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. 2023 Aug 18;230:271–287. doi: 10.3897/phytokeys.230.87675

Hedyotislongiramulis (Rubiaceae), a new species from south China

Yi-Da Xu 1,2,3, Ying Zhang 1,2,3, Rui-Jiang Wang 1,3,✉
PMCID: PMC10458045  PMID: 37637214

Abstract

Hedyotislongiramulissp. nov. (Rubiaceae) is described from Guangdong Province, China. It is similar to H.caudatifolia but differs in having puberulent, more or less tetragonal and decussately sulcate juvenile stems, waxy leaf surface, short inflorescence peduncles, high length ratio of corolla lobe to tube, and subglobose capsules. The phylogenetic analysis reveals that H.longiramulis is sister to H.pubirachis. Dimorphism concerning pollen size was observed in the heterostylous flowers. The complete chloroplast genome of the new species comprises a typical quadripartite structure of 153,616 bp in length, with two inverted repeats of 25,457 bp, a large single-copy of 85,050 bp and a small single-copy of 17,652 bp. It contains 112 unique genes, including 79 protein-coding genes, 29 tRNA genes, and four rRNA genes, the GC content of the chloroplast genome is 32.4%. The new species is provisionally evaluated as “Least Concern” because it is common and well-protected in two Provincial Nature Reserves.

Key words: Chloroplast genome, Hedyotis-Oldenlandia complex, phylogeny, taxonomy

Introduction

The genera Hedyotis L. and Oldenlandia L. are two taxonomically disputed genera and usually considered as a taxonomic complex in the tribe Spermacoceae of Rubiaceae. These two genera include more than 500 species distributed in tropical and subtropical regions worldwide (Dutta and Deb 2004). Taxonomical treatment of several genera within the tribe, especially regarding to generic delimitation, has much been debated (e.g., Lamarck 1792; Willdenow 1797; Bremekamp 1952; Dutta and Deb 2004). Recent phylogenetic analyses proved that the Hedyotis-Oldenlandia complex was polyphyletic and a narrow generic delimitation was then proposed accordingly (Guo et al. 2013; Gibbons 2020). Currently, Hedyotis s. str. is characterized by having an erect and robust herbaceous or shrubby habit, homo- or heterostylous flowers, triangular or ovate stipules with serrate marginal glands and tipped colleters, mostly diplophragmous capsules (loculicidal dehiscence first and then septicidal dehiscence along the septum) and fruticosa-type seeds (dorsiventrally flattened, lenticular with irregularly narrow wing-like margin). The distribution center of Hedyotis s. str. is the Asian-Pacific region (Terrell and Robinson 2003).

With the rapid development of high-throughput sequencing technologies, whole chloroplast genome dataset is increasingly used for simulating phylogenetic relationships (Liu et al. 2018; Song et al. 2019; Charr et al. 2020; Rono et al. 2020; Zhang et al. 2021). However, all of the present molecular phylogenetic analyses on the Hedyotis-Oldenlandia complex are based on a handful of nuclear or chloroplast DNA markers. Therefore, a more reliable phylogenetic relationship with robust support based on the whole chloroplast genome dataset is strongly anticipated. But unfortunately, for Hedyotis s. str., only the whole chloroplast genome dataset of H.ovata Thunb. ex Maxim. is available (MK203877) up to now (Zhang et al. 2019).

During a field collection in Guangdong Ehuagnzhang Provincial Nature Reserve, we found a sub-shrubby species of Hedyotis s. str. with purplish and puberulent young stems and long axillary branches. It is similar to H.caudatifolia Merr. & F.P.Metcalf with respect to its erect subshrubby habit, ovate to lanceolate leaf shape, and long lateral branches bearing several terminal and axillary inflorescences, but conspicuously differs by its puberulent, more or less tetragonal and decussately sulcate juvenile stems. After detailed morphological comparison and phylogenetic analysis, we confirm that this species is a hitherto undescribed one.

Materials and methods

Morphological examination

Morphological data of the new species was observed on living individuals and herbarium specimens deposited at IBSC and CANT (herbarium code follows https://sweetgum.nybg.org/science/ih/).

For micromorphology, scanning electron microscopy (SEM, JSM-6360LV) was applied under 15.00 kV accelerating voltage. Pollen grains were put in 70% alcohol, washed by an ultrasonic cleaner (WIGGENS UA10MFD, 100W, 59KHZ) for 5 min, and then centrifuged at 8000 rpm for 5 min. After this, we removed the supernatant and added 70% alcohol to the sediment. These steps were repeated three times. Finally, the pollen suspension was dropped on the sample stubs with conductive double sided adhesive carbon tapes. The pollen samples were gilded by sputter coater (LEICA EM ACE600, 10 μm, 20 mA) once dried in room conditions. Seed samples were cleaned using the same method as for pollen grains and then transferred to sample stubs for gilding after drying. Leaf material was cleaned by brushing lightly and rinsing gently in warm water and then transferred to sample stubs after drying.

Pollen terminology for description followed Hesse et al. (2009), seed terminology followed Neupane et al. (2015), and foliar epidermal terminology followed David (1974).

Conservation assessment

The conservation assessment was undertaken according to the guidelines for assessing the conservation status of species (IUCN 2022). Estimation of the extent of occurrence (EOO) and area of occupancy (AOO) were performed in GeoCAT (Bachman et al. 2011) with 2 × 2 km grid cells.

Genomic DNA extraction and sequencing

Leaf material for DNA extraction was dried in silica gel. Total DNA was extracted using the modified cetyltrimethylammonium bromide (CTAB) protocol (Doyle 1991). Primers for polymerase chain reaction (PCR) are listed in Table 1, and the methods for PCR followed Guo et al. (2011). PCR products were purified and sequenced by Sangon Biotech Limited Company (Shanghai, China). For whole genome sequencing, the DNA samples were sent to Beijing Genomics Institute (Shenzhen, China) for genomic library construction and de novo sequencing (paired-end, PE=150 bp) using the BGISEQ-500. Raw reads were filtered and trimmed using SOAPnuke v.1.5.6 with software parameters “-n 0.01 -l 20 -q 0.3 -A 0.25 --cutAdaptor -Q 2 -G --polyX 50 --minLen 150”.

