3 urn:lsid:arphahub.com:pub:F7FCE910-8E78-573F-9C77-7788555F8AAD PhytoKeys PK 1314-2011 1314-2003 Pensoft Publishers 10.3897/phytokeys.135.38531 38531 Research Article Alismatales Hydrocharitaceae Phylogeny Taxonomy Cenozoic Asia Ottelia fengshanensis, a new bisexual species of Ottelia (Hydrocharitaceae) from southwestern China Li Zhi-Zhong 1 2 Wu Shuang 3 Zou Chun-Yu 4 Liu Yan 4 https://orcid.org/0000-0001-8931-9656 Hu Guang-Wan 1 5 https://orcid.org/0000-0001-7728-7976 Lehtonen Samuli 6 Wang Qing-Feng 1 5 qfwang@wbgcas.cn Chen Jin-Ming 1 jmchen@wbgcas.cn CAS Key Laboratory of Aquatic Botany and Watershed Ecology, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, CN-430074, China Wuhan Botanical Garden, Chinese Academy of Sciences Wuhan China University of Chinese Academy of Sciences, Beijing, CN-100049, China University of Chinese Academy of Sciences Beijing China Guangxi Association for Science and Technology, CN-530022, China Guangxi Association for Science and Technology Guangxi China Guangxi Institute of Botany, Chinese Academy of Sciences, Guilin, CN-541006, China Guangxi Institute of Botany, Chinese Academy of Sciences Guilin China Sino-Africa Joint Research Center, Chinese Academy of Sciences, Wuhan, CN-430074, China Sino-Africa Joint Research Center, Chinese Academy of Sciences Wuhan China Herbarium, Biodiversity Unit, University of Turku FI-20014 Turku, Finland University of Turku Turku Finland

Corresponding author: Qing-Feng Wang (qfwang@wbgcas.cn); Jin-Ming Chen (jmchen@wbgcas.cn)

Academic editor: Peter Boyce

 2019 30 10 2019 135 1 10 17B55881-8856-55AE-9977-FC1859D704AF 3531142 25 07 2019 10 09 2019 Zhi-Zhong Li, Shuang Wu, Chun-Yu Zhou, Yan Liu, Guang-Wan Hu, Samuli Lehtonen, Qing-Feng Wang, Jin-Ming Chen This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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.

 karst bisexual flowers molecular phylogeny Wuhan Botanical Garden, CAS Citation

Li Z-Z, Wu S, Zou C-Y, Liu Y, Hu G-W, Lehtonen S, Wang Q-F, Chen J-M (2019) Ottelia fengshanensis, a new bisexual species of Ottelia (Hydrocharitaceae) from southwestern China. PhytoKeys 135: 1–10. https://doi.org/10.3897/phytokeys.135.38531

 Introduction

OtteliaPersoon (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. acuminataDandy (1934: 132) have been recorded from the area with narrowly endemic distribution in karst rivers or lakes. Among these, just three species, O. alismoidesPersoon (1805: 273), O. balansaeDandy (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 (Cook et al. 1984, Cook and Urmi-Konig 1984, 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.

10.3897/phytokeys.135.38531.figure1 3531144 DC0407F3-62ED-5ABA-9F95-F4831DCDA9B1

Distribution record of Ottelia fengshanensis Z.Z.Li, S.Wu & Q.F.Wang (red triangle) from Fengshan county, Guangxi province, China.

https://binary.pensoft.net/fig/351411 Material and methods 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).

10.3897/phytokeys.135.38531.figure2 3531146 0A515E75-D385-5B06-8C7E-FC5ABA2B9F85

Ottelia fengshanensis Z.Z.Li, S.Wu & Q.F.Wang. A Habitat B flowering plant C bisexual flower with red-green sepals D individual and leaf: triplinerved with conspicuous cross veins E fruit: Hexagonal-cylindric with tiny wings F the character of pollens by SEM G spathe H seeds.

https://binary.pensoft.net/fig/351412

The voucher information and GenBank accession numbers for the sequences of internal transcribed spacer (ITS) and three cp regions (trnS-trnG, rbcL and trnK5’ intron) in the present study.

