1 Preprint: Pisuttimarn P, Simões ARG, Petrongari FS, Simão-Bianchini R, Barbosa JCJ, de Man I, Martins Fonseca H, Janssens SB, Patil SB, Shimpale VB, Pornpongrungrueng P, Leliaert F & Chatrou LW. 2023. Distimake vitifolius (Convolvulaceae): reclassification of a widespread species in view of phylogenetics and convergent pollen evolution.
Botanical Journal of the Linnean Society 202(3): 363–388 https://doi.org/10.1093/botlinnean/boac077
Distimake vitifolius: phylogenetic evidence to establish the generic placement of a widespread SE Asian species of Convolvulaceae
Ponprom Pisuttimarn1, Ana Rita Giraldes Simões2*, Fernanda Satori-Petrongari3, Rosângela Simão-Bianchini3, Juliana Cruz Jardim Barbosa3, Ine De Man4, Luiz Henrique Martins Fonseca4, Steven B. Janssens5, Sujit B. Patil6, Vinod B. Shimpale6, Pimwadee Pornpongrungrueng1, Frederik Leliaert5, and Lars W. Chatrou4.
1Applied Taxonomic Research Center, Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, THAILAND
2Herbarium, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AE, UK
3Núcleo de Curadoria do Herbário, Instituto de Botânica (São Paulo), Av. Miguel Stéfano 3687, Água Funda 04301–902, São Paulo, SP, BRAZIL
4Systematic and Evolutionary Botany Lab, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, BELGIUM
5Meise Botanic Garden, Nieuwelaan 38, 1860 Meise, BELGIUM
6Department of Botany, The New College, Kolhapur, Maharashtra, INDIA
*Author for correspondence: [email protected]
2 Summary. Camonea vitifolia (Convolvulaceae) is a common and widespread species in Southeast Asia. With recent re-delimitation of the genus Merremia, a large number of species were classified into other genera based on molecular, morphological and palynological evidence. This species was, then, placed in genus Camonea, as suggested by tentative molecular phylogenetic results and the presence of 6-zonocolpate pollen.
However, new and more detailed molecular, morphological and palynological data suggest that the species is, in fact, better classified in Distimake. The species is here formally transferred to Distimake with a discussion of the new evidence which has steered towards the newly proposed generic placement.
Keywords. climber, distribution, Merremia, molecular phylogeny, morning glory, morphology, palynology, taxonomy
Introduction
Convolvulaceae is a family of climbers, herbs, and shrubs (rarely trees), with approximately 60 genera and 1900 species, occurring throughout tropical and warm temperate regions. It is distinguished by alternate leaves lacking tendrils, and sympetalous corollas with five conspicuous midpetaline bands. The inflorescence is fundamentally cymose, and the fruit is often a dehiscent 4-seeded capsule, although several other types of fruits also occur (Staples & Brummitt, 2007). It is the only family of Lamiids, Asterids (APG IV, 2016), to have seeds showing physical dormancy (Baskin et al., 2000; Jayasuriya et al., 2008). A molecular synapomorphy of Convolvulaceae is the absence of the rpl2-intron, unique in the Asteridae (Stefanović et al., 2002).
The classification of Convolvulaceae (Staples & Brummitt, 2007) is based on a comprehensive molecular phylogeny of the family (Stefanović et al., 2002; Stefanović et al., 2003), where twelve tribes have been recognized and morphologically characterized (Aniseieae, Cardiochlamyeae, Convolvuleae, Cresseae, Cuscuteae, Dichondreae, Erycibeae, Humbertieae, Ipomoeeae, Jacquemontieae, Maripeae and Merremieae).
Tribe Merremieae for a long time was a problematic group within the family, for the lack of support for its monophyly and the demonstrated polyphyly of its largest genus, Merremia Hall.f. Morphologically, it was coarsely circumscribed by characters that were not exclusive to the tribe (white to yellow flowers, biglobose stigma and non-spinulose pollen), and Merremia itself not being clearly morphologically circumscribed generated great uncertainty about the generic placement of some species, when molecular phylogenetic data were not available. A molecular phylogenetic study of the family
3 (Stefanović et al., 2002) efficiently demonstrated the frailties in the systematics of this group and highlighted the need for additional evidence to resolve the classification at tribal and generic level. Later, increased taxon sampling shed light on phylogenetic relationships in the tribe Merremieae (Simões et al., 2015; Simões & Staples, 2017) resulting in the segregation of most species of Merremia sensu lato into several new and re-delimited genera, in a total of 10 genera recognized: Merremia s.s., Daustinia Buril &
A.R.Simões, Decalobanthus Ooststr., Distimake Raf., Operculina Silva Manso, Camonea Raf., Hewittia Wight. & Arn., Hyalocystis Hall. f., Xenostegia Austin & Staples, and Remirema Kerr (Simões & Staples, 2017).
Camonea Raf. is a widely distributed genus in tropical Asia; with a single species (C. umbellata) being widespread in tropical America and Africa, and some regions of Australia, Vietnam, Taiwan and Polynesia. In its current circumscription, it includes five species - C. bambusetorum (Kerr) A.R.Simões & Staples, C. kingii (Prain) A.R.Simões &
Staples, C. umbellata (L.) A.R.Simões & Staples, C. pilosa (Houttuyn) A.R.Simões & Staples and C. vitifolia (Burm. f.) A.R.Simões & Staples - and it is morphologically characterized as follows: perennial twiners, prostrate creepers or rarely woody climbers; leaves simple, entire, or palmately lobed with two firm outgrowths (paired auricles or pseudostipules) at the base of the petiole (absent in C. vitifolia); corolla with a tuft of hairs at the apex of the midpetaline bands (glabrous in C. vitifolia); anthers longitudinally dehiscing with curly or curved apex, or completely spirally dehiscing; pollen 6-zonocolpate; fruit a four- valved capsule; seeds pubescent, with long, green, brown or golden hairs either covering the entire surface or concentrated along the ridge, edges and hilum or short hairs, (glabrous in C. vitifolia). The exclusive distinguishing traits are the presence of pseudostipules at the base of the leaf insertion, and 6-zonocolpate pollen grains, which were not documented in any other taxa throughout the family (Staples, 2010a; Staples, 2010b; Simões et al., 2015; Simões & Staples, 2017; Chao et al., 2017; Staples, 2018).
