Analysis of Putative Hybrid between Anaphalis longifolia and A.javanica (Asteraceae) in Mount Talang, West Sumatra based on Anatomical Characteristics

The putative natural hybrid between Anaphalis longifolia and Anaphalis javanica from Talang Mountain in West Sumatra has been discovered. The aim of this research is to analyze the anatomical characteristics of the leaf and stem to clarify the status of this putative natural hybrid. The anatomical characteristics of leaves and stem were analyzed by making cross sections and paradermal sections using Johansen’s method. The results show seven characteristics of this putative natural hybrid (the stomata density, length, width, and length/width ratio; the number of cortical cell layers, the range of cortex length, and the range of cortex width) that were close to A. longifolia, one characteristic that was close to A. javanica (the structure of palisade), one characteristic that was intermediate (the structure of spongy layer), and one characteristic that was a combination of both of them (the trichome morphotype). All of the foregoing characteristics can be used to support the identification of a natural hybrid of A. longifolia and A. javanica.


Introduction
Anaphalis (known as Edelweis) is a member of the Asteraceae family. In the tropics, Anaphalis grows at altitudes of 1,600 to 3,600 meters above sea level in hostile conditions. It can survive in soil with a low nutrient content because Anaphalis can be associated with mycorrhizal [1]. Mainland Sumatra has only two types of Anaphalis: Anaphalis longifolia and Anaphalis javanica [2]. Taxonomic analysis of Anaphalis from Mount Talang in West Sumatra also identified these two types of Anaphalis [3]. However, some specimens could not be classified because the morphological structure showed characteristics from both A. longifolia and A. javanica and appeared to be a putative natural hybrid. Morphological characters of putative natural hybrid showed from both A. longifolia and A. javanica are distance between nodes and segments, achene width, pattern of leaf growth, leaf margin, trichomes on the underside of the leaves, and color on the tip of the phyllaries. The distance between nodes and segments, achene width, and the pattern of leaf growth are characteristic of A. javanica. However, the leaf margin, the trichomes on the underside of the leaves, and the color on the tip of the phyllaries are more typical of A. longifolia.
Morphological structure comparison is often used to find evidence of hybridization [3][4][5][6][7][8][9]. Morphologically, hybrids typically display the intermediate of parental characteristics, but it should be noted that a few morphological intermediates may develop through convergent evolution or environmental selection, not through hybridization [6]. However, as environmental factors also influence morphology this is not always a completely accurate method. So, it is very difficult to confirm the hybrids strictly from morphological evidence. Recently, with the use of anatomical methods, a large number of natural hybrids have been confirmed by research. Additional data like anatomical features has been used to establish the status of a putative hybrid in the cases of Nothofagus obliqua (Mirb.) Oerst and N. glauca (Phil.) Krasser [10]; Potamogeton crispus and P. praelongus [11]; Cohniella ascendens and C. brachyphylla [12]. This more accurate dual approach using anatomy established the status of the putative natural hybrid of Anaphalis discovered by the authors.

Materials and Methods
The investigation was conducted from February to September 2014 and took place in the Laboratory of Plant Structure and Development, Department of Biology, Andalas University, Padang. Samples of fresh leaves and stems from A. longifolia, A. javanica, and the putative hybrid plants were collected from Talang Mountain. A total of nine (9) individuals were collected: three (3) individuals were A. longifolia, three (3) individuals were A. javanica, and three (3) individuals were hybrids. Leaf materials and stems were prepared from mature plants.
Fresh paradermal leaf sections were used for analysis. Transverse sections of leaves and stems were prepared using Johansen's method. These specimens were fixed for 24 hours in FAA solvent (glacial acetic acid: formalin: alcohol 96%: aquadest = 5:10:50:35) and then dehydrated with Johansen solvent [13]. The specimens were then infiltrated with a soft paraffin solution and solid paraffin. The preparation was then cut with a rotary microtome into 12 µm sections and stained with Safranin-Fastgreen.
The transverse sections were used to study the type and position of stomata, the type of trichomes, the morphotype of the trichomes cells, the palisade structure and position, and the spongy layer structure of the leaves were studied using the transverse sections. Paradermal sections furnished information on the length of stomata, its width, density, and the width/length ratio. Subsequently, the transverse stem sections were used to determine the number of cortical cell layers and the range of values for length and width of the cortical cells.

Results and Discussion
Of the fourteen anatomical characteristics measured, ten characteristics of the putative hybrid plants differed from A. longifolia and A. javanica. These include both quantitative and qualitative characteristics as shown in Table 1.
Seven of the quantitative anatomical characteristics found in the putative hybrid plants can be classified as characteristic of A. longifolia. From three qualitative anatomy characteristics that were found, one was close to A. javanica; one was intermediate between that of A. longifolia and A. javanica, and the other was a combination of both putative parent plants.
The anatomical characteristics of the hybrid plants that were close to A. longifolia included the stomata density, its length, width, ratio, the number of cortical cell layers, the range of cortex length and width (Table 1, Figure 1 and Figure 2). One anatomical characteristic of the putative hybrid plants close to A. javanica was the palisade structure (Table 1). Like A. javanica, the structure was less dense and neat. This differs from the palisade structure of A. longifolia which is tight, cohesive, and neat; there are no spaces between the cells within the palisade layer ( Figure 3).
The intermediate anatomical characteristic that was found in the hybrid plants was the spongy structure ( Table 1). The spongy structure in the hybrid plants showed only narrow spaces between the cells. A. longifolia has a tighter, more cohesive, and neater spongy structure with no spaces between the cells while A. javanica has much larger spaces between the cells within the spongy layer ( Figure 3) than the putative hybrid.
The morphotype characteristics of the trichomes in the putative hybrid plant appear to be a combination of both A. longifolia and A. javanica. A. longifolia has multicellular-unicellular-capitate glandular trichomes, while A. javanica has a unicellular-capitate glandular pattern (Table 1 and Figure 4). The putative hybrid exhibits both of these morphologies.
Previous studies of naturally-occurring hybrids have investigated the relationship between the anatomical characteristics of putative hybrids and the putative parent species. Putative Nothofagus leoni hybrids have a tendency to show anatomical similarities with one putative parent and characteristics that were of an intermediate character between the two putative parent plants. The researchers found that five out of ten characteristics of the putative hybrid were close to N. glauca, three characteristics resembled N. obliqua, while the other two were identified as intermediate characteristics [10]. A more recent study by the researchers also found that five out of nine anatomical characteristics of the putative hybrid of Cohniella were close to C. ascendens; while one of the characteristics was close to C. brachyphylla [12].     meters above sea level, while A. javanica grows between 2,330-2,797 meters above sea level. The wider distribution of A. longifolia suggests this species has more dominant features that enable the plant to survive natural selection [15]. This reflects the observation that N. glauca also dominated its habitat having a wider distribution than N. oblique hence the anatomical characteristics present in its putative hybrid reflected this with a closer resemblance to N. glauca [10].

Conclusions
The characteristics of stomatal density, length, width, length/width ratio; trichome morphotype; palisade structure; spongy structure; the number of cortical cell layers; and the range of values for cortex length and width can be used to support the identification of this plant as a natural hybrid between A. longifolia and A. javanica.