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Determining relevant petiole anatomy characters to delimit Eupolypods I families

Tan JMP, Buot Jr. IE. 2020. Determining relevant petiole anatomy characters to delimit Eupolypods I families. Biodiversitas 21: 2721-2726. The delineation of the families under eupolypods I has been quite challenging. With that, characters that are stable and taxonomically significant are necessary. The study attempted to use petiole anatomy characters to study the taxonomy of the eupolypods I families. Fresh eupolypods I representatives were collected from the field and were fixed with formalin-acetic acid alcohol (FAA). The petioles were cut and processed into permanent mounts using alcohol dehydration series and following standard microtechnique protocols. The petiole anatomy characters of the 21 representative species of eupolypods I were first studied of their stabil ity and subjected to multiple correspondence analysis (MCA) to determine the most taxonomically relevant character. The MCA biplot showed that four petiole anatomy characters were useful to describe and delineate families of eupolypods I. These petiole anatomy characters included, xylem shape, vascular bundle shape, and number of vascular bundles.


INTRODUCTION
Up to the present, the taxonomic placement of species under eupolypods I families is unstable. Due to lumping and shifting of families, delineating families is becoming challenging, particularly concerning characters unique to the clade or family (Christenhusz and Chase 2014). Fortunately, anatomical structures provide strong evidence in terms of delineating larger groups such as families and aid in distinguishing genera of uncertain taxonomic status. It is also significant in identifying individual species particularly to specimens without floral parts. Numerous studies showed the importance of petiole anatomy to seed plants. One such study was that of Samiyarsih et al. (2019) on Catharanthus roseus variability. Another one was the work of Pitoyo et al. (2018) in their study of Colocasia esculenta from southeastern part of Central Java, Indonesia.
Petiole anatomy was also found to be useful in systematic studies of ferns. In fact, each species was found to exhibit unique petiole anatomical characteristics that can be used to delineate one species from the other (Resmi et al. 2016). Sofiyanti et al. (2019) were able to characterize the four common ferns of Pekanbaru, Riau Province, Indonesia using petiole anatomy. The study clearly illustrated that the species share the same amphicribral concentric vascular bundles but differ in size within species. Petiole anatomy was very helpful also in the study of Tan et al. (2020) to describe, delineate, and classify species of eupolypods I.
Numerous authors have considered the structure of the vascular bundles as basis for differentiating various taxonomic groups. Martínez and Vilte (2012) were able to generate a classification of the twelve species of Pteris using shape and number of vascular bundles. Diverse morphological states of the vascular bundles include the shape of xylem, shape, and number of vascular bundles. Another study (Hernandez-Hernandez et al. 2012) showed the different shapes of vascular bundles of the 89 fern species and that it correlated with the presence of circumendodermal band.
The circumendodermal band is composed of distinct cells surrounding the vascular bundles and endodermis which are distinct with or without stains (Hernandez-Hernandez 2009). This band originated from parenchyma cells which protect the phloem (Russow 1872). The term 'circumendodermal band' was attributed to this group of cells on account of its proximity to the endodermis. Considering the position of the circumendodermal band, there could be two possible functions, namely (Hernandez-Hernandez et al. 2012); (i) protection against microorganisms and other pathogens; and (ii) protection against stresses.
Hence, petiole anatomy characters are indeed stable and can be utilized in taxonomic studies. According to Metcalfe and Chalk (1950), it is of great importance to rely on anatomical structures that are taxonomically well established. Actually, there are many petiole anatomy characters but we need to be very strategic so that we will utilize only the most stable ones. The objectives of this study therefore, were: (i) to determine the key taxonomic characters of the eupolypods I petiole anatomy and (ii) to identify those stable characters that delimit families of eupolypods I using multiple correspondence analysis.

Selection and preparation of the fresh Eupolypods representative specimens
A total of 21 representatives of eupolypods I were studied. Twenty specimens were fresh samples from the field ( Zhang and Zhang (2015) for the purpose of this study.
The petioles of each specimen were cut into three parts: basal region (next to the rhizome), middle region (half of the petiole), and distal region (next to the blade) and were fixed and preserved in formalin-acetic acid alcohol (FAA) (Johansen 1940). Free-hand sectioning technique was used, and the specimens underwent the alcohol dehydration series and permanently mounted using Canada Balsam. Permanent slides were deposited in the Plant Biology Division Herbarium, Institute of Biological Sciences, University of the Philippines Los Baños, Laguna, Philippines. Cross-section of the petioles were observed using Olympus light compound microscope. Images were documented, and scales of each section were obtained through ImageJ software, generating scales through pixels of the photo.

