Two new combinations in Oreocharis (Gesneriaceae) based on morphological, molecular and cytological evidence

Abstract The newly-circumscribed genus Oreocharis is recently enlarged by incorporating ten other genera with high floral diversity. In this study, our morphological, molecular and cytological evidence supports our adding two species from other two different genera (Boeica and Beccarinda) to Oreocharis. The special corolla shape (campanulate or flat-faced) and related short filament of these two new combinations, Oreocharis guileana and O. baolianis, further enrich the diversity of floral characters of the enlarged Oreocharis. Meanwhile, some supplementary and amended descriptions of these two species are made here. Our morphological, molecular and geographical data indicate that O. guileana is related to O. pilosopetiolata to a certain extent. For O. baolianis, however, our current dataset does not allow conclusions on the species relationship within Oreocharis.


Introduction
Recent phylogenetic studies on the Old World Gesneriaceae have greatly advanced our understanding of species relationships and generic delimitations in this plant group (Wang et al. 2010;Möller et al. 2011a). One of the typical cases is the re-circumscription of Oreocharis Bentham. Molecular phylogenetic analyses indicate that species of the original Oreocharis are phylogenetically intertwined with species of ten other small or monotypic genera (Möller et al. 2011b). After considering both the phylogenetic conclusion and the morphological evaluation, Möller et al. (2011bMöller et al. ( , 2014 formally transferred all species of nine genera (i.e. Ancylostemon Craib, Bournea Oliver, Dayaoshania Wang, Deinocheilos Wang, Isometrum Craib, Opithandra Burtt, Paraisometrum W.T. Wang, Thamnocharis Wang and Tremacron Craib) and rosette species of Briggsia Craib (except B. longipes and B. mihieri) to Oreocharis. This expansion not only made the Oreocharis a large genus within subfamily Didymocarpoideae (tribe Trichosporeae, subtribe Didymocarpinae) of the Gesneriaceae (Weber et al. 2013), but also made it one of the florally-diverse groups. Species of the newly-circumscribed Oreocharis all represent rosette herbaceous plants with spirally-arranged leaves, scapose inflorescences and loculicidally dehiscent capsules; however, their floral characters are extremely variable (Möller et al. 2011b). For instance, the characters corolla shape and colour, stamen number, placement of fertile stamens and anther coherence and shape are highly varied in this genus. The expansion of the morphological boundary of Oreocharis calls for reconsideration of the generic status of some problematic species; in particular, species whose generic placements are heavily based on flower characters, such as Boeica guileana B.L. Burtt and Beccarinda baolianis Q.W. Lin.
Boeica guileana was first described in 1977 by Burtt based on cultivated plants without description of mature fruits. The species has been regarded as endemic to Hong Kong for a long time (Burtt 1977;Wang et al. 1998;Li and Wang 2004); however, it was later discovered in Shenzhen, southern China (Wei et al. 2010). Based on corolla shape (short tube and spreading limb) and morphological resemblance to two existing species (Boe. brachyandra Ridl. and Boe. nutans Ridl.), Burtt (1977) assigned the plant to Boeica Clarke. However, Boe. guileana is distinctly different from most of other Boeica species by its rosette herbaceous habit, spirally-arranged leaves and longitudinally dehiscent anthers. Thus, in the original protologue, Burtt (1977) emphasised that the inclusion of Boe. guileana (S China) and two Peninsular Malaysian species (Boe. brachyandra and Boe. nutans) into Boeica is questionable. The generic status of all these three species is furthermore doubted by Weber and Skog (2007).
Beccarinda baolianis was newly described in 2016 from southern China, based on two specimens (Lin 2016). At present, only one population of this species can be found at its type locality (Lin 2016). Based on floral characters, such as the campanulate corolla and four stamens and capsule at a 90° angle to the pedicel, Lin (2016) assigned this species to Beccarinda Kuntze and compared it with Be. minima K.Y. Pan. However, our field observations found that the 90° angle between capsule and pedicel is not a stable character in the population. In addition, the inconspicuous rhizome and rosette habit of this species allow us to assume that it may only be distantly related to Beccarinda. Moreover, other important characters, i.e. the way of dehiscence in anthers and fruits, were not even described by Lin (2016). In the original protologue, Lin (2016) excluded this species from the original Oreocharis for its ovate anthers with apically confluent thecae, but this character does exist in the enlarged Oreocharis.
Summarising, the generic placements of Boe. guileana and Be. baolianis need reconsideration. Therefore, this study aims to clarify the generic status of these two species and discuss their species relationships in the enlarged Oreocharis using cytological, molecular and morphological analyses.