Table 1.

Primers used for PCR in the present study.

DNA region Primer name Sequence References
ITS P17 5’-CTACCGATTGAATGGTCCGGTGAA-3’ Popp and Oxelman 2001
26S-82R 5’-TCCCGGTTCGCTCGCCGTTACTA-3’
petB-petD PIpetB1365F 5’-TTGACYCGTTTTTATAGTTTAC-3’ Löhne and Borsch 2005
PIpetD738R 5’-AATTTAGCYCTTAATACAGG-3’
rps16 rps16F 5’-GTGGTAGAAAGCAACGTGCGACTT-3’ Oxelman et al. 1997
rps16R3 5’-CGATAGACGGCTCATTGGGATA-3’
trnH-psbA trnH-05 5’-CGCGCATGGTGGATTCACAATCC-3’ Tate and Simpson 2003
psbA3 5’-GTTATGCATGAACGTAATGCTC-3’ Sang et al. 1997
trnL-F TabC 5’-CGAAATCGGTAGACGCTACG-3’ Taberlet et al. 1991
TabF 5’-ATTTGAACTGGTGACACGAG-3’
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Chloroplast genome assembly and annotation

A total of 2 Gb clean reads were obtained and assembled using GetOrganelle v.1.7.3.5 (Jin et al. 2020). With reference to H.ovata (GenBank: MK203877), the genome was first annotated using GeSeq (https://chlorobox.mpimp-golm.mpg.de/geseq.html) (Tillich et al. 2017) and PGA (Qu et al. 2019), and then manually adjusted using Geneious v.11.0.3. A circular map of the chloroplast genome was drawn using OGDRAW v.1.3.1 (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html) (Greiner et al. 2019).

Molecular phylogenetic analyses

Twenty-three morphologically similar and sympatric Hedyotis taxa, as well as two accessions of the new species (see Table 2), were selected as ingroup operational taxonomic units (OTUs) for molecular phylogenetic analyses. Two Spermacoceae species, Dentellarepens (L.) J.R.Forst. & G.Forst. from Australia and Pentodonpentandrus Vatke from Zambia were chosen as outgroup OUTs (see Table 2).

Table 2.

Taxa, vouchers, localities, and GenBank accession numbers of ITS, petD, rps16, trnH-psbA and trnL-F sequences for phylogenetic analysis.

Taxon Voucher (herbarium) ITS petD rps16 trnH-psbA trnL-F
Dentellarepens J.R.Forst. & G.Forst Australia: Andersson 2262 (GB) AM939440 EU557693 AF333370 / EU543091
Hedyotisacutangula Champ. ex Benth. China: unknown BW21 (CUHK) HQ148749 / HM752907 HM640307 HM752822
Hedyotisacutangula Champ. ex Benth. China: Ruijiang Wang HA-02 (IBSC) JX111197 JX111085 JX111241 JX111160 JX111316
Hedyotiscantoniensis F.C.How ex W.C.Ko China: Ruijiang Wang et al. 1250 (IBSC) JF699912 JF700061 JX111247 JF699773 JX111322
Hedyotiscaudatifolia Merr. & F.P.Metcalf China: Ruijiang Wang et al. 1229 (IBSC) JF699915 JF700064 JX111255 JF699776 JX111328
Hedyotiscaudatifolia Merr. & F.P.Metcalf China: Ruijiang Wang et al. 1269 (IBSC) JF699916 JF700065 JX111256 JF699777 JX111329
Hedyotiscommunis W.C.Ko China: Bo Li LB0172 (IBSC) JX111208 JX111094 JX111257 JX111167 JX111330
Hedyotisconsanguinea Hance China: Ruijiang Wang 1254 (IBSC) JF699923 JF700071 JX111258 JF699783 JX111331
Hedyotiseffusa Hance China: Ruijiang Wang et al. 1268_1 (IBSC) JF699933 JF700083 JX111262 JF699790 JX111335
Hedyotisexserta Merr. China: Guobin Jiang and Xinxin Zhou 1124 (IBSC) MT345066 MT347606 MT792387 MT792403 MZ514116
Hedyotisinterrupta G.B.Jiang & R.J.Wang China: Guobin Jiang and Xinxin Zhou 1136_2 (IBSC) MT345072 MT347612 MT792393 MT792409 MZ514117
Hedyotisloganioides Benth. China: Ruijiang Wang 1253-1 (IBSC) JF699910 JF700059 JX111246 JF699771 JX111320
Hedyotislongiexserta Merr. & F.P.Metcalf China: Mingdeng Yuan et al. YS60 (IBSC) MW396581 MW405435 MW405424 / MZ514123
Hedyotislongipetala Merr. China: Ruijiang Wang 1334 (IBSC) JX111216 JX111102 JX111268 JX111175 JX111342
Hedyotislongiramulis Y.D.Xu & R.J.Wang China: Yida Xu and Fan Su AP0138 (IBSC) MZ326005* MZ425928** MZ425928** MZ425928** MZ425928**
Hedyotislongiramulis Y.D.Xu & R.J.Wang China: Dan Liang et al. WP1366 (IBSC) MZ411390* MZ403800* MZ417507* MZ403809* MZ417501*
Hedyotismatthewii Dunn China: Ruijiang Wang et al. 1251 (IBSC) JF699900 JF700049 JX111243 JF699761 JX111318
Hedyotisnankunshanensis R.J.Wang & S.J.Deng China: Ruijiang Wang et al. 1688 (IBSC) JN975969 JN975964 OQ723460* OQ723461* OQ723462*
Hedyotisnanlingensis R.J.Wang China: Mingdeng Yuan et al. YS228 (IBSC) MW396579 MW405437 MW405426 MZ514110 MZ514124
Hedyotisovata Thunb. ex Maxim. China: Guobin Jiang et al. 1508 (IBSC) MZ326003 MZ403799 MZ343053 MZ403807 MZ403793
Hedyotispuberulifolia Y.D.Xu & R.J.Wang China: Ruijiang Wang and Yida Xu 6216 (IBSC) MW169047 MW196744 OQ723463* OQ723464* OQ723465*
Hedyotispubirachis Y.D.Xu & R.J.Wang China: Yida Xu and Fan Su AP0147 (IBSC) MW264177 MW266052 MZ447121 MZ447124 MZ447126
Hedyotispulcherrima Dunn China: Ruijiang Wang 1233-1 (IBSC) JF699946 JF700096 JX111274 JF699801 JX111348
Hedyotistaishanensis G.T.Wang & R.J.Wang China: Yida Xu et al. WP1330 (IBSC) MZ479676 MZ514102 MZ514103 MZ514108 MZ514121
Hedyotistenuipes Hemsl. China: Ruijiang Wang 1234_1 (IBSC) JF699960 JF700110 JX111280 JF699812 JX111354
Hedyotisxanthochroa Hance China: Ruijiang Wang 1361 (IBSC) JX111227 JX111110 JX111286 JX111183 JX111361
Hedyotisxinyiensis X.Guo & R.J.Wang China: Ruijiang Wang 1182 (IBSC) JF699970 JF700120 JX111288 JF699820 JX111362
Hedyotisyangchunensis W.C.Ko & Zhang China: Ruijiang Wang 1270-1 (IBSC) JF699972 JF700122 JX111290 JF699821 JX111364
Pentodonpentandrus Vatke Zambia: Dessein et al. 598 (BR) AM939528 EU557759 EU543066 / EU543154
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*indicates that the sequences are newly obtained by PCR sequencing. **indicates that the sequences are newly obtained by whole genome sequencing.

