Hydrostachys flabellifera (Hydrostachyaceae), a new species from Madagascar

Abstract Hydrostachys flabellifera, a new species of Hydrostachyaceae found in a stream in Manandriana, Madagascar, is described and illustrated herein. It is similar to H. verruculosa and H. laciniata in morphology, but can be distinguished from them by its leaves with sparsely arranged, flabelliform and palmately parted emergences, obvious rachis and the pattern of segments arranged on the male bracts. Molecular phylogenetic analysis of the nuclear ribosomal internal transcribed spacer (ITS) dataset provides a robust support for it as a new species as well.


introduction
Hydrostachys Thouars (1806: 2) is the sole genus in the family Hydrostachyaceae (Tul.) Engler (1894: 136) with about 22 known species. Fourteen of them are endemic to Madagascar (Phillipson et al. 2018) and the remaining species are native to southern and tropical Africa. Hydrostachys has been used in traditional medicine and probably could be a potential candidate for use in chemotherapy to fight against cancer (Ranarijaona et al. 2014). The plants of Hydrostachys, which are aquatic herbs living in fastmoving streams or rivers, are well adapted to turbulent aquatic environments with their roots and discoidal rhizome adhering to the rocks. Hydrostachys are annual or perennial, submerged or partially submerged in the rainy season, flowering in the dry season. Their simple or pinnate leaves emerge from the rhizome, and petiole, rachis and subdivisions are often covered with diverse forms of emergences, including verrucae, scales and lobules (modified leaf blade lobes), which give the plant the appearance of a fern or lycopodium. Hydrostachys are dioecious or seldom monoecious, with highly reduced and unisexual flowers borne on the spike, the spikes usually emerging from the rhizome, sepals and petals are absent; the fruit is a capsule with numerous tiny seeds (Perrier 1952;Cusset 1973;Stannard 1997;Verdcourt 1986;Erbar and Leins 2004).
Hydrostachys are highly modified aquatic plants and the taxonomic placement of this enigmatic genus has confounded botanists for two hundred years. Due to their similar habitat and highly modified morphological characters, the genus was once placed in Podostemaceae (Tulasne 1849). However, this placement was rejected thanks to evidence from embryology, inflorescence morphology (Jäger-Zürn 1965;Rauh and Jäger-Zürn 1966) and biochemistry (Scogin 1992). Phylogenetic studies showed unstable placements, based on different DNA markers and taxon sampling (Les et al. 1997;Soltis et al. 2000;Burleigh et al. 2009). Currently, Hydrostachyaceae is treated as a distinctive family in Cornales with a phylogenetic long branch in most studies (Albach et al. 2001;Xiang et al. 2002;Fan and Xiang 2003;Xiang et al. 2011;Fu et al. 2019). Despite the uncertain placement at the order level, the inter-species identification is much clearer.
During a field investigation in Madagascar in 2017, a Hydrostachys population was found in Manandriana which appeared similar to H. verruculosa A. Juss. (1837: tab. 91) andH. laciniata Warming (1899: 152). However, after carefully comparing the collection with all available specimens of Hydrostachys and consulting relevant literature (Perrier 1952;Cusset 1973), we observed that its leaf structure is different from that of all known species in this genus and that this plant is wholly new to science. Hence, we describe it herein as a new species.

