Drepananthus khaosoi sp. nov. (Annonaceae) from southern Thailand, with molecular phylogenetic reconstructions

Molecular phylogenetic analyses

The ingroup was composed of 32 accessions: 30 accessions belonging to Canangeae (three of Cananga, eight of Cyathocalyx, 18 of Drepananthus (including the unidentified accession, Drepananthus sp.) and one of Lettowianthus) and two belonging to Tetramerantheae (one of Cleistopholis Pierre ex Engl. and one of Mezzettia Becc.). Two accessions of Drepananthus (D. pruniferus and the unidentified accession) were newly sequenced in this study. Outgroups consisted of Meiocarpidium oliverianum (Baill.) D.M.Johnson & N.A.Murray (Meiocarpidioideae) and Annickia pilosa (Exell) Setten & Maas (a representative of Malmeoideae). Voucher information of all accessions, including GenBank accession numbers are shown in Appendix 1. Up to six plastid DNA regions (matK and rbcL exons; trnL intron; atpB-rbcL, psbA-trnH and trnL-trnF intergenic spacers) were used. The methods for DNA extraction, amplification and sequencing used in the present study, including primer information, followed Chaowasku & al. (2018Chaowasku T., DamthongdeeA., JongsookH., NgoD.T., LeH.T., TranD.M. & SuddeeS.2018. Enlarging the monotypic Monocarpieae (Annonaceae, Malmeoideae): recognition of a second genus from Vietnam informed by morphology and molecular phylogenetics. Candollea73: 261–275., 2020) and Chaowasku (2020Chaowasku T., AongyongK., DamthongdeeA., JongsookH. & JohnsonD.M.2020. Generic status of Winitia (Annonaceae, Miliuseae) reaffirmed by molecular phylogenetic analysis, including a new species and a new combination from Thailand. European Journal of Taxonomy659: 1–23.). Sequences were edited using the Staden package (Staden & al. 2000StadenR., BealK.F. & BonfieldJ.K.2000. The Staden Package, 1998. InMisenerS. & KrawetzS.A. (eds.), Bioinformatics methods and protocols: 115–130. Humana Press, Totowa. [Methods in Molecular Biology 132].) and then aligned using MUSCLE (Edgar 2004EdgarR.C.2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research32: 1792–1797.) in MEGA11 (Tamura & al. 2021TamuraK., StecherG. & KumarS.2021. MEGA11: molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution38: 3022–3027.). The alignments were subsequently checked manually and adjusted where necessary based on the similarity criterion (Simmons 2004SimmonsM.P.2004. Independence of alignment and tree search. Molecular Phylogenetics and Evolution31: 874–879.). In some psbA-trnH intergenic spacer sequences, there was an inversion of 15 continuous nucleotides and this was reversed complementarily to be alignable to the remaining sequences, following Pirie & al. (2006PirieM.D., ChatrouL.W., MolsJ.B., ErkensR.H.J. & OosterhofJ.2006. ‘Andean-centred’ genera in the short-branch clade of Annonaceae: testing biogeographical hypotheses using phylogeny reconstruction and molecular dating. Journal of Biogeography33: 31–46.). The total 3,767-nucleotide alignment plus seven binary-coded indel characters were included in the analysis. Indel coding followed the simple method of Simmons & Ochoterena (2000SimmonsM.P. & OchoterenaH.2000. Gaps as characters in sequence-based phylogenetic analyses. Systematic Biology49: 369–381.), with emphasis on less homoplastic and non-autapomorphic indel structures.

Parsimony analysis was performed in TNT version 1.5 (Goloboff & Catalano 2016GoloboffP.A. & CatalanoS.A.2016. TNT version 1.5, including a full implementation of phylogenetic morphometrics. Cladistics32: 221–238.). All characters were equally weighted and unordered. The setting of collapsing rules was set to “max. length = 0”. Incongruence among chloroplast DNA regions was assessed by analyzing each region individually to detect if there was any significant topological conflict (e.g., Wiens 1998WiensJ.J.1998. Combining data sets with different phylogenetic histories. Systematic Biology47: 568–581.). Most parsimonious trees were generated by a heuristic search of the combined data, with 9000 replicates of random sequence addition, saving 10 trees per replicate and using the tree bisection and reconnection (TBR) branch-swapping algorithm. Clade support was measured by symmetric resampling (SR; Goloboff & al. 2003GoloboffP.A., FarrisJ.S., KällersjöM., OxelmanB., RamírezM.J. & SzumikC.A.2003. Improvements to resampling measures of group support. Cladistics19: 324–332.). A default change probability (P = 33) was used. Two hundred thousand replicates were run, each with four replicates of random sequence addition, saving four trees per replicate. A clade with SR ≥ 85%, 70–84% or 50–69% was considered strongly, moderately or weakly supported, respectively.