Five DNA markers (ITS, petD, rps16, trnH-psbA and trnL-F) were employed to reconstruct the phylogenetic trees. Sequences were aligned using MAFFT v.7.017 (Katoh et al. 2002) and then concatenated together in Geneious. Maximum Likelihood (ML) analyses were accomplished with IQ-TREE v.2.0 (Nguyen et al. 2015). The best-fit nucleotide substitution model of GTR+F+R2 was selected by using ModelFinder (Kalyaanamoorthy et al. 2017). Bayesian inference (BI) analyses were accomplished with MrBayes v.3.1.2 (Ronquist et al. 2012). GTR+G+I was selected to be the best-fit nucleotide substitution model by MrModeltest v.2.3 (Nylander 2004). The sampled species along with their voucher information and GenBank accession numbers are listed in Table 2.

Results

A new species based on morphological and molecular evidence

Morphology

During our examination of herbarium material, we found that Hedyotislongiramulis was often misidentified as either H.caudatifolia or H.communis W.C.Ko because of the subshrubby habit, the ovate to lanceolate leaves and the triangular stipules. A detailed morphological comparison is therefore provided to elucidate the differences among them (Table 3).

Table 3.

Diagnostic characters of Hedyotislongiramulis, H.pubirachis (sister species in molecular analysis), H.caudatifolia and H.communis (two morphologically similar species).

Characters H.longiramulis H.caudatifolia H.communis H.pubirachis
Stem more or less tetragonal and decussately sulcate at juvenile internodes, puberulent terete or slightly flattened, glabrous terete or slightly flattened, glabrous terete with inconspicuous ridges, glabrous
Leave surface waxy on both side glabrous on both side glabrous on both side glabrous on both side
Petiole length (mm) 5–15 on main stem and 2–5 on lateral branches 3–15 subsessile 3–10
Stipules triangular, densely puberulent abaxially triangular, glabrous abaxially narrowly triangular, glabrous abaxially triangular to broadly ovate, glabrous abaxially
Inflorescences growing on lateral branches, terminal and axillary in the upper nodes growing on lateral branches, terminal and axillary in the upper nodes growing on main stem and on lateral branches, strictly axillary growing on main stem and on lateral branches, terminal and axillary in the upper nodes
Peduncle length (cm) 0.5–2.0 2.0–10.0 0.5–2.5 2.5–7.0
Calyx lobes length (mm) ca. 0.9 0.8–1.0 2–3 ca. 0.5
Calyx lobes shape ovate-triangular with blunt or rounded apex triangular with acute apex narrowly triangular with acute apex broadly triangular
Ratio of calyx lobe length to its basal width ca. 1:1 1–1.5:1 2.5–3:1 ca. 0.8:1
Corolla tube length (mm) 3.5–3.8 3.0–4.0 4.0–5.0 2.8–3.3
Corolla lobe length (mm) 3.5–3.8 2.0–2.7 2.5–4.0 2.0–2.2
Length ratio of corolla lobe to tube 0.9–1.0 ca. 0.8 0.6–0.8 ca. 0.7
Capsule shape subglobose ellipsoid-oblong or ellipsoid obovoid or subglobose ellipsoid to subglobose
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Molecular analysis

BI and ML analyses based on the combined nuclear ITS and four plastid markers (petD, rps16, trnH-psbA and trnL-F) result in the same tree topology. The two accessions of the new species form a monophyletic clade that is sister to H.pubirachis Y.D.Xu & R.J.Wang with robust support (PP = 1, BS = 98) (Fig. 1). The two species share common characters, such as subshrubby habit and ovate to lanceolate leaf shape, but differ in other characters. A comparison of the morphological characters is given in Table 3.

Figure 1.

Figure 1.

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Phylogenetic relationships of Hedyotis based on combined nuclear ITS and four plastid markers (petD, rps16, trnH-psbA and trnL-F). Bootstrap values (BS≥50%, right) and Bayesian Posterior Probabilities (PP≥0.5, left) are labeled above the branches. Field collection numbers are labeled after species names.

Taxonomic treatment

. Hedyotis longiramulis

Y.D.Xu & R.J.Wang sp. nov.

8545C705-65AD-5BE6-AB0C-91E0A73FC442

urn:lsid:ipni.org:names:77325483-1

Figs 2 , 3 鹅凰嶂耳草 (é Huáng Zhàng ěr Căo)

Figure 2.

Figure 2.

