Comparative Morpho-Anatomical Studies of Two Philippine Endemic Species of Amyema Tiegh. (Loranthaceae)

Amyema are epiphytic hemiparasitic plants on different types of woody host plants and are abundant in temperate, subtropical, and tropical regions. In Marilog Forest Reserve, Southern Philippines, two Philippine endemic species of Amyema were recorded, viz., Amyema curranii (Merr.) Danser and A. seriata (Merr.) Barlow. In this study, these two species were compared and examined for their morphology and anatomy. Data revealed that the two Amyema species are morphologically distinct, with A. curranii having lanceolate leaves, pink flowers, and red fruits, whereas A. seriata has obovate leaves, red flowers, and yellow fruits. For the morpho-anatomy, A. curranii has a single-layered epidermis, paracytic stomata, collateral open vascular bundles, the Eustele type of stele with pith at the center, and the inferior free central type with a hairy ovary wall. Meanwhile, A. seriata has a pinkish, single-layered epidermis, paracytic stomata, collateral open vascular bundles, a eustele type of stele with the presence of pith at the center, and an inferior free central ovary type. As a result, employing these species’ gross morphology and anatomy could scrutinise future evaluations and taxonomic placements.


INTRODUCTION
Loranthaceae, also known as Showy Mistletoes, are perennial flowering plants that are known for their vivid inflorescence and enigmatic appearance (Devkota 2015). One of the genera in this family is Amyema, an epiphytic hemiparasitic plant (partially parasitic) on the xylem tissues of their host (Kuijt 1969;Calder & Bernhardt 1983;Kuijt 2009;Arruda et al. 2012), inhabiting a wide range of host plants (Devkota et al. 2011;Ogunmefun et al. 2015), and acquiring water, nutrients, 140 and sugar while having the capability of photosynthesis (Norton & Carpenter 1998). Amyema is the second genus with high diversity in Loranthaceae, after Psittacanthus, which is the most specious (Kujit 2009).
The Marilog Forest Reserve in Marilog District, Davao City, has a total land area of 63,000 ha. This area has a rich diversity of flora, which makes it one of the priority areas for biodiversity studies by Conservation International. Recently, several inventory studies on vascular flora in the area have been conducted (e.g., Acma et al. 2021;Coritico et al. 2022;Rufila et al. 2022 Acma et al. (2021). Of these, the two Amyema species were studied and examined for their gross morphology and anatomy.

Botanical Fieldworks
Botanical field collections were carried out from August to November 2019. Necessary permits were obtained from the local people such as Gratuitous Permit (GP) from the Department of Environment and Natural Resources (DENR). The specimens of A. curranii were collected from Busay Garden Resort near the base of Mt. Malambo (a minor peak), while A. seriata was collected in Mt. Antayapan and Sitio Tribal Village, both in Brgy. Datu Salumay, Marilog District, Davao City, Philippines (Fig. 1). Repeated transect walks and opportunistic sampling were employed to survey and collect specimens. Moreover, characteristics of habitat, vegetation type, and documentation of host plants and anthropogenic disturbances in the areas were recorded.

Identification of the Specimens
Specimens available at the Journal Storage (JSTOR) were used for the comparison, identification, and classification of Amyema species. For the purpose of verifying the morphological and anatomical characteristics of the plants, references from books, journals, and online databases, such as Pelser and Barcelona (2013) and The Global Plant List and Co's Digital Flora of the Philippines by Pelser et al. (2011) forward, were utilised.

Plant Measurements and Descriptions
The morphological characters of A. curanii and A. seriata were examined, documented, and described. Five plants were treated for each species. The lengths of the vegetative parts of living specimens were measured using a tape measure. Measurable features, such as plant height, stem length, length and width of leaf, length and diameter of the petiole, length and diameter of the haustorium, length of flowers and fruits, and detailed floral parts, were described and documented. The terminology of Wilson and Calvin (2006) was used in this study.