Maximum likelihood analysis was carried out in IQTREE version 2.1.3 (Minh & al. 2020MinhB.Q., SchmidtH.A., ChernomorO., SchrempfD., WoodhamsM.D., von Haeseler A. & LanfearR.2020. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Molecular Biology and>Evolution37: 1530–1534.) under partition models (Chernomor & al. 2016ChernomorO., von Haeseler A. & MinhB.Q.2016. Terrace aware data structure for phylogenomic inference from supermatrices. Systematic Biology65: 997–1008.) implemented with the “-p” command, whereas Bayesian Markov Chain Monte Carlo (MCMC; Yang & Rannala 1997YangZ. & RannalaB.1997. Bayesian phylogenetic inference using DNA sequences: a Markov Chain Monte Carlo method. Molecular Biology and Evolution14: 717–724.) phylogenetic analysis was performed in MrBayes version 3.2.7a (Ronquist & al. 2012RonquistF., TeslenkoM., van der MarkP., AyresD.L., DarlingA., HöhnaS., LargetB., LiuL., SuchardM.A. & HuelsenbeckJ.P.2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology61: 539–542.) via the CIPRES Science Gateway version 3.3 (Miller & al. 2010MillerM.A., PfeifferW. & SchwartzT.2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. InProceedings of the Gateway Computing Environments Workshop (GCE): 1–8. IEEE, Piscataway.). The aligned data matrix was divided into five partitions based on identity of DNA regions (the trnL intron and adjacent trnL-trnF intergenic spacer were combined as a single partition = trnL-F). The most suitable model of sequence evolution for each DNA partition was chosen by the Akaike Information Criterion (AIC; Akaike 1974AkaikeH.1974. A new look at the statistical model identification. IEEE Transactions on Automatic Control19: 716–723.) scores, using jModelTest version 2.1.10 (Darriba & al. 2012DarribaD., TaboadaG.L., DoalloR. & PosadaD.2012. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods9: 772–772.), with the following selections: +F, +G (nCat 4), ML optimized (base tree for likelihood calculations) and Best (base tree search). The General Time Reversible (GTR; Tavaré 1986TavaréS.1986. Some probabilistic and statistical problems in the analysis of DNA sequences. Lectures on Mathematics in the Life Sciences17: 57–86.) substitution model with a gamma distribution for among-site rate variation (Γ) was selected for three partitions (atpB-rbcL, psbA-trnH and trnL-F), the GTR substitution model without Γ for one partition (matK) and the Hasegawa-Kishino-Yano (HKY; Hasegawa & al. 1985HasegawaM., KishinoH. & YanoT.1985. Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. Journal of Molecular Evolution22: 160–174.) substitution model with Γ for the remaining partition (rbcL). In the maximum likelihood analysis, the model “JC2+FQ+ASC” was selected by the corrected AIC scores for the binary indel partition. Clade support was evaluated by a non-parametric bootstrap resampling (BS; Felsenstein 1985FelsensteinJ.1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution39: 783–791.) with 2000 replicates. A clade with BS ≥ 85%, 70–84% or 50– 69% was considered strongly, moderately or weakly supported, respectively. In the Bayesian analysis, the setting “coding = variable” was applied for the binary indel partition (employed under a simple F81-like model without Γ). Four independent runs, each using four MCMC chains, were simultaneously executed; each run was set for 10 million generations. The default prior settings were used except for the prior parameter of rate multiplier (“ratepr” [= variable]). The temperature parameter was set to 0.08. Trees and all parameter values were sampled every 1000^th generation. Convergence was assessed by checking the standard deviation of split frequencies of the runs with values < 0.01 interpreted as indicative of a good convergence and by checking for adequate effective sample sizes (ESS > 200) using Tracer version 1.7.1 (Rambaut & al. 2018RambautA., DrummondA.J., XieD., BaeleG. & SuchardM.A.2018. Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Systematic Biology67: 901–904.). The first 25% of all trees sampled were removed as burn-in and the 50% majority-rule consensus tree was generated from the remaining trees. A clade with posterior probabilities (PP) ≥ 0.95, 0.9–0.94 or 0.5–0.89 was considered strongly supported, weakly supported or unsupported, respectively.

Phylogenetic relationships and morphological comparisons

The parsimony analysis generated 120 most parsimonious trees with 510 steps. The consistency and retention indices (CI and RI) were 0.85 and 0.87, respectively. There was no strong conflict (SR ≥ 85%) among the analyses of each chloroplast DNA region. As shown on the phylogenetic tree (Fig. 1), the ingroup (Ambavioideae s.s.) as well as the tribe Tetramerantheae was monophyletic with maximum support. Recovered as a sister group of Tetramerantheae, the tribe Canangeae received strong support (SR = 99%, BS = 100%, PP = 1). In Canangeae, Lettowianthus was sister to a strongly supported (SR = 99%, BS = 100%, PP = 1) clade composed of the maximally supported Cananga, the strongly supported (SR = 99%, BS = 100%, PP = 1) Cyathocalyx and the moderately to strongly supported (SR = 91%, BS = 76%, PP = 1) Drepananthus.Cyathocalyx and Drepananthus appeared to be sister groups with no support to weak support (SR = 61%, BS = 64%, PP = 0.66). The unidentified accession of Drepananthus (= Drepananthus sp.) belonged to an unsupported to weakly supported (SR < 50%, BS < 50%, PP = 0.93) clade that also included four other members: D. biovulatus (Boerl.) Survesw. & R.M.K.Saunders, D. hexagynus (Miq.) Survesw. & R.M.K.Saunders, D. pubescens and D. ridleyi. Similar to Surveswaran & al. (2010SurveswaranS., WangR.J., SuY.C.F. & SaundersR.M.K.2010. Generic delimitation and historical biogeography in the early‐divergent ‘ambavioid’ lineage of Annonaceae: Cananga, Cyathocalyx and Drepananthus. Taxon59: 1721–1734.), the phylogenetic relationships in Drepananthus herein depicted were also largely unresolved.

Fig. 1 Fifty percent majority-rule consensus phylogram derived from Bayesian inference. Parsimony symmetric resampling (SR) percentages on the left; maximum likelihood bootstrap (BS) percentages in the middle; Bayesian posterior probabilities (PP) on the right; ** = < 50%; scale bar unit = substitutions per site. 

On the basis of morphological comparisons, the unidentified Drepananthus species is most similar to D. ridleyi (native to Peninsular Malaysia, Singapore and Borneo; Turner 2018TurnerI.M.2018. Annonaceae of the Asia-Pacific region: names, types and distributions. Gardens’ Bulletin Singapore70: 409–744.). The two can be distinguished by several features as shown in Table 1. Drepananthus sp. also somewhat resembles D. pubescens (native to Peninsular Malaysia and Sumatra; Scheffer 1881SchefferR.H.C.C.1881. Sur quelques plantes nouvelles ou peu connues de l’Archipel Indien. Annales du Jardin botanique de Buitenzorg2: 1–31.; Turner 2018TurnerI.M.2018. Annonaceae of the Asia-Pacific region: names, types and distributions. Gardens’ Bulletin Singapore70: 409–744.), but a number of traits separate them (Table 1). Based on these findings, we consider the unidentified Drepananthus species deserves recognition as a new species, which is described below (= D. khaosoi sp. nov.). Although the plastid DNA regions sampled in this study fail to provide a resolved phylogenetic hypothesis in Drepananthus and a sister group of the new species, morphological comparisons show it is clearly distinct. Clarifying the relationships of this new species will nonetheless require a more comprehensive phylogenetic analysis, possibly using high-throughput sequencing data (e.g., Couvreur & al. 2019CouvreurT.L.P., HelmstetterA.J., KoenenE.J.M., BethuneK., BrandãoR.D., LittleS.A., SauquetH. & ErkensR.H.J.2019. Phylogenomics of the major tropical plant family Annonaceae using targeted enrichment of nuclear genes. Frontiers in Plant Science9: 1941.).

The difference in pericarp thickness between D. ridleyi and D. khaosoi sp. nov. could be associated with different dispersers. In Drepananthus, terrestrial mammals, including fruit bats and large frugivory birds such as hornbills are likely to play an important role in seed dispersal because the monocarps are usually medium-sized and display various colors at maturity, including red (Wang 2004WangR.J.2004. Systematics and phylogeny of Cyathocalyx (Annonaceae). Ph.D. dissertation, the University of Hong Kong, Hong Kong.). Further studies are needed to verify how the seeds of the new species are dispersed.

Taxonomic treatment

Drepananthus khaosoi Damth. & Chaowasku, sp. nov. Type: Thailand, Narathiwat Province, Sukhirin District, elevation c. 150 m, 16 Mar. 2021, Chanthamrong & Baka 58 (holotype: CMUB [CMUB003998901]; isotypes: BK, CMUB, QBG), fl. & fr. Figs. 2–4.

Fig. 2 Drepananthus khaosoi Damth. & Chaowasku, sp. nov.: a, inflorescences and flowers; b, flower with petal blades removed, bottom view, particularly showing abaxial side of sepals; c, outer petal claw (above): abaxial (left) and adaxial (right) sides; inner petal claw (below): abaxial (left) and adaxial (right) sides; d, flower with petals, one sepal and stamens removed, side view, particularly showing adaxial side of sepals and carpels on torus; e, stamen (middle: adaxial side; right: abaxial side) and carpel (left); f, fruit with monocarps [all, Chanthamrong & Baka 58 (CMUB)]; photos: A. Baka (a, f)]. 

Fig. 3 Holotype of Drepananthus khaosoi Damth. & Chaowasku, sp. nov. [Chanthamrong & Baka 58 (CMUB)]. 

Fig. 4 Enlargement of another specimen [isotype] of Drepananthus khaosoi Damth. & Chaowasku, sp. nov., showing dried monocarps without constrictions between seeds [Chanthamrong & Baka 58 (CMUB)]. 

Diagnosis.––The new species is morphologically most similar to D. ridleyi, but differs from it by having larger leaf blade, cordate to rounded-subcordate (vs. cuneate, occasionally ± obtuse) leaf base, subglobose to ellipsoid (vs. ellipsoid-cylindrical to cylindrical) monocarps which are not constricted between seeds (vs. somewhat constricted between seeds) when dry, wider monocarps, shorter monocarp stipe, thicker pericarp and different seed arrangement (interdigitated vs. uniseriate).

Description.—Trees, c. 15 m tall; young twigs puberulous-tomentose with erect and appressed hairs. Petioles 12⎼23 mm long, ± tomentose with erect and appressed hairs, slightly grooved above. Leaf blades elliptic-obovate to obovate, 24⎼30.6 × 11⎼15.4 cm, subcoriaceous-coriaceous, almost glabrous (except secondary veins, which are puberulous with erect and appressed hairs) above, puberulous with erect and appressed hairs below, base cordate to rounded-subcordate, often asymmetrical, apex caudately blunt-acuminate (acumen 7⎼16 mm long); midrib sunken above, ± tomentose with erect hairs, raised below, puberulous-tomentose with erect and appressed hairs; secondary veins 13⎼18 per side, prominent below, angle with midrib 48°⎼60° (at middle part of blade). Inflorescences 2- to 4-flowered, terminal, developing to ± leaf-opposed; peduncle inconspicuous, with one minute bract; rachis inconspicuous (when present), with minute bracts; pedicel c. 6 mm long, tomentose with erect and appressed hairs, bearing 1 cup-shaped bract, placed at pedicel midpoint or higher. Sepals free, ovate to broadly ovate, 5⎼9 × 5⎼6.5 mm, outside and margin tomentose with erect and appressed hairs, inside puberulous with appressed hairs, margin tomentose with erect and appressed hairs, apex blunt-acuminate. Outer petals linear (overall), portion above constriction 60⎼88 × 2.5⎼3 mm, both sides and margin tomentose with mostly appressed hairs, constriction tomentose with mostly appressed hairs on outside and margin, inside tomentose with mostly erect hairs, portion below constriction 6.5⎼7 × 5.5⎼7 mm, shortly clawed towards base, outside and margin tomentose with mostly appressed hairs, inside almost glabrous, apex of outer petals obtuse-rounded; inner petals linear (overall), portion above constriction 52⎼80 × 2⎼2.5 mm, both sides and margin tomentose with mostly appressed hairs, constriction tomentose with mostly appressed hairs on outside and margin, inside tomentose with erect hairs, portion below constriction 5⎼6 × 4 mm, outside tomentose with mostly appressed hairs, margin (plus adjacent areas on outside) almost glabrous, inside glabrous, apex of inner petals obtuse. Torus ± depressed hemispherical, tomentose-villous with erect hairs on areas surrounding each carpel socket and areas between stamens and carpels. Stamens 67⎼69 per flower, 1.6⎼2 mm long, connective apex truncate or with a slanted orientation and prolongation (outermost and innermost stamens). Carpels 11⎼14 per flower, 2.5⎼3 mm long; stigmas ± elongated ellipsoid; ovaries tomentose-villous with appressed hairs; ovules 5 per ovary, uniseriate. Fruits each consisting of up to 7 monocarps which are subglobose to ellipsoid, 18⎼27 × 17⎼20.3 mm, not constricted between seeds when dry, rather smooth, short-puberulous with erect and appressed hairs, stipe nearly 0 to 1.5 mm long; fruiting pedicel up to 10 mm long. Seeds 3–5 per monocarp, with interdigitated arrangement, ± flattened D-shaped, 17⎼17.5 × 11.5⎼12 mm, smooth, shiny, raphe slightly grooved to flat, hilum ± elliptic, aril absent.

Phenology.—Flowering and fruiting material was collected in March.

Distribution and habitat.—This species is so far endemic to Narathiwat Province, southern Thailand. It occurs in disturbed evergreen forests surrounded by rubber and fruit tree plantations, c. 30 m from a stream.

Field notes.—Flowers strongly fragrant, petals pale yellow when mature.

Provisional conservation status.—Only five individuals of the new species in a single location were encountered. The area surveyed is about 6 km². We also explored nearby areas, but no more individuals were found. Unfortunately, two of the five individuals were cut recently and the cleared area has been used for agricultural purposes. On the basis of this information, we provisionally assess the conservation status of the new species as Critically Endangered: CR B2ab(iii,v) based on IUCN Standards and Petitions Committee (2022IUCN 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 [accessed: Apr. 2023].).

Etymology.—As a noun in apposition, the specific epithet is derived from Khao Soi, a traditional northern Thai noodle soup claimed to be one of the best soups in the world, in allusion to the similar appearance between petals of the new species (Fig. 2a) and Khao Soi noodles.

Notes.—As evidenced by a number of species described based on recently collected gatherings, especially in the family Annonaceae (e.g., Jongsook & al. 2020JongsookH., SamerpitakK., DamthongdeeA. & Chaowasku T.2020. The non-monophyly of Dasymaschalon dasymaschalum (Annonaceae) revealed by a plastid DNA phylogeny, with D. halabalanum sp. nov. from Thailand and D. argenteum comb. nov.Phytotaxa449: 265–278.; Bunchalee & al. 2021BunchaleeP., LeeratiwongC. & JohnsonD.M.2021. Two new species and a new record of the genus Polyalthia (Annonaceae) from Peninsular Thailand. Phytotaxa510: 239–250.; Leeratiwong & al. 2021LeeratiwongC., ChalermglinP. & SaundersR.M.K.2021. Goniothalamus roseipetalus and G. sukhirinensis (Annonaceae): two new species from Peninsular Thailand. PhytoKeys184: 1–17.; Wiya & al. 2021WiyaC., AongyongK., Damthongdee A., Baka A. & Chaowasku T. 2021. The genus Phaeanthus (Annonaceae, Miliuseae) in Thailand: P. piyae sp. nov. and resurrection of P. lucidus, with molecular phylogenetic analyses. Taiwania66: 509–516.; Damthongdee & al. 2023DamthongdeeA., KhunarakN., KaeokulaS., SaengphoC., WiyaC., Ue-areeP., BakaA., AongyongK. &amp; Chaowasku T.2023. Molecular phylogenetic and morphological support for the recognition of Friesodielsia lalisae (Annonaceae), a new species from S Thailand. Willdenowia53: 45–55.), the discovery of Drepananthus khaosoi once again stresses the importance of Narathiwat Province, southern Thailand as one of the most underexplored areas in Thailand.