Camonea vitifolia presents the 6-zonocolpate pollen that is a synapomorphy for the genus but does not show the pseudostipules at the leaf insertion. It also differs significantly from the remaining species of Camonea by the leaves being usually palmately five-angled or lobed (instead of entire to shallowly lobed at the leaf base in the remaining species); the sepals are lanceolate and appressed to the corolla (instead of elliptic, with obtuse apex, and strongly convex); the corolla is entirely glabrous (an evident tuft of hairs at the apex of the midpetaline bands, in the other Camonea species); glands are present on the outer surface of the corolla (absent in the other species); anthers are longitudinally dehiscing
4 with curly or curved at the apex (opposed to spirally dehiscing); the fruit is indehiscent and chartaceous with four-locules (opposed to the coriaceous, dehiscing by four-valves, capsule in Camonea); and the seeds are glabrous, differing from other species in the genus which have long hairy seeds (Fig. 1).
Owing to the morphological discordance between C. vitifolia and the remaining species in the genus, and the unconvincing results of previous phylogenetic analyses, the placement of this species in Camonea has remained uncertain. Thus, some of these features make it morphologically closer to Distimake, a genus newly described as segregation of Merremia sensu lato. As currently circumscribed, Distimake Raf. comprises 44 species, occurring in tropical America (29 spp.), West Africa (11 spp.), India (1 sp.), SE Asia and Australia (3 spp.). Species of Distimake are herbaceous climbers (rarely lianas or erect shrubs), with palmately five- to seven-lobed or compound leaves (rarely simple or reduced to scale or absent); calyx mostly with flat sepals (not convex) appressed to the corolla tube, accrescent in fruit; corolla often white or pale yellowish, with or without a dark red or purple center, entirely glabrous, drying with dark lines in midpetaline bands;
anthers spirally dehiscing; pollen 3-zonocolpate (rarely 12-zonocolpate in D. tuberosus and D. quinatus), fruit usually a four-valved capsule or indehiscent, chartaceous, with four lobes, calyx greatly accrescent in fruit, later the sepals reflexing; seeds glabrous (less commonly shortly velvety puberulent). Importantly, this species has some unique features, which are neither present in Camonea nor Distimake, such as an asymmetric funnel-shaped corolla (with a swollen tube base), and the characteristic vine-shaped leaves (palmately five-lobed) that have inspired the epithet vitifolia (Simões & Staples, 2017).
Yet, this systematic debate is not new - the generic placement of Camonea vitifolia has a long trail of uncertainty, due to the mosaic of morphological characters that are present in other species, and its unique morphological features.
Camonea vitifolia was originally described as Convolvulus vitifolius Burm.f. in Fl.
Indica: 45, tab. 18, fig. 1, (1768), based on the following characteristics: 'foliis palmatis quinquelobis glabris dentatis, caule pi[loso]' (leaves palmately five-lobed, glabrous, with pilose stem). In 1826, the English botanist, Robert Sweet, placed the species in Ipomoea (Hortus Britannicus: 289), although without any justification. Hallier (1893) transferred this species to Merremia, proposed a sectional division for Merremia and placed Merremia vitifolia in section Streptandra, together with Merremia tridentata (currently accepted name: Xenostegia tridentata), and other species that are now included in the genus Distimake: Merremia quinquefolia (D. quinquefolius), M. quinata (D. quinatus), M. tuberosa
5 (D. tuberosus), M. aegyptia (D. aegyptius) and M. dissecta (D. dissectus). The classification of Merremia vitifolia was followed by Van Ooststroom & Hoogland (1953) in their Convolvulaceae treatment for Flora Malesiana, which is one of the most comprehensive taxonomic works for Merremia sensu lato. All species that Van Ooststroom & Hoogland (1953) placed in this section have a wide distribution in the Neotropics and Africa, except for M. quinata and M. vitifolia, the only two species included in this section which are restricted to Southeast Asia. In the same work, Van Ooststroom & Hoogland (1953) placed the species Merremia umbellata (which two subspecies are currently segregated into Camonea umbellata and Camonea pilosa) in the monotypic section Xanthips.
Before any phylogenetic studies were conducted, Ferguson et al. (1977) published a palynological survey of Merremia and Operculina, which aimed mainly at improving the understanding of the sectional divisions within Merremia, by documenting and classifying for the first time the pollen variation of the group. The only species that they reported to have 5- to 6-zonocolpate pollen were Camonea umbellata, C. kingii and C.
vitifolia. Ferguson et al. (1977) suggested that Merremia vitifolia was sufficiently distinct from the other species in section Steptrandra (currently Xenostegia and several species now placed in Distimake) and could “well deserve its own section”.
A morphological cladistic analysis of the family (Austin, 1998), resulted in the re- alignment of the sections within Merremia, one of which was the ‘Merremia vitifolia allied species', which comprised the species M. vitifolia, M. aegyptia and M. dissecta, in agreement with the previously delimited section Streptandra by Hallier (1893) and Van Ooststroom & Hoogland (1953).
Jenett-Siems et al. (2005) studied the distribution of tropane alkaloids across 18 species of Merremia, to provide potentially taxonomically informative characters for the infrageneric division of the genus. They considered three groups, one of which defined by the absence of tropanes, in which they included only two species (Merremia medium (L.) Hallier f. and Merremia tridentata (L.) Hallier f.), although these species had already been transferred from Merremia, to Xenostegia (Austin & Staples, 1980). For the remaining species, they defined two groups: 1) with simple tropanes only, and 2) with simple tropanes as well as merresectines. In the first group Merremia s.s. (M. emarginata, M.
gemella, M. hederacea); Decalobanthus (D. peltatus); Camonea (C. umbellata); Distimake (D. aureus and D. tuberosus) as well as the currently unplaced Merremia pterygocaulos were included. The second group included mostly species of Distimake (D. aegyptius, D.
cissoides, D. dissectus, D. guerichii, D. quinatus, D. quinquefolius, D. kentrocaulos), plus
‘Merremia vitifolia’.
