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.

Aquatic plants, Cornales, endemic, new taxa, taxonomy

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 fast-moving 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) and H. 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.

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. GenBank 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).

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 observe 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.

We would like to thank the University of Antananarivo and the Parc Botanique et Zoologique de Tsimbazaza for arranging the field expedition and thank Ministère de l’Environnement et du Développement Durable of Madagascar for issuing the research permit (31/17/MEEF/SG/DGF/DSAP/SCB.Re). This work was financially supported by grants from the National Natural Science Foundation of China (31970211) and Sino-Africa Joint Research Center, CAS (SAJC201614). We appreciate Shuai Peng and Cai-Fei Zhang for revising the manuscript and our guides and team members for the assistance in the fieldwork and also appreciate the constructive comments and suggestions from reviewers and editors.