortto long-creeping rhizome covered by clathrate scales; laminas are simple, entire and mostly naked; and sori are borne in single rows on either side of the midrib, and covered by clathrate paraphyses (Hennipman et al., 1990; Verdcourt, 2001). Until now, the generic circumscription of Lepisorus has remained controversial because it sometimes included or excluded its related taxa, namely Paragramma and Platygyria Ching & S.K. Wu. In addition, Platygyria, which is closely related to Lepisorus s.l. sometimes merged with the genus Neocheiropteris Christ. 1 Department of Botany, Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand. E-mail: wannachaichatan@gmail.com 2 Corresponding author. E-mail: bthawees@gmail.com 3 Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada. E-mail: bernard.baum@agr.gc.ca


Introduction
Lepisorus (J.Sm.)Ching s.l.(including Paragramma T. Moore) is the fern genus of Polypodiaceae, which comprised approximately 30 species (Verdcourt, 2001) or 70 species (Lin, 2000) naturally occurring in the tropical and subtropical Old World and extending northwards to the Far East of Russia with one species in Hawaii (Verdcourt, 2001).Lepisorus s.s.(excluding the Paragramma), however, was first treated by J. Smith in 1846(in Zink, 1993) as a section of a highly heterogeneous Drynaria, and Ching (1933) raised the section Lepisorus to generic rank.The common features of Lepisorus s.l. are epiphytic, epilithic or terrestrial ferns with short-to long-creeping rhizome covered by clathrate scales; laminas are simple, entire and mostly naked; and sori are borne in single rows on either side of the midrib, and covered by clathrate paraphyses (Hennipman et al., 1990;Verdcourt, 2001).
Until now, the generic circumscription of Lepisorus has remained controversial because it sometimes included or excluded its related taxa, namely Paragramma and Platygyria Ching & S.K. Wu.In addition, Platygyria, which is closely related to Lepisorus s.l.sometimes merged with the genus Neocheiropteris Christ.
The Chinese fern genus Platygyria was erected based on the P. waltonii (Ching) Ching & S.K. Wu (Ching and Wu, 1980) and the characters of sporangium were used as the important defining characters.So, five species, namely Platygyria sinuata Ching & S.K. Wu, P. inaequibasis Ching & S.K. Wu, P. variabilis Ching & S.K. Wu, P. kongtingensis Ching & Y.X.Lin and P. muliensis Ching & S.K. Wu were recognized (Ching and Wu, 1980;Ching et al., 1983).Likewise, Zhang et al. (2003) agreed to keep Platygyria at the genus level and treated P. kongtingensis and P. muliensis as two synonyms of P. variabilis.However, there were other two treatments of the genus Platygyria.The first involved reducing Platygyria under Neocheiropteris (Ching, 1933;Hennipman et al., 1990), while the second involved merging Platygyria with Lepisorus s.s.(Yu and Lin, 1997).Therefore, the merging of Platygyria with either Lepisorus or Neocheiropteris, or its acceptance as a distinct genus needs to be assessed.
As the controversial generic circumscription or position of these taxa shown above, until now, there has been no taxonomic study aimed at clarifying these problems.Therefore, the objectives of the present work were, 1) to investigate the phenetic relationship and use the result to determine the suitability of the generic circumscription or position of the Lepisorus and the other three related genera, i.e.Neocheiropteris, Paragramma and Platygyria, and 2) to determine the important morphological or anatomical characters that can be used to distinguish these taxa.With the aforesaid objectives in mind, both cluster analysis (CA) and discriminant analysis (DA) were performed based on 53 qualitative and quantitative characters examining 487 herbarium specimens.

Materials and Methods
Plant materials: In the present study, about 2500 herbarium specimens collected from around the world and housed at the herbaria in Europe (BM, E, L, K and P) and Asia (BCU, BKF, BK, PE, KUN, PYU and TI) were studied (Herbarium abbreviations according to Holmgrens and Holmgrens, 2008).A total of 487 complete specimens were selected for examination constituting the Operational Taxonomic Units (OTUs).(Some representative specimens are listed in Appendix I.A complete list is available upon request from the corresponding author.)The specimens that were included in this study belonged to Lepisorus s.s. and its related three genera, namely Neocheiropteris, Paragramma and Platygyria.These specimens included specimens of the type species of each genus.Most specimens were identified by examining type specimens or identifications were made by consulting literature, e.g.Ching (1933), Tagawa and Iwatsuki (1989), Zink (1993), Shieh et al. (1994), Hovenkamp (1998), Verdcourt (2001), and Zhang et al. (2003).