Morphological observation
The previous descriptions of Boe. guileana and Be. baolianis were based on only one or two specimens from a single location; thus, the described characters do not reflect the whole range of variation and other characters were not described at all. For example, the dehiscent way of fruit of both of these two species had not been mentioned in the original protologue. To revise and amend the morphological descriptions and clarify the morphological affinities between these two species and its congeners, detailed morphological studies were made on living plants in the field (Shenzhen and Fujian, China) and on plants cultivated in greenhouses (South China Botanical Garden, SCBG and Gesneriad Conservation Center of China, GCCC). Furthermore, dried specimens were investigated. Checking of specimens was undertaken at IBSC, IBK, SZG and FAFU. Additionally, high-resolution images of specimens were critically checked using the web service of E (http://data.rbge.org.uk/search/herbarium/) and HK (http:// www.herbarium.gov.hk/index.aspx).

Molecular taxon sampling
In the recent classification of Gesneriaceae, Boeica and Beccarinda were deposited in subfamily Didymocarpoideae, tribe Trichosporeae, subtribe Leptoboeinae and the enlarged Oreocharis was deposited in subtribe Didymocarpinae (Weber et al. 2013). To verify the generic placements of Boe. guileana and Be. baolianis, we sampled a total of 82 ingroup samples, comprising almost all genera of the subtribe Leptoboeinae and 24 out of 34 genera in subtribe Didymocarpinae. Two species, Rhynchoglossum obliquum Blume and Stauranthera grandifolia Benth., were selected as outgroup, according to the phylogeny of the Old World Gesneriaceae (Möller et al. 2011a).

DNA extraction, PCR and sequencing
The plastid trnL-F and nuclear ribosome internal transcribed spacer (ITS) sequences were used for the phylogenetic analysis. Most of these DNA sequences were acquired from GenBank and the sequences of six species, Boe. guileana, Boe. ornithocephalantha F. Wen, T.V. Do & Y.G. Wei (Wen et al. 2016), Be. minima, Be. baolianis, Oreocharis pilosopetiolata L.H. Yang & M. Kang (Yang et al. 2015) and O. benthamii var. reticulata Dunn, were newly amplified and sequenced in this study. DNA was extracted from dried leaves using a modified CTAB procedure described by Doyle and Doyle (1987). The polymerase chain reaction (PCR) amplification procedures and the PCR primers of these regions were described in Kong et al. (2017). Newly-amplified sequences were deposited in GenBank. The GenBank accession numbers used in this study are listed in Suppl. material 1.

Sequence alignment and phylogenetic analysis
Sequence matrices of trnL-F and ITS were separately aligned using the programme MUSCLE implemented in the software MEGA7 (Kumar et al. 2016) with minor manual adjustments. A combined ITS and trnL-F matrix was generated using Se-quenceMatrix (Vaidya et al. 2011). The incongruence length difference test (Farris et al. 1994) was implemented in PAUP*4.0B10 (Swofford 2002) to assess potential congruence between ITS and trnL-F. Phylogenetic analyses were performed on a highperformance computer cluster available in the CIPRES Science Gateway 3.3 (www. phylo.org, Miller et al. 2015). Bayesian Inference (BI) in MRBAYES 3.2.6 (Ronquist et al. 2012) and Maximum Likelihood (ML) in RAxML8.2.10 (Stamatakis 2014) were calculated for each of trnL-F, ITS and the combined sequence matrix. Best-fitting models for MrBayes were obtained separately for trnL-F and ITS spacers through AIC in Smart Model Selection (SMS) by its web server (www.atgc-montpellier.fr, Lefort et al. 2017) and were GTR+G and GTR+I+G, respectively. A total of 10,000,000 generations were run in two independent analyses, each with four Markov Chain Monte Carlo (MCMC) chains. One tree was sampled every 1,000 generations and the first 2,000 trees discarded as burn-in. Posterior probabilities (PP) obtained from the analysis were used to indicate the credibility of various branches. The ML tree was evaluated by non-parametric bootstrapping (1,000 replications) with the thorough bootstrap option of RAxML under the general time-reversible (GTR) model with a gamma model (Γ) of the rate of heterogeneity for each of trnL-F, ITS and the combined sequence matrix.