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Hedyotislongiramulis Y.D. Xu & R.J. Wang A habit B habitat C inflorescences D part of stem (right) and its transverse section E adaxial (left) and abaxial (right) surgaces of leaf F Stipules G long-styled flower (left) and its longitudinal section (right) H short-styled flower (left) and its longitudinal section (right) I infructescence J diplophragmous capsule K dorsal (left) and ventral (right) view of seeds.

Figure 3.

Figure 3.

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Micromorphology of seed, pollen and leaf epidermis of Hedyotislongiramulis using SEMA–C ventral view, dorsal view, and surface ornamentation of seeds, respectively D, G, E, H, F, I equatorial view, polar view, and reticulate ornamentation of pollen grains, respectively J–L leaf epidermis, adaxial and abaxial surfaces, and stomatal apparatus, respectively A–C, J–LYi-Da Xu & Fan Su AP0138D–FRui-Jiang Wang & Yi-Da Xu 6540, long-styled flower G–IRui-Jiang Wang & Yi-Da Xu 6541, short-styled flower.

Type.

China. Guangdong Province: Yangchun City, Bajia Town, Guangdong Ehuangzhang Provincial Nature Reserve, roadsides, 21°52'N, 111°25'E, elev. 643 m. April 9, 2021, Y.D. Xu & R.J. Wang 6540 (holotype: IBSC [IBSC0865777!]; isotype: IBSC [IBSC0865778!]).

Diagnosis.

The species is similar to H.caudatifolia in having a subshrubby habit, ovate to lanceolate leaves, and long lateral branches with several terminal and axillary inflorescences, but differs from it by having puberulent, more or less tetragonal and decussately sulcate juvenile stems (versus glabrous and terete in H.caudatifolia), waxy leaf surface (versus non-waxy in H.caudatifolia), shorter peduncles (0.5–2.0 cm versus 2.0–10.0 cm in H.caudatifolia), a higher length ratio of corolla lobe to tube (0.9–1.0 versus approximately 0.8 in H.caudatifolia), and subglobose capsules (versus ellipsoid-oblong or ellipsoid in H.caudatifolia).

Description.

Perennial woody subshrubs, 40–120 cm tall. Stem more or less tetragonal and decussately sulcate at juvenile internodes, becoming terete with age, purplish, puberulent, branched at upper part. Leaves opposite, 5–16 × 1.5–4 cm on main stem and 1.0–6.5 × 0.3–1.5 cm on lateral branches, ovate to lanceolate, coriaceous, dark green adaxially, greyish-green or sometimes purplish abaxially, both surfaces waxy, apex acute or subacute, base cuneate or shortly decurrent; petiole 5–15 mm long on main stem and 2–5 mm long on lateral branches, waxy or puberulent; midrib depressed adaxially and prominent abaxially, secondary veins usually 5–6 on each side, sometimes indistinct adaxially; stipules 4–10 × 3–6 mm, triangular, apex acute to acuminate, margin sparsely glandular serrate, puberulent abaxially. Inflorescences growing on long lateral branches, terminal and axillary in the upper nodes, 1.5–3.5 cm long, cymose or paniculate-cymose; inflorescence axes tetragonal, sulcate; peduncles 0.5–2.0 cm long; bracts ca. 1 mm long, subulate. Flowers heterostylous, pedicels 0.9–2.0 mm long. Hypanthium ca. 1 mm long, obconic to subglobose; lobes 4, ca. 0.9 × 0.9 mm, ovate-triangular, blunt or rounded at apex. Corolla white or purplish, tube 3.5–3.8 mm long, glabrous abaxially and densely or sparsely pubescent adaxially; lobes 4, 3.5–3.8 × 1.8–2.2 mm, ovate-triangular; stamens 4, anthers ca. 0.9 mm long; stigma bilobed, ca. 0.5 mm long, subglobose, papillate. Long-styled flowers: stamens included, filaments ca. 0.6 mm long, adnate to the middle part of corolla tube; style ca. 7.6 mm long, exserted, glabrous. Short-styled flowers: stamens exserted, filaments ca. 2.8 mm long, adnate to the throat of corolla tube; style ca. 2.7 mm long, included, glabrous. Fruits capsular, ca. 2.0 mm in diameter, subglobose, glabrous, dehiscent diplophragmously; seeds several, ca. 1 mm long, cymbiform, with reticulate surface. (Fig. 3A–C.)

Distribution and habitat.

Hedyotislongiramulis is only known from Yangchun City of Guangdong Province, China. It grows mainly in damp places under broad-leaved forests, sometimes on roadsides at the elevation of 500–700 m. The associated species are mainly (Hance ex Benth.) Krass. (Melastomataceae), Melastomasanguineum Sims (Melastomataceae), Dunniasinensis Tutcher (Rubiaceae), Dicranopterisampla Ching & P.S.Chiu (Gleicheniaceae) and Selaginelladoederleinii Hieron. (Selaginellaceae).

Phenology.

Flowering from late March to July, fruiting from August to October.

Etymology.

The specific epithet “longiramulis” of the new species refers to its long lateral branches bearing many inflorescences.

Palynology.

The pollen grains of Hedyotislongiramulis are monads, isopolar, spheroidal, 3-colporate; the tectum is a double microreticulum, with a psilate suprareticulum and a microechinate infrareticulum. The pollen size is 22.5 (20.2–25.1) × 21.5 (19.0–22.8) μm with P/E value 1.04 in long-styled flowers (Fig. 3D–F) and 27.1 (25.2–29.3) × 27.1 (25.1–28.7) μm with P/E value 1.00 in short-styled flowers (Fig. 3G–I).

Foliar epidermal anatomy.

The epidermal cells on the upper (Fig. 3J) and lower (Fig. 3K) surface of leaves of H.longiramulis are irregularly polygonal, randomly arranged and have striated and papillate surface ornamentation, with the striations thickened at the middle of the periclinal walls, and the papillae conical, with granular ornamentation on the surface. The anticlinal walls are straight in epidermis cells of the upper leaf surface and undulate in those of the lower leaf surface.