Morphological and anatomical characters: Fifty-three morphological and anatomical characters were examined for each of the 487 specimens.Measurement was carried out by using a Keiba digital caliper No. 111-101HB or specimens were measured under Stereomicroscope (Zeiss Stemi DV4) and light microscopes (Olympus CH30).Of these characters, 26 were quantitative including four ratio characters (Appendix II), and 27 were qualitative characters scored as binary or multi-state characters (Appendix III).These characters and their states were used to construct a data matrix.
Phenetic analysis: The phenetic relationships among the taxa were investigated by two types of multivariate analysis: cluster analysis (CA) and canonical discriminant analysis (DA).The CA was performed by using an unweighted pair-group method with arithmetic average (UPGMA) clustering implemented in the Multivariate Statistical Package (MVSP), Version 3.13 (Kovack Computing Services) to place individual specimen into groups.Because the characters submitted to analysis were both quantitative and qualitative, the Gower similarity coefficient (GSC) was calculated (Gower, 1971) and clustered by the group-average method of the MVSP program.
A subset of characters that maximized differences among the groups determined by CA or other groups (i.e.Lepisorus s.s., Paragramma, Platygyria and Neocheiropteris) that were recognized by previous pteridologists as a distinct group were selected by stepwise discriminant analysis.Prior to performing discriminant analyses, the data matrix was modified, i.e. characters that did not satisfy the assumption of normal distribution were transformed by taking them with the natural logarithm.The canonical discriminant analyses was performed by using the CLASSIFY procedure in SPSS/PC for Windows, release 10.0 (Anonymous, 1999).

Results and Discussion
Cluster Analysis (CA): The UPGMA dendrogram that constructed using GSC measure showed three discrete groups (Fig. 1) at GSC 0.75.Group 1 included Neocheiropteris palmatopedata and Group 2 comprised the four species of Platygyria.In addition, Group 3 was the largest group consisting of Lepisorus s.s., N. ensata and the two species of Paragramma.At Gower similarity coefficient 0.71, these fern taxa were divided into two groups, i.e.Group 1 and a group composed of Groups 2 and 3 (Fig. 1).Group 1 is distinct from the rest mainly by the combination characters of pedatifid lamina, presence of large veins at the lamina base and lamina width (more than 120 mm) as shown in the key to the genera below.The result suggested that N. palmatopedata was far distinct from the rests while the genus Platygyria was more closely related to the genus Lepisorus and Paragramma than N. palmatopedata.In addition, genus Lepisorus, N. ensata and Paragramma are closely related to each other than the rest.
Canonical Discriminant Analysis (DA): DA was divided into two analyses based on the number of prior groups obtained: 1) four groups, including Lepisorus s.s., Neocheiropteris, Paragramma and Platygyria, all of which were assigned based on previous recognized genera (such as Christ, 1905;Ching, 1940;Copeland, 1947;Pichi Sermolli, 1977;Ching and Wu, 1980;Zhang et al., 2003); and 2) three groups, including Groups 1, 2 and 3, which were obtained from CA. Overall, 26 quantitative characters were used in these analyses with a purpose to test their groupings.Once the stepwise analysis had been performed for all four groups, the linear discriminant function classification showed that 97.3% of the specimens had been correctly classified.The nature of the differences between the entries were shown by the pooled within canonical structure wherein canonical variable 1 was 97.3% correlated with the 26 quantitative characters and explained 86.2% of the total variance, which was highly associated with three characters (Table 1).Canonical variable 2 was 81.6% correlated with the quantitative characters and explained 9.7% of the total variance, which was highly associated with four characters.Canonical variable 3 was 68.1% correlated with the quantitative characters and explained 4.2% of the total variance, which was highly associated with seven characters (Table 1).The stepwise analysis was carried out for the three groups, i.e. these groups were split by the UPGMA dendrogram using GSC at 0.75.The nature of the differences between the entries was shown by the pooled within the canonical structure wherein canonical variable 1 was 97.2% correlated with the 26 quantitative characters and explained 89.7% of the total variance, which was highly associated with three characters (Table 2).Canonical variable 2 was 81.6% correlated with the quantitative characters and explained 10.3% of the total variance which was highly associated with seven characters (Table 2).The linear discriminant function classification (Table 3) obtaining from the program showed that the specimens had been 100% correctly classified; obviously, therefore, this function could be used for further identification of these ferns.To identify an unknown specimen, one needs to multiply each character score by its coefficient in each column, compute the total for each column, the column with the highest total is the group to which the specimen belongs.