Determining delimiting taxonomic characters of Eupolypods I
The data obtained from petiole anatomy were analyzed. Stable characters were determined by examining each species collected from different environments, particularly, substrates, gradients, and altitude. Those characters that remained constant were chosen and further analyzed using Multiple Correspondence Analysis, FactoMineR package (MCA) (Sebastien et al. 2008). MCA is equivalent to Principal Component Analysis for nominal data and can be used for qualitative variables (Le Roux and Rouanet 2010).

Stable and distinct petiole anatomy characters of the nine families of Eupolypods I
Eupolypods I families exhibited unique petiole anatomy characters such as diverse stelar shape, xylem shape, vascular bundle shape, vascular bundle number, circumendodermal band, and adaxial grooves (Figure 1). Each family showed similarities and differences in terms of the presence circumendodermal band, number of vascular bundles, and xylem strand shape. For instance, Davalliaceae species showed the presence of circumendodermal band, U-shaped vascular bundles, and arc-shaped xylem strand shape (except D. hymenophylloides with V-shaped xylem) (Figures 1.A-C). Also, Dryopteridaceae species showed similar arc-shaped xylem but out of the three species examined, Bolbitis heteroclita and Polystichum horizontale showed the presence of circumendodermal band except for These characters were screened based on stability across varying environments. It was observed that xylem shape, vascular bundle shape, and number of vascular bundles of species from different habitat, substrate, and elevation, remained constant across varying environmental conditions. Table 1 presented petiole characters of the same species collected from different elevation, collection sites, and habits so as to clearly illustrate the stable and unstable petiole anatomy characters. Despite differences in microenvironmental states, species showed similar petiole anatomy characters such as xylem shape, vascular bundle shape, and number of vascular bundles (Table 1).
This indicated that these character states are more or less stable. Transverse section of Microsorum heterocarpum collected from 352 m asl and 1230 m asl, showed similar C-shaped xylem, U-shaped vascular bundle, and 3 to 4 vascular bundles (Table 1). If this petiole anatomy character were unstable, the one collected from 352 m asl should have possessed a different petiole anatomy character from the one collected from 1230m asl. Further, the transverse sections of Lomariopsis lineata which were collected from Mt. Makiling (lithophyte) and Mt. Mantalingahan (epiphyte), showed similar S-shaped xylem, U-shaped vascular bundle, and 2-5 vascular bundles (Table 1). These remarkable observations illustrated that vascular bundles are constant and stable characters (Table  1), and hence, can be useful taxonomic and systematic characters.
After analysis of Table 1, the following petiole anatomy characters were found constant even if collected from varying environmental conditions: stellar type, xylem strand shape, shape of vascular bundles, and number of vascular bundles. Based on the studies of Ogura (1972), Lin and De Vol (1977), Hernandez-Hernandez et al. (2012), and Noraini et al. (2012), these characters are taxonomically and systematically significant. Also, the architecture of vascular bundles was conserved for a long time starting from the Upper Jurassic to the Cretaceous period (Hacke and Sperry 2001), making it a very stable character.