The leaves of H.longiramulis are hypostomatic, with the stomata randomly orientated over most of the lower surface. The stomata are paracytic, ca. 56.5 (51.4–63.4) × 42.8 (37.1–52.1) μm in size (Fig. 3L).

Additional specimens examined

(paratypes). China. Guangdong Province: Yangchun City, Guigang Town, Baichong Provincial Nature Reserve, roadside, 13 Sept. 1990, Nian Liu et al. 424 (IBSC); ibid., 18 May 1991, Nian Liu et al. 1735 (IBSC). Yangchun City, Bajia Town, Guangdong Ehuangzhang Provincial Nature Reserve, mountain land and valley, 24 Oct. 1957, Kui Liang 69692 (CANT); ibid., 23 Oct. 1957, Bao-Han Liang 89654 (CANT); ibid., 11 Oct. 1990, Nian Liu et al. 866 and 899 (IBSC); ibid., 11 May 2001, Hua-Gu Ye et al. 5629 (IBSC); ibid., 7 Apr. 2019, Xin-Xin Zhou et al. ZXX0026 (IBSC); ibid., 12 Aug. 2020, Dan Liang et al. WP1366 (IBSC); ibid., 10 Sept. 2020, Yi-Da Xu & Fan Su AP0138 (IBSC); ibid., 9 Apr. 2021, Rui-Jiang Wang & Yi-Da Xu 6541 (IBSC).

Conservation status assessment.

So far 10 subpopulations of Hedyotislongiramulis were found in Yangchun City (AOO 40 km2, EOO 758 km2), Guangdong Province, and their habitats are well protected. About 60 mature individuals were found in each of these subpopulations (within 2 × 2 km grid cells). We therefore estimated that there are at least 600 mature individuals in this area. According to the criteria D1 of IUCN Red List Categories and Criteria (IUCN 2022), the species can be assessed as “Vulnerable”. However, many other subpopulations of this species may be found in similar habitat nearby the vouchers’ localities in the nature reserves. Considering that this species has no economic uses and that there are no plausible threats since it occurs in two protected reserves, we recommend to evaluate it as “Least Concern”.

Characteristics of the chloroplast genome

The size of the complete chloroplast genome of H.longiramulis is 153,616 bp (GenBank: MZ425928, Fig. 4) with a typical quadripartite structure, including a small single-copy region (SSC, 17652 bp), a large single-copy region (LSC, 85050 bp), and a pair of inverted repeat regions (IRs, 25457 bp). It contains 112 unique genes, and the GC content is 32.4% (Table 4). The rps19, ycf1, ndhF, rpl2 and trnH genes were found nearby the IR/Single-Copy (SC) region boundaries. Compared with H.ovata, the IR of H.longiramulis contracted to include only 4 bp of the 5’ end of rps19 (vs. entirely included and occurring twice in IRs of H.ovata), and excludes the entire ndhF and 100 bp of the intergenic region (vs. including 32 bp of the 3’ end of ndhF in H.ovata) (Fig. 5). Detailed characteristics and statistics of the chloroplast genomes are listed in Tables 4, 5.

Figure 4.

Figure 4.

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Chloroplast genome map of Hedyotislongiramulis. The thick lines on the outer complete circle identify the inverted repeat regions (IRa and IRb). The arrows indicate the transcription directions of the genes inside and outside of the circle. Genes belonging to different functional groups are color-coded. The dark gray in the innermost track corresponds to the GC content, the light gray to the AT content.

Table 4.

Characteristics of the chloroplast genomes of Hedyotislongiramulis and H.ovata.

Characteristics H.longiramulis GenBank: MZ425928 H.ovata GenBank: MK203877
Size (bp) Total 153,616 154,560
LSC 85,050 84,579
SSC 17,652 17,865
IR 25,457 26,058
Number of unique genes Total 112 112
Protein-coding genes 79 79
rRNA genes 4 4
tRNA genes 29 29
GC% Total 32.4 32.6
LSC 35.9 36.0
SSC 32.4 32.6
IR 43.5 43.4
protein-coding sequences (CDS) 38.4 38.9
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Figure 5.

Figure 5.

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Sequence comparison of the IR/SC boundaries between Hedyotislongiramulis and H.ovata.

Table 5.

Genes encoded in the chloroplast genome of Hedyotislongiramulis.

Category Group of genes Names of unique genes
Self-replication tRNA genes trnA-UGC, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-GCC, trnH-GUG, trnI-CAU, trnI-GAU, trnK-UUU, trnL-CAA, trnL-UAA, trnL-UAG, trnM-CAU, trnN-GUU, trnP-UGG, trnQ-UUG, trnR-ACG, trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC, trnV-UAC, trnW-CCA, trnY-GUA
rRNA genes rrn4.5, rrn5, rrn16, rrn23
Ribosomal small subunit rps2, rps3, rps4, rps7, rps8, rps11, rps12, rps14, rps15, rps16, rps18, rps19
Ribosomal large subunit rpl2, rpl14, rps16, rpl20, rpl22, rpl23, rpl32, rpl33, rpl36
DNA-dependent RNA polymerase rpoA, rpoB, rpoC1, rpoC2
Photosynthesis Photosystem I psaA, psaB, psaC, psaI, psaJ, ycf3, ycf4
Large subunit of rubisco rbcL
Photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
NADH dehydrogenase ndhA, ndhB, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
Cytochrome b/f complex petA, petB, petD, petG, petL, petN
ATP synthase atpA, atpB, atpE, atpF, atpH, atpI
Other genes Maturase matK
Subunit of acetyl-CoA carboxylase accD
Envelope membrane protein cemA
Protease clpP
C-type cytochrome synthesis ccsA
Conserved open reading frames ycf1, ycf2
Peseudogene Translation-related gene infA
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Discussion

Similar to other Hedyotis species described previously (Wang et al. 2018; Jiang and Wang 2019; Xu and Wang 2021; Jiang and Wang 2021), the pollen grains of H.longiramulis are dimorphic between long-styled and short-styled flowers, i.e., the pollen of the short-styled flowers is larger than that of the long-styled flowers. This pattern was also found in other Rubiaceae with dimorphic flowers, e.g., Damnacanthus C.F.Gaertn. (Naiki and Nagamasu 2003) and Arcytophyllum Schult. & Schult.f. (Wolff and Liede-Schumann 2007).

