Oreocharis jasminina (Gesneriaceae), a new species from mountain tops of Hainan Island, South China.

A new species of Gesneriaceae, Oreocharis jasminina S.J.Ling, F.Wen & M.X. Ren from Hainan Island, south China, is highlighted and described. The new species is distinguished by its actinomorphic corolla, narrow floral tube and ovate anthers hidden in the floral tube. The new species also showed clear geographic and altitudinal isolation from the three currently-recognised Oreocharis species on the Island. Molecular phylogenetic analysis, based on nuclear ITS1/2 and plastid trnL-trnF sequences, supported the delimitation of the new species, which forms a single lineage with all the other Oreocharis species from Hainan Island. The roles of geographic and floral isolation in the evolution of the new species and its affinities are discussed. Shao-Jun Ling, Shu-Ping Guan, Fang Wen, Yu-Min Shui, Ming-Xun Ren.

Introduction

The Bentham was recently re-circumscribed to a large genus by including ten more genera and over 135 species, based mainly on molecular phylogenetic studies (Möller et al. 2011, 2016; Xu et al. 2017; Möller 2019; Wen et al. 2019). The enlarged genus was predominantly distributed in China with some species in India, Myanmar, Thailand and Vietnam (e.g. Li and Wang 2005; Möller and Clark 2013; Möller et al. 2018). Regardless of the limited differences in habit and fruit structure, shows a strikingly-high diversity in floral syndromes (Li and Wang 2005; Wei 2010; Möller and Clark 2013).

As one of the globally-important biodiversity hotspots, Hainan Island harbours about 4000 seed-plant species, of which ca. 500 are endemics (Francisco-Ortega et al. 2010) and which are concentrated in the south-central mountains. , in Hainan Island especially, includes a high ratio of species endemism, eight out of the total of 24 species being endemic (Ling et al. 2017a). Currently, three taxa of are recorded on Hainan Island and all of them are Hainan-endemic and monophyletic, i.e. Chun, Pan and Merrill (Li and Wang 2005; Ling et al. 2020), while each of these species shows considerable variations in morphological traits (Wei 2010; Ling et al. 2020a).

During several fieldwork trips in the past three years, we found that some populations of on mountain tops in Hainan Island showed obvious differences in various morphological characters. After careful literature studies (Pan 1987; Li and Wang 2005; Wei 2010) and morphological and molecular examinations, we are convinced that populations from the mountain tops of Mt. Yingge and Mt. Limu represent a new species, which we report and describe here.

Materials and methods

Morphological observations

The field study and conservation on were undertaken by two of the authors (SJL and MXR) over a long period of time, especially focusing on the Hainan-endemic species (Ling et al. 2017a, b; Xing et al. 2018; Li et al. 2019). Morphological observations and measurements were carried out, based on living plants during fieldwork. All available specimens of species, stored in the herbaria in China (PE, KUN, IBK and IBSC), were examined. We also downloaded all specimens from JSTOR Global Plants (http://plants.jstor.org), and Chinese Virtual Herbarium (http://www.cvh.ac.cn) to compare detailed morphological traits between the proposed new species with the currently-accepted species of . Specifically, we compared morphological traits of the possible new species with all the three currently-recognised species from Hainan Island, i.e. , and . The specimens of new species were collected over the past two years and deposited in the herbarium of Hainan University (HUTB) and Kunming Institute of Botany, Chinese Academy of Sciences (KUN).

Taxonomic sampling, DNA extraction and molecular markers

The leaf samples of , , and the putative new species were collected in the field and dried in a vascular bag with silica gel. Total genomic DNA extraction was conducted using CTAB methods (Doyle and Doyle 1987). One nuclear ribosomal DNA (nrDNA) sequence, the ITS region comprising spacer 1, the 5.8S gene and spacer 2 (White et al. 1990) and one chloroplast DNA (cpDNA) intron-spacer region trnL-trnF (Taberlet et al. 1991) were used in this study. Laboratory procedures followed Ling et al. (2020) and newly-acquired sequences were deposited in GenBank (Table 2).

Table 2.

List of Hainan taxa and outgroup used in the phylogenetic analysis, including respective Genbank accession and voucher numbers.

