Sinocurculigo, a New Genus of Hypoxidaceae from China Based on Molecular and Morphological Evidence

Background The monocot family Hypoxidaceae consists of nine genera with nearly 200 species. They occur mostly in the Southern Hemisphere with only a few species in the Northern Hemisphere, of which three genera, Hypoxis, Molineria, and Curculigo, with eight species are distributed in China. Recently, we have found a hypoxid-like plant in China that is quite different in floral structure from any of the three genera and even of the known taxa in Hypoxidaceae. Methodology/Principal Findings In addition to morphological analysis, we performed maximum parsimony, maximum likelihood, and Bayesian inference analyses based on fragments of the chloroplast matK and rbcL genes of 60 taxa in 12 families representing all major clades of the Hypoxidaceae alliance. Results showed that Hypoxidaceae is monophyletic and and that the new plant belongs to it, forming a distinct clade within the family Hypoxidaceae as a sister of Molineria. Phylogeny of the Hypoxidaceae family was constructed based on a combined matrix of the chloroplast rbcL, trnS-G, and trnL-F regions of 59 taxa in Hypoxidaceae and its alliance. Findings of the molecular investigation is consistent with those of the morphological analysis. Conclusions/Significance Based on the results of our molecular and morphological analyses in the present study, we propose a new genus, Sinocurculigo.


Introduction
The economic importance of the monocot family Hypoxidaceae lies mostly in their use as traditional medicines. Some species of this family could be beneficial in the treatment of diseases, including HIV and certain tumors [1][2][3][4][5]. It occurs mostly in the Southern Hemisphere with only a few species in the Northern Hemisphere. The family consists of nine genera with nearly 200 species [6][7][8][9], of which three genera, Hypoxis, Curculigo, and Molineria, and eight species occur in China [8,9]. General flower structure of the family is characterized by six perianthial segments in two wheels, six stamens, and a trimerous gynoecium [8,10]. The classification of Hypoxidaceae has been variously treated by different authors based on morphological and DNA data. Fox example, Thompson [11] separated the southern African genera, placing Hypoxis and Rhodohypoxis in one group and Empodium, Spiloxene, and Pauridia in another. Rudall et al. [6] confirmed that Hypoxidaceae is monophyly composed of nine genera by cladistic analyses of rbcL DNA sequences. Nordal [12], based on geographical and basic morphological information, suggested two primary groupings of the family: one centering around the Indian Ocean with genera Curculigo, Hypoxidia, and Molineria; another occurring mainly in southern Africa with Empodium, Pauridia, Rhodohypoxis, Saniella, Spiloxene, and Hypoxis. Hypoxis was regarded as sister to the southern African genera because of its much wider distribution. In 2011, Kocyan et al. [7] reconstructed the phylogenetic relationships of the family by using four plastid DNA regions and identified three well-supported major clades: the Curculigo, Hypoxis and Pauridia-Empodium clades. These clades comprise a complex assemblage of the genera in the family. Recently, we have discovered a new member of the family in southern China that is quite different in floral features from any of the known taxa in Hypoxidaceae [13]. Clarification requires both molecular and morphological analyses and consideration of phytogeography.

Morphological Analysis
The new hypoxid entity was collected from Taishan in southern Guangdong province, China ( Figure S1). At the beginning of our discovery of this hypoxid, a minute comparison between our hypoxid plant and many members of Hypoxidaceae revealed that the new plant is characterized by sepals distinguishable from petals and long filaments arising from style base and adnate to the perianth segments with their basal parts (1-1.5 mm long). It has an obscure 3-ridged style with two rows of glandular hairs along the uppermost part of each obscure ridge. These hairs then come together at the top of the style, forming a terminal stigma composed of three inverted U-shaped hairy piles. The plant had unilocular ovary with three parietal placentas and fruit contains numerous seeds which are papillate on the surface (Figure 1). These features distinguish this taxon from all hypoxid genera currently known to us.
The new plant is no doubt a very remarkable hypoxid that is difficult to place in any known genera of Hypoxidaceae. Although it presents a certain similarity in habit and floral morphology to Empodium, Curculigo and Molineria, none of them has such a distinctive stigma, ovary and seed structure.

Observation of Pollination Mechanism
The inflorescence of the new plant is characterized by only one flower opening at a time. After the flower is fully open, the perianth segments begin to move back while the style begins to curve at base. In the closed flower, the glandular-hairy stigma touches the opened anthers and allows adherence of the pollen from them in order to complete pollination ( Figure 2). The flowers observed have all fruited and borne numerous seeds.