The phylogenetic analysis shows that H.longiramulis is sister to H.pubirachis (Fig. 1), but it can be distinguished from this species by the puberulent stems and stipules (versus glabrous in H.pubirachis), the waxy leaf surface (versus non-waxy in H.pubirachis) and the inflorescences growing on long lateral branches (versus inflorescences on the main stem and lateral branches in H.pubirachis) (Table 3).

Comparing to chloroplast genome of the new species to that of H.ovata, we found that there was a 300 bp contraction that occurred in the IR regions of H.longiramulis excluding almost entirely the rps19 gene from the IR/LSC boundaries (Fig. 5). However, we currently can’t predict the fluctuation tendency in this genus due to insufficient chloroplast genomic data. We suggest that the complete chloroplast genome would be informative and would help resolve infrageneric relationships within the genus.

Key to the 24 Hedyotis species sampled in this study

1 Stem terete or slightly flattened 2
– Stem tetragonal or sulcate, or at least so when juvenile 13
2 Leaves ovate to ovate-triangular; inflorescences 1-flowered or 2–4-flowered and fasciculate H.pulcherrima
– Leaves lanceolate, ovate-lanceolate, or lanceolate-elliptic; inflorescences cymose or paniculate cymose 3
3 Stipules more or less puberulent abaxially 4
– Stipules glabrous abaxially 6
4 Leaves densely puberulent on both sides H.puberulifolia
– Leaves glabrous on both sides or only puberulent on midrib adaxially 5
5 Position of inflorescences strictly axillary H.loganioides
– Position of inflorescences terminal and axillary in upper nodes H.tenuipes
6 Position of inflorescences strictly axillary H.communis
– Position of inflorescences terminal and axillary in upper nodes 7
7 Inflorescences showing dichasial branching at sub-axes 8
– Inflorescences showing monochasial branching at sub-axes 10
8 Inflorescence axes terete H.cantoniensis
– Inflorescence axes more or less 4-angled or sulcate 9
9 Peduncles hollow, slightly sulcate; corolla tubes ca. 2.5 mm long H.nankunshanensis
– Peduncles solid, 4-angled and sulcate; corolla tubes 3.0–4.0 mm long H.caudatifolia
10 Inflorescence axes 4-angled and sulcate H.pubirachis
– Inflorescence axes terete 11
11 Leaves narrowly elliptic to lanceolate; stipules triangular H.nanlingensis
– Leaves ovate, broadly elliptic or lanceolate; stipules broadly triangular 12
12 Leaves ovate to lanceolate; capsules oblong-ellipsoid H.longiexserta
– Leaves ovate to broadly elliptic; capsules subglobose H.effusa
13 Stems more or less puberulent or scabrous 14
– Stems glabrous 17
14 Leaves base broadly rounded or amplexicaul; leaves densely pilose on both sides H.xanthochroa
– Leaves base cuneate, narrowly cuneate or shortly decurrent; leaves glabrous, waxy or puberulent on both sides 15
15 Leaf surface waxy on both sides; inflorescences growing at lateral branches H.longiramulis
– Leaves glabrous to puberulent on both sides; inflorescences growing at terminal and upper axillary of main stem 16
16 Flowers not enclosed by two ovate leaflike bracts; corolla white or purplish abaxially; corolla tubes shorter than 3 mm H.matthewii
– Flowers enclosed by two ovate leaflike bracts; corolla purple abaxially; corolla tubes longer than 15 mm H.yangchunensis
17 Inflorescences axillary 18
– Inflorescences terminal and axillary in upper nodes of stem 19
18 Stipules broadly triangular, margins not reflexed, apex apiculate to aristate H.interrupta
– Stipules ovate or triangular, margins becoming reflexed, apex acute to acuminate H.acutangula
19 Corolla purple abaxially; corolla tubes longer than 5 mm 20
– Corolla white or purplish abaxially; corolla tubes shorter than 3 mm 21
20 Leaves lanceolate, narrowly lanceolate or narrowly elliptic, scabrous H.exserta
– Leaves ovate, glabrous H.ovata
21 Flowers homostylous; corolla tubes pilosulous adaxially 22
– Flowers heterostylous; corolla tubes pubescent adaxially 23
22 Leaves narrowly lanceolate or lanceolate; corolla lobes longer than tube H.longipetala
– Leaves narrowly elliptic, elliptic or lanceolate; corolla lobes nearly equal to tube in length H.matthewii
23 Stipules broadly triangular, glabrous abaxially H.consanguinea
– Stipules triangular, pubescent abaxially 24
24 Inflorescences at terminal and upper axillary of main stem; peduncles shorter than 5 cm H.xinyiensis
– Inflorescences at terminal of main stem; peduncles longer than 5 cm H.taishanensis
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Conclusion

The new species of Hedyotislongiramulis is described based on the combination of morphological and molecular evidence. In addition, the micromorphological characters of seed, pollen and leaf epidermal features were illustrated.

Supplementary Material

XML Treatment for Hedyotis longiramulis

Acknowledgments

We are grateful to Dan Liang, Guo-Bin Jiang, Jiang-Ping Shu, Ya-Nan Guo and Fan Su for field assistance, Xiao-Ying Hu for SEM observation at SCBG; We also would like to extend our thanks to the director Yong-Ju Chen, the deputy director Shi-Biao Luo and Professor Xi-Po Lin of the Guangdong Ehuangzhang Provincial Nature Reserve for providing accommodation during our field investigation there.

Citation

Xu Y-D, Zhang Y, Wang R-J (2023) Hedyotis longiramulis (Rubiaceae), a new species from south China. PhytoKeys 230: 271–287. https://doi.org/10.3897/phytokeys.230.87675

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was supported by Guangdong Provincial Key R&D Program “the Conservation and Utilization of the Important Strategic Wild Plant Resource in Guangdong province (grant no. 2022B1111040003)”.