6 The two molecular phylogenetic studies that have sampled Merremieae and its largest genus Merremia s.l. (Stefanović et al., 2002; Simões et al., 2015) have agreed for the most part, but they disagreed in the placement of Camonea vitifolia. Stefanović et al.
(2002) retrieved the species in Distimake (sister to D. aegyptius and D. dissectus), with 97% bootstrap support in parsimony analysis, whereas Simões et al. (2015) inferred a sister group relationship between C. vitifolia and a clade of C. bambusetorum and C. pilosa although without significant support (0.91 PP in Bayesian inference, unsupported in maximum likelihood analysis, and 52% bootstrap support in parsimony analysis). While the first study seemed to corroborate the previous hypothesis based on morphology and phytochemistry, the second curiously corroborated the palynological study of Ferguson et al. (1977). The two molecular studies used different sets of chloroplast markers, and Simões et al. (2015) additionally used a nuclear region (ITS). The study of Stefanović et al. (2002) was aimed at family level, and only sampled nine of the c. 120 species of tribe Merremieae, against 57 in the study of Simões et al. (2015). The difference in the placement of Camonea vitifolia could be explained by the additional data in Simões et al.
(2015) (i.e. different chloroplast markers, and the introduction of a nuclear region), as well as the much expanded taxon sampling.
Simões & Staples (2017), in the face of the available evidence, decided to transfer Merremia vitifolia to Camonea, as Camonea vitifolia, weighing heavily the palynological character (presence of 6-zonocolpate pollen), and the biogeographical links (Camonea being a mostly SE Asian genus, and Distimake being a mostly an American and African genus), in addition to the molecular phylogenetic results which, although with weak support values, were the most comprehensive representation of tribe Merremieae.
However, the species was red flagged for further investigation, namely additional palynological and molecular phylogenetic studies, given the morphological incongruence and the lack of support for the monophyly of Camonea in the study of Simões et al. (2015).
A more recent molecular phylogenetic study, with samples of Camonea and Distimake from India (Tamboli et al., 2021) suggested its placement in Distimake, as sister to D.
rhyncorhiza and D. quinatus, both Asian species of Distimake but without any statistical support in Bayesian phylogenetic inference (Fig. 2).
The present study aims to 1) test the monophyly of Camonea, with sampling of both SE Asian and American material of its most widespread species; 2) test the generic placement of Camonea vitifolia in the light of expanded molecular sampling of the genera that have been implied in this taxonomic conundrum — Distimake and Camonea — and with support of additional morphological and palynological evidence.
7 Material and Methods
Morphological studies and collection of leaf tissue. Fieldwork was conducted between 2015 and 2018. In Southeast Asia, specimens were collected in several field trips throughout Thailand, Cambodia, Laos and Vietnam, and deposited in herbarium KKU;
these collections incremented the sampling and morphological data for the SE Asian taxa of Camonea vitifolia, other Camonea species, and widespread species of Distimake with occurrence in Asia. In Brazil, fieldwork covered regions of the Atlantic Forest in the SE of the country, as well as Cerrado in the Central Region of the country; specimens were collected and deposited in herbarium SP. In all expeditions, fresh leaf tissue was collected in silica gel for DNA extraction; and morphological and ecological information was annotated. Morphological observations and DNA sampling also relied much on the examination of herbarium specimens; the following herbaria were consulted: AAU, BCU, BK, BKF, BM, BO, BR, CMU, FOF, HN, HNL, HNU, K, KEP, KKU, L, MEXU, MG, P, PSU, QBG, SING, and SP.
Gene sampling. Sampling of previously published sequence data, and DNA extraction and sequencing were aimed at compiling a supermatrix, composed of sequences taken from Simões et al. (2015) and Tamboli et al. (2021), supplemented with six sequences first published here (Appendix I). The dataset consisted of 82 ingroup species and two outgroup species (Aniseia martinicensis (Jacq.) Choisy and Iseia luxurians (Moric.) O'Donell), for which the following markers were sequenced: ITS (48 species), trnL intron (69 species), matK (66 species), and rps16 (68 species). Six species were represented by multiple accessions. A single marker had been sequenced for each accession, and by including multiple accessions we have certified that these six species were represented by various markers.
Molecular phylogenetic analyses. Total genomic DNA was extracted from approximately 0.1 g of dried leaf material (silica gel-dried or herbarium specimens) using a modification of the CTAB micro-extraction method (Doyle & Doyle, 1987), in which the aqueous phase from the chloroform precipitation was cleaned using Qiagen DNeasy kit and the protocol as described in Carine et al. (2004). Plastid DNA barcoding region matK (matK390f-matK1326r, Cuénoud et al., 2002) was amplified using PCR conditions, as
8 described in Hollingsworth et al. (2009). The plastid rps16 (rps16x2F2-trnK(UUU), Shaw et al., 2007) and the trnL-F intron (trnLC-trnLD, Taberlet et al., 1991) regions were amplified using PCR cycling conditions described in Shaw et al. (2007). The internal transcribed spacer region of nuclear ribosomal DNA (17SE-26SE, Sun et al., 1994; also called AB101-AB102 by Douzery et al., 1999) was amplified with betaine (1.2 mol/L) added to prevent the formation of secondary structures, following the protocol of Carine et al. (2004). Sequences were obtained by the Sanger dideoxy sequencing method at Centro de Pesquisa Sobre Genoma Humano e Células Tronco, University of São Paulo, and at Macrogen Europe, Inc. (Amsterdam, The Netherlands). Newly generated DNA sequences were edited using Sequencher ® version 5.4.6 (DNA sequence analysis software, Gene Codes Corporation, Ann Arbor, MI USA).