Analyses of Phylogenetic Position
A morphological comparison of this hypoxid with the extant members of Hypoxidaceae showed that it is more or less related to Empodium and, to a lesser degree, to Molineria and Curculigo [11,13]. We integrated a detailed molecular matrix in order to place the plant in an appropriate phylogenetic position. The phylogeny of related families and genera was constructed based on a combined matrix of 3,055 nucleotides sequences of the rbcL and matK genes of one family of Magnoliopsida, one family of Piperopsida, one family of Caryophyllopsida, 10 families of Liliopsida, and one family of Ranunculopsida, including a total of 60 taxa (Tables S1, S2). The aligned length, the numbers of variable sites and parsimony informative sites, tree statistics for the maximum parsimony (MP) analysis, and the best-fit model selected by Modeltest are presented in Tables 1 and 2. After Bayesian inference of the phylogeny, majority of consensus phylogeny trees demonstrated monophyly of hypoxid plant and the other families with a posterior probability of 64% ( Figure 3).
Results indicate that 13 clades were absolutely distinguished, with a posterior probability of 100% ( Figure 3 and Figures S2,  S3). The 13 clades respectively correspond to 12 families and their evolutionary sequence. Hypoxidaceae is an independent clade (PP100%), in which Sinocurculigo is located together with the known genera of this family. Based on the combination of matK and rbcL gene sequence, the Hypoxidaceae clade is divided into three subclades (PP100%). Individual results of matK and rbcL are similar to those of their combination ( Figures S4, S5, S6, S7, S8, S9).

Phylogeny of Hypoxidaceae
Analysis of single sequence data. Phylogenetic trees based on the analysis of rbcL and trnS-G produced similar topological structures (Figures S10, S11, S12, S13, S14, S15). The trees showed that the 10 genera of this family are grouped into three clades: the first clade (Pauridia -Empodium clade) includes all the species of Spiloxene, Pauridia, and Saniella, and two species of Hypoxis (Hypoxis glabella and H. occidentalis); the second clade (Curculigo clade) includes Molineria, Curculigo, Hypoxidia, and the new genus, Sinocurculigo; and the third clade (Hypoxis clade) includes most species of Hypoxis and Rhodohyoxis. The phylogenetic tree of trnL-F (Figures S16, S17, S18) showed some differences from those of rbcL and trnS-G. The 10 genera can be grouped into four clades, of which the second and third clades are the same as the above. The first clade can be divided into two distinct clades: the Pauridia clade includes Spiloxene, Pauridia, Saniella, and two species of Hypoxis; and the genus Empodium forms another clade. The three datasets produce rather similar topological structures, especially in the main clades. The aligned length, the numbers of variable sites and parsimony informative sites, tree statistics for the maximum parsimony (MP) analysis, and the best-fit model selected by Modeltest are presented in Tables 3 and 4.

Discussion
To our knowledge, this is the first study to report a member of the family Hypoxidaceae to have heterochlamydeous perianth, elongate filaments arising from style base and adnate to perianth segments with their basal parts, a unique stigma composed of three inverted U-shaped hairy piles, a unilocular ovary with three parietal placentas, and numerous papillate seeds. These features differ sharply from those found in Empodium, that is an African genus characterized by having a single ebracteate flower and smooth seeds with a persistent outgrowth on surface [7]; but its allies, Curculigo and Molineria, have a trilobed stigma, a trilocular ovary, and non-papillate seeds; and in Hypoxis, the stigma is usually composed of three lobes or stripes, though in H. angustifolia the stigma can differ in shape in different individuals [14]. The glandular-hairy stigma of the Taishan hypoxid is a feature that makes self-fertilization possible, which distinguishes it from all other hypoxids. Findings of this study significantly improve our understanding of the hypoxid evolution in stigma structure and reproductive mechanisms.
Molecular evidence agrees well to the conclusion drawn from morphological features. Analyses of a combined data set with parsimony and Bayesian methods revealed that the Hypoxidaceae is highly monophyletic and that the Taishan hypoxid represents an independent lineage parallel to the genera Curculigo and Molineria in the family. The Taishan hypoxid is treated as a new genus, Sinocurculigo, in the present study.
The flowers of Hypoxidaceae usually use pollen to attract insects to facilitate pollination [7]. Self-pollination has never been found in this family. We have observed honeybees visiting the flowers of Curculigo sinensis, C. orchioides, and Molineria capitulata in Guangdong, but failed to see any insect visiting the flowers of Sinocurculigo. Sinocurculigo appears to adapt to intra-flower pollination, a derivative mechanism in the family.
Hypoxidaceae has been proposed as a potential sister family to Orchidaceae [15][16][17][18]  N The new genus is akin to Curculigo and Molineria, from which it differs by having an entire glandular-hairy stigma, sepals distinguishable from petals, filaments much longer than anthers and arising from style base and adnate to the base of perianth segments, unilocular ovary with three parietal placentas, and densely papillate seeds. N Description. Rhizomes long. Leaves plicate, basal. Inflorescence erect, short, densely several-flowered; flowers subopposite; pedicel very short; perianth segments 6, in two whorls, free; sepals distinguishable from petals; stamens 6, inserted at base of style; filaments much longer than anthers, adnate to perianth segments with their basal part for 1-2 mm; anthers nearly basifixed; ovary inferior, unilocular with three parietal placentas, without an apical beak; style obscurely 3-ridged, dilated at base, with six rows glandular hairs on its uppermost part and then coming together apically forming an entire, hairy stigma; fruit containing numerous papillate seeds.      (6100) [19], Cymbidium sinense (Jackson ex Andr.) Willd., and C. ensifolium (L.) Sw. [20] ( Figure S1).