Species	trnL-trnF	ITS1/2	Voucher Number
O. dasyantha Chun (Mt. Bawang)-1	MK587993	MK587954	S.J.Ling & M.X. Ren 2015011803 (HUTB)
O. dasyantha Chun (Mt. Bawang)-2	MK587994	MK587954	S.J.Ling & M.X. Ren 2015011804 (HUTB)
O. dasyantha Chun (Mt. Jianfeng)-1	MK587995	MK587955	S.J.Ling 2015102201 (HUTB)
O. dasyantha Chun (Mt. Jianfeng)-2	MK587996	MK587955	S.J.Ling 2015102202 (HUTB)
O. dasyantha Chun var. ferruginosa Pan (Mt. Jianfeng)-1	MK587954	MK587956	S.J.Ling 2015102203 (HUTB)
O. dasyantha Chun var. ferruginosa Pan (Mt. Jianfeng)-2	MK587954	MK587957	S.J.Ling 2015102204 (HUTB)
O. flavida Merrill (Mt. Qixian)	MK587947	MK587990	S.J.Ling 2018112901 (HUTB)
O. flavida Merrill (Mt. Wuzhi)	MK587989	MK587943	S.J.Ling 2018112902 (HUTB)
O. jasminina (Mt. Yingge)-1	MK587987	MK587948	S.J.Ling 2018112601 (HUTB)
O. jasminina (Mt. Yingge)-2	MK587988	MK587950	S.J.Ling 2018112602 (HUTB)
O. jasminina (Mt. Limu)-1	MK587981	MK587949	S.J.Ling 2018112603 (HUTB)
O. jasminina (Mt. Limu)-2	MK587982	MK587953	S.J.Ling 2018112604 (HUTB)
O. sinohenryi (Chun) Mich.Möller & A.Weber	HQ632913	HQ633009	M.Möller MMO 07-1150 (E)

Alignments and phylogenetic analyses

According to Möller et al. (2011), Chen et al. (2014) and Ling et al. (2020), (Chun) Mich.Möller & A.Weber which had the closest phylogenetic relationships with the Hainan taxa was used as outgroup with sequences (Genbank with accession numbers HQ632913 and HQ633009). The original chromatograms from both directions of the ITS1/2 and trnL-trnF sequences were evaluated using Bioedit (Hall 1999) for base confirmation and contiguous sequences editing, then we manually aligned sequences, where necessary, using MEGA v.6.5 (Kumar et al. 2008) and ambiguous positions were excluded from the alignments. The ITS1/2 and trnL-trnF were concatenated to a single matrix after a congruency test by PAUP* 4.0a164 (Swofford 2003). Bayesian Inference (BI) analysis was conducted using MrBayes version 3.1.2 (Huelsenbeck and Ronquist 2001) and Maximum Likelihood (ML) analysis was performed using MEGA v.6.5 (Kumar et al. 2008). Both procedures followed the Ling et al. (2020), based on the combined ITS1/2 and trnL-trnF sequences.

Results

Phylogenetic reconstruction

The combined ITS1/2 and trnL-trnF datasets were 640 and 818 bp long, amongst which 64 and 17 were polymorphic sites and 27 and 6 were parsimony-informative sites, respectively. The aligned dataset was 1458 bp long and a total number of 81 polymorphic sites were measured, of which 33 were parsimony-informative sites. There was no significant incongruence, based on the incongruence length difference (ILD) test between the ITS1/2 and trnL-trnF (p > 0.05).

Molecular phylogeny recognised the individuals from different mountains and these were grouped as separate lineages. The putative new species from Mt. Limu and Mt. Yingge is accepted as a new species with PP (posterior probability) = 1 and BS (bootstrap value) = 100% (Fig. 1). All the species from Hainan Island form a single lineage with relatively-high support (Fig. 1).

Figure 1.

Molecular phylogeny of Hainan taxa with outgroup , based on the combined nuclear ribosomal DNA (nrDNA) sequence ITS1/2 and chroloplast gene trnL-trnF data matrices. Posterior probability (PP) and Bootstrap value (BS) are showed above branches.

Taxonomic treatment

S.J.Ling, F.Wen & M.X. Ren sp.nov.