Newly generated sequences and previously published sequences were aligned using the MAFFT (Katoh & Standley, 2013) plugin in Mesquite version 3.61 (Maddison & Maddison, 2019). Alignments were manually adjusted in Mesquite following guidelines by Kelchner (2000). The alignments of ITS and rps16 contained several indels and were aligned by subsequent iterations of manual adjustment of the alignment, Bayesian inference (see below), and inspection of phylogenetic trees for deviating branch lengths or phylogenetic position of species. Regions in ITS and rps16 that could not be aligned unambiguously were excluded prior to analysis. Presence / absence of eleven indels in the alignment of rps16 was coded as binary characters following the simple indel coding method of Simmons & Ochoterena (2000). The four alignments, matK (597 positions), rps16 (998 positions), trnL-F (391 positions) and ITS (590 positions), were analyzed separately using Bayesian phylogenetic inference, performed with MrBayes 3.2.7 (Ronquist et al., 2012), available at the CIPRES portal in San Diego, CA, USA (http://www.phylo.org/portal2, Miller et al., 2010). During all analyses, DNA substitution models and phylogenetic parameters (topology, branch lengths etc.) were estimated simultaneously using a reversible jump Markov chain Monte Carlo sampler (so-called model-jumping, Huelsenbeck et al., 2004), allowing among-site rate heterogeneity (Γ). Initially, the MCMC chain was run for 20 million generations, with four simultaneous runs and four chains per run, with default values for the acceptance rates of the proposal mechanisms and for the temperature of the heated chains and sampling every 1000th generation. Convergence diagnostics were assessed using the sump command in MrBayes, and the R package Convenience (Fabreti & Höhna, 2021), discarding the first 10% of samples as burnin. The latter package allows the assessment of convergence of the most difficult parameter (phylogenetic trees) as this is a discrete
9 parameter unlike all other (continuous) parameters that are estimated during Bayesian inference. Fabreti & Höhna (2021) derive a threshold value of 625 for the effective sample size (ESS) from the choice to adopt a standard error of the mean with a width ≤ 1% of the width of the 95% probability interval of the true distribution. Bayesian inference with a chain length of 20 million generations converged for all continuous parameters (i.e. ESS values > 625), but not for the splits. We subsequently ran longer analyses (incrementally adding 20 million generations), with the temperature of the hot chains lowered from the default value of 0.1 to 0.05 to reduce outliers in estimated split frequencies. A Markov chain of 100 million generations resulted in convergence of the splits, with the exception of four splits for which the similarity in frequencies among the runs was only just rejected (see supplementary Figure 1). The ESS values of the continuous parameters amounted to several tens of thousands, and we deemed these convergence statistics to be sufficient.
After having reached convergence, maximum clade credibility trees were generated using the LogCombiner and TreeAnnotator utilities in BEAST (Drummond & Rambaut, 2007).
Maximum clade credibility trees for the individual markers were compared to detect possible incongruences among the topologies. As no well-supported incongruences were encountered, the alignments were subsequently concatenated for further joint analysis.
The total length of the alignment was 2587 positions and consisted of five partitions (the four DNA sequence alignments and the indel partition), that each were allowed to have their own substitution model. For the indel partition, the Mk model (Lewis, 2001) was applied. All settings of the Bayesian analyses, and methods for inspecting convergence, were as described above, including the reversible jump Markov chain Monte Carlo sampler to account for uncertainty in the DNA substitution model.
Palynological analyses. Palynological data results from work developed by Ana Rita Simões at the Jodrell Laboratory, Royal Kew Botanic Gardens, UK (2010) and Ponprom Pisuttimarn at the Department of Biology, Faculty of Science, Khon Kaen University, Thailand (2019). Unopened mature buds were removed from herbarium specimens.
Acetolysis was carried out using Erdman’s technique (1952), with slight modifications.
Buds were soaked in wetting agent, and pollen was dissected out of the anthers and washed in deionized water, to which a few drops of lactic acid (Pisuttimarn: 10% KOH) were added to prevent expansion of the pollen grains. The pollen was acidified using glacial acetic acid and then acetolyzed according to the Erdtman acetolysis technique (Erdtman, 1960) for two minutes. A short acetolysis time was required to prevent the pollen collapsing. Pollen was pipetted onto SEM stubs from 95% ethanol and allowed to
10 air dry. Stubs were sputter coated with platinum (Pisuttimarn: gold particles) and examined using a Hitachi S-4700 cold field emission scanning electron microscope at an accelerating voltage of 2kV (Pisuttimarn: LEO 1450 VP scanning electron microscope, at an accelerating voltage of 15 kV). For LM, acetolyzed pollen was suspended in 50%
glycerol, centrifuged and decanted, and pollen was mounted in glycerine jelly to make slides. Slides were examined using a Nikon Labophot light microscope (Pisuttimarn:
Olympus CH30 light microscope) using normal brightfield optics and measurements were made of the length of the polar and equatorial axes (P and E) in optical section from ten grains using a calibrated eyepiece graticule. For light microscopy analysis, acetolyzed pollen was washed with benzene, centrifuged, and decanted, silicone oil was added to the sample and left overnight to allow benzene to evaporate, and pollen was mounted in silicone oil to make the permanent slides. Additional palynological evidence was retrieved from specialized literature (Ferguson et al., 1977; Sengupta, 1972) (Table I).
Terminology follows Punt et al. (2007) and Hesse et al. (2009).
Species distribution modeling. A selection of the analyzed specimens of Camonea vitifolia from across its distribution range were georeferenced (Table II). Geographical coordinates were extracted when available, or calculated by approximation, if sufficiently precise information was given in the collector’s notes, using Google Maps. The geographic data were plotted onto DIVA-GIS (Hijmans et al., 2001) for visualization of the current species distribution, adding a geopolitical world map (DIVA-GIS, Hijmans et al., 2001). To account for the bias in little collected areas of SE Asia, or the fact that many distribution points were discarded for incorrect/incomplete information, a species distribution model based on climatic envelope was applied, to infer the potential current distribution of the species. For this, the BioClim algorithm from DIVA-GIS was modelled, using 19 Bioclimatic variables, which rely mostly on temperature and precipitation, from the WorldClim database (Hijmans et al., 2005, available at http://www.worldclim.org).
Results
Phylogenetic inference. The Bayesian analysis of the concatenated dataset showed good mixing among the chains, as indicated by the acceptance rates of swaps between adjacent chains, produced by MrBayes. As indicated in the methods section, all MrBayes runs converged on the same posterior distributions of parameters, including topologies.
Molecular phylogenetic analyses of nuclear and cpDNA demonstrate that Camonea is monophyletic with high support (PP = 1.00), with the exception of Camonea vitifolia (Fig.