Delimiting petiole anatomy characters of the Eupolypods I families
The four petiole anatomy characters, namely, type of stele, xylem shape, vascular bundle shape, and number of vascular bundles were subjected to MCA, in order to determine which were the delineating characters. Based on the MCA biplot, the distinct petiole anatomy characters delimiting eupolypods I families were grouped together. Meanwhile, those characters such as dictyostele, V-shaped vascular bundles, and U-shaped vascular bundles that were common to two or more families were separated from other families ( Figure 2). Those species under one family were also grouped together. This implied that characters that were clumped nearest to the families were those distinct and unique characters that can be used to identify and distinguish the specific families ( Figure 2).
It is very clear that three petiole anatomy characters, including xylem shape, vascular bundle shape, and number of vascular bundles were found to delimit each family. The following characters were the petiole anatomy characters found to be useful in distinguishing families of eupolypods I based on the Multiple Analysis Biplot (MCA): Davalliaceae-arc-shaped xylem and one to five vascular bundles; Didymochlaenaceae-arc-shaped xylem, semi-circle, and three vascular bundles; Dryopteridaceae-arcshaped xylem, U-shaped and three vascular bundles; Hypodematiaceae-two vascular bundles; Lomariopsidaceae-S-shaped xylem, two to five vascular bundles; Nephrolepidaceae-C-shaped xylem and three to five vascular bundles; Oleandraceae-arc-shaped xylem, Vshaped and two to three vascular bundles; Polypodiaceae-C-shaped xylem, U-shaped and three to four vascular bundles; and Tectariaceae-hook-shaped xylem and three to seventeen vascular bundles.
The number and the shape of the vascular bundle and xylem strands distinguished the family. The three distinct characters observed for Polypodiaceae (U-shaped vascular bundles with C-shaped xylem strand, and 3-4 vascular bundles) corresponded to the findings of Hennipman (1990) (Figure 3.E). This study also illustrated that Nephrolepidaceae (Figure 3.G) has C-shaped xylem and 3-5 vascular bundle characters while Lomariopsidaceae (Figure 3.H) has S-shaped xylem strand and 2-5 vascular bundles in U-shaped. Until now, there is an ongoing debate about the relationship of Nephrolepidaceae and Lomariopsidaceae. PPG I (2016) considered both families as sister families, however, Liu et al. (2013), Kuo et al. (2011), and Testo and Sundue (2016) disagree. Through molecular data, they considered Nephrolepidaceae as sister to a larger clade including Polypodiaceae, Tectariaceae, Davalliaceae, and Oleandraceae congruent to the results of the study of Kramer (1990). Results of this study using petiole anatomy characters or evidence proved that Nephrolepidaceae and Lomariopsidaceae are not sister families supporting Liu et al. (2013), Kuo et al. (2011), and Testo and Sundue (2016) but contradicting the claims of PPG I (2016).    Davalliaceae can be identified using the following characters: protostele and dictyostele, arc-shaped xylem, and 1-5 vascular bundles (Figure 3.A). This exactly agrees with the result of Noraini et al. (2012), Kramer (1990), and Kato and Mitsuta (1980). Meanwhile, Hypodematiaceae sets apart from the rest of the families under eupolypods I by having only two vascular bundles (Figure 3.C) which eventually fused as it reached the distal region of the petiole supporting the findings of Khare and Shankar (1987). This character as seen in Figure 3 is nearest and closely related to Hypodematiaceae making it a unique and distinct character. Currently, the taxonomic position of Hypodematium is still uncertain. It was moved from one family to another until PPG I moved it to its own family Hypodematiaceae (Tan et al. 2020).
Based on petiole anatomy characters, both Didymochlaenaceae and Dryopteridaceae shared the same arc-shaped xylem, but Didymochlaenaceae differs by having three semi-circle shaped vascular bundles. On the other hand, Dryopteridaceae showed three U-shaped vascular bundles that delimit the family Dryopteridaceeae from other families under eupolypods I similar to the findings of Kramer (1990) (Figure 3.B).
It can be observed that Didymochlaenaceae is related to Dryopteridaceae. Previously, molecular data considered Didymochlaena as a member of Dryopteridaceae (Ching 1965;Tryon and Tryon 1982;Kramer et al. 1990). However, recent molecular studies confirmed that it merits its own family Didymochlaenaceae and a member of eupolypods I (Zhang and Zhang 2015). The petiole anatomy results showed that Didymochlaenaceae, whose taxonomic placement has always been a subject of controversy, merits its own family and not part of Dryopteridaceae which conforms to the findings of PPG I.
Finally, the petiole anatomy of family Oleandraceae with its representative genus, Oleandra is illustrated in Figure 3.D. Results showed that Oleandraceae can be uniquely characterized as having 2-3 U shaped vascular bundles which conform with the study of Kramer (1990). The results indicated support to the current circumscription of Oleandra in its own family Oleandraceae.
The distinctiveness and species delineation of Oleandra and Oleandraceae are still uncertain and difficult (Smith et al. 2006). In fact, this family has been previously linked to other families such as Davalliaceae, Nephrolepidaceae, and Dryopteridaceae (Tan et al. 2020). The circumscription of this family has been challenging as many of the species are similar in appearance (Kramer 1990).
In conclusion, the multiple correspondence analysis generated three stable and significant petiole anatomy characters, delineating eupolypods I families, namely; xylem shape, shape of the vascular bundle, and number of vascular bundles. This finding implied that these characters are most relevant in the classification and identification of eupolypods 1 families and species. Similar anatomical features may have high potentials in characterizing and delineating other groups of ferns too.