Halophila Balfourii Solereder (Hydrocharitaceae)—An Overlooked Seagrass Species

Halophila balfourii Solereder has long been treated as a synonym of Halophila stipulacea (Forrsk.) Asch., although it was named more than a century ago. Microscopic (optical microscope and scanning electron microscope) studies on all available herbarium materials of these two species have reconfirmed that the unique papillose leaf epidermis is only presented in H. balfourii but not in H. stipulacea. The pattern of seed testa reticulate is significantly different between these two species. Furthermore, H. balfourii is predominately restricted to the Rodriguez and Mauritius Islands while membranous leafed H. stipulacea is widely distributed in the Red Sea, the Indian Ocean and the Mediterranean Sea as well as East Africa coasts. Based on distinctive characteristics of the leaf and seed coat, and its geographic distribution, it is recommended to reinstate H. balfourii as an independent species and not as a synonym of H. stipulacea.


Introduction
Halophila plants are small in size and their reproductive structures, such as flowers and fruits, are simple and rarely collected. As a result, the identification characteristics of Halophila species are rather limited and are mainly based on gross vegetative morphology [1,2]. Consequently, the nomenclatural confusion has been continuously present and the names used in the literature may often be changed or misapplied. Although Tomlinson [3] stressed that sufficient microscopic detailed features of Halophila's vegetative anatomy could be applied allowing a clear separation of taxa, such valuable characteristics have hardly been used.
Balfour [7] found two Halophila species, H. ovalis and H. stipulacea, growing on the reefs of the Rodriguez Islands while he was participating in "The Transit of Venus Expeditions, 1874-75". Balfour [8] reported in great detail the vegetative morphology, anatomy and reproductive biology of these two Halophila species. Through a microscopic study on these Halophila species, Balfour commented and illustrated specifically that H. stipulacea from the Rodriguez Is. had a remarkable leaf surface with a papillose or tuberulated appearance ( [8] Plate lX, Figure 21). Ascherson and Gürke [9] and Ascherson [10] did not discuss this unusual leaf epidermal morphology when they redescribed H. stipulacea. On the other hand, based on this unique morphological characteristic (the papillose or tuberulated appearance), Solereder [11] introduced a new species name, Halophila balfourii, for the H. stipulacea from the Rodriguez Is. without a formal species description. However, Ostenfeld [12], Ostenfeld and Meerestraser [13], den Hartog [1] and Simpson [14] continued treating H. balfourii as well as H. bullata as synonyms for H. stipulacea and this classification has been maintained in the recent seagrass taxonomic reviews [2,15]. Leaves of H. stipulacea have been collectively described as cartilaginous to membranous; surface glabrous, papillose or with minute hairs, occasionally bullate [1,2]. In the meanwhile, H. stipulacea had exhibited the migrating capability to expand into the Mediterranean Ocean from the Red Sea after the opening of the Suez Canal [16,17] and then, recently, to the Eastern Caribbean [18][19][20][21][22].
Waycott et al. [23] initiated a molecular genetic survey on the Halophila taxonomy by using the H. stipulacea materials from the Mediterranean Ocean and concluded that H. stipulacea is a distinct Halophila species. Nguyen et al. [24] had also molecularly identified H. stipulacea from the Chilika Lake, India (19 • 43 N; 85 • 19 E). Unfortunately, no molecular genetic or microscopic study has been undertaken on the species status of Halophila balfourii since the species was named more than a century ago.
The present microscopic examination on all available herbarium materials of H. stipulacea, H. balfourii and H. bullata from a wide geographic area aims to reconfirm the papillose epidermal appearance of H. balfourii in order to reinstate the taxonomic status of H. balfourii as an independent species instead of as a synonym to the H. stipulacea.