11 3). This species is nested in Distimake, the monophyly of which is strongly supported (PP
= 0.99). The monophyly of three individuals of Camonea vitifolia, each represented by a single marker, is moderately supported (PP = 0.80) The significant support for the nodes mentioned, and for the node uniting Camonea with the clade consisting of Operculina, Xenostegia, Hewittia, Hyalocystis and two species of Merremia (PP = 1.00), decisively excludes Camonea vitifolia from Camonea and puts it in Distimake.
Pollen morphology. Evidence from the current investigation showed that pollen of Camonea vitifolia varies from 5- to 6-zonocolpate, supporting previous studies where at least the 6-zonocolpate pattern was documented (Ferguson et al., 1977). As previously discussed by Simões et al. (2017), the 6-zonocolpate is shared with other species of Camonea, and this was thought to be a synapomorphy for the genus since no other species in subfamily Convolvuloideae are known to possess this pollen type. In Distimake, this pollen type is also not documented. However, species of Distimake do not exclusively have 3-zonocolpate pollen as other aperture patterns are present. The species of Distimake to which C. vitifolia is most closely related have 9- and 12-zonocolpate pollen (D. quinatus and D. tuberosus, respectively) (Fig.4). This demonstrates, therefore, that 6-zonocolpate pollen has evolved independently in both Distimake and Camonea, which our studies are the first to suggest. Thus, in the case of Distimake, some clades are palynologically more diverse, with apertures ranging from 3 to 12-zonocolpate. This further means that the 6- zonocolpate is not a synapomorphy of genus Camonea as previously assumed.
Morphological characters. The morphological characteristics of the sampled species of Camonea and Distimake were investigated including C. pilosa, C. umbellata, C. vitifolia, D.
rhynchoriza, D. quinatus, D. cissoides and D. tomentosa (Table II; Fig. 5). The comparisons with key species of Distimake reveals a number of shared morphological characters which support the new placement in this genus: palmately lobed or compound leaves, absence of pseudostipules, calyx appressed to the corolla; chartaceous or membranous capsule;
and glabrous or minutely hairy seeds. With Camonea, D. vitifolius still shares the 6- zonocolpate, which is not present in the other species of Distimake. The morphological observations reveal that, despite the 6-zonocolpate pollen not being a good synapomorphy for Camonea, the fact that C. vitifolia is excluded from Camonea supports that the presence of pseudostipules at the base of the leaf insertion is, indeed, a good one, since C. vitifolia was the only species in which it was absent. From the point of view of the morphological delimitation of Distimake, C. vitifolia adds new variation, which was not
12 previously documented in the genus, particularly the entire plant being yellowish hirsute;
the palmately five-lobed with angulate lobes (grapevine-shaped) leaves; and the markedly asymmetric corolla with a gibbous base. However, we have also documented the presence of a slightly curved tube base in Distimake rhynchorrhizus (endemic from Western Ghats, India).
Taxonomic treatment
The presence of several species of Camonea throughout the distribution range of D. vitifolius (from the Indian subcontinent, to Malesia, widely spread in continental Asia), where few species of Distimake are recorded, could raise confusion towards understanding the characters that separate the two genera and how to correctly identify its species. To support floristic studies in the region, we here provide an identification key of the species involved.
Key to species of Camonea and Distimake occurring in SE Asia
1. Stem sericeous or glabrous, never covered with glandular trichomes. Leaves simple, entire to shallowly 3-lobed at the base, narrowly cordate to lanceolate in outline.
Pseudostipules at the petiole insertion. Flower buds rounded at the apex. Flowers bearing a tuft of hairs at the apex of the midpetaline bands ... 2 (Camonea) 1’. Stem densely hirsute or glabrous, with or without glandular hairs. Leaves 5–7 compound or deeply lobed, broadly cordate in outline. Absence of pseudostipules at the petiole insertion. Flower buds acute. Flowers completely glabrous 3 (Distimake) 2. Inflorescences umbelliform, densely congested. Flowers bright yellow. Capsule rounded. Seeds not angular, densely hairy throughout ...
... C. umbellata (Fig. 5a.) 2’. Flowers solitary or in 1–3 monochasia, lax. Flowers pale yellow or white, Capsule ovoid. Seeds angular, densely hairy along the margin ... C. pilosa (Fig. 5b.) 3. Leaves compound, palmately 5-7 divided. Corolla pure white to pale yellow, with or without a purple throat, funnelform with symmetric base ... 4 3’. Leaves entire, 5–7 palmately lobed. Corolla pale to bright yellow, never with a purple throat, funnelform with a gibbous, asymmetric base. ... 5 4. Stem and leaves “sticky” (glandular). Calyx covered by enlarged foliaceous bracts.
Sepals subequal, ovate to ovate-lanceolate, apex long-acuminate, densely glandular,
13 and sparsely hirsute. Corolla pure white, rarely with a dark red corolla tube ...
... D. cissoides (Fig. 5c.) 4’. Stem and leaves “not sticky” (not glandular). Bracts minute, not foliaceous, shorter than the calyx. Sepals strongly unequal, outer ones shorter and obtuse; mucronulate;
glabrous. Corolla white or pale yellow, never with a dark red corolla tube ...
... D. quinatus (Fig. 5d.) 5. Stem glabrous. Leaf lobes deeply and irregularly dissected and entire at the margin.
Corolla slightly curved at the base ... D. rhyncorhiza (Fig. 5e.) 5’. Stem densely yellowish to whitish hirsute. Leaves grapevine-shaped, margins coarsely
dentate to crenate. Corolla markedly asymmetric and gibbous at the base
... D. vitifolius (Fig. 5f.)
Distimake vitifolius (Burm.f.) Pisuttimarn & Petrongari, comb. nov.
(IPNI: https//www.ipni.org/urn:lsid:ipni.org:names:77163302-1)
Camonea vitifolia (Burm.f.) Simões & Staples, Bot. J. Linn Soc 183: 583 (2017). Merremia vitifolia (Burm.f.) Hallier f., Bot. Jahrb. Syst. 16: 552 (1893). Ipomoea vitifolia Blume, Bijdr.
