Ottelia fengshanensis, a new bisexual species of Ottelia (Hydrocharitaceae) from southwestern China

Abstract Ottelia fengshanensis, a new species (Hydrocharitaceae) from southwest China is here described and illustrated. Comparing its morphological features to putative close relatives O. guanyangensis, it has 3–4 flowers (vs. 2–5) each spathe, hexagonal-cylindric fruit, white styles (vs. yellow), green leaves (vs. dark green) and fruit tiny winged (vs. winged obviously). Molecular phylogenetic investigation of four DNA sequences (ITS, rbcL, trnK5’ intron and trnS-trnG) and the Poisson Tree Processes model for species delimitation (PTP) analysis, further resolves O. fengshanensis as a new species that is close to O. guanyangensis with distinct support.


Introduction
Ottelia Persoon (1805:1) has about 22 species and is widely distributed in the tropical, subtropical and temperate regions. In comparison with other genera within the family Hydrocharitaceae, Ottelia is morphologically complex and variable, e.g. the leaf type of the genus is exceptionally erratic even within an individual depending on the developmental stage, as well as within the varieties or populations (Li et al. 2018). The flower sexuality varies within species and flowers can be either bisexual or unisexual. Southwestern China possesses complex terrain and various ecosystems and is a center of diversity for Ottelia species (Chen et al. 2017, Zhai et al. 2018. To date, six species and three varieties of O. acuminata Dandy (1934: 132) have been recorded from the area with narrowly endemic distribution in karst rivers or lakes. Among these, just three species, O. alismoides Persoon (1805: 273), O. balansae Dandy (1934: 137) and O. guanyangensis Z.Z. Li, Q.F. Wang & S. Wu (2018: 294) are bisexual and can only be found in specific karst regions, except for the widespread species O. alismoides , Li 1981.
In 2017-2018, we found and reported a new bisexual species O. guanyangensis in Guilin City, China (Li et al. 2018). We deemed that there are some previously undetected potentially new Ottelia species in Guangxi province's karst steams (Fig. 1). We made further aquatic plant investigations in Guangxi province, China, in 2018. From the Fengshan County, we found once again a species with bisexual flowers which generally appeared to be like O. balansae. Based on investigations of herbarium specimens in GXMG, HIB, IBSC, KUN and PE, and literature review, only three bisexual species of Ottelia are known from China. These are O. guanyangensis, a species described in 2018 (Li et al. 2018), O. balansae, and O. alismoides, the latter two recorded from "Flora of China". Compared to the recorded three bisexual species, it was interesting that the population from Fengshan county had some unique flowers (e.g. white styles and over three flowers each spathe) and leaf traits (e.g. triplinerved with obvious cross-  veins). We transplanted several individuals to the greenhouse at Wuhan Botanical Garden, Chinese Academy of Sciences, to observe the growth. Here we formally describe and discuss this taxon as a new species based on careful morphological observations and molecular phylogeny.

Morphological study
The morphological characteristics of the new species were collected during fieldwork in July 2018. We randomly selected 10 individuals, took pictures of each part and measured the characteristics of flowers, leaves and fruits (Fig. 2). The pollen grains of new species were gold-coated, and photographed using a Hitachi S-800 SEM system at Wuhan Botanical Garden, CAS. Simultaneously, we collected voucher specimens and several fresh leaves were dried using silica gel for DNA extraction. For further detailed morphological analysis, we transplanted five living individuals to a greenhouse at Wuhan Botanical Garden. We also observed the characteristics of flowers, leaves and fruits of these two bisexual species in our greenhouse for further comparative analysis (Table 1).

Phylogenetic analysis
Total genomic DNA of one sample, collected from Fengshan county, Hechi city, Guangxi province, was extracted following Li et al. (2018). One nuclear DNA region (ITS) and three chloroplast DNA regions (trnS-trnG, rbcL and trnK5' intron) were sequenced; the primers and PCR protocols followed Li et al. (2018). The same sequence regions from other species were downloaded from the NCBI ( Table 2). The sequence alignments were made using MAFFT with default settings (Kuraku et al. 2013). The best nucleotide substitution model was detected using jModeltest 2.1.4 (Darriba et al. 2012) with the Akaike Information Criterion (AIC). The Maximum Likelihood (ML) analysis was made using IQtree with 5000 bootstrap replicates (Nguyen et al. 2003). The Bayesian Inference (BI) was analyzed by MrBayes v.3.2.6 (Ronquist and Huelsenbeck 2015), with 20,000,000 generations and four chains run with sampling after every 2000 generations. The first 25% of generations were discarded and a majority rule consensus tree (> 50%) was computed from the remaining trees. In order to test molecular support for species delimitation in Ottelia, the Poisson Tree Processes model for species delimitation (PTP) was applied to the tree with the following parameters: 500,000 generations; thinning: 100; burnin: 0.1 and seed: 123 (Zhang et al. 2013).

Results and discussion
The Four sequence regions (ITS, trnS-trnG, rbcL and trnK5' intron) were aligned and concatenated into a 3623 bp sequence. 605 variable nucleotides were detected. Two clades were displayed with high support (BS= 70, PP= 0.7). PTP analysis further recognized four species with O. fengshanensis having the highest support (0.678). Based on phylogenetic analyses, O. fengshanensis was resolved as sister to O. guanyangensis with high support (BS= 100, PP= 1.0) and only distantly related to O. balansae, which clusters together with O. acuminata (Fig. 4), and based on PTP analysis, O. balansae was not supported as a species, but was more likely to be treated as a bisexual variety of O. acuminata. In combination, the morphological and molecular phylogenetic analyses support that O. fengshanensis is a distinct species closely related to O. guanyangensis, a species also distributed in Guangxi province.
Ottelia possesses complex floral traits and may have bisexual and unisexual flowers. Based on the previous studies (He 1991, Chen et al. 2012) bisexual flowers have evolved multiple times in Ottelia. Here we report a new bisexual species O. fengshanensis and verify that bisexual flower indeed has multiple origins in Ottelia. Ottelia fengshanensis probably has a common ancestor with the unisexual O. acuminata var. songmingensis. Besides, we also suggest that O. balansae should be treated as a variety of O. acuminata. This point has also been put forward by Yu Ito et al. (2019). It will also help us have a better understanding of the diversity and evolution of sex evolution in Ottelia.
Distribution and habitat. Ottelia fengshanensis is known from a single population in Fengshan County, Guangxi Province, China. The species inhabits a karst river less than 1.5 m in depth. Due to the complex underground river system in the karst region, it is probable that the species occurs in nearby areas as well.
Conservation status. Only one population of new species was found at Fengshan County, Guangxi Province, China. Although it might be distributed in adjacent karst rivers. Until now, approximately 50-100 individuals were found in a single population. However, there is not enough information on population size and dynamics. According to the IUCN Red List Categories and Criteria (IUCN 2017), we suggested that the species be evaluated as Data Deficient (DD).
Phenology. The new species was found in flower from April to November. Etymology. The epithet is derived from the name of Fengshan County, which is the only known locality of occurrence.