Chromosome preparations
Four species, Boe. guileana, Boe. stolonifera K.Y. Pan, Be. baolianis and Be. tonkinensis (Pellegr.) B.L. Burtt, were investigated cytologically. The plants for chromosome studies were collected from the field and cultivated in Gesneriad Conservation Center of China (GCCC). Actively growing root tips were collected and pretreated with 2 mM 8-hydroxyquinoline at 20 °C for about 2 hrs, then fixed with Farmer's solution (absolute alcohol: glacial acetic acid 3:1) at 4 °C for about 2 hrs. After hydrolysis for 30 min in 1 M HCl at room temperature, followed by washing through several changes of distilled water, the roots were transferred to carbol fuchsin for about 2 hrs.

Morphological affinities
The results of morphological observation indicated that both of Boe. guileana (Fig. 1A-H) and Bec. baolianis (Fig. 2) should be deposited into the enlarged Oreocharis. Both of these two species possessed these characters: rosette herbaceous habit (Figs 1A, E, 2A-C), longitudinally dehiscent anther (Figs 1D, 2G) and loculicidal dehiscent capsule (Figs 1G, H, 2J), which showed distinct relationships to Boeica or Beccarinda and the equivalent characters of these two genera were discussed below. Our morphological comparisons showed Boe. guileana was similar to Ore. pilosopetiolata in the texture, shape and margin of leaf and the pubescence on most part of the plants. Nevertheless, Bec. baolianis showed a special combination of characters in the enlarged Oreocharis, such as small plant size, campanulate corolla and short filaments. The morphological affinities of Bec. baolianis are discussed below. Additionally, we amended or supple- mented the description of some characters (such as dehiscent way of anther and fruit, corolla lobes margin and calyx) of these two species here and the detailed amended descriptions were given below.

Matrix characteristics
The aligned trnL-F and ITS datasets were 966 and 824 characters long, thereof, 195 and 138 were parsimony-uninformative and 139 and 388 parsimony-informative characters, respectively. The combined dataset was 1790 characters long with 527 (29.4%) parsimony-informative characters. The incongruence length different (ILD) test showed no significant incongruence between the ITS and trnL-F (p = 0.53).