Author contributions

Methodology: YZ. Supervision: RJW. Writing - original draft: YDX.

Author ORCIDs

Yi-Da Xu https://orcid.org/0000-0001-9812-4770

Ying Zhang https://orcid.org/0000-0002-8352-1543

Rui-Jiang Wang https://orcid.org/0000-0002-4985-8281

Data availability

All of the data that support the findings of this study are available in the main text.

References

  1. Bachman S, Moat J, Hill AW, de la Torre J, Scott B. (2011) Supporting Red List threat assessments with GeoCAT: Geospatial conservation assessment tool. ZooKeys 150: 117–126. 10.3897/zookeys.150.2109 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bremekamp CEB. (1952) The African species of Oldenlandia L. sensu Hiern & K. Schumann. Verhandelingen der Koninklijke Nederlandse Akademie van Wetenschappen, Afd. Natuurkunde 48(2): 1–297. [Google Scholar]
  3. Charr JC, Garavito A, Guyeux C, Crouzillat D, Descombes P, Fournier C, Ly SN, Raharimalala EN, Rakotomalala JJ, Stoffelen P, Janssens S, Hamon P, Guyot R. (2020) Complex evolutionary history of coffees revealed by full plastid genomes and 28,800 nuclear SNP analyses, with particular emphasis on Coffeacanephora (Robusta coffee). Molecular Phylogenetics and Evolution 151: e106906. 10.1016/j.ympev.2020.106906 [DOI] [PubMed]
  4. David LD. (1974) Approaches to the identification of angiosperm leaf remains. Botanical Review 40(1): 1–157. 10.1007/BF02860067 [DOI] [Google Scholar]
  5. Doyle J. (1991) DNA protocols for plants. In: Hewitt GM, Johnston AWB, Young JPW. (Eds) Molecular Techniques in Taxonomy.Springer Berlin Heidelberg, Berlin, 283–293. 10.1007/978-3-642-83962-7_18 [DOI]
  6. Dutta R, Deb DB. (2004) Taxonomic Revision of Hedyotis L. (Rubiaceae) in Indian Sub-continent. Botanical Survey of India, 211 pp.
  7. Gibbons KL. (2020) Hedyotis, Oldenlandia and related genera (Rubiaceae: Spermacoceae) in Australia: New genera and new combinations in an Asian-Australian-Pacific lineage. Taxon 69(3): 515–542. 10.1002/tax.12236 [DOI] [Google Scholar]
  8. Greiner S, Lehwark P, Bock R. (2019) OrganellarGenomeDRAW (OGDRAW) version 1.3.1: Expanded toolkit for the graphical visualization of organellar genomes. Nucleic Acids Research 328(5979): 710–722. 10.1093/nar/gkz238 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Guo X, Simmons MP, But PPH, Shaw PC, Wang RJ. (2011) Application of DNA barcodes in Hedyotis L. (Spermacoceae, Rubiaceae). Journal of Systematics and Evolution 49(3): 203–212. 10.1111/j.1759-6831.2011.00130.x [DOI] [Google Scholar]
  10. Guo X, Wang RJ, Simmons MP, But PPH, Yu J. (2013) Phylogeny of the Asian Hedyotis-Oldenlandia complex (Spermacoceae, Rubiaceae): Evidence for high levels of polyphyly and the parallel evolution of diplophragmous capsules. Molecular Phylogenetics and Evolution 67(1): 110–122. 10.1016/j.ympev.2013.01.006 [DOI] [PubMed] [Google Scholar]
  11. Hesse H, Halbritter H, Zetter R, Weber M, Buchner R, Frosch-Radivo A, Ulrich S. (2009) Pollen Terminology: An Illustrated Handbook. Springer Wien, New York, 261 pp. [Google Scholar]
  12. IUCN Standards and Petitions Committee (2022) Guidelines for using the IUCN Red List Categories and Criteria. Version 15.1. Prepared by the Standards and Petitions Committee. https://nc.iucnredlist.org/redlist/content/attachment_files/RedListGuidelines.pdf
  13. Jiang GB, Wang RJ. (2019) Hedyotistonggulingensis (Rubiaceae): A new species from Hainan, China. Phytotaxa 406(3): 191–198. 10.11646/phytotaxa.406.3.5 [DOI] [Google Scholar]
  14. Jiang GB, Wang RJ. (2021) Two new species of Hedyotis L. (Rubiaceae) from Hainan, China. Nordic Journal of Botany 39(6): e02934. 10.1111/njb.02934 [DOI]
  15. Jin JJ, Yu WB, Yang JB, Song Y, dePamphilis CW, Yi TS, Li DZ. (2020) GetOrganelle: A fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biology 21(1): 241–271. 10.1186/s13059-020-02154-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS. (2017) ModelFinder: Fast model selection for accurate phylogenetic estimates. Nature Methods 14(6): 587–589. 10.1038/nmeth.4285 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Katoh K, Misawa K, Kuma K-I, Miyata T. (2002) MAFFT: A novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research 30(14): 3059–3066. 10.1093/nar/gkf436 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lamarck JBPAM. (1792) Hedyotis. In: Lamarck JBAPM, Poiret JLM. (Eds) Tableau Encyclopédique et Méthodique des Trois Règnes de la Nature.Botanique (Vol. 1(2)). Chez Panckoucke, Paris, 269–272. 10.5962/bhl.title.218 [DOI]
  19. Liu WZ, Kong HH, Zhou J, Fritsch PW, Hao G, Gong W. (2018) Complete chloroplast genome of Cercischuniana (Fabaceae) with structural and genetic comparison to six species in Caesalpinioideae. International Journal of Molecular Sciences 19(5): e1286. 10.3390/ijms19051286 [DOI] [PMC free article] [PubMed]
  20. Löhne C, Borsch T. (2005) Molecular evolution and phylogenetic utility of the petD group II intron: A case study in basal angiosperms. Molecular Biology and Evolution 22(2): 317–332. 10.1093/molbev/msi019 [DOI] [PubMed] [Google Scholar]
  21. Naiki A, Nagamasu H. (2003) Distyly and pollen dimorphism in Damnacanthus (Rubiaceae). Journal of Plant Research 25(1): 105–113. 10.1007/s10265-002-0075-1 [DOI] [PubMed] [Google Scholar]
  22. Neupane S, Dessein S, Wikstrom N, Lewis P, Long CL, Bremer B, Motley T. (2015) The Hedyotis-Oldenlandia complex (Rubiaceae: Spermacoceae) in Asia and the Pacific: Phylogeny revisited with new generic delimitations. Taxon 64(4): 299–322. 10.12705/642.8 [DOI] [Google Scholar]
  23. Nguyen LT, Schmidt HA, Haeseler A, Minh BQ. (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32(1): 268–274. 10.1093/molbev/msu300 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nylander JAA. (2004) MrModeltest v2: Program distributed by the author. Evolutionary Biology Centre, Uppsala University.
  25. Oxelman B, Liden M, Berglund D. (1997) Chloroplast rps16 intron phylogeny of the tribe Sileneae (Caryophyllaceae). Plant Systematics and Evolution 206: 393–410. 10.1007/BF00987959 [DOI] [Google Scholar]
  26. Popp M, Oxelman B. (2001) Inferring the history of the polyploid Sileneaegaea (Caryophyllaceae) using plastid and homoeologous nuclear DNA sequences. Molecular Phylogenetics and Evolution 20(3): 474–481. 10.1006/mpev.2001.0977 [DOI] [PubMed] [Google Scholar]
  27. Qu XJ, Moore MJ, Li DZ, Yi TS. (2019) PGA: A software package for rapid, accurate, and flexible batch annotation of plastomes. Plant Methods 15(1): 50–61. 10.1186/s13007-019-0435-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rono PC, Dong X, Yang JX, Mutie FM, Oulo MA, Malombe I, Kirika PM, Hu GW, Wang QF. (2020) Initial complete chloroplast genomes of Alchemilla (Rosaceae): Comparative analysis and phylogenetic relationships. Frontiers in Genetics 11: e560368. 10.3389/fgene.2020.560368 [DOI] [PMC free article] [PubMed]
  29. Ronquist F, Teslenko M, Mark PVD, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539–542. 10.1093/sysbio/sys029 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sang T, Crawford DJ, Stuessy TF. (1997) Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae). American Journal of Botany 84(9): 1120–1136. 10.2307/2446155 [DOI] [PubMed] [Google Scholar]
  31. Song Y, Chen Y, Lv JZ, Xu J, Zhu SF, Li MF. (2019) Comparative chloroplast genomes of Sorghum species: Sequence divergence and phylogenetic relationships. BioMed Research International 5046958: 1–11. 10.1155/2019/5046958 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Taberlet P, Gielly L, Pautou G, Bouvet J. (1991) Universal primers for amplification of three noncoding regions of chloroplast DNA. Plant Molecular Biology 17(5): 1105–1109. 10.1007/BF00037152 [DOI] [PubMed] [Google Scholar]
  33. Tate JA, Simpson BB. (2003) Paraphyly of Tarasa (Malvaceae) and diverse origins of the polyploid species. Systematic Botany 28(4): 723–737. [Google Scholar]
  34. Terrell EE, Robinson H. (2003) Survey of Asian and Pacific species of Hedyotis and Exallage (Rubiaceae) with nomenclatural notes on Hedyotis types. Taxon 52(4): 775–782. 10.2307/3647351 [DOI] [Google Scholar]
  35. Tillich M, Lehwark P, Pellizzer T, Ulbricht-Jones ES, Fischer A, Bock R, Greiner S. (2017) GeSeq – versatile and accurate annotation of organelle genomes. Nucleic Acids Research 45(W1): W6–W11. 10.1093/nar/gkx391 [DOI] [PMC free article] [PubMed]
  36. Wang GT, Zhang Y, Liang D, Chen YQ, Wang RJ. (2018) Hedyotistaishanensis (Rubiaceae): A new species from Guangdong, China. Phytotaxa 367(1): 38–44. 10.11646/phytotaxa.367.1.4 [DOI] [Google Scholar]
  37. Willdenow KL. (1797) Caroli a Linné Species Plantarum Exhibentes Plantas Rite Cognitas ad Genera Relatas, cum Differentiis Specificis, Nominibus Trivialibus, Synonymis Selectis, Locis Natalibus, Secundum Systema Sexuale Digestas (Tomus 1). Impensis G. C. Nauk, 1568 pp. 10.5962/bhl.title.60064 [DOI]
  38. Wolff D, Liede-Schumann S. (2007) Evolution of flower morphology, pollen dimorphism, and nectar composition in Arcytophyllum, a distylous genus of Rubiaceae. Organisms, Diversity & Evolution 7(2): 106–123. 10.1016/j.ode.2006.02.005 [DOI] [Google Scholar]
  39. Xu YD, Wang RJ. (2021) Hedyotispuberulifolia (Rubiaceae), a new species from Guangdong, China. Phytotaxa 502(2): 160–168. 10.11646/phytotaxa.502.2.4 [DOI] [Google Scholar]
  40. Zhang XF, Wang JH, Zhao KK, Fan WW, Wang HX, Zhu ZX, Wang HF. (2019) Complete plastome sequence of Hedyotisovata Thunb. ex Maxim (Rubiaceae): An endemic shrub in Hainan, China. Mitochondrial DNA. Part B, Resources 4(1): 675–676. 10.1080/23802359.2019.1572467 [DOI] [Google Scholar]
  41. Zhang Y, Wang ZF, Guo YN, Chen S, Xu XY, Wang RJ. (2021) Complete chloroplast genomes of Leptodermisscabrida complex: Comparative genomic analyses and phylogenetic relationships. Gene 791: e145715. 10.1016/j.gene.2021.145715 [DOI] [PubMed]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

XML Treatment for Hedyotis longiramulis
phytokeys.230.87675-treatment1.xml (17.6KB, xml)

Data Availability Statement

All of the data that support the findings of this study are available in the main text.

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