964314ED-2817-544D-80C7-534A26EF51D5

urn:lsid:ipni.org:names:77211189-1

Figs 2 , 3

Figure 2.

sp. nov. A Habitat B habit C face view of corolla D lateral view of corolla E opening flower showing stamens and staminode F stamens and staminode G pistil and sepals H fruit pods I adaxial leaf surface J abaxial leaf surface.

Figure 3.

sp. nov. (all drawings based on the holotype S.J.Ling 20181126–01 in HUTB, drawn by S.P. Guan). A Habit B face view of corolla C lateral view of corolla D opening corolla showing pistil and stamens E pistil F stamens and staminode.

Diagnosis.

has the closest phylogenetic relationship with , and with very high support values, all being Hainan-endemic and monophyletic. can be easily distinguished from them by having: (1) a long and narrow floral tube (both and have conical floral tubes, has campanulate-tubular floral tube); (2) yellow and actinomorphic corolla (both and are zygomorphic with orange-red to yellow corolla, is actinomorphic with orange corolla); (3) didynamous stamens with ovate anthers hidden in the floral tube (both and have exposed didynamous stamens with ovate anthers, has four equivalent stamens with horseshoe-shaped anthers included in the floral tube) (Table 1, Fig. 4).

Table 1.

Diagnostic morphological characters of sp. nov. and all the three currently-recognised species in Hainan Island.

Characters	Oreocharis jasminina sp. nov.	O. dasyantha	O. dasyantha var. ferruginosa	O. flavida
Corolla colour	yellow	orange-red to yellow	orange-red to yellow	orange
Corolla tube	narrowly tubular,1.7–2.2 cm long 1.8–2.2 cm × 3–4.5 mm	conical,1.6–2.4 cm long 0.9–2 cm × 6–7 mm	conical, ca.1.6 cm, tube 9–1.1 mm	campanulate-tubular,1.7–1.9 cm long 1.6–1.8 cm × 6–8 mm
Corolla symmetry	actinomorphic	zygomorphic	zygomorphic	actinomorphic
Leaf blade shape	ovate to broadly ovate, rarely elliptic or obovate	ovate-elliptic to broadly ovate	ovate-elliptic to broadly ovate	ovate-elliptic to broadly ovate, rarely broadly elliptic
Leaf base shape	cordate to rounded	oblique, cuneate to subrounded or cordate	sometimes oblique, cuneate to subcordate	oblique, subrounded
Leaf base margin	nearly entire to shallowly crenate, apex rounded	serrulate or crenate-serrate, apex acute to rounded	crenate-serrate	shallow crenate
Stamens	included, didynamous, staminode 1	exposed, didynamous, staminode absent	exposed, equivalent, staminode absent	included, equivalent, staminode 1
Anthers	ovate, 2-loculed, dehiscing transversely	broadly oblong, 2-loculed, dehiscing longitudinally	broadly oblong, 2-loculed, dehiscing longitudinally	horseshoe-shaped,1-loculed, dehiscing transversely
Filaments	pubescent	pubescent	pubescent	glabrous
Pistil	ca. 9 mm long	ca. 22mm long	ca. 22mm long	ca.9 mm long

Figure 4.

Three formerly-recognised taxa in Hainan Island. (A, B), (C, D) and (E, F).

Type.

China. Hainan: Qiongzhong County (琼中县), Limu Mountain, 1350 m a.s.l., on moist rocks, 26 Nov 2018, S.J.Ling 2018112601 (holotype: HUTB!; Isotypes: HUTB!, KUN!).

Description.