Taxon Individual code Locality Voucher no. Acceession No.
ITS rbcL trnK5’ intron trnS-trnG
O. acuminata var. jingxiensis 01 Jingxi, Guangxi HIB-Otte010 MG751780 MH257624 MH257642 MH257660
12 Debao, Guangxi HIB-Otte009 MG751781 MH257628 MH257646 MH257664
19 Du’an, Guangxi HIB-Otte012 MG751782 MH257630 MH257648 MH257666
O. acuminata var. crispa 10_1, 10_2 Luguhu, Yunan HIB-Otte011 MG751784/MG751785 MH257626/MH257627 MH257644/MH257645 MH257662/MH257663
O. acuminata var. acuminata 9 Heqing, Yunan HIB-Otte003 MG751786 MH257625 MH257643 MH257661
15 Jianchuan, Yunan HIB-Otte006 MG751787 MH257637 MH257655 MH257673
30 Caohai, Guizhou HIB-Otte014 MG751788 MH257633 MH257651 MH257669
O. acuminata var. lunanensis 16 Shilin, Yunnan HIB-Otte008 MG751789 MH257629 MH257647 MH257665
O. acuminata var. songmingensis 21_1, 21_2 Songming, Yunnan HIB-Otte007 MG751790/MG751791 MH257631/MH257632 MH257649/MH257650 MH257667/MH257668
O. balansae 29 Huaxi,Guizhou HIB-Otte005 MG751792 MH257634 MH257652 MH257670
O. emersa 41 Guigang, Guangxi HIB-Otte004 MG751794 MH257638 MH257656 MH257674
O. cordata 40 Haikou, Hainan HIB-Otte001 MG751795 MH257639 MH257657 MH257675
O. alismoides 42 Changping, Fujian HIB-Otte002 MG751796 MH257640 MH257658 MH257676
O. guanyangensis 32 Guanyang, Guangxi HIB-Otte015 MG751797 MH257635 MH257653 MH257671
34 Guanyang, Guangxi HIB-Otte016 MG751798 MH257636 MH257654 MH257672
O. fengshanensis 35 Fengshan, Guangxi HIB-lzz51 MK531550 MK531552 MK531553 MK531551
B. japonica Wuyishan, Fujian HIB-Bly001 MG751799 MH257641 MH257659 MH257677
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).

Morphological characters comparison among Ottelia fengshanensis, Ottelia guanyangensis and Ottelia balansae.

Characters Ottelia fengshanensis Ottelia guanyangensis Ottelia balansae
Flowers bisexual bisexual bisexual
Sepals 1.0–1.5 cm, red green 1.0–1.5 cm long, red brown 2.0–2.5 cm long, green
Stamens 3;filaments 3.0–5.0 mm long 3; filaments 5.0–7.0 mm 3; filaments 4.0–5.0 mm
Ovary 5–10 cm long, hexagonal-cylindric to cylinder 4–5 cm long, hexagonal-cylindric 3.5–5.0 cm long, triangularcylindric
Styles 3, bifid to base,white 3, bifid nearly to base,yellow 3.5–5.0 cm long, yellow
Spathe 3-4 (3) flowered 2–5 flowered 3–11 flowered
Leaf shape Linear or oblong, 30–70 × 8–14 cm,base rounded, apex acute or obtuse;petiole 8.0–10.0 cm long linear, 15–50 × 2.5–4.0 cm, base rounded, apex acute, petiole 8.0–13.0 cm long oblong or ovate, 20–40 × 6.0–8.0 cm, base truncate, rounded, or cordate, apex acute or rounded, petiole ca. 20 cm long
Texture green, opaque, thick ca. 0.8 mm dark green, opaque, thick ca. 1.2 mm green, translucent, thick ca. 0.5 mm
Venation trinerved with obvious cross veins, distance 4.0–6.0 cm to base, longitudinal veins 9 trinerved with obvious cross veins, distance 4.0–6.0 cm to base, longitudinal veins 9 basal veins, longitudinal veins 7
Fruit hexagonal-cylindric, winged unobviously hexagonal-cylindric, winged narrowly elliptic, unwinged
Seed fusiform, ca. 1.0 mm long fusiform, ca. 1.5 mm long cylindric to fusiform, ca. 3.0 mm long
Pollen spheroidal, inaperturate, ca. 40 × 40 μm spheroidal, inaperturate, ca. 35 × 45 μm spheroidal, inaperturate, ca. 49 × 53 μm
Flowering time April to November April to October June to November
Results and discussion

The comparison among three bisexual species, O. fengshanensis, O. guanyangensis and O. balansae, is presented in Table 2. The new species had unique features, including the number of flowers, white styles, trinerved venation with distinct cross veins and longer leaf shape.