The ordination plot on the two canonical axes obtained from the four groups analysis (Fig. 2) showed that canonical axis 1 divided these plants into two main groups, one group included Lepisorus s.s., Paragramma and Neocheiropteris, and the other consisting solely of Platygyria.However, canonical axis 2 was able to separate N. palmatopedata from the rest.Therefore, these two axes could divide these ferns into three groups.Furthermore, these results were similar when the plants were divided into three groups based on the result of CA (not shown).

Circumscription of Lepisorus and Paragramma
As far as the taxonomic position or circumscription of the Paragramma is concerned, there are two different forms of recognition so far, i) the form that maintains them as a distinct genus (Ching, 1940;Copeland, 1947;Pichi Sermolli, 1977) and ii) the form that combines the Paragramma with Lepisorus s.s.(Holttum, 1954;Tagawa and Iwatsuki, 1989;Hennipman et al., 1990;Hovenkamp, 1998).The key characters that Copeland (1947) used to distinguish Paragramma from his Pleopeltis (i.e.including Lepisorus s.s.) were the combination of its soral shape and the presence of lamina scales.In Copeland's key to genera of Polypodiaceae, as well as in his description, he showed that Paragramma had elongated, oblong or linear-oblong sori and that its lamina was not covered by peltate scales, while his Pleopeltis generally had round or elongate sori or fused sori, but the elongate-sori species had peltate scales on the lamina.
It was observed from this study that Paragramma longifolia and P. balteiformis had round sori together with elongate sori.Paragramma longifolia, however, had glabrous lamina while few clathrate scales occurred on the lamina of P. balteiformis.Moreover, both elongate sori and few scales on the lower surface of lamina could have occurred in some Lepisorus species, for example L. angustus Ching, L. subconfluens Ching and L. scolopendrium Tagawa.So, the combination of lamina scales and soral shape could not be used to separate Paragramma from Pleopeltis.Accordingly, Paragramma could not also be separated from Lepisorus by using these characters.
The results of both CA and DA strongly indicated that Paragramma and Neocheiropteris ensata were not distinct from Lepisorus s.s., so the genus Paragramma should be treated as a synonym of the genus Lepisorus.Likewise, N. ensata should be treated as a species of the genus Lepisorus.Furthermore, Fraser-Jenkins (1997) noted that N. ensata is a misapplied name of N. ovata (Fée) Fras.-Jenk.Bosman et al. (1998), however, recognized N. ensata as Microsorum ensatum (Thunb.)H. Itô, but also noted that their justification was based on a single specimen from Malesia.They also added that M. ensatum is probably a hybrid between a species of Lepisorus and a true Microsorum species.
The results from this study, therefore, strongly support the recognition of Tagawa and Iwatsuki (1989), Hennipman et al. (1990) and Hovenkamp (1998) that the circumscription of the genus Lepisorus must include Paragramma.In addition, the new finding is that N. ensata should be transferred to the genus Lepisorus.

Circumscription of Neocheiropteris and Platygyria
For Platygyria, three taxonomic positions have been recognized, i.e. combining with Lepisorus s.s.(Yu and Lin, 1997; C.R. Fraser-Jenkins, personal communication), treating it under Neocheiropteris (Ching, 1933;Hennipman et al., 1990) and maintaining the status of distinct genus (Ching and Wu, 1980;Zhang et al., 2003).Firstly, the Platygyria was proposed as a genus of Polypodiaceae by Ching and Wu in 1980 wherein the characters used to define this taxon were the globose sporangium and the very broad annulus consisting of scarcely indurate cell walls.Fraser-Jenkins (1997) had an opinion that P. variabilis should belong to Lepisorus clathratus (C.B.Clarke) Ching and the rest of Platygyia appeared to belong to Phymatopteris Pic.Serm.because according to him the type-species, Platygyia waltonii, is in fact a Phymatopteris, another genus of the Polypodiaceae.However, the genus Phymatopteris is not recognized by Hennipman et al. (1990) and Smith et al. (2006).It was suggested that annulus characters are not constant (C.R. Fraser-Jenkins, personal communication), while Zhang et al. (2003) determined that they are rather stable.After studying Platygyria in comparison with Lepisorus, Neocheiropteris and Paragramma, it was found that the annulus characters were not only important in separating Platygyria from Neocheiropteris, but also from Lepisorus and Paragramma.However, when herbarium specimens were examined, we found that some specimens having both sporangia which were globose, having very broad annulus (≥ 105 µm) and few indurate cells of annulus (i.e.indurate cells of annulus 1-5 or less than 1/5 of annulus or absent) and sporangia which were flat or slightly flat, having narrow annulus (<100 µm) and prominent indurate cells (i.e. more than half of annulus are indurate cell).It was found from this study that these specimens were mixed with specimens of Lepisorus clathratus and were placed in the folder of Lepisorus clathratus complex.However, they should be separated from L. clathratus and put into the genus Platygyria.