Fl. Ned. Ind. 13: 709 (1825). Ipomoea vitifolia (Burm.f.) Sweet, Hort. Brit.: 289 (1826), nom. illeg. Convolvulus vitifolius Burm.f., Fl. Indica: 45, t. 18, f. 1. (1768). Type: Garzin s.n., Java, without locality (G-Burman!).
Ipomoea vitifolia var. angularis (Burm.f.) Choisy in Prodr., A. P. de Candolle 9: 361 (1845).
Ipomoea angularis (Burm.f.) Choisy, Mém. Soc. Phys. Genève 6: 454 (1833). Convolvulus angularis Burm.f., Fl. Indica: 46, t. 19, f. 2. (1768). Type: Pryon s.n., Java (G-Burman!)!)
Large twiners or prostrate creepers, all parts spreading yellowish or whitish hirsute or glabrous. Stems purplish, rounded or slightly terete, the older ones striate, 2–4 m, glabrous or patently hirsute with white or fulvous hairs. Leaves simple, orbicular in outline, 5–19 by 4–16 cm, cordate at the base, palmately 3–7 lobed; lobes broadly triangular to lanceolate, more or less acuminate or acute to obtuse at the apex and mucronate, mostly not contracted at the base or sometimes slightly so, coarsely dentate to crenate, or subentire, sparsely to densely yellowish hairy on both sides, more densely beneath than above, or glabrous above; petiole 2–15 cm, occasionally longer, patently hairy or glabrous. Peduncles axillary, 1–3 or several flowered, shorter or longer than the
14 petiole, 1–15 cm or more, patently hirsute. Pedicels 4–20 mm, hirsute like the peduncles, thickened towards the apex, clavate in fruit. Bracts subulate, 0.5–2 mm. Floral buds narrow, ovoid, acute. Sepals subequal, oblong to ovate-oblong, obtuse or acute, mucronulate, the outer ones more or less hirsute, glabrescent, the inner ones glabrous, all with glandular pellucid dots, 9–20 mm long, in fruit to 20–25 mm and then thick, sub- leathery, whitish inside and with many glandular pits. Corolla funnel-shaped, 3–6 cm long, glabrous, bright-yellow, paler towards the base; the limb with five obtuse lobes, midpetaline bands distinctly 5-nerved, tube distinctly curved at the base, gibbous.
Filaments 4–10 mm, basal insertion sparsely papillose. Anthers spirally coil at the apex at dehiscence, opening lengthwise. Ovary glabrous. Nectary disc conical. Stigma biglobose, white. Capsule subglobose, 12-16 mm high, papery, straw-colored, 4-valved. Seeds 4 or less, broadly elliptic-trigonous 5.5–7 mm long, dark brown to black, glabrous.
DISTRIBUTION. Indian subcontinent, Sri Lanka, China, Thailand, Cambodia, Vietnam, and throughout Malesia, as far as East Timor, but not documented in Australia (Van Ooststroom & Hoogland, 1953; Staples, 2015; Staples, 2018; Simões et al., 2011) (Fig. 6).
HABITAT. Both in regions with a feeble and with a rather strong dry season, in open grasslands, thickets, and hedges, along fields, in teak-forests, along edges of secondary forests, on riverbanks and waysides, from sea-level to c. 900 m (Van Ooststroom and Hoogland, 1953); often forming large mats of stems on the ground in areas where there is nothing on which to climb (Staples, 2015).
VERNACULAR NAMES. Akar lulang bulu, nlan raya, Mal. Pen., areuj kawojang, S, ginda pura utan, katapong, tampar kidang, ojod kotong, katong, samber kidang, J, dewuln, pos sepoh, subuln, Md, rabet bulu, Kangean, taradju, tjambulu-bulu, kai-kai mamia, Celebes, takwaha, Sula, kalalakmit, Sulu, lakmit, Tag. (Van Ooststroom and Hoogland, 1953).
USES. It is used for poulticing, and an infusion is drunk for high fever (Burkill in Van Ooststroom and Hoogland 1953).
CONSERVATION STATUS. Least Concern (Staples, 2015).
Conclusion
Distimake vitifolius is a widely distributed species in India, continental SE Asia and Malesia. Its ambiguous generic placement has been of great concern and urgency to resolve, from the point of view of taxonomic, conservation, biogeographic and evolutionary studies of the flora of the region, as well as of family Convolvulaceae. Recent advances in molecular phylogenetic studies of Distimake and Camonea, with significant
15 expansion of the taxonomic and geographic sampling, have now brought to light a new understanding of the circumscription of both genera, and the correct placement for this long doubtful species, which should now be known as Distimake vitifolius.
This new classification thus impacts a number of assumptions that should be considered in future studies of Convolvulaceae: 1) the presence of pseudostipules at the base of the petiole constituting a reliable diagnostic character for Camonea; 2) a stronger presence of Distimake in SE Asia, now amounting to three species native to this region (D.
rhyncorhiza, endemic from Western Ghats, India; D. quinatus, occurring throughout SE Asia to Northern Australia; and now also D. vitifolius, spreading from the Indian subcontinent to the Malay Archipelago), out of 44 currently recognized in the genus; 3) a biogeographical connection between Central America and SE Asia, put into evidence by the close relationship between D. vitifolius, D. rhyncorhiza, D. quinatus and D. tuberosus;
4) the independent evolution of 6-zonocolpate pollen; 5) a noticeable increase in pollen aperture patterns, in the clade of D. vitifolius/D. tuberosus, from 6 to 12-zonocolpate, important to explore from the perspective of pollen evolutionary studies.