Perennial herb, rhizomatous, leaves basal; 4.0–10.0 cm long, 2–3 mm in diameter, densely pale brown villous or woolly; leaf blade ovate to broadly ovate, rarely elliptic or obovate, 6–11 × 4–8 cm, adaxially densely grey to brown pubescent, abaxially sparsely to densely grey or grey-brown pubescent, sparsely brown villous along veins which are adaxially sunken and abaxially ridged, lateral veins 6–7 on each side of midrib, base often cordate to rounded, margin nearly entire to shallowly crenate, apex rounded. Cymes axillary, 2–3, inflorescence 3–10-flowered; Peduncle 9–16 cm long, sparsely pale grey villous; bracts 2, linear to narrowly triangular, outside densely villous, apex acuminate, cuneate to triangular, margin entire; pedicel 1.5–2.2 cm long, densely pale brown villous to woolly. Calyx 5-lobed, divided to base, lobes green, narrowly lanceolate, 9–11 × ca. 2 mm, apex acuminate, margin entire, outside villous, inside glabrous. Corolla yellow, 1.7–2.2 cm long, outside pubescent; tube thin tubular, 1.8–2.1 cm × 3–4.5 mm, limb barely 2-lipped, adaxial lip shallowly 2-lobed from near base, abaxial 3-lobed slightly equal. Stamens 4, 8–9 mm long, included, adnate to corolla 4–5 mm from base; filaments slender, pubescent; anthers ovate, 2-loculed, dehiscing transversely; staminode 1, adnate to corolla 2–4 mm from base, ca. 2 mm. Disc ca. 1 mm high, entire. Pistil ca. 7 mm long; ovary cylindrical, ca. 5 mm long, glabrous. Stigma 2, equal, suborbicular. Capsula linear, 3–4 cm long, glabrous to sparsely puberulent.

Phenology.

flowers from September to December and fruits from November to January.

Distribution and habitat.

is currently only found in cloud forests on the mountain tops of Mt. Limu and Mt. Yingge, in the middle of Hainan Island. The habitat of is on the moss layer on wet rocks under cloud forests.

Etymology.

The specific epithet refers to the yellow and narrowly tubular corolla of this new species.

Vernacular name.

迎春花马铃苣苔 (Yíng Chūn Huā Mǎ Líng Jù Tái) is the Chinese name for , the first three characters meaning ‘winter jasmine’, indicating its similar floral syndromes to Lindl. The last four characters are the Chinese name for .

Conservation status.

is, so far, known only from the two locations with about 800–1000 individuals. The populations are under threat due to the restricted and fragmented habitat. Therefore, we propose that should be considered as ‘Vulnerable’ (VU), according to the IUCN Red List Categories and Criteria (IUCN 2012).

Discussion

Our former study showed the new species and the three other Hainan-endemic taxa are homologous, indicating these species in Hainan Island had a common origin (Ling et al. 2020). The new species is only found on mountain tops higher than 1200 m in two mountains, Mt. Limu and Mt. Yingge, located at the middle of Hainan Island. These mountain tops likely formed island-like habitats because the deep and wide valleys interrupted gene flows, resulting in population differentiation and speciation (Shen et al. 2017; Ling et al. 2017a, b; Xing et al. 2018). Such ‘sky islands’ may be the main reason for the origin and maintenance of this Hainan-endemic alpine species (Robin et al. 2015; Ling et al. 2017a).

The new species also shows a clear geographic isolation from the three currently-recognised taxa on Hainan Island. The new species was only found in Mt. Limu and Mt. Yingge in the middle of the island, while and are restricted to the west side of the Island and was only found in the east side (Fig. 5). They are isolated by a large river, the Changhua River (the second largest river on Hainan Island). Li et al. (2019) found that the geographic isolation by the Changhua River is a driving force for the great population differentiation in the two Hainan-endemic species, (Merr.) Yan Liu and (Merr. et Chun) W. T. Wang. Thus, the geographic isolation by rivers or valleys may also play a key role in the evolution of and other Hainan-endemic taxa. However, the relative contributions of such geographic isolation and altitudinal differentiation are still in need of further experimental examination.

Figure 5.

Geographic distribution of sp. nov. and the three congeners on Hainan Island. ★ sp. nov. ∆ ◊ □ .

Floral symmetry is widely recognised as a key trait in pollination and taxonomy. Normally, the zygomorphic corolla possesses higher pollen-transfer efficiency than the actinomorphic corolla (Sargent 2004). has yellow actinomorphic corolla with a long and narrow floral tube, differing from and (both have zygomorphic corolla). Although has an actinomorphic flower, its campanulate corolla with four equivalent stamens and horseshoe-shaped anthers make it distinct from the new species (Table 1).