Materials and Methods
All necessary permits for our field studies were obtained. The locations for our field studies are not private lands but protected areas controlled by the Forestry Bureau of Guangdong Province, China. We have obtained a valid permit from this authoritative organization. Field observations did not damage any plant, animal, or insect. Although the species of Hypoxidaceae are not endangered plants, Sinocurculigo taishanica is a rare plant facing threat and needing protection.

Materials
A total 53 species in ten genera of Hypoxidaceae were analyzed and six representatives of Asteliaceae, Blandfordiaceae, and Lanariaceae were chosen as outgroup. The new hypoxid was sampled in this study and others were accessed from GenBank. In addition, 60 genera representing 12 closely related families (e.g. Orchidaceae, Palmas, Nelumbonaceae, Amaryllidaceae, Asparagaceae, Liliaceae, Caryophyllaceae, Piperaceae, Araceae, and Iridaceae) were accessed from GenBank and were treated as an ingroup in order to test the monophyly of Hypoxidaceae and to interpret its genetic relationships. One species of Magnoliaceae was chosen as outgroup [21,22]. For detailed information regarding the assessment, see Supplementary Tables S1 and S2.

Amplification and Sequencing
Total DNA was extracted from fresh material or silica-gel-dried plant tissue with a Multisource Genomic DNA Miniprep Kit (Axygen Biosciences) following the manufacturer's instructions. The amplification reaction included total DNA, primers, Ex-Taq buffer, and Ex-Taq DNA polymerase (Takara Bio). The polymerase chain reaction (PCR) profile consisted of an initial 5 min pre-melt stage at 95uC, then 30 cycles of 30 s at 95uC (denaturation), 30 s at 46-52uC (annealing temperature was determined by primer), and 1-3 min at 72uC (extension time was determined by length of the target DNA region), followed by a final 10-min extension at 72uC.
Amplification of the rbcL region was performed using the primer pairs rbcL-1F and rbcL-1352R [23]. The trnL-F region was amplified with primers c and f [24]. For matK sequences, amplification was performed using the primer pair matK-19F and trnK-2R [25]. The spacer region between trnS-trnG was amplified using the S and G primers [26]. To check the quality of the amplified DNA, PCR products were run on 1.5% agarose gels. Gels with target products were excised, purified using DNA Gel Extraction Kits (Axygen Biosciences), and sequenced by BGI Americas Corporation.
Sequence editing and assembly. Both forward and reverse sequences and electropherograms were edited and assembled with DNASTAR (http://www.dnastar.com/). DNA sequences were  Table 4. Best-fit model and parameter for intra-Hypoxidaceae analysis datasets.  aligned with MEGA5.05 [27] under the Muscle model and then adjusted manually with MEGA5.05 [27]. Aligned sequences are available from the corresponding author upon request. Data analyses. For the family-level analysis, the datasets included rbcL, matK, and their combination. Under the family analysis, the datasets included rbcL, trnS-G, trnL-F, and their combination. Insertions, deletions, and some unavailable sequences were treated as missing. Phylogenetic analyses were performed under maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI). The best fit model for each dataset was selected by Modeltest 3.7 [28] under the Akaike Information Criterion (AIC) (Tables S1, S2).
MP analyses were performed using PAUP* version 4.0b10 [29]. All characters were equally weighed and unordered. Test settings included 1,000 replications of random addition sequence and heuristic search with tree bisection-reconnection (TBR) branch swapping. Tree length, consistency indices (CI), and retention indices (RI) are given in Table 1. The ML analysis was computed by RAxML version 7.2.8 with 100 bootstrap replicates using settings described by Stamatakis et al. [30]. BI analysis was performed using MrBayes 3.1.2 [31]. The best-fit model for each dataset was selected by Modeltest 3.7. In the combined datasets, the models were based on the best fit model for each individual dataset. The following settings were applied: sampling frequency = 100; temp = 0.1; burn-in = 10,000; and the number of Markov chain Monte Carlo (MCMC) generations = 4,000,000. The first 10,000 trees were discarded as burn-in to ensure that the chains are stationary. Majority-rule consensus tree was constructed on those trees sampled after generation 1,000,000.

Nomenclature
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