Acknowledgements
Dr. Ponprom Pisuttimarn thanks the Science Achievement Scholarship of Thailand (SAST), and Study and Research in Abroad Scholarship Fiscal Year of 2018, Graduate School, Khon Kaen University (Thailand). Dr. Ana Rita G. Simões thanks Fundação Ciência e Tecnologia Grant SFRH/BD/45924/2008 (2009–2013) and CAPES (Brazil) Grant BJT 88881.067993/2014-01 (2015–2018). The authors also thank all curators and staff at the consulted herbaria, who facilitated access to the collections, and molecular sampling when appropriate: AAU, BCU, BK, BKF, BM, BO, BR, CMU, FOF, HN, HNL, HNU, K, KEP, KKU, L, MEXU, MG, P, PSU, QBG, SING, and SP. Thanks are also due to the managers and staff of the labs where molecular work was conducted, i.e. Dra Marília Gaspar (Departamento de Fisiologia and Bioquímica, IBT São Paulo, Brazil), Dr. Jefferson Prado and Dra Regina Hirai (Departamento de Curadoria do Herbário, IBt São Paulo, Brazil), Mr. Pieter Asselman (Systematic and Evolutionary Botany lab, Ghent University, Belgium). In addition, the authors thank Dr. Carol Furness and Dr. Hannah Banks at the Jodrell Laboratory (Royal Botanic Gardens, Kew) and Mr. Boonsong Kongsook at Applied Taxonomic Research Center (Department of Biology, Faculty of Science, Khon Kaen University), for help with acetolysis and imaging of pollen grains.
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22 Figures and Tables
Fig. 1. Key morphological characters of Camonea vitifolia. A Climbing habit; B 5-7 palmately lobed, grapevine shaped leaves; C Stem densely hirsute with long yellowish trichomes; D Bright yellow flower, slightly 5-angled; E Corolla with asymmetric base, slightly gibbous, and showing lanceolate, appressed sepals; F Stamens with curled apex of the anthers; G Capsule, 4-valved, chartaceous with accrescent calyx; H. Seeds, scale bar
= 2 mm.
23 Fig. 2. Summary of previous molecular phylogenetic hypotheses, based on Bayesian Inference analyses; significantly supported clades (posterior probabilities ≥ 0.95) are marked with a black circle.
24 Fig. 3. Maximum clade credibility tree after Bayesian inference of concatenated nuclear (ITS) and cpDNA (matK, trnL-F, rps16) sequences of Merremieae and tribe Ipomoeeae.
Significantly supported nodes (posterior probabilities ≥ 0.95) are marked with a black circle.
25 Fig. 4. Comparison of pollen aperture patterns. A-D Distimake vitifolius, showing 6- zonocolpate (A-B) and 5-zonocolpate pollen (C-D). A Polar view. B Semi-equatorial view.
C polar view. D equatorial view. E-F Camonea pilosa, showing 6-zonocolpate pollen. E Polar view. F Equatorial view. G-H Distimake rhyncorhiza, showing 12-pantocolpate pollen. G Semi-polar view. H Equatorial view. Scale bar = 10 µm.
26 Fig. 5. Morphological variation in species of Distimake and Camonea, for comparison with Distimake vitifolius. A-C Camonea pilosa, A Leaves entire, oblong-lanceolate, with slightly lobed or sagittate base; B Corolla broadly funnel-shaped, sepals mostly equal in size, convex, ovate-elliptic in outline; C Fruit a 4-valved capsule, with each valve longitudinally splitting; seeds densely pilose. D-F Distimake quinatus D Leaves 5-7 palmately compound, broadly ovate in outline; E Corolla funnel-shaped with a distinct tube, slightly contorted
27 at the base; sepals unequal, inner ones shorter, oblong to lanceolate, with a shortly mucronate apex, appressed to the base of the corolla tube; F Fruit a 4-valved capsule, each valve entire and not further splitting. G-I Distimake rhyncorhiza, G Leaves entire, 5- 7 palmately lobed, margins deeply dissected; H Corolla funnel-shaped; sepals subequal, oblong to lanceolate, with acuminate apex, appressed to the base of the corolla tube. I Fruit a 4-valved capsule.J-L – Distimake tuberosus. J Leaves entire, 5-7 palmately lobed. K Corolla funnel-shaped; sepals subequal, oblong to lanceolate, with acuminate apex, appressed to the base of the corolla tube; L Fruit a chartaceous 4-valved capsule, tardily dehiscing, or sometimes indehiscent; calyx much enlarged in fruit.
28 Fig. 6. Potential distribution of Distimake vitifolius across the Indian subcontinent, Asia and Australia, inferred from bioclimatic niche modelling, showing areas where the species may occur despite not having been collected; red dots indicate distribution records used to model the potential distribution; climatically suitable regions for this species are colour coded from least optimal (light green) to excellent (red); grey areas are those where the species is not expected to occur.
29 Table I. Palynological characterization of species of Camonea and Distimake, for comparison with D. vitifolius.
Species/Specimens Country of origin Pollen measurements (µm)
P/E Exine thickness (µm) Source Polar axis (P) Equatorial axis (E)
5-zonocolpate Distimake vitifolius
Pisuttimarn 223 (KKU) Thailand (50-)56.3(-62) (63-)66.2(-69) 0.85 4.6
6-zonocolpate Camonea kingii
Kerr 10427 (K) Thailand (57-)62.7(-69) (73-)76.7(-80) 0.89 5.6 Ferguson (1977)
Clarke 24775 (K) India (80-)85.20(-112) (49-)74.60(-110) 1.21 4.3
Camonea umbellata
Savory & Keay 25002 (K) Thailand (64-)70(-74) (72-)73(-79) 0.97 5.8 Ferguson (1977)
Glaziou 13025 (K) Brazil (55-)63.7(-76) (62-)73.1(-79) 0.87 6 Ferguson (1977)
Cuezzo & de la Sota 1508 (LIL), 18009 (LIL) - 55-76 62-79 - 5-6 Telleria & Daners (2003)
Subbarao 22624 (MH) India - (54-)60(-62) - - Sengupta (1972)
Camonea pilosa
Mitchell 6377 (BRI) Australia (60-)70.20(-84) (62-)71.40(-84) 0.98 6
Williams 85236 (BRI) Australia (55-)61.50(-69) (54-)58.00(-62) 1.07 4.4
Distimake vitifolius
Garrett 1473 (K) Thailand (56-)60.3(-65) (62-)70.2(-73) 0.86 5.2 Ferguson (1977)
Wood s.n. (CAL) - (62-)67(-72) (48-)55(-60) - - Sengupta (1972)
Pisuttimarn 35 (KKU) Thailand (50-)52.1(-54) (58-)60.1(-67) 0.87 3.6
Vinod+Sujit – more data?
12-zonocolpate Distimake rhyncorhiza Vinod + Sujit – more data?