Morphological characters distinguish O. fengshanensis from the three bisexual species. For O. alismoides, there is only one flower in each spathe and it is easy to distinguish from the new species. However, O. guanyangensis and O. balansae, which are distributed in Guangxi province and Guizhou province respectively, are closest to the new species. The critical diagnostic characters of O. fengshanensis include having white styles, longer leaf shape and number of flowers in each spathe. Moreover, these three species are also isolated geographically, O. fengshanensis was only found in Fengshan county, but O. guanyangensis was found in Guilin city. O. balansae was only recorded in Guizhou province based on a recent survey. Karst terrain will play an important role in species divergence in this lineage.

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.

Description of the new species Plantae Alismatales Hydrocharitaceae 559D0913-8825-5336-83BB-9B591BEF2421 Ottelia fengshanensis urn:lsid:ipni.org:names:77202741-1 Z.Z.Li, S.Wu & Q.F.Wang sp. nov. Fig. 3 Description.

The new species is similar to Ottelia guanyangensis in having bisexual flowers, three stamens, but differs through having (3)-4 flowers in each spathe (vs. 2–5), white styles (vs. yellow), green leaves (vs. dark green) and by fruits which are tiny winged (vs. obviously winged).

 Type.

CHINA. Guangxi, Hechi City, Fengshan County, elev. 507 m, 24°34'20"N, 107°10'17"E 11 September 2018, Z. Z. Li & S. Wu-Otte51 (holotype HIB-lzz51!).

Annual or perennial herb. Rhizome, short. Leaves entirely submerged, dark green and opaque, linear or oblong, 30–70 × 8–14 cm, base rounded, apex acute or obtuse; longitudinal veins 9; midrib conspicuous, stretched to the apex, becoming trinerved with obvious cross-veins at a distance of 5–7 cm from the base; petiole smooth, green, 8.0–10.0 cm long, the base expanded into a sheath. Spathe oblate, ca 3. 0 × ca. 3.5 cm, warty along edges or smooth, longitudinally ribbed and winged on the lateral margins, containing 3–4 (3) flowers; flowers bisexual; sepals reddish green, 1.0–1.5 × ca. 0.5 cm, with longitudinal ribs; petals white with yellow base, obovate, ca. 2.0 × ca. 2.0–2.5 cm, with longitudinal pleats; stamens 3, opposite to sepals, anthers elliptic, connective obscure, filaments 3.0–5.0 mm long; glands 3, 0.5–1.0 × 0.5–1.5 mm, opposite to petals, pale yellow. Ovary hexagonal-cylindric to the cylinder, 5–10 cm long, with 3 carpels; styles 3, white, slender and hairy, 1.2–1.5 cm long, stigma bifid, divided to base; stigmas 6, liner and hairy, ca. 8 mm long. Fruit a hexagonal-cylindric capsule, with 6 inconspicuous wings, dark green, with persistent calyx, 4.0–9.0 cm × ca. 6.5 mm, always longer than spathe. Seeds numerous, fusiform, ca. 1.0 mm long, both ends hairy. Pollen, subglobose, ca. 40μm in diam, with spiny granules.

10.3897/phytokeys.135.38531.figure3 3531148 4E7F9A75-E4D3-5969-8F8B-71477D3C3B23

Illustration of Ottelia fengshanensis Z.Z.Li, S.Wu & Q.F.Wang. Drawn by Shuai-Jie Li.

https://binary.pensoft.net/fig/351413 10.3897/phytokeys.135.38531.figure4 3531150 03D7E1B1-BEE0-5836-980D-01898855E7F1

Phylogenetic tree and PTP analysis of O. fengshanensis sp. nov. and Blyxa japonica as an outgroup. Posterior probabilities (PP > 0.70) and bootstrap values (BS > 70) based on Bayesian Inference and maximum likelihood (ML) analysis are shown above the branches.

https://binary.pensoft.net/fig/351845 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.

 Other specimens examined (paratypes).

CHINA. Guangxi, Hechi City, Fengshan County, elev. 507 m, 11 September 2018, Z. Z. Li & S. Wu Otte 056 (HIB!)

 Acknowledgments

This study was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB31000000), Wuhan Botanical Garden (CAS) (No. Y655261W03), and the Special Funds for the Young Scholars of Taxonomy of the Chinese Academy of Sciences (ZSBR-013).

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