Comparative Microscopic Studies on the Leaf and Seed Surfaces in H. stipulacea and H. balfourii
The results of microscopic studies on herbarium materials confirm that the H. stipulacea plants with bullated leaves, and occasionally with hairs, are restricted to the Red Sea; while the H. stipulacea plants with membranous leaves without hairs ( Figure 1A) are widely distributed in the Red Sea, Mediterranean Ocean, East Africa and the Indian Ocean. On the other hand, plants with the papillose leaves of H. balfourii have come from the Rodriguez and Mauritius Islands (eleven collections) with only one collection coming from southern India (Palk Bay, 9 • 35 N; 79 • 15 E). It should be noted that the other three collections from the Palk Bay examined in this study were identified to be H. stipulacea.
The individual squamous epidermal cells in these Halophila stipulacea leaves can easily be identified ( Figure 1B). The epidermal cells of the papillose leaves have an enlarged protrusion which obstructs the view to the base of epidermal cells ( Figure 1C), thus each epidermal cell outline cannot be distinguished easily from the surface of the leaf ( Figure 1D). Furthermore, cross sections of the membranous leaves of H. stipulacea possess a smooth leaf surface with a squamous appearance of epidermal cells ( Figure 1E). Conversely, those of the papillose leaves of H. balfourii exhibit an undulated surface due to a pyramid appearance of epidermal cells ( Figure 1F). This special feature of the leaf epidermal cells is unique to this species, when compared to all known seagrass species (Kuo, per. obs.). In addition, the pattern of seed testa reticulate is quite different in these two Halophila species; in H. stipulacea it appears to be square ( Figure 1G) and in H. balfourii it shows a rectangular shape ( Figure 1H).
The present microscopic study confirms Balfour's earlier observations that the unique papillose leaves only belong to H. balfourii and not to H. stipulacea. This study further demonstrates that seed testa appearances are also quite different between these two species. In addition, H. balfourii is predominately restricted to the Rodriguez and Mauritius Islands. Thus, it is recommended that H. balfourii should have an independent species status and not be a synonym of H. stipulacea. Since there is no formal species description of H. balfourii existing, a proper taxonomic description is provided below.
It is anticipated that the present microscopic results will stimulate and encourage more molecular genetics, ecological and biological studies to be carried out on this little-known H. balfourii species in the near future.
It is anticipated that the present microscopic results will stimulate and encourage more molecular genetics, ecological and biological studies to be carried out on this little-known H. balfourii species in the near future.   Distribution: Currently, Halophila balfourii is confined to the Rodriguez and Mauritius Islands. It is very possible that Iyengar 133 A (K, NHM) from Pamban, India was not a true locality. Ramamurthy et al. [25] did not mention the papillose leaves of H. stipulacea from the Coromandel Coast. H. balfourii also is not present at Kenya coast, as reported by Issac previously [26].

Redescription of Halophila Balfourii
Habitats: The species had been collected from less than 1 to 6 m in water depth. Biology: The limited herbarium specimens indicate that both male and female flowers occur in October and fruits are produced from October to December in Mauritius.

Material and Methods
Herbarium samples of Halophila stipulacea (157 collections) and H. balfourii (12 collections) at major herbaria e.g., BSI (4), C (9), K (21), L (17), NHB (27), NY (5), P (18), S (7), UC (2), US (6), W (6), and at Tel Aviv University (80) and the Hebrew University of Jerusalem (10) were studied. The geographic distribution of these herbarium specimens is presented in the Table 1. Leaves and seeds were chosen and imaged by microscope. Up to 60% of these herbarium specimens were imaged and measured for root lengths, rhizome internodes, petiole length, leaf length and width as well as the number of cross veins. When available, reproductive structure was measured and noted. Botanical drawings of reprehensive specimens were also made.
Only five out of 157 herbarium specimens had exhibited 'bullata' appearance (=H. bullata), apart from this feature, there are no other morphological differences from H. stipulacea. Therefore, these bullata specimens have been treated as H. stipulacea in this study.
Most of the membranous and bullated leaved H. stipulacea are easily identified under the dissecting microscope, however, those leaves which were covered by microscopic epiphytes were examined by SEM to confirm that these specimens were not H. balfourii. On the other hand, all twelve H. balfourii collections have been further confirmed by the SEM examination of their special papillose characters.
More than twenty selected H. stipulacea (with bullate, membranous and hairy appearances) representing all their geographic distributions, along with all twelve H. balfourii collections, were prepared for SEM imaging. It should be noted that both surfaces of each leaf were studied using a Philips Scanning Electron Microscope 505 at 15 kV.
Three samples each from H. stipulacea and H. balfourii were prepared for histology studies. Transverse sections (2 µm in thickness) on glycol methacrylate embedded samples of selected leaves were stained with 0.5% toluidine blue O (pH 4.4) and photographed using a Zeiss research light microscope.
Three seeds of H. stipulacea (all from Elate Gulf, Tel Aviv herbarium) and two seeds of H. balfourii from Rodriguez (K) were examined using the Zeiss Scanning Electron Microscope 505 at 15 kV.
Funding: This research received no external funding.