Distimake tuberosus
Chase 5536 (K) Mozambique (75-)79(-86) - - 6 Ferguson (1977)
Wood 3463 (K) South Africa (82-)90.6(-98) - - 5.6 Ferguson (1977)
Distimake quinatus
Kerr 4825 (K) Thailand (70-)77.6(-87) - - 5.4 Ferguson (1977)
30 Table II. Morphological, palynological and geographic characterization of species of Camonea and Distimake, for comparison with Distimake vitifolius.
Species Camonea
umbellata C. pilosa Distimake
vitifolius D. rhyncorhiza D. quinatus D. cissoides D. tuberosus Geographic
distribution range
Native in C and S America,
Introduced in Tropical Africa and SE Asia
Native in SE Asia Native in SE Asia Native in India Native in SE Asia Native in C and S America,
introduced in SE Asia
Native in C America and Mexico, introduced in Tropical areas as ornamentals Habit Perennial
herbaceous, twining climber or prostrate
Perennial herbaceous, twining climber or prostrate
Perennial herbaceous, twining climber or prostrate
Perennial herbaceous, twining climber or prostrate
Perennial herbaceous, twining climber or prostrate
Perennial herbaceous, twining climber or prostrate
Perennial woody twining, climber
Type of trichomes
Glandular, multicellular, peltate shape, inconspicuous,
~20 µm; non- glandular, multicellular, simple, filiform,
~200 µm
Glandular, multicellular, peltate shape, inconspicuous, 20 µm; non- glandular, multicellular, simple, filiform, 200 µm
Glandular, multicellular, peltate shape, inconspicuous,
~30 µm; non- glandular, multicellular, simple, filiform,
~900 µm
Glandular, multicellular, peltate shape, inconspicuous,
~20 µm; non- glandular, multicellular, simple, filiform,
~1000 µm
Glandular, multicellular, peltate shape, inconspicuous,
~30 µm;
Glandular, multicellular, clavate shape,
~150–200 µm;
non-glandular, multicellular,
Glandular, multicellular, peltate shape, inconspicuous,
~20 µm
31 simple, filiform,
~1–3 mm
Stem
indumentum
Pubescent to glabrous
Pubescent to glabrous
Hirsute Glabrous Hirsute to
glabrous
Dense glandular and hirsute- pilose, yellowish
Glabrous
Leaf Simple, cordate to shallowly 3- lobed at the base, entire margin
Simple, cordate to shallowly 3- lobed at the base, entire margin
Simple,
palmately lobed, (3) 5–7 lobed, dentate margin
Simple, deeply palmate 5–7 lobed, irregularly dissected margin
Palmately compound, 5 leaflets subequal, elliptic, entire margin
Palmately compound, ,5 leaflets, unequal, elliptic, dentate margin
Simple, deeply palmate (5) 7 lobed, entire margin
Leaf
indumentum Pubescent to
glabrous Pubescent to
glabrous Hirsute Hispid at adaxial and glabrous at abaxial
Hirsute to
glabrous Dense, glandular and pilose, yellowish
Glabrous
Pseudostipules at leaf
insertion
Present Present Absent Absent Absent Absent Absent
Corolla colour Bright yellow White or pale yellow, sometimes bright yellow at midpetaline bands
Pale to bright
Yellow Bright yellow White or pale yellow, sometimes bright yellow at midpetaline bands and corolla tube
White, rarely with a dark red corolla tube
Bright Yellow
32 Corolla shape Funnelform,
symmetric, with a conical base
Funnelform and
symmetric Funnelform, asymmetric, with a gibbous base
Funnelform and
symmetric Funnelform and
symmetric Funnelform and
symmetric Funnelform and symmetric
Sepal shape in
flower Equal, convex, elliptic to rounded, outer sepals glabrous to pubescent
Subequal,
convex, obovate, outer sepals glabrous to pubescent
Subequal, flat, oblong or ovate- oblong, obtuse or acute, outer sepals hirsute
Unequal, flat, narrowly elliptic to lanceolate, outer sepals glabrous
Unequal, flat, obtuse, outer sepals shorter glabrous
Subequal, flat, ovate to ovate- lanceolate, outer sepals shorter densely glandular and sparsely simple hair
Subequal, flat, outer lanceolate, outer sepals glabrous
Sepal shape in
fruit Slightly accrescent, uncovered in fruit, broadly rounded
Slightly accrescent, uncovered in fruit, broadly elliptic to ovate
Greatly
accrescent, all enlarged in fruit, ovate, glossy and pitted inside
Slightly accrescent, reflexed in fruit, lanceolate
Slightly accrescent, uncovered in fruit, foliaceous, oblong
Slightly accrescent, all enlarged in fruit, foliaceous, lanceolate
Greatly
accrescent, all enlarged in fruit, ovate, glossy
Fruit Capsule with exocarp chartaceous, globose, 4- valved dehiscence
Capsule with exocarp chartaceous, ovoid, 4-valved dehiscent
Capsule with exocarp membranous, globose, slightly 4-lobed, tardily 4-valved
dehiscent
Capsule with exocarp chartaceous, ovoid, 4-valved dehiscent
Capsule with exocarp chartaceous, ovoid, 4-valved dehiscent
Capsule with exocarp membranous, oblate, shallowly 4-lobed, tardily 4-valved
dehiscent
Capsule with exocarp membranous, globose, slightly 4-lobe, tardily 4- valved dehiscent
Seed Trigonous, dark brown to black, ovoid, whole seed tomentose with short
Trigonous, dark brown to black, obovoid, whole seed green,
Trigonous, dark brown to black, broadly
Trigonous, brown, ellipsoid, whole seed glabrous
Trigonous, black, oblongoid, crustose at the
Trigonous, black, broadly ellipsoid, whole
Trigonous, dark brown to black, ovoid, pubescent or glabrescent with short black
33 yellowish hair at
seed margin brown or golden
long hair ellipsoid, whole
seed glabrous apex and along
the ridges seed grey or
white floccose hairs at seed margin
Pollen 6-zonocolpate 6-zonocolpate (5-) 6-
zonocolpate 12-pantocolpate 12-zonocolpate 3-zonocolpate 3-zonocolpate