Materials and methods
The description of the new species is based on field notes and observations of field pictures, dried specimens and FAA-fixed (formalin/acetic acid/alcohol) materials. Specimens were collected from Manandriana, Madagascar (20°14'S, 47°06'E) and deposited at the herbaria of Parc Botanique et Zoologique de Tsimbazaza (TAN) and Wuhan Botanical Garden, Chinese Academy of Sciences (HIB). Some leaves and spikes were fixed and conserved in formalin/acetic acid/alcohol (FAA) fixatives. Detailed characteristics of the bracts and emergences were observed and measured on the fixed materials by using a stereomicroscope (Nikon Stereo Microscope SMZ25). Terminology was referenced in several books and literature (Perrier 1952;Cusset 1973;Verdcourt 1986;Simpson 2010;Beentje and Williamson 2016). The herbarium abbreviations follow Index Herbariorum (http://sweetgum.nybg.org/science/ih/). Physical specimens of Hydrostachys, deposited at BM, E, K and TAN, were examined. High-resolution digital specimen images from B, BNRH, BR, GH, MA, P and US were checked via JSTOR Global Plants (https:// plants.jstor.org) and GBIF (https://www.gbif.org). The distribution map was produced by QGIS3 (available from: https://qgis.org/).
The nuclear ribosomal ITS was used as the DNA marker, with 13 samples included in the phylogenetic analysis. All Hydrostachys sequences were newly generated, while three taxa from Nyssa and one from Triphyophyllum were treated as outgroups. Gen-Bank accession numbers are available in Table 1. Genomic DNA was extracted from dry specimens using Mag-MK Plant Genomic DNA extraction kits (Sangon Biotech, Shanghai). Primers for polymerase chain reactions (PCR) were referred to White et al. (1990). PCR products were sequenced by Sangon Biotech using the 3730xl DNA Analyzer and Geneious v.11.1.5 (available from: http://www.geneious.com/) was used for DNA assembling and manually editing. The dataset was aligned by MAFFT v.7.294 (Katoh and Standley 2013), then trimmed by trimAl v.1.2 (Capella- Gutierrez et al. 2009). The Maximum Likelihood tree was inferred using IQ-TREE v.2.0.6 (Minh et al. 2020) with default parameters and ultrafast bootstrap approximation was assessed with 1000 replicates. The consensus tree was visualised and annotated by ggtree v.2.2.1 (Yu et al. 2017). Dataset, scripts and command lines in the phylogenetic analysis are available in Github (https://github.com/xuzhun1008/Hydrostachys_flabellifera_paper.git). Description. A hydrophyte herb. Rhizomes discoid, 3-8 mm in diameter; 7-12 leaves emerging from the rhizome. Leaves simple, 3-12 cm long, the upper part slightly curved when rising from water, the base enlarged with stipule; stipule ovateelliptical, basal half dorsally attached on petiole, apex sometimes with a tail ca. 1.5 mm; centre bud enclosed by stipules of inner leaves; emergences spirally arranged on rachis and stretching out into loose layers, denser towards the apex of the leaf and gradually reduced to the base. Rachis obvious, 1-2 mm in diameter, white to light green. Petioles indistinct. Emergences (modified leaf blade lobes) 1-6 mm long, flabellate, basal ones reduced into scale-like, upper ones palmately parted, lobes cuneiform, secondly divided into rectangular to linear terminal lobes; the flat of emergences almost perpendicular to the axis; the apex of emergence slightly rolling up, ciliate at the end, cilia gathering into tufts after rising from water; emergences green to mauve at the pinnacle, the rest dark green. Male spikes 5.4-8.0 cm long, peduncles 4.7-6.2 cm long, covered with few small scale-like emergences. Bracts 1-2.2 mm × 1-2.2 mm, rhombic, dark green; segment I (the terminal segment) acute, flanked by 1-2 tiny lobules on each side; segments II (lobules at the dorsal side of bract) acute or obtuse, 2 rows, each row with 3-5 separated lobules, lobules ca. 0.3 mm high. Stamen sessile, anther oblate, with two divergent thecae dehiscing longitudinally. Female spike not found.
Etymology. The epithet refers to the flabellate shape of emergences on leaves. Distribution and ecology. Only one population was found on rocks in a stream in Manandriana, Madagascar (20°14'S, 47°06'E), at an elevation of 1400 m (Fig. 3).
Conservation status. Hydrostachys flabellifera is currently only known from one location with a very small population. Additionally, all species of Hydrostachys are highly dependent on the moving aquatic environment which is threatened by water pollution, natural system modifications, energy production and mining, all of which could drive the taxon to Critically Endangered (CR) or Extinct (EX) in a very short time (IUCN 2020). Following Guidelines for IUCN Red List Categories and Criteria (2020), H. flabellifera should be categorised as Vulnerable (VU D2).  Phylogenetic analysis. Hydrostachys flabellifera was placed in a robust clade together with H. stolonifera and H. imbricata (Fig. 4) with a high bootstrap support (BS = 96%), while they share limited morphological characteristics. H. multifida, considered as a clade in morphology, is not a monophyletic group, although with low bootstrap support.

Discussion
Hydrostachys are mostly annual, only a limited number of species with stolons are perennial (Perrier 1952), like H. monoica and H. stolonifera. In this case, we did not ob- serve any structure, like stolons, that could help H. flabellifera live for many more years. Therefore, H. flabellifera probably is annual, but continuous observation is needed.
Hydrostachys flabellifera closely resembles H. verruculosa and H. laciniata in having simple leaves, but can be easily recognised by several characters. The leaf emergences of the latter two species are densely arranged and overlapping and they constitute a thick cylindrical leaf with indistinct rachis. In contrast, the rachis of H. flabellifera are thin and obvious and the emergences stretch out into layers, sparsely arranged and significantly reduced at the lower leaves. Their segments patterns on male bract are also obviously different. H. laciniata was once treated as a form of H. verruculosa (Perrier 1952) after it was published as a new species (Warming 1899). Cusset (1973) also recognised it as a distinct species and further presented a diagram to compare it with H. verruculosa and that diagram clearly showed the differences in the bracts and the emergences on the leaf. After examining the type specimens of these two species, we accepted the treatment of Cusset (1973) and continued to compare the new species with these two species. Combining with morphology, phylogenetic results also provide solid evidence for the newly-discovered species. Hydrostachys flabellifera, H. stolonifera and H. imbricata form a robust clade, but share limited morphological characters. H. stolonifera and H. imbricata are much larger than H. flabellifera in size and they are definitely different in leaf types. H. distichophylla var. hildebrandtii is similar to H. flabellifera in morphology, but they are located in two distinct clades. The phylogenetic position of H. flabellifera would be much clearer when more taxa are included in the analysis. The combined results from phylogenetic analysis and detailed comparisons of morphological characteristics amongst H. flabellifera, H. verruculosa, H. laciniata, H. distichophylla var. distichophylla and H. stolonifera are listed in Table 2.
Hydrostachys are adaptable to diverse aquatic environments, from clean mountain streams to muddy rivers. These species can be distinguished by the type of leaves, emergences, spikes, also the bract shape and segments arrangement are valuable identification characteristics. Due to different statuses between fresh plant and pressed specimens, greater attention to detail is needed when comparing and describing these species in different conditions. Based on our empirical research, in some specific cases, it is tricky to connect the living plant to the corresponding dry specimens. We highly recommend combining field investigations and herbarium examinations to obtain the full knowledge of this aquatic family.