Floral shape was expected to be a vital factor in generating floral isolation and evolutionary shifts (Castellanos et al. 2004; Muchhala 2007). Generally, the floral shape has a strong connection with the expected pollinators in , for example, bees or hummingbirds for tubular flowers, bats for campanulate flowers and subcampanulate flowers having generalised pollination systems (Martén-Rodríguez et al. 2009). has thin-tubular corolla (Fig. 1), differing from , (both are conical corolla) and (campanulate-tubular corolla), indicating a possible pollination mechanism associated with the long-tongued butterflies and moths. Such distinctive morphological differences indicate different pollination adaptation and clear reproductive isolation amongst these taxa, suggesting should be treated as a new species.

1	Anthers horseshoe-shaped, 1-loculed, dehiscing transversely	O. flavida
–	Anthers broadly oblong, 2-loculed, dehiscing longitudinally	2
2	Stamens included, floral tube thin tubular, corolla yellow	O. jasminina
–	Stamens exposed, floral tube conical, corolla orange-red	3
3	Leaf blade adaxially grey pubescent, base oblique, subrounded to cordate, margin serrulate; petiole to 14.5 cm, densely pale brown villous; cymes 1–3(or 4)-flowered; corolla 1.7–2.4 cm, tube 1.1–2 cm	O. dasyantha
–	Leaf blade adaxially grey to brown pubescent and villous, base sometimes oblique, cuneate to subcordate, margin crenate-serrate; petiole to 6 cm, densely pale brown woolly; cymes 3–8-flowered; corolla ca. 1.6 cm, tube 9–11 mm	O. dasyantha var. ferruginosa

The specimens we checked in this study.