Discussion
Traditionally, the classification of the Old World Gesneriaceae is heavily based on floral characters (e.g. Burtt 1977;Wang et al. 1998). However, recent phylogenetic studies reveal that the diversity of floral characters within this family has a complex evolutionary background, i.e. multiple convergences and parallel changes (Wang et al. 2010;Möller et al. 2011b;Weber et al 2011;Lu et al. 2017). Therefore, wrong generic placements have been made for some species based on the homoplasious floral traits. For example, symmetrical corolla was regarded as an original character in the Old World Gesneriaceae (Wang et al. 1998) and several genera have been established by this character such as Bournea Oliv. (Hooker 1893), Tengia Chun (Chun 1946) and Thamnocharis Wang (Wang 1981); but the recent phylogenetic studies indicate that the symmetrical corolla convergently evolve in different alliances of Old World Gesneriaceae (Wang et al. 2010) and all these three genera have been incorporated into other genera (Möller et al. 2011b;Weber et al. 2011 Wang et al. 1998;Weber and Skog 2007) and Beccarinda (long rhizomatous, caulescent or stoloniferous habit; Burtt 1955;Wang et al. 1998;Weber and Skog 2007) species. The anthers of Boe. guileana (Fig. 1D) and Be. baolianis (Fig.  2G) are longitudinally dehiscent which is different from other Boeica and Beccarinda species (poricidally-or transversely-dehiscent anthers; Burtt 1955Burtt , 1977Wang et al. 1998;Weber and Skog 2007). In addition, fruits of both Boe. guileana (Fig. 1G, H) and Be. baolianis (Fig. 2J) are loculicidally dehiscent, which is a character compatible to the enlarged Oreocharis (Möller et al. 2011b). In other Boeica species, fruits dehisce septicidally or loculicidally ( Fig. 1J; Weber and Skog 2007) and in other Beccarinda species, the fruits dehisce septicidally ( Fig. 1I; Weber and Skog 2007). The somatic chromosome numbers of Boe. guileana and Be. baolianis were determined as diploid with 2n = 34 (Fig. 4A, B), which is consistent with previous findings for species within the enlarged Oreocharis (Ratter 1963;Ratter and Prentice 1964;Fussell 1958;Wang and Gu 1999;Lu et al. 2002;Zhou et al. 2003;Tan et al. 2011). The somatic chromosome numbers of Boeica and Beccarinda species studied here were 2n = 20 (Fig. 4C-D) which is also consistent with previous studies (Ratter and Prentice 1964), except one case of Boe. brachyandra was recorded as 2n = +/-22 (Kiehn et al. 1998). Ultimately, our phylogenetic analyses prove with high support rate (Fig. 3) that Boe. guileana and Be. baolianis belong to the enlarged Oreocharis clade. Additionally, the further phylogenetic analysis, based on some additional plastid sequences, revealed the above relationship (pers. Yu-Min Shui).
Distribution and habitat. Oreocharis guileana was once recognised as an endemic species and only recorded at Ma On Shan, Hong Kong. However, recent field works reveal that it can be found from several sites in Shenzhen, such as Wu Tong Shan, Pai Ya Shan, Tian Xin Shan, Dakeng reservoir and Xigong village (Fig. 5). The plants grow on moist rock surfaces under evergreen broad-leaved forests, at an altitude of 300-900 m.
Note. Burtt (1977) thought that Oreocharis guileana is morphologically similar to Boe. brachyandra and Boe. nutans by its rosette habit, short corolla tube and spreading limb. However, as mentioned above, the currently taxonomic status of these two Boeica species is doubtful and need further studies to confirm it. Additionally, the distinctively disjunctive distribution between Oreocharis guileana and Boe. brachyandra and Boe. nutans may indicate a distant relationship between them (Oreocharis guileana is a species occurring in S China, but Boe. brachyandra and Boe. nutans are distributed on the Malayan Peninsula). Although with low support, our phylogenetic results show that Oreocharis guileana and O. pilosopetiolata have a sister relationship (Fig. 3). These two species have similar leaf blade (size, shape, texture and margin) and indumentum on most of organs and it is difficult to distinguish these two species without flowers. Additionally, the related adjacent geographic distribution also indicates the close relationship between these two species (Fig. 5).
Distribution and habitat. Currently, Oreocharis baolianis has only been found at its type locality, Sidu Twon, Changting County, Fujian Province, China (Fig. 5). Plants grow on moist rock surfaces under evergreen broad-leaved forests, at an altitude of ca. 600 m.
Note. The results of our morphological comparisons show a special status of Oreocharis baolianis in the enlarged Oreocharis. It is difficult to find a congeneric species with small plant size, campanulate corolla and short filaments. At present, we reservedly consider morphological affinities exist between O. baolianis, O. guileana and O. pilosopetiolata by their similar leaf blade texture, shape and margin and pubescence on most of organs and, especially, the similar pistil (short ovary densely covered with villous) between O. baolianis (Fig. 2I) and O. guileana (Fig. 1F). Although, the combined molecular data do not support a close phylogenetic relationship between O. baolianis and the latter two species, we cannot rule out the above speculation due to the general low resolution in the backbone of the phylogeny of Oreocharis (Fig. 3). In fact, the analysis of ITS indicate that these three species form a monophyletic clade (data not shown). Further field and molecular works are needed to verify this hypothesis and to discuss the deep species relationships in the enlarged Oreocharis.
Additional specimens examined. The same place as type locality, 2 June 2015, Q.W. Lin