Anatomical Analysis
Anatomy was done in the laboratory of the Department of Biology, Central Mindanao University, Musuan, Bukidnon. The procedure of Johansen (1940) for anatomical studies was followed. The free-hand technique was used to cut small pieces (about 1 mm long transverse sections) of the various plant parts. A clearing technique was done on the young leaves to study the venation pattern, stomatal type, and epidermal composition. Close-up views of the anatomy were taken using light and stereo microscopes.

RESULTS AND DISCUSSION
A. Gross Morphology a. Amyema curranii (Fig. 2) Amyema curranii stems creep around the host plant; leaves are lanceolate to oblanceolate, 10 cm-18 cm long. Inflorescence: pink, umbel-shaped, pedunculate inflorescence with a yellowish corolla tip. 10 to 15 flowers with at least 5 cm-5.5 cm long; fruits ovate, berry, aggregate, green when young, red when mature. It has a very close resemblance to A. incarnatiflora (Elmer) Danser, which has a pinkish, umbel-shaped, pedunculate inflorescence. They differ in the tip of the corolla, where A. incarnatiflora has a pinkish coloration from the base to the tip, while A. curanii has a yellowish corolla tip. Further, A. curranii has dimorphic leaves (Table 1). b. Amyema seriata (Fig. 3) Amyema seriata stems erect and mostly attached to the haustorium; leaves obovate to oblanceolate, 10-13 cm long. Amyema seriata stems erect and mostly attached to the haustorium; leaves obovate to oblanceolate, 10 cm-13 cm long. Inflorescence pedunculate, umbelled, and red, with dark red at the tip of the corolla, ca. 2.5 cm-3 cm long, with at least 6 to 8 flowers attached; fruits 2 cm long, berry-shaped, ovate, aggregated, green when young, and red when mature. A. seriata closely resembles A. celebica (Tiegh.) Danser, except for the latter having a red umbel, pedunculate inflorescence, and obovate leaves. They also differ at the tip of the corolla, where A. celebica has a yellowish tip and an outward fold when fully bloomed. It also has a pedicel attached to each flower (Table 1).
A. seriata has a single layer of leaf epidermis that is pinkish in colour and has paracytic stomata. Palisade mesophyll cells 4 layers-5 layers, spongy mesophyll cells 5 layers-6 layers, vascular bundle collateral open. Petiole epidermis, single layer, pinkish colour, cortex (10 layers-11 layers), vascular bundle collateral open. Stem epidermis has single layer, cortex 8 layers-10 layers; eustele stem has pith at the centre; haustorium is solitary and attached to the secondary xylem. The ovary is inferior, free central, and smooth ( Fig. 5; Table 2).
According to Costa and Ceccantini (2015), the host plants should have a distinct thick-walled latewood as compared to that of the haustoria of parasitic plants. Haustorium evolution was complex in the aerial Loranthaceae, with multiple origins for each basic haustorial type (Wilson & Calvin 2006). They also tend to accumulate a large portion of the water reservoir in the woods, which can harm or kill the host plants (Hawksworth 1983). Some host plants also tend to resist the haustorium formation of Amyema by forming wound periderm or by means of changes in the host plant's tissue (Yan, 1993). This can be shown in Figs. 4E and 5E, where both Amyema species have not fully penetrated the entire latewood or secondary xylem of the host plant.

CONCLUSIONS
The flowers of the two Amyema species differ in colour, with A. curranii having pink flowers and A. seriata having red flowers. They also differ in haustorial attachment, where A. curranii has several haustoria as it is creeping, while A. seriata has a solitary attachment, and ovary wall, where A. curranii has the presence of hairs, whereas A. seriata is smooth.

FUTURE SCOPE
Conservation and protection of the Marilog Forest Reserve should be implemented due to the increasing forest and habitat disturbances caused by several anthropogenic activities that place these plants at risk of vulnerability or depletion.
Anatomical studies using other techniques are also suggested to carefully record the differences in their parts.