Voucher Number	Species	Voucher Number	Species
PE00030859	Oreocharis rhytidophylla	bm000041491	Oreocharis aurantiaca
IBSC0004917	Oreocharis henryana	gh00353683	Oreocharis dentata
A00353713	Oreocharis magnidens	k000858130	Oreocharis maximowiczii
bm000041734	Oreocharis benthamii	e00087519	Oreocharis tubicella
e00067455	Oreocharis cavaleriei	e00087520	Oreocharis nemoralis
a00025113	Oreocharis flavida	IBSC0004912	Oreocharis aurea
bm000041721	Oreocharis georgei	p04060117	Oreocharis forrestii
KUN1219176	Oreocharis cordato-ovata	e00135096	Oreocharis amabilis
e00087535	Oreocharis dasyantha	e00135074	Oreocharis bodinieri
bm000041708	Oreocharis cinnamomea	gh00353695	Oreocharis benthamii var. reticulata
PE00030854	Oreocharis tubiflora	p04060171	Oreocharis delavayi
IBSC0004920	Oreocharis xiangguiensis	CSFI028502	Oreocharis brachypodus
PE19401111	Oreocharis amabilis	PE02052999	Oreocharis heterandra
IBSC0550960	Oreocharis sericea	IBSC0550860	Oreocharis cordatula
IBSC0550891	Oreocharis georgei	IBSC0550875	Oreocharis elliptica
GZTM0075588	Oreocharis primuloides	JIU63907	Oreocharis speciosa
PE02052990	Oreocharis argyreia var. angustifolia	PE02053568	Oreocharis eximia
IBK00054784	Oreocahris auricula	PE01909883	Oreocharis mileensis
KUN1219104	Oreocharis hekouensis	WUK0494363	Oreocharis saxatilis
PE02053062	Oreocharis concava	PE01486523	Oreocharis rosthornii
PE02106072	Oreocharis begoniifolia	KUN1385365	Oreocharis nanchuanica
KUN1385575	Oreocharis urceolata	HITBC106680	Oreocharis longifolia
PE00030861	Oreocharis rotundifolia	IBSC0548683	Oreocharis chienii
PE02053433	Oreocharis acaulis	KUN1385156	Oreocharis bullata
PE02241281	Oreocharis burttii	PE02053072	Oreocharis cinerea
KUN1220227	Oreocharis convexa	IBK00054466	Oreocharis cotinifolia
PE00155697	Oreocharis craibii	WUK0350789	Oreocharis crenata
IBSC0550709	Oreocharis dalzielii	PE02106079	Oreocharis dinghushanensis
IBSC0551649	Oreocharis esquirolii	PE02052984	Oreocharis fargesii
IBSC0649611	Oreocharis flabellata	PE02053533	Oreocharis gamosepala
PE02052812	Oreocharis giraldii	PE02106025	Oreocharis glandulosa
PE02052995	Oreocharis humilis	PE01548041	Oreocharis jiangxiensis
PE02021009	Oreocharis lancifolia	FJSI004239	Oreocharis leiophylla
PE02053066	Oreocharis leucantha	IBSC0550069	Oreocharis lungshengensis
IBSC0551655	Oreocharis mairei	PE02053564	Oreocharis minor
WUK0160594	Oreocharis muscicola	IBSC0548476	Oreocharis notochlaena
PE02106041	Oreocharis obliqua	PE02052801	Oreocharis obliquifolia
PE02088092	Oreocharis obtusidentata	PE02053064	Oreocharis pankaiyuae
PE01270485	Oreocharis primuloides	WUK0213194	Oreocharis pumila
PE02053576	Oreocharis pinnatilobata	KUN1241303	Oreocharis primuliflora
PE02053532	Oreocharis rhombifolia	PE00030693	Oreocharis ronganensis
PE00030747	Oreocharis sichuanensis	IBSC0550081	Oreocharis sichuanica
IBK00054319	Oreocharis sinensis	IBK00207093	Oreocharis sinohenryi
PE02053579	Oreocharis stenosiphon	IBSC0548730	Oreocharis stewardii
PE02053570	Oreocharis trichantha	HEAC0016525	Oreocharis villosa
PE02053561	Oreocharis wangwentsaii	PE02053077	Oreocharis wanshanensis
Y. M. Shui et al. B2014-299 (KUN)	Oreocharis synergia	Y.M.Shui et al. N699 (KUN)	Oreocharis ninglangensis
PE-02114626	Oreocharis duyunensis	IBSC0825078	Oreocahris ovata
KUN1219115	Oreocharis acutiloba	PE00030682	Oreocharis agnesiae
PE00140281	Oreocharis billburttii	PE02025205	Oreocharis elegantissima
IBSC0649550	Oreocharis latisepala	PE00030685	Oreocharis parva
Z.K. Wu et al.C2016055 (KUN)	Oreocharis parvifolia	PE02025202	Oreocharis pinfaensis
IBSC0548691	Oreocharis shweliensis	PE01909893	Oreocharis tongtchouanensis
Y.M.Shui, Y.K.Sima & W.H.Chen B2013-258 (KUN)	Oreocharis crispata	Y.M.Shui et al. 91309 (KUN)	Oreocharis jinpingensis
Bo Pan & M. Q. Han HMQ859 (IBK)	Oreocharis purpurata	Yun-Hong Tan 3308 (HITBC)	Oreocharis tsaii
Averyanov, L., Hiep, N.T., Khang, N.S., Thang, N.D. & Qui, L.D. CPC 7019 (KUN)	Oreocharis blepharophylla	Jia-Mei Li and Yao-Guang Zhang 1606151 (HEAC)	Oreocharis zhenpingensis
Bo Pan & Jia-Jia Wei et al. GY002 (IBK)	Oreocharis curvituba	C.Z. Yang et al. 35042620140913001 (FNU)	Oreocharis striata
Y.M. Shui et al. B2013-551 (KUN)	Oreocharis longituba	Averyanov, L., Hiep, N.T., Khang, N.S., Thang, N.D. & Qui, L.D. CPC 7175 (KUN)	Oreocharis argyrophylla
Y.M. Shui et al. B2013-550	Oreocharis grandiflora	T.V. Do 57 (VNMN)	Oreocharis caobangensis
L.H. Yang et al. YLH197 (IBSC)	Oreocharis pilosopetiolata	Li-Hua Yang et al. YLH285 (IBSC)	Oreocharis uniflora
Ying Guo C2015005 (KUN)	Oreocharis panzhouensis	L. E. Yang 60 (KUN)	Oreocharis rubrostriata
Yan Liu and Wei-Bin Xu 08018 (IBK)	Oreocharis dayaoshanioides	Yun-Hong Tan 6925 (HITBC)	Oreocharis glandulosa
PE 02053063	Oreocharis farreri	Lin Qin-Wen et al. 0016 (FAFU)	Oreocharis baolianis
IBSC0548624	Oreocharis guileana	IBK00054993	Oreocharis dasyantha var. ferruginosa