In addition, the position of the stomium is an important character to distinguish the Platygyria from the rest.According to Wilson (1959), the annulus was the whole ring of cells horizontally encircling the capsule and interrupted at the point of attachment to the stalk.Most ferns had a row of indurate cells for the annulus, and this row were also interrupted by thin wall cells of epistomium, stomium and hypostomium.The stomium in most ferns could occur between the epi-and hypostomium, but it could not occur on the row of indurate cells of annulus.In Platygyria, however, the annulus cells are homogeneous or slightly homogeneous, and most or all annulus cells had thin walls.For these reasons, the position of the stomium in Platygyria could not be of constant occurrence as in other ferns and can be present throughout or slightly throughout the annulus.
The tuft of hairs dorsally attached to the rhizome scales has been given much weight by Ching (1933) and Tagawa and Iwatsuki (1989) as a characteristic of their Neocheiropteris s.l.Examinations of this characteristic found that these hairs could also be found in Neocheiropteris palmatopedata, N. ensata and Platygyria waltonii, but they were not found in the other Platygyria species.In addition, they could be found in some Lepisorus species, i.e.L. kawakami Tagawa, L. macrosphaerus Ching, L. marginatus Ching and L. monilisorus (Hayata) Tagawa.Thus, this characteristic could not be considered as a diagnostic characteristic of Neocheiropteris.
Moreover, according to both CA and DA, Platygyria, Lepisorus s.l. and Neocheiropteris were split into three distinct groups (Figs 1 and 2).These results were supported by the recognition of Ching and Wu (1980) and Zhang et al. (2003) in maintaining Platygyria as a distinct taxon.Therefore, the characteristics of the Platygyria were globose sporangia, very broad annulus (≥ 105 µm) and few indurate cells of annulus (i.e.indurate cell of annulus 1-5 or less than 1/5 of annulus or absent).In addition, the circumscription of Platygyria should include the species or specimens that have similar characters.
Neocheiropteris palmatopedata, the type species and one of the two representatives of the genus Neocheiropteris in this study should be kept under Neocheiropteris.The striking characters that have never been used as key characters to distinguish it and the other taxa are pedatifid laminas and the presence of large veins at the lamina base.Also, the lamina width is between 147-376 mm while less than 100 mm lamina width found in the rest taxa.Thus the result is also supported by CA and DA.
Previously, Lepisorus s.s.(Ching, 1933(Ching, , 1940)), Paragramma (Ching, 1940;Copeland, 1947;Pichi Sermolli, 1977) and Platygyria (Ching and Wu, 1980;Zhang et al., 2003) were recognized as separate genera by some pteridologists.Also, Neocheiropteris was recognized as a distinct genus from the genera above (Christ, 1905).The results were examined in terms of morphology and anatomy, and the two multivariate analyses of which can be proven to recognize that Platygyria was a distinct taxon from Lepisorus, Neocheiropteris and Paragramma.Moreover, N. palmatopedata should be put into a different group.On the other hand, the circumscription of Lepisorus should include N. ensata and Paragramma.
This study found ten important quantitative characters that could be used for splitting Lepisorus (including N. ensata and Paragramma), N. palmatopedata (here it is recognized as a monotypic genus) and Platygyria by including the annulus width, sporangium length, length of the fertile portion of lamina, lamina width, sorus length, ratio of lamina length and lamina tip length, ratio of lamina length and stipe length, number of sclerenchyma strand in rhizome, phyllopodia length, and sorus width.Some of these characters, including annulus width and lamina width, and some useful qualitative characters i.e. lamina indentation, prominent large vein at lamina base, occurrence of indurate cells and stomium position were used to construct a key to genera as below: