Introduction

Premna L. consists of about 130 species distributed in the tropics and subtropics of Asia, Africa, and Australia1,2,3,4,5,6. The genus was first described by Linnaeus7 based on the two species: P. serratifolia L. and P. integrifolia L. (now synonymized under the former one). The generic name was derived from the Greek word ‘Premnon’ which means ‘stump of the tree’, likely alluding to the short and twisted tree trunks of the type species. The genus could be distinguished from other Lamiaceae genera by its terminal inflorescences, mostly small zygomorphic to sub-actinomorphic flowers, a two-lipped calyx with 4–5 lobes or unlobed, a typically two-lipped, five-lobed corolla with dense white hairs on the inner surface, and drupaceous fruits3,4,8.

Several species of Premna are widely recognized in Asia for their medicinal applications, attributed to their active phytochemical constituents and diverse bioactivities9. For example, local people in northern Thailand boil the stems and leaves of P. tomentosa Willd. to cure lumbago10. This species also holds significance in traditional Indian medicine, as it contains flavonoids, tannins, alkaloids, and terpenoids, compounds associated with anti-hyperlipidemic, hepatoprotective, anticancer, antioxidant, and antibacterial properties11. In western India, various vegetative parts of P. barbata Wall. ex Voigt are employed by local populations to treat a wide range of conditions, including joint and back pain, diarrhea, wound healing, throat infections, bacterial diseases, fever, herpes simplex infections, and arthritic pain12. Several studies have explored the antibacterial and mosquito larvicidal activities of essential oil extracts from various species, including P. acutata W. W. Sm., P. cambodiana Dop, P. mollissima Roth, P. odorata Blanco, P. puberula Pamp., P. serratifolia, and P. tomentosa11,13,14. Among these, the essential oils derived from the leaves of P. odorata have demonstrated notable anti-aging properties15.

After the transfer from Verbenaceae to Lamiaceae2,16, Premna has been shown to be monophyletic5,6,17,18 and its phylogenetic position within Lamiaceae has been well explored as a member of the subfamily Premnoideae in previous large-scale molecular phylogenetic studies5,19,20. Recently, several new species of Premna have been reported from China, Myanmar, and Vietnam, such as P. bhamoensis Y. T. Tan & B. Li, P. caridantha Y. H. Tan & B. Li, P. vietnamensis B. Li, and P. grandipaniculata Y. H. Tan & B. Li17,21,22,23. The nomenclatural changes have been also made with consideration of on Chinese and Vietnamese species24,25,26,27. Nevertheless, classification and identification within the genus Premna are considered highly complex within the Lamiaceae and require further clarifications5.

Premna serratifolia, a species widely distributed across southern and southeastern Asia, Northern Australia, and western Africa, has been traditionally utilized by people in Malesian regions as a vegetable and for its medicinal properties. Various parts of the plant are used to treat ailments such as fever, stomach-ache, diarrhea, shortness of breath, and breast milk production3. Furthermore, this species produces diverse essential oils with bioactive compounds that demonstrate potential therapeutic applications14. Despite its ecological and pharmacological importance, the delimitation of P. serratifolia has faced significant taxonomic challenges due to morphological variation across its extensive geographical range, particularly in the shape and size of leaves and inflorescences3. More than 50 taxa have been placed in synonymy with P. serratifolia4, reflecting confusion in its taxonomic history. However, some synonymized species such as P. paniculata H. R. Fletcher, and P. punctulata C. B. Clarke, exhibit apparently distinct morphological and ecological characteristics that differentiate them from P. serratifolia, warranting further investigation for their taxonomic identities. The taxonomic status of P. cordifolia Roxb. is also uncertain, as this accepted species was previously regarded as invalidly published and later synonymized under P. serratifolia in a recent taxonomic treatment3. This ambiguity highlights the need for a comprehensive re-evaluation to clarify its taxonomic identity. The taxonomic complexity surrounding P. serratifolia underscores the need for comprehensive studies to clarify its species boundaries and phylogenetic relationships. Such efforts will not only enhance our understanding of this genus but also contribute to the preservation and sustainable utilization of its valuable resources.

In Thailand, the first comprehensive taxonomic work of Premna was carried out by Fletcher28, recognizing 24 species (30 taxa). Subsequently, Moldenke29 listed 28 species (36 taxa) without detailed morphological description. However, there were marked differences in species recognition between these two taxonomic treatments, highlighting the need for further clarification and revision of the genus in Thailand. For example, Moldenke29 notably excluded P. coriacea C. B. Clarke, P. herbacea Roxb., P. nana Collett & Hemsl., and P. racemosa Wall. ex Schauer [= P. interrupta Wall. ex Schauer in this study] in his work, while Fletcher’s taxonomic treatment was lacking P. benglaensis C. B. Clarke, P. chevalieri Dop, and P. esculenta Roxb. After reporting three new records for Thailand: P. punctulata, P. rabakensis Moldenke, and P. stenobotrys Merr.30, Leeratiwong et al.8 published the first synopsis of Premna in Thailand based on the above-mentioned works, recognizing 23 species. However, several following studies have introduced nomenclatural changes that significantly impact species recognition within the genus in Thailand. For example, when revising Premna for the Flora Malesiana, de Kok3,4 treated four species listed in Thailand as synonyms of P. serratifolia, including P. cordifolia, P. octonervia Merr. & F. P. Metcalf, P. paniculata, and P. punctulata, and synonymized P. hamiltonii J. L. Ellis and P. pyramidata Wall. ex Schauer under P. odorata, and P. tomentosa, respectively. These treatments have contributed to taxonomic uncertainty regarding total number of species in Thailand, complicating species identification. Furthermore, our preliminary findings further suggest the taxonomic status of some taxa in Thailand, including P. cordifolia, P. coriacea, P. interrupta var. smitinandii Moldenke, and P. octonervia, requires reassessment. Therefore, these discrepancies highlight the challenges in achieving consensus on Premna taxonomy and underscore the need for a comprehensive taxonomic revision to clarify both the P. serratifolia complex and the status of these taxa in Thailand.

Phylogenetic studies on Asian Lamiaceae have utilized chloroplast regions as molecular markers to delimit species complexes and to infer interspecific relationships, such as genera Coleus Lour., Chelonopsis Miq., Clerodendrum L., Isodon (Benth.) Schrad. ex Spach, Paraphlomis Prain, Phlomoides Moench, Plectranthus L'Hér., Pogostemon Desf., and Vitex L.19,31,32,33,34,35,36,37,38. Previous taxonomic ambiguities within these genera have been clarified through morphological characteristics and molecular analyses based on multi-locus approach of chloroplast regions, such as matK, ndhF, rbcL, rpl32-trnL, rps16, rpsF, rpsR2, trnL, trnL-F, trnS-G5,19,31,34,35,39,40. In Premna, only few phylogenetic analyses have demonstrated that three to five chloroplast regions allow the resolution of phylogenetic placements and constructing the relationships across both regional and larger scales5,17. However, most of these analyses have been based on species primarily sampled from China and Vietnam. Integrating molecular and morphological evidence is essential for resolving species delimitation within the P. serratifolia complex and clarifying the overall phylogenetic relationships of Premna in Thailand.

The present study aims to: (1) establish a comprehensive phylogenetic framework with a focus on Premna in Thailand using four chloroplast markers (ndhF, rbcL, rps16, and trnL-F); (2) revise the species delimitation within the P. serratifolia complex in Thailand based on morphological, ecological investigations and molecular analyses; (3) clarify taxonomic status of P. cordifolia in Thailand (4) reassess the taxonomic status and typification of certain species in Thailand; and (5) determine the number of Premna occurring in Thailand and provide a revised identification key.

Results

A total of 3797 nucleotides were analyzed from four chloroplast markers (1015 for ndhF, 1279 for rbcL, 795 for rps16, and 708 for trnL-F) after being concatenated and aligned. The topologies generated from the ML and BI analyses were slightly different (Fig. 1, Supplementary Fig. S1). In the BI analysis, P. menglaensis B. Li was placed in different position in comparison to the ML analysis within subclade I. Minor conflicts were also observed in the different analyses: P. ligustroides Hemsl. and P. vietnamensis within subclade III (Fig. 1, Supplementary Fig. S1). However, these minor conflicts were only observed in otherwise similar subclades. Given the overall branch support and robustness of the topology, the BI tree was selected here for interpreting relationships within Premna in Thailand with the posterior probability (PP) inferred from BI analysis and the bootstrap value (BS) from ML analysis marked above branches, respectively (Fig. 1).

Fig. 1
Fig. 1
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Bayesian 50% majority-rule consensus tree of Premna with emphasis on Thai species based on concatenated four chloroplast regions dataset: ndhF, rbcL, rps16, and trnL-F. Bayesian inference posterior probability (PP) and maximum likelihood bootstrap support (BS) and are shown in order above the branches, respectively. The bold lines indicate more than 0.95 PP and bold font refers to species presented in Thailand. A dash (–) indicates a different clade obtained from maximum likelihood analysis. Labelled clades (I and II) and subclade (I to III) are recognized in this study.

All species of Premna in this study formed a monophyletic clade with full supports [1.00/100 – 1.00 from BI posterior probabilities (PP) and 100% from ML bootstrap (BS), the values are listed in the same order below] (Fig. 1). Within Premna, two primary clades were formed. Clade I (1.00/97) consists of P. mekongensis W. W. Sm., P. punicea C. Y. Wu, and P. tenii C. Pei with unresolved relationships. Clade II contains the remaining species with P. tomentosa and P. serrata H. R. Fletcher unresolved with respect to a large clade (1.00/99) which could be divided into three subclades. Though relationships of the three subclades were unresolved, each of them obtained moderate to high support: subclades I (1.00/72) and III (1.00/64) accommodates seven and six species found in Thailand, respectively, while subclade II (1.00/77) consists of two species: P. herbacea and P. nana.

A taxonomic note of Premna in Thailand was achieved through the investigation of herbarium specimens, morphological and ecological characters, as well as examinations on type specimens. These findings, combined with molecular analyses, have successfully resolved the P. serratifolia complex in Thailand, as summarized in Table 1 and shown in Figs. 1–5 (see Taxonomic note of species within the P. serratifolia complex in Thailand and Supplementary Table S1). Additionally, our study has validated the taxonomic status of P. coriacea, P. interrupta var. smitinandii, and P. octonervia in Thailand (see Taxonomic notes below).

Table 1 Morphological comparison between P. serratifolia and the taxa previously treated as its synonyms, based on specimen examinations and data from earlier publications8,28,30,50,58,80.
Full size table

Discussion

Phylogenetic reconstruction

By using multi-locus approach of four chloroplast markers, this study provided the first phylogenetic relationships with emphasis on species of Premna in Thailand (bolded species, Fig. 1). Our findings are strongly consistent with previous studies based on chloroplast regions and plastome analyses, confirming that the genus Premna is a monophyletic within the subfamily Premnoideae and forms a sister relationship with Gmelina L.5,6,17,20,32,41,42. However, the phylogenetic positions of some species remain unresolved due to polytomy, such as P. ligustroides, P. serrata, and P. tomentosa. These unresolved relationships have been commonly observed in previous phylogenetic analyses within Premna5,17 and other taxa in many Lamiaceae genera, such as Leucas R.Br., Otostegia Benth., Phlomis L., Phlomoides, Salvia L., Stachys L.37,43,44,45. This may be the result of insufficient data, as few chloroplast markers have been used. Therefore, further studies employing more comprehensive approaches, such as plastid genome, a target enrichment or whole genome analyses, are necessary to explore these relationships (e.g. Zhao et al.6,46, Satthaphorn et al.47). In this study, phylogenetic relationships based on four concatenated chloroplast markers resolved in two clades (Fig. 1). In following, phylogenetic positions and morphological characteristics are discussed.

CLADE I—This highly supported clade includes species distributed in China and Vietnam, namely P. mekongensis, P. punicea, and P. tenii, which are characterized by morphological synapomorphies such as cymes in a dense capitate inflorescence and calyces distinctly 5-lobed. This phylogenetic placement is consistent with morphology-based classification48. Further molecular study is required to test whether all species with the similar traits as recorded in Flora of China49 and adjacent regions form a monophyly.

CLADE II —The well-supported clade consists of a clade comprising subclades I to III (red labelled in Fig. 1), with the unresolved relationships among P. serrata and P. tomentosa. These two latter species possess distinct morphological characteristics from all species in Thailand. Premna serrata has serrate leaf margin and distinct calyx lobes (1–1.8 mm long)8,28. Premna tomentosa, previously recognized as P. pyramidata by Chen and Gilbert49 and Leeratiwong et al.8, exhibits stellate or dendritic hairs on both leaf surfaces, a unique trait distinguishing from other Premna (vs. non-stellate or dendritic in other species). The presence of this characteristic has been documented in several morphological studies3,4,8,22,49. In addition, this clade incorporated several species that have been not included in previous phylogenetic studies, such as P. annulata H. R. Fletcher, P. cordifolia, P. repens H. R. Fletcher, P. scandens Roxb., P. serrata, P. tenii, P. tomentosa, and P. trichostoma Miq.

Three unresolved subclades (subclades I to III) within Clade II were identified based on phylogenetic positions (with high ML and full BS supports, Fig. 1) and morphological characteristics. These subclades include several species in Thailand, as well as members of the P. serratifolia complex in Thailand. Although clear morphological characteristics have been not identified to support the assignment of infrageneric ranks across all subclades, we observed certain habits and morphological affinities among particular species to discuss with their phylogenetic positions.

Subclade I includes nine species of Premna which are shrub or woody climber, distributed in Myanmar, China, and Thailand. These species are mostly inhibited in hill evergreen or limestone forests8. Of these, five species forming a monophyletic group within this subclade (P. bracteata Wall. ex C. B. Clarke, P. garrettii H. R. Fletcher, P. interrupta, P. scandens, and P. trichostoma) have shared clavoid fruits and a thin, fleshy pericarp layer, whereas other species exhibit more or less globose fruits with thicker pericarp3,4,8,49,50. These fruit characteristics, along with endocarp morphology, have been validated as important diagnostic features for species identification and grouping of Premna in Thailand50 and align with the results of our chloroplast-based analysis. This subclade also involves P. cordifolia, a species though to be a synonym under P. serratifolia (subclade III) in the Malesian treatment3. The presence of P. cordifolia in this subclade, integrated with morphological evidence, greatly support the distinction between these taxa, as congruent with earlier treatments8,50,51. Additional samples for this subclade exhibiting the synapomorphy is necessary to further establish a robust foundation for a potential morphology-based infrageneric classification. Subclade II consists of two herbaceous species: P. herbacea and P. nana. Both species display a herbaceous suffrutescent habit with a woody rootstalk and softly woody to herbaceous stems which can be found in dry places, particularly in deciduous forest across southeast Asia8,49,52.The close relationships of these two species are consistent with the previous phylogenetic analyses by Li et al.5 and Hai et al.17. Premna nana was treated as a synonym of P. herbacea in the revision by Chen and Gilbert49. However, subsequent studies and our findings have supported the recognition of P. nana as a distinct species based on morphological characteristics (see identification key below) and molecular phylogenetic evidence3,4,5,8,17.

Subclade III contains Premna representatives which are diverse in their habits and morphological characteristics. Most species are shrubs, scandent shrubs, or woody climbers, with the exception of P. mollissima, P. rabakensis, and P. szemaoensis C. Pei, which are tree species. Species belonging to this clade occupy wide range of habitats such as coastal, evergreen, hill evergreen, or limestone forests8. Distinct morphological traits among species were observed such as the presence or absence of an interpetiolar ridge on stems, type of hairs on leaf surfaces, the size and shape of the calyx lobes. Some species within this subclade are also endemic, including P. annulata (Thailand), P. szemaoensis (China), and P. vietnamensis (Vietnam)8,17,49. Three accessions of P. serratifolia sampled from different sides of peninsular Thailand recovered a monophyletic group also present within this subclade (0.77/92) as well as its previously synonymized species, P. punctulata3. Our results showed that P. punctulata was unresolved with respect to P. serratifolia, exhibiting a relatively long branch, which reflects a greater degree of genetic change53. However, this pattern confirms that these taxa are not fully represented as genetically discrete entities54. This result is consistent with previous phylogenetic studies, which likewise reported a distinct phylogenetic position for P. punctulata separate from P. serratifolia5,17. Our findings thus reaffirm this separation, further supported by morphological and ecological evidence (see taxonomic note below).

Taxonomic notes

Note on P. serratifolia complex in Thailand

As part of the preparation of Premna for the Flora of Thailand, the taxonomic status of three names, including P. cordifolia, P. paniculata, and P. punctulata, has remained ambiguous in relation to P. serratifolia, which were thought to be synonyms in the previous study3. However, our integrated morphological and ecological investigation, and chloroplast-based phylogenetic analyses support the recognition of these as distinct species, prompting a revision of their taxonomic identities (Table1, Fig. 1). Notably, P. serratifolia is confined to coastal habitats, whereas the other species are restricted to evergreen forests. The relevant taxonomic literature, comparative morphology and ecological descriptions, and taxonomic notes for each species are provided. A list of examined specimens is included in Supplementary File, with their mapped distributions shown in Fig. 6.

Premna cordifolia

Roxb., [Hort. Bengal.: 95. 1814, nom. nud.], Fl. Ind. 3: 78. 1832; Schauer in DC., Prodr. 11: 632. 1847; Miq., Fl. Ned. Ind. 2: 895. 1858; C. B. Clarke in Hook. f., Fl. Brit. India 4(12): 572. 1885; King & Gamble, J. Asiat. Soc. Bengal, Pt. 2, Nat. Hist. 74: 818. 1909; H. J. Lam, Verbenaceae Malayan Archipel.: 111. 1919; Ridl., Fl. Malay Penins. 2: 621. 1923; Dop in Lecomte et al., Fl. Indo-Chine 4(7): 816. 1935; H. R. Fletcher, Bull. Misc. Inform. Kew 1938: 418. 1938; Kochummen in Ng, Tree Fl. Mal. 3: 307. 1978; Phuong in N.T. Ban et al., Fl. Vietnam 3: 298. 2005; Leerat. et al., Trop. Nat. Hist. 9(2): 119. 2009.—Gumira cordifolia (Roxb.) Kuntze, Rev. Gen. Pl., 2: 507. 1891. Type: Roxburgh illustration no. 1462 (lectotype K!, selected by Daniel & Rajendran55; isolectotype CAL!) (Fig. 2).

Fig. 2
Fig. 2
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Premna cordifolia. (A, B) habit and habitat; (C) flowering branch; (D) inflorescence; (E) flowers; (F) fruits. Photos A, C–F by C. Leeratiwong and B by J. Satthaphorn.

Description: Scandent shrub or woody climber. Branches with an interpetiolar woody ridge, brown to reddish-brown pubescent. Leaves decussate, mostly cordate or cordate-ovate, rarely oblong-ovate, 5–15 by 3–7 cm, apex caudate or acuminate, rarely retuse, base cordate or rounded, margins entire; adaxial surface glabrous, shining, midrib pubescent; abaxial surface glabrous, with yellowish-brown or yellow sessile glands, midrib sparsely brown pubescent, nerve axils of lower half of abaxial leaf surface glabrous to sparsely pubescent; petiole 1–4 cm long. Inflorescence corymbose, 4–8 cm long; pedicels 0.5–1.5 mm long. Calyx pale green or green, (1.5–)2–3 mm long, unequally 2-lipped; tube 1.2–2.7 mm long; posterior lip entire or subentire; anterior lip 2-lobed, lobes 0.1–0.6 mm long. Corolla yellowish-white or white, 2-lipped, 5–7 mm long; tube 2–3.5 mm long, distally sparsely pubescent outside, densely white villous distally inside; posterior lip ovate to elliptic, 2.5–3.5 mm long; anterior lip with middle lobe slightly larger, broadly oblong or oblong-ovate, 1.2–1.5 mm long, with yellow patch on middle lobe; lobes with villose hairs on base. Stamens long exserted, filaments 1.5–3.5 mm long; anthers 0.3–0.6 mm long. Ovary ovoid to obovoid, 1–1.2 mm long, glabrous with sparse sessile glands at apex; stigma branches 0.15–0.25 mm long. Fruits black, broadly obovoid, 4–7 mm long, glabrous with sessile glands.

Distribution: Thailand (Songkhla, Yala, Narathiwat), Vietnam.

Ecology: In open areas or partly shaded primary or secondary evergreen forests, dry evergreen forests, hardwood forests, granite bedrock; elevation up to 380 m. Flowering and fruiting March to July.

Vernacular: ya yo (ยายอ).

Note: Premna cordifolia was considered as a ‘nomen nudum’ and listed as a synonym under P. serratifolia in de Kok’s3 treatment for Flora Malesiana. This corresponds to its first publication by Roxburgh56 in Hortus Bengalensis, which was invalid due to the absence of a description and type specimen. However, the name was later validated by Roxburgh57 in Flora Indica, although the origin of the type locality remains uncertain due to Roxburgh’s drawing was based on the cultivated specimen55. Therefore, P. cordifolia is a validly published and accepted name.

Although the previous treatment3 lacked the explanation for this inclusion, we presumed that specimens initially thought to be P. cordifolia (non Roxburgh) are referrable to P. serratifolia based on their similar morphological characteristics. However, our morphological investigation and ecological observations (Table 1) demonstrate that the original concept of P. cordifolia as published by Roxburgh57 is clearly distinct from P. serrtifolia. Specimens of P. cordifolia (sensu Roxburgh) examined from Thailand are listed in the Supplementary File, and additional selected specimens from Vietnam were also investigated to revise its distribution range (Clemens 3147 [K K001083294!], Clemens 4452 [K K001083319!], Evrard 44 [K K001083320!], Poilane 40706 [K K001083318!]).

Morphologically, P. cordifolia (sensu Roxburgh) differs from P. serratifolia in having cordate or rounded leaf bases (vs. mostly cuneate in P. serratifolia), glabrous vein axils on the lower half of the abaxial leaf surface (vs. densely pubescent), a corolla with a large yellow patch on the inside base of the middle lobe of anterior lip (vs. without a yellow patch), and a longer corolla, 5–7 mm long (vs. 2–5 mm long) (Figs. 2, 5). The study of fruit and endocarp morphology by Satthaphorn et al.50 demonstrated that P. cordifolia produces significantly larger fruits (5.32–6.65 × 4.25–5.66 mm) compared to those of P. serratifolia (3.33–4.89 × 2.58–2.99 mm). The endocarp protrusion of P. cordifolia is type II characterized by sharpened and tapered (thorn-like) protruding structure while P. serratifolia bears type I endocarps with rounded, swollen (saccate-like) protruding structure. Ecological observations and herbarium label data further indicate that P. cordifolia typically occurs in open or shaded primary or secondary evergreen forests, whereas P. serratifolia is usually found in exposed coastal and mangrove habitats (Figs. 5A, 6). Our phylogenetic analyses further support the phylogenetic distinction between P. cordifolia and P. serratifolia, placing them in well-supported separate subclades (Fig. 1). We therefore confirm that P. cordifolia (sensu Roxburgh)57 occurs in Thailand and clearly treated as a distinct species in the Flora of Thailand.

Premna cordifolia can be distinguished from other species regarded in the P. serratifolia complex in Thailand, namely P. paniculata and P. punctulata which are reinstated as distinct species in this study. One key diagnostic feature of P. cordifolia is the presence of interpetiolar ridges on the stem, a character absents in both P. paniculata and P. punctulata. The inflorescence of P. cordifolia is longer (4–8 cm) than that of P. paniculata (0.7–2 cm), though it overlaps in length with P. punctulata. In terms of floral morphology, P. cordifolia exhibits both a yellow patch on the corolla and hairs on the corolla tube, while these features are absent in P. paniculata. Fruit size also provides a distinguishing character, with P. cordifolia bearing larger fruits (5.32–6.65 × 4.25–5.66 mm) compared to P. paniculata (3.22–4.20 × 2.33–3.82 mm). Additionally, the shape of the endocarp protrusions differs: P. cordifolia possesses thorn-like protrusions, whereas P. punctulata exhibits a saccate-like form. Phylogenetic analysis reveals that P. cordifolia occupies a distinct position in subclade I, separate from P. punctulata, which is placed in subclade III. This integrative evidence, encompassing morphological and molecular data, supports the recognition of P. cordifolia (sensu Roxburgh) as a species distinct from both P. paniculata and P. punctulata.

Premna paniculata

H. R. Fletcher, Bull. Misc. Inform. Kew 1938: 201. 1938 & 421. 1938; Leerat. et al., Trop. Nat. Hist. 9(2): 135. 2009 & Thai Forest Bull. (Bot.) 44(2): 122. 2016. Type: Thailand, Prachuap Khiri Khan (Pak Tawan), elev. ca 20 m, 1 August 1931, A.F.G. Kerr 20,536 (lectotype BK BK257574!, selected by Leeratiwong et al.58; isolectotypes BM BM000950181!, E E00284177!, LL LL00375155!, K K001098063!, SING SING0068002!) (Fig. 3).

Fig. 3
Fig. 3
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Premna paniculata. (A, B) habit with fruiting branch; (C) close-up fruits. All photos by C. Leeratiwong.

Description: Scandent shrub to shrub, 2–4 m tall. Branches without an interpetiolar woody ridge, glabrous or brown to reddish-brown pubescent. Leaves decussate, elliptic or ovate-elliptic, 2–11 by (1–)1–5 cm, apex acute or acuminate, base cuneate, margins mostly entire or rarely slightly serrate distally; adaxial surface mostly glabrous, base and margin with sparse brown appressed hairs; abaxial surface glabrous or sometimes pubescent with sparse brown appressed hairs, with or without sparse yellow to brown sessile glands, nerve axils glabrous; petiole 2–5 mm long. Inflorescence corymbose, 0.7–2 cm long; pedicels 0.2–1.5 mm long. Calyx campanulate, 2–2.5 mm long, obscurely and equally 2-lipped; tube 1.5–2 mm long; posterior lip 3-lobed, lobes 0.3–0.8 mm long; anterior lip 2-lobed, lobes 0.4–1 mm long; fruiting calyx 2–3 mm long. Corolla greenish-white or white, 2-lipped, 3–5 mm long; tube 2–3 mm long, glabrous outside, white villous distally inside; posterior lip 1.2–1.5 mm long; anterior lip with middle lobe larger, 1–1.5 mm long, without yellow patch on middle lobe; lobes glabrous on both sides. Stamens long exserted, filaments 1–1.5 mm long; anthers 0.2–0.4 mm long. Ovary glabrous, with or without sparse sessile glands at apex; stylar branches 0.1–0.3 mm long. Fruits green when young, broadly obovoid, 4–6 mm long, glabrous, sparse sessile glands at apex.

Distribution: Endemic to Thailand (Phetchaburi, Prachuap Khiri Khan).

Ecology: In open areas of secondary dry evergreen forests; elevation 20–150 m. Flowering and fruiting June to August.

Vernacular: sak khi kai prachuap (สักขี้ไก่ประจวบ).

Note: Premna paniculata was listed as a synonym of P. serratifolia in the treatment of de Kok3. This inclusion was not explicitly justified, as the species was merely listed under P. serratifolia without further explanation. We presume that de Kok3 considered the type specimens of P. paniculata to represent morphological variation within P. serratifolia. Nevertheless, P. paniculata is geographically restricted to south-western Thailand. Based on comprehensive morphological analyses and ecological evidence, we recognize P. paniculata as a distinct species and reinstate its taxonomic status in this study. This conclusion is further supported by earlier studies8,28,50,58.

The type specimen of P. paniculata (Kerr 20,536 [K K001098063!]) together with three additional specimens (Leeratiwong 16–545 [PSU 01683!], Kerr 20,536 [K K001098063!], Niyomdham 2978 [BKF SN101085!]) can be morphologically distinguished from P. serratifolia by several diagnostic characters. These include the absence of an interpetiolar woody ridge at the stem nodes (vs. presence of an interpetiolar woody ridge in P. serratifolia), glabrous nerve axils on the lower half of the abaxial leaf surface (vs. densely hairy), a glabrous corolla tube externally (vs. distally pubescent), a shorter inflorescence (0.7–2 cm long vs. (0.5–)2–6 cm long), and a posterior calyx lip that is apically 3-lobed (vs. mostly entire to subentire, or rarely 2–3-lobed) (Figs. 3, 5). Endocarp morphology, as documented by Satthaphorn et al.50, further supports the taxonomic separation of these species: P. paniculata exhibits type II endocarps protrusion (thorn-like), whereas P. serratifolia bears type I endocarps protrusion (saccate-like). Additionally, these two species occupy distinct ecological niches, with P. paniculata confined to secondary dry evergreen forests, endemic to Thailand, in contrast to P. serratifolia, which is typically found in coastal or mangrove habitats in wider regions (Fig. 6). Although P. paniculata and P. punctulata are both found in evergreen forest habitats, they can be readily distinguished based on several morphological characters (Table 1). The leaf base of P. paniculata is cuneate, in contrast to the rounded or slightly oblique-rounded base observed in P. punctulata. On the abaxial leaf surface, the nerve axils of P. paniculata are glabrous, whereas those of P. punctulata are sparsely pubescent. Inflorescence length further differentiates the two species, with P. paniculata exhibiting notably shorter inflorescences (0.7–2 cm) compared to the longer ones in P. punctulata (6–20 cm). The posterior calyx lip of P. paniculata is distinctly apically 3-lobed, whereas it is entire to subentire in P. punctulata. Additionally, P. paniculata is also characterized by the absence of a yellow patch on the middle lobe of the anterior corolla lip, but a feature presents in P. punctulata. The endocarp protrusion type also serves as a key diagnostic trait: P. paniculata possesses thorn-like (type II) protrusions, while P. punctulata exhibits saccate-like (Type I) protrusions, as documented by Satthaphorn et al.50. These consistent morphological differences support the recognition of P. paniculata and P. punctulata as distinct species.

We attempted to include P. paniculata in the molecular analyses to clarify its taxonomic placement. However, DNA extraction proved highly challenging due to the limited availability of suitable material. Specimens available for study were mature leaves, and DNA isolated using both CTAB and a commercial extraction kit yielded poor-quality products that consistently failed to amplify. This difficulty is likely attributable to the high levels of polyphenols, polysaccharides, and other secondary metabolites typically accumulated in mature leaf tissues, which are known to interfere with DNA purity and inhibit enzymatic reactions59,60,61,62. Consequently, future studies should prioritize the use of young leaf material for P. paniculata to improve DNA quality and ensure successful molecular analyses.

To clarify the taxonomic status of P. paniculata, the combined evidence from morphological characteristics and ecological information as outlined above provides sufficient resolution to support the recognition as a distinct species from both P. punctulata and P. serratifolia. Accordingly, P. paniculata, which was previously treated as a synonym of P. serratifolia, is here reinstated.

Premna punctulata

C. B. Clarke in Hook.f., Fl. Brit. India 4: 575. 1885; King & Gamble, J. Asiat. Soc. Bengal, Pt. 2, Nat. Hist. 74: 817. 1909; H. J. Lam, Verbenaceae Malayan Archipel.: 121. 1919; Ridl., Fl. Malay Penins. 2: 620. 1923; Kochummen in Ng, Tree Fl. Malaya 3: 307. 1978; Keng, Con. Fl. Singapore: 194. 1990; Leerat. et al., Trop. Nat. Hist. 8(1): 9. 2008 & 9(2): 135. 2009.—Premna serratifolia sensu de Kok, Fl. Malesiana 23. 327. 2019, pro parte. Type: Malaysia, Malacca, 6 June 1865, Maingay Kew Distribution 1200 (holotype K K000645986!; isotype NY NY00137951!) (Fig. 4).

Fig. 4
Fig. 4
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Premna punctulata. (A) habit with flowering branch; (B) inflorescence; (C) closed-up inflorescence; (D) fruit. Photos A by J. Satthaphorn and B–D by C. Leeratiwong.

Description: Woody climber or rarely scandent shrub. Branches without an interpetiolar woody ridge, glabrous or brown to reddish-brown pubescent. Leaves decussate, ovate to broadly elliptic, 9–15 by 5–9 cm, apex acuminate, acute or rarely retuse, base rounded or slightly oblique-rounded, margins entire; adaxial surface glabrous, midrib sparsely pubescent; abaxial surface brown to reddish-brown pubescent, nerve axils sparsely pubescent, with brown sessile glands; petiole 3–6 cm long. Inflorescence corymbose, 6–20 cm long; pedicels 0.5–1.5 mm long. Calyx pale green to green, 1.5–2 mm long, equally and obscurely 2-lipped; tube 1.5–2 mm long; posterior lip entire to subentire (rarely 2–3-lobed); anterior lip 2-lobed, lobes 0.1–0.15 mm long; fruiting calyx 1–2.5 mm long. Corolla greenish-white or white, 2-lipped, 5–6 mm long; tube 2.7–3.5 mm long, glabrous or sparsely pubescent hairs distally outside, with densely white villous distally inside; posterior lip 1.5–1.7 mm long; anterior lip with middle lobe larger, 1.8–2 mm long, with yellow patch on middle lobe; lobes glabrous on both sides. Stamens long exserted, filaments 2–3 mm long; anthers 0.4–0.5 mm long. Ovary sparsely pubescent and with sessile glands at apex; stigma branches 0.15–0.3 mm long. Fruits black, broadly obovate, 3–7 mm long, glabrous, with or without sessile glands.

Distribution: Thailand (Phatthalung, Songkhla, Narathiwat), Malaysia.

Ecology: In primary to secondary evergreen or dry evergreen forests, along the waterfall; elevation up to 150 m. Flowering March to May, fruiting August.

Vernacular: ย่านใบหอม (yan bai hom) (Songkhla).

Note: Premna punctulata was treated as a synonym of P. serratifolia by de Kok3. However, there was no discussion or justification for this synonymization in the previous study. We infer that this decision was likely based on morphological similarities, as the type specimen of P. punctulata (Maingay 1200 [K K000645986!]) had been subsequently determined as P. serratifolia. Although the phylogenetic analyses presented here placed P. punctulata and P. serratifolia in an unresolved clade in both BI and ML trees (Fig. 1), the relatively long branch length separating P. punctulata suggests considerable genetic divergence53. This molecular distinction aligns with the consistent morphological and ecological difference observed between these two taxa.

Premna punctulata is morphologically distinguished from P. serratifolia by the absence of an interpetiolar woody ridge, a longer corolla, the presence of yellow patches on the middle lobe of the anterior lip of the corolla, and a hairy ovary, whereas P. serratifolia possesses an interpetiolar woody ridge, has a shorter corolla, lacks yellow corolla patches, and has a glabrous ovary (Figs. 4, 5). This taxonomic uncertainty was previously addressed by Satthaphorn et al.50, and demonstrated that the fruit width of P. punctulata is clearly distinct from that of P. serratifolia (3.79–5.69 mm wide vs 2.58–2.99 mm wide, respectively). Furthermore, the two taxa occupy different habitats: P. punctulata occurs in primary to secondary evergreen or dry evergreen forests, whereas P. serratifolia is restricted to coastal and mangrove environments.

Fig. 5
Fig. 5
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Premna serratifolia. (A) habit and habitat; (B) habit; (C) branches with leaves and fruits; (D) inflorescence; (E) closed-up inflorescence; (F) young fruits; (G) mature fruits. All photos by J. Satthaphorn.

The separation of these two taxa is further supported by recent phylogenetic studies conducted by Li et al.5 and Hai et al.17, as well as morphological evidence provided by Leeratiwong et al.8,30 and Satthaphorn et al.50. Based on the combination of consistent morphological distinctions, ecological differentiation, and the relatively long branch length separating P. punctulata from P. serratifolia in our phylogenetic analyses, P. punctulata is here reinstated as a distinct species.

Premna serratifolia L.

Mant. Pl. 2: 253. 1771; Roxb., Fl. Ind. 3: 77. 1832; Schauer in DC., Prodr. 11: 632. 1847; Kurz, Forest Fl. Burma 2: 262. 1877; Munir, J. Adelaide Bot. Gard. 7: 13. 1984; S.L. Chen & M. G. Gilbert in C. Y. Wu & P. H. Raven, Fl. China 17: 26. 1994; A. Rajendran & P. Daniel, Indian Verbenac.: 284. 2002; Leerat. et al., Nat. Hist. J. Chulalongkorn Univ. 9(2): 138. 2009; de Kok, Kew Bull. 68(1): 17. 2013; de Kok in Bramley, Bramley, Fl. Malesiana 23: 327. 2019.—Premna obtusifolia var. serratifolia (L.) Moldenke, Phytologia 28(4): 403. 1974.—Premna obtusifolia f. serratifolia (L.) Moldenke, Phytologia 36(5): 438. 1977; Moldenke & Moldenke in Dassanayake & Fosberg, Rev. Handb. Fl. Ceyl. 4: 342. 1983.Type: India, Hermann Herb. Linn. 782.4 (holotype LINN!) (Fig. 5).

Cornutia corymbosa Burm.f., Fl. Ind.: 132, t. 411, f. 1. 1768, nom. illeg. (non Lam).—Premna integrifolia L., Mant. Pl. Altera: 252. 1771, nom. superfl.—Premna corymbosa (Burm.f.) Schauer in DC., Prodr. 11: 632. 1847, nom. illeg. (non Rottler & Willd.).—Gumira integrifolia Kuntze, Revis. Gen. Pl. 2: 507. 1891, nom. superfl. Type: Sri Lanka [Ceylon], Hermann 2:1 (lectotype BM-HERM BM000621491!, designated by Verdcourt63).

Gumira domestica Hassk., Flora 25, 2, Beibl.: 26. 1842.—Gumira integrifolia Hassk., Cat. Hort. Bot. Bogor.: 135. 1844, nom. superfl. Type: Rumphius, Herb. Amboin. 3: 209, t 134. 1743.

Premna obtusifolia R. Br., Prodr. Fl. Nov. Holland.: 512. 1810.—Premna integrifolia L. var. obtusifolia (R.Br.) C. Pei, Mem. Sci. Soc. China 1: 75. 1932.—Premna corymbosa (Burm.f.) Schauer var. obtusifolia (R.Br.) H. R. Fletcher, Notes Roy. Bot. Gard. Edinburgh 19: 178. 1936. Type: Australia, Prince of Wales Island, Brown s.n. (= Bennett 2324) (holotype BM BM000019556!).

Premna spinosa Roxb., [Hort. Beng.: 46. 1814, nom. nud.] Fl. Ind. 3: 77. 1832; Walp., Report. Bot. Syst. 4: 93. 1845. Type: Roxburgh illustration no. 961 (holotype K!).

Premna foetida Reinw. ex Blume, Bijdr. 14: 816. 1826.—Gumira foetida (Reinw. ex Blume) Hassk., Cat. Hort. Bot. Bogor. 1844; H. J. Lam, Verbenaceae Malayan Archipel.: 153. 1919. Syntypes: Indonesia, Java Reinwardt s.n. (L L0699518!, L0699519!).

Premna gaudichaudii Schauer in DC., Prodr. 11: 631. 1847.—P. obtusifolia var. gaudichaudii (Schauer) Moldenke, Phytologia 27: 69. 1973. Type: Archipelago, Mariannae, 1830, Gaudichuad s.n. (holotype G-DC, n.v.).

Premna sambucina Wall. [Cat. No. 1775, nom. nud.] ex Schauer in DC., Prodr. 11: 631. 1847; Kurz, For. Fl. Burma 2: 261. 1877.—P. corymbosa var. sambucina (Wall. ex Schauer) Moldenke, Known Geogr. Distr. Verbenac. Avicenn.: 68. 1942. Type: India, Moalmyn, 1827, Wallich, Cat. No. 1775 (holotype G-DC, n.v.; isotype K K001114145!).

Premna integrifolia L. var. angustior C. B. Clarke in Hook.f., Fl. Brit. India 4: 574. 1885.—Premna angustior (C. B. Clarke) Ridl., Fl. Malay Penins. 2: 619. 1923.—Premna corymbosa (Burm. f.) Schauer var. angustior (C. B. Clarke) H. R. Fletcher, Notes Roy. Bot. Gard. Edinburgh 19: 178. 1936.—Premna obtusifolia R.Br. var. angustior (C. B. Clarke) Moldenke, Phytologia 5: 87. 1954. Syntypes: Malaysia, Malacca, Griffith 6030 (K K000645926!, K000645927!).

Premna integrifolia L. subsp. truncatolabium H. J. Lam, Verbenaceae Malayan Archipel.: 142. 1919. Type: Papua New Guinea, Kaiser Wilhelmsland, Sepik, Lederman 6572 (lectotype K K000670846!, designated by De Kok3).

Premna integrifolia L. var. minor Ridl., Fl. Malay Penins. 2: 619. 1923.—Premna obtusifolia R. Br. var. minor (Ridl.) Moldenke, Phytologia 5: 88. 1954.—Premna corymbosa (Burm. f.) Schauer var. minor (Ridl.) H. R. Fletcher, Notes Roy. Bot. Gard. Edinburgh 19: 178. 1936. Type: Malaysia, Pahang, Pekan, Ridley s.n. (holotype SING, n.v.).

Description: Scandent shrub or shrub, 0.2–5 m high. Branches with an interpetiolar woody ridge, glabrous or brown to reddish-brown pubescent. Leaves decussate, elliptic, ovate or obovate, (0.5–)2–15 by (0.3–)1.2–10 cm, apex mostly acute or obtuse, rarely acuminate or retuse, leaf base usually cuneate, rarely slightly cordate or rounded, margins entire or serrate distally; adaxial surface glabrous, margins with sparse brown hairs; abaxial surface glabrous or sparse brown pubescent, nerve axils densely pubescent on lower half, with yellow to brown sessile glands; petiole (0.4–)0.8–5.5 cm long. Inflorescence corymbose, 1.7–15 cm long; pedicels 0.2–1.5 mm long. Calyx green, (1–)1.5–2.5 mm long, unequally 2-lipped; tube 1–1.8 mm long; posterior lip mostly entire to subentire or rarely 2–3-lobed, lobes 0.05–0.1 mm; anterior lip 2-lobed, lobes 0.3–1 mm long; fruiting calyx 2–3 mm long. Corolla creamy-white or white, 2-lipped, (2–)3.5–5 mm long; tube (1.8–)2.5–3 mm long, pubescent or sparsely so, with yellow sessile glands at apex outside, densely white villous distally inside; posterior lip 1.2–1.6 mm long; anterior lip with lobes subequal; middle lobe 1.2–2 mm long, without yellow patch on middle lobe; lobes glabrous or sparsely pubescent, with sparse sessile glands outside, glabrous inside. Stamens long exserted, filaments 1–3 mm long; anthers 0.3–0.6 mm long. Ovary 0.5–1 mm long, glabrous, with or without sessile glands; stigma branches 0.05–0.15 mm long. Fruits black, broadly obovoid, 3–8 mm long, glabrous.

Distribution: Thailand (Krungthep Mahanakhon (Bangkok), Samut Sakhon, Chon buri, Rayong, Chantaburi, Trat, Chumphon, Ranong, Suratthani, Phangnga, Phuket, Krabi, Trang, Satun, Songkhla, Narathiwat). Widely distributed throughout the coasts and the islands of tropical and subtropical Asia, Africa, Australia and the Pacific.

Ecology: In open, sandy soil or wetland areas of beach forests and mangrove forests, or strand vegetation along the coastal areas and islands, in tidal zone; elevation from sea level up to 80 m. Flowering and fruiting all year round.

Vernacular: Khet nam man ((เค็ดน้ำมัน), cha lueat (ช้าเลือด) (Trat), man kai (มันไก่), sam pra nga (สามประงา)), sam pra nga bai (สามประงาใบ), akkhi thawan thale (อัคคีทวารทะเล).

Note: Premna serratifolia is a widely distributed species across Southeast Asia. In recent taxonomic revisions in specific regions, numerous synonyms have been associated with this species, particularly from type specimens originating in the Malay Archipelago3. In our study, we focus on defining the species circumscription of P. serratifolia in Thailand, providing a comprehensive morphological description while excluding the concepts of the reinstated species P. paniculata and P. punctulata. The distinguishing morphological features are summarized in Table 1.

Ecologically, P. serratifolia typically inhabits beach forests or coastal habitats, clearly differing from the moist evergreen forest habitats associated with the previously synonymized species (Figs. 5, 6). Field observations indicate that the leaves of P. serratifolia are notably thick leaves and cuticle, likely as an adaptation to protect against salt spray and strong wind from the sea64,65,66. This species may be actively adapted its seed germination facilitated by monsoon climate, mobile substrates, and salinized64,67, while other species may require different mode of seed dispersal and germination. In addition, phylogenetic analysis places three accessions of P. serratifolia, sampled from the opposite sides of peninsular Thailand, within subclade III of Clade II (Fig. 1) which provide the information of phylogenetic placement with its related species.

Fig. 6
Fig. 6
Full size image

Distribution map of P. cordifolia, P. paniculata, P. punctulata, and P. serratifolia in Thailand. Numbers near locations refer to the collections cited in Supplementary File.

Although the species circumscription of P. serratifolia in Thailand and its morphologically related species have been clarified in this study, further investigation across a broader geographic range is still required to assess the extent of morphological variation and confirm species delimitation at a monographic scale.

Taxonomic note on Premna octonervia in Thailand

Premna octonervia was listed as a distinct species in the synopsis for Thailand by Leeratiwong et al.8 based on multiple collections from peninsular Thailand, including Kerr 13309 (K K001084832!), Puudjaa et al. 251 (K K00108481!), Chana et al. 251 (BKF SN064958!, KKU 15247!), Leeratiwong 04–53 (BKF SN232004!, KKU 15246!, PSU 0013431!), and Leeratiwong 05–251 (BKF SN232005!, PSU 0013430!). Our re-investigation showed that these specimens is consistent with the original description of P. mollissima rather than P. octonervia. Meanwhile, the actual identity of P. octonervia can be attributed to P. serratifolia, as previously suggested by de Kok3. The dried specimens exhibit an ebony color and a lower density of hairs on the leaf surfaces, slightly distinguishing from those found in other regions, where dried specimens are typically yellowish-brown to yellowish-black and have moderately to densely hairy leaf surfaces. This difference may be attributed to environmental influences affecting the secondary compounds within southern Thailand population.

Taxonomic note on Premna coriacea in Thailand

The specimens identified as P. coriacea (non C. B. Clarke) and P. coriacea var. villosa (non Rajendran & Daniel) were included to Thai account by Leeratiwong et al.8. Our morphological revision supports these two taxa are P. scandens. The actual taxa of P. coriacea (lectotype Law s.n., K K000884638!) and P. coriacea var. villosa (lectotype s.coll. 2912; selected here, K K000884633!) are restricted to the Indian subcontinent and do not occur in Thailand.

In the protologue of P. coriacea var. villosa (C. B. Clarke) A. Rajendran & P. Daniel [= P. villosa C. B. Clarke in Flora of British India by Hooker68], two specimens were cited in the original publication without the designation of the holotype. These two specimens (s.coll. 2912, [K K000884633!] and Beddome s.n. [K K000884632!]) could be considered as syntypes. We therefore selected the specimen of s.coll. 2912 [K000884633] to be the lectotype of the synonymized taxon due to the specimen being cited in the protologue as well as having complete vegetative and reproductive parts.

Taxonomic note on Premna interrupta in Thailand

Premna interrupta Wall. ex Schauer, in A. P. de Candolle, Prodr. 11: 633. 1847.—Premna interrupta var. smitinandii Moldenke, Phytologia 8: 163. 1962, syn. nov.

Leeratiwong et al.8 recognized P. interrupta var. smitinandii based on the presence of villose hairs on the abaxial leaf surface (vs. glabrous leaf in typical variety). This variety was established based on the single collection of Smitinand & Alsterlund 6783 [holotype LL LL00375145!; isotypes K K001084815!, L L0414245!, L0414256!]. After investigation of various specimens belonging to P. interrupta and the type specimen of P. interrupta var. smitinandii, we found that the density of the indumentum on an abaxial leaf surface of P. interrupta var. interrupta is variable among different populations from absent to densely hairy, and leaf maturity. Therefore, we do not recognize P. interrupta var. smitinandii here.

An updated accounted and revised key to species in Thailand

Our taxonomic treatment and notes have resulted in nomenclatural changes for Premna in Thailand, as summarized in Table 2. A total of 21 species are recognized in Thailand, and a revised key to the species is provided below.

Table 2 The updated account of Premna in Thailand.
Full size table

Key to species of Premna in Thailand

  1. 1

    Leaf surfaces with stellate and dendroid hairs..P. tomentosa

  2. 1′

    Leaf surfaces without stellate and dendroid hairs..2

  3. 2

    Calyx deeply lobed, with 1– 1.8 mm long..P. serrata

  4. 2′

    Calyx shallowly lobed or entire, with shorter than 1 mm long..3

  5. 3

    Herb to undershrub with large woody rhizome..4

  6. 3′

    Tree, shrub (0.2–)2–5 m high, scandent shrub or woody climber..5

  7. 4

    Calyx 1.5–2.5 mm long without glandular hairs; corolla 3–5 mm long; style less than 3.5 mm long, inserted; abaxial surface of leaves glabrous to sparsely scabrid and pubescent with eglandular hairs..P. herbacea

  8. 4′

    Calyx 3–4 mm long with glandular hairs; corolla 5–7 mm long; style 3.5–6 mm long, exserted; abaxial surface of leaves densely villous sometimes mixed with glandular or pubescent hairs..P. nana

  9. 5

    Fruits clavoid (narrowly club-shaped)..6

  10. 5′

    Fruits narrowly obovoid to broadly obovoid..10

  11. 6

    Inflorescence a spike-like thyrsi.. P. interrupta

  12. 6′

    Inflorescence a corymbose thyrsi..7

  13. 7

    Ovary pubescent; abaxial leaf surface densely villous..8

  14. 7′

    Ovary glabrous; abaxial leaf surface glabrous or sparsely to moderately pubescent..9

  15. 8

    Calyx lips entire or one lip with two lobes, the other entire or subentire; fruit 4–6 mm long, moderately or sparsely pubescent..P. garrettii

  16. 8′

    Calyx lips with two lobes; fruit 2–4 mm long, glabrous..P. siamensis.9Stigma branches 0.5–1 mm long; flowers usually sessile or rarely pedicellate..P. trichostoma

  17. 9

    Stigma branches 0.5–1 mm long; flowers usually sessile or rarely pedicellate..P. trichostoma

  18. 9′

    Stigma branches 0.2–0.4 mm long; flowers only pedicellate..P. scandens

  19. 10

    Corolla red or pinkish-red..P. stenobotrys

  20. 10′

    Corolla white, greenish-white, yellowish-white, yellow, pale yellow, creamy white or greenish-yellow..11

  21. 11

    Both leaf surfaces with reddish-brown to orangish-red sessile glands mixed with yellow to brown sessile glands..P. rabakensis

  22. 11′

    Both leaf surfaces with yellow to brown sessile glands, without reddish-brown to orangish-red sessile glands..12

  23. 12

    Branch nodes with an interpetiolar woody ridge..13

  24. 12′

    Branch nodes without an interpetiolar woody ridge..17

  25. 13

    Calyx apically purple, fruits narrowly obovoid..P. annulata

  26. 13′

    Calyx apically green, fruits broadly obovoid..14

  27. 14

    Posterior lip of calyx mostly 3-lobed, rarely 2-lobed or subentire..15

  28. 14′

    Posterior lip of calyx mostly entire to rarely 2–3-lobed..16

  29. 15

    Adaxial leaf surface and midribs with villous hairs; corolla with yellow patch at the inside base of middle lobe of anterior lip..P. odorata

  30. 15′

    Adaxial leaf surface glabrous, midribs pubescent; corolla without yellow patch at the inside base of middle lobe of anterior lip..P. pubescens

  31. 16

    Corolla 5–7 mm long, with yellow patch on inside base of middle lobe of anterior lip; leaf base cordate or rounded; nerve axils of lower half of abaxial leaf surface glabrous to sparsely pubescent..P. cordifolia

  32. 16′

    Corolla 2–5 mm long, without yellow patch on inside base of middle lobe of anterior lip; leaf bases usually cuneate; nerve axils of lower half of abaxial leaf surface densely pubescent..P. serratifolia

  33. 17

    Inside of middle lobe of anterior lip covered with villous hairs along base to apex..P. mollissima

  34. 17′

    Inside of middle lobe of anterior lip glabrous or only covered with villous hairs at the base, other parts glabrous or pubescent..18

  35. 18

    Leaf margin deeply lobed..P. repens

  36. 18′

    Leaf margin entire or shallowly lobed..19

  37. 19

    Branches and abaxial leaf surface yellowish-brown villous; fruits apically pubescent, 7–10 mm long..P. fulva

  38. 19′

    Branches and abaxial leaf surface glabrous or brown to reddish-brown pubescent; fruits glabrous, shorter or equal to 7 mm long..20

  39. 20

    Abaxial leaf surface without hairs on nerve axils; posterior lip of calyx 3-lobed; inflorescences 0.7–2 cm long..P. paniculata

  40. 20′

    Abaxial leaf surface with hairs on nerve axils; posterior lip of calyx entire to subentire or rarely 2–3-lobed; inflorescences 6–20 cm..P. punctulata

Conclusion

Species of Premna in Thailand included in this study constitute a monophyletic group based on four chloroplast markers (ndhF, rbcL, rps16, and trnL-F). Two clades are recognized based on phylogenetic relationships and morphological affinities. Our findings have resolved the taxonomic confusion of previously synonymized species within the P. serratifolia complex. Premna paniculata, and P. punctulata are reinstated to the species level and P. cordifolia is validly published as a distinct species, included in the account for Thailand. Premna coriacea does not occur in Thailand, and P. octonervia and P. interrupta var. smitinandii are not recognized here. Premna coriacea var. villosa, a taxon previously thought to be found in Thailand, is lectotypified. Consequently, 21 species have been recognized in the account being prepared for the Flora of Thailand. Further work is required to increase samples to construct a more inclusive sample of species Premna to provide the basis for an infrageneric classification and biogeographical study for a better understanding the natural history of Premna.

Methods

Morphological and ecological investigation

This study is based on field collections from natural habitats during field works in Thailand during 2022 and 2025. All experimental research, field studies involving wild plants, and the collection of plant materials were conducted in strict accordance with relevant institutional, national, and international regulations and guidelines. All plant specimens newly collected for this study were obtained from natural areas located outside officially designated protected zones. Fresh leaves were collected and dried with silica gel, and voucher specimens were deposited at herbarium of Prince of Songkla University (PSU) for further morphological investigations. Herbarium specimens were examined as well as digital image databases from the following herbaria: BKF, BM, CAL, E, K, KKU, L, LL, LINN, NY, PSU, and SING (acronyms follow the Index Herbariorum (Thiers, 2025)). Some type specimens were also examined thorough the Global Plants on JSTOR (https://plants.jstor.org/) and the Global Biodiversity Information Facility (https://www.gbif.org). The ecological information was obtained from the specimen labels and was noted from natural populations during field works. Formal plant identification in this study was performed by J. S., B. L., and C. L.

Taxon sampling and datasets

The samples for phylogenetic analyses of Premna in Thailand were based on the taxonomic account of Leeratiwong et al.8,58. Eighteen out of 21 species of Premna in Thailand were included in this study, covering approximately 86% species diversity of the genus according to Leeratiwong et al.8. Of this total number of species in Thailand, we excluded P. coriacea [sensu Leeratiwong et al.8 (non C. B. Clarke)] and P. octonervia due to this species being treated as a synonym of P. scandens and P. serratifolia, respectively (see Taxonomic notes). Additional species from neighboring floristic regions were also included in the analyses (i.e., P. bracteata, P. ligustroides, P. menglaensis, P. mekongensis, P. punicea, P. szemaoensis, P. tenii, and P. vietnamensis). Totally, 30 accessions of Premna were sampled from either herbarium specimens or freshly collected silica-dried leaf materials as well as the GenBank database (Supplementary Table S1). Two outgroups from the genus Gmelina, belonging to the subfamily Premnoideae, were selected based on the recent classification and previous molecular studies (e.g. Li et al.5, Zhao et al.6, Rose et al.20), consisting of G. elliptica Sm. and G. philippensis Cham.

Our study selected four chloroplast regions (cpDNA) for constructing phylogenetic reconstruction of Premna in Thailand, including ndhF, rbcL, rps16, trnL-F. This selection of each marker was based on selected previous phylogenetic analyses widely used across different genera within Lamiaceae, and species of Premna which have been sequenced available in GenBank database5,17,19,34.

DNA extraction, PCR amplification and sequencing

The genomic DNA from silica dried leaf tissue was extracted by the 2 × CTAB method of Doyle and Dolye69 and DNA from herbarium specimens was isolated by using DNEasy® Plant Mini Kit (QIAGEN®, Valencia, California, USA), following the manufacturer’s protocols. The extracted DNA was dissolved in TE buffer and preserved at -20 ̊C for further application and was checked for quality using a spectrophotometer (Thermo Scientific NanoDrop™ 1000, USA) and agarose. The Polymerase Chain Reaction (PCR) was performed to amplify four chloroplast markers, using primers nhdF70, rbcL71, rps1672, and trnL-F73. The reaction contains 2 µL sample DNA, 5 µL ExcelTaq™ 5X PCR Master Dye Mix (SMOBIO Technology, Inc., Taiwan), 1 µL of each primer in a final reaction volume of 25 µL (Bio-Rad PCR Thermal Cycler, Model T100, Thailand). The uniform primer conditions were adapted from Li et al.5 and Hai et al.17 (an initial denaturation at 94 °C for 5 min, 35 cycles of 30 s denaturation at 94 °C, 1 min primer annealing at 52 °C, 2 min extension at 72 °C, with a final extension of 5 min at 72 °C). PCR products were visualized using agarose gel electrophoresis and were sequenced by Macrogen Inc. (South Korea), using the same primers for the PCR amplification.

Alignment and phylogenetic analyses

Sequences were assembled and edited using Geneious Prime® 2024.0.374 (Biomatters Ltd., Auckland, New Zealand [http://www.geneious.com/]) and were verified using the BLAST (Basic Local Alignment Search Tool) algorithm. The sequences were then aligned by MAFFT v7.49075 to analyze biological data. The aligned dataset was determined as the best-fitting nucleotide substitution model using jModelTest software version 2.1676. The concatenated datasets from four chloroplast markers were analyzed using Maximum likelihood (ML) and Bayesian Inference (BI) methods with gaps treated as simple indels. ML analysis was performed using the phylogenetic IQ-TREE 277 with 1000 ultrafast bootstraps using the ‘-B’ option. Support for nodes was estimated using 1000 replicates for ultrafast bootstrapping78. BI was analyzed using MrBayes version 3.2.279. Four Markov Chain Monte Carlo (MCMC) chains were run for 10 million generations and sampled every 10,000 generations. The first 25% was discarded as burn-in. The phylogenetic trees from both analyses were visualized using FigTree version 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). The consensus tree representing the overall relationships was based on a Bayesian 50% majority-rule consensus tree.