Molecular phylogeny of Hiptage (Malpighiaceae) reveals a new species from Southwest China

Abstract Hiptage is an Asia-endemic genus of Malpighiaceae currently placed in the tetrapteroid clade, representing one of the seven inter-continent dispersions from New to Old World. A molecular phylogeny based on sequences of the internal transcribed spacer (ITS) region was recovered for the first time for the genus. Our results showed that the most recent common ancestor of Hiptage probably originated in the South Indo-China Peninsula and diversified in this region. Based on phylogenetic evidence and relevant morphological traits, we propose a new species; Hiptage incurvatum is characterised by mericarps with arcuate anterior lateral wings, two large glands on the dorsal sepals, and small glands on the remaining sepals. The new species is from Mt. Cangshan, Dali City (25°35'N, 100°02'E) in North Yunnan, Southwest China and is notable for its occurrence at high altitude, 1400 m (the highest distribution currently known for the genus). The implications of this unusual species for the dispersal and evolution of the genus are discussed.


Introduction
Hiptage Gaertn. is a genus of Malpighiaceae currently comprising ca. 40 species (Anderson et al. 2006;Chen and Funston 2008;Ren 2015;Yang et al. 2018). Its species typically grow as woody lianas in the margins of evergreen and seasonal rainforests, in river valleys, or on limestone hills of South and Southeast Asia (Sirirugsa 1991;Srivastava 1992;Chen and Funston 2008). The genus is characterised by its many-flowered thyrse bearing mirror-image flowers with nectar secreting sepal glands, and a single mericarp per flower bearing three lateral wings (Anderson et al. 2006;Ren et al. 2013;Zhang et al. 2016). Mirror-image flowers are a sexual polymorphism in which the style is deflected away from the floral axis, resulting in mirror images between the left-styled flower and right-styled flowers, facilitating cross-pollination (Jesson and Barrett 2002;Ren et al. 2013). Currently, the genus is placed in the tetrapteroid clade, one of the ten major lineages recovered for Malpighiaceae by Davis and Anderson (2010). Hiptage was represented in that phylogeny by six species and recovered as sister to Flabellariopsis R.Wilczek, an African endemic, and together both genera form a poorly supported clade, sister to the Neotropical genus Carolus W.R.Anderson (Davis and Anderson 2010). However, the monophyly of Hiptage has never been properly tested by the inclusion of its type species (Jacobs 1955;Davis and Anderson 2010;Zhang et al. 2016).
During recent field studies addressing the pollination ecology of Hiptage in North Yunnan, two populations of an unusual morphotype of Hiptage benghalensis were discovered near Pingpo Town, in Mount Cangshan near Dali City. After molecular and morphological analyses, based on the nuclear internal transcribed spacer (ITS) region and on the comparison of living and herbarium specimens (including type specimens of all currently accepted names in the genus), we concluded that these abovementioned populations represent an undescribed species of Hiptage. We present a molecular phylogeny sampling 17 of 39 species of Hiptage, including a discussion on the systematics and biogeography of the genus, besides the formal description of the new species and an updated key for the genus in China.

Molecular analysis
We sampled most species of Hiptage occurring in the Philippines, Thailand, Vietnam, Singapore and Southwest China, to explore the phylogenetic relationships of the suspected new species (Table 1). The sequences of nuclear ribosomal Internal Transcribed Spacer (ITS) region of 17 species (with some species with multi accessions) of Hiptage were generated and analysed. The ITS sequences of two Americanendemic species of Mascagnia (i.e., M. australis and M. divaricata) were obtained from GenBank and used as outgroups. Total genomic DNA was extracted from dried leaf material following a modified CTAB method (Doyle and Doyle 1987). All polymerase chain reactions (PCR) were carried out in 25 μl volumes consisting of 1 μl sample DNA, 12.5 μl 2 × Taq PCR master Mix (Aidlab Biotechnologies Co. Ltd), 1 μl each primer (10 μmol/ml), and a final volume adjusted to 25 μl with double distilled water. The ITS region was amplified with the primers ITS17SE and ITS26SE (Sun et al. 1994). We used an amplification profile with an initial denaturation of 5 min at 94 °C, followed by 35 cycles of 40 seconds at 94 °C, 20 seconds at 69 °C, 1 min at 72 °C, and a final 10 min extension at 72 °C. The PCR products were sequenced from both directions using an ABI3730XL sequencer.
The original chromatograms from both directions of the ITS sequences were evaluated with PhyDE (Müller et al. 2010) for base confirmation and contiguous sequences editing. All sequences were aligned manually in MEGA v.7 (Kumar et al. 2016). Ambiguous positions were excluded from the alignments. The Akaike Information Criterion (AIC), which allows non-nested models to be evaluated, was used as a selection criterion (Kumar et al. 2016), and the GTR + I + G model was used in both ML and BI analyses. Maximum Likelihood (ML) analysis was performed with optimal substitution models suggested by MEGA v.7 to carry out 1000 bootstrap (BS) replicates analyses. Bayesian inference (BI) was performed with MrBayes v.3.1 (Ronquist and Huelsenbeck 2003) with a Markov chain Monte Carlo (MCMC) simulations were run for 10 000 000 generations and sampled every 1000 generations. The first 2500 trees (25% of total trees) were discarded as burn-in. The remaining trees were summarised in a 50% majority-rule consensus tree, and the posterior probabilities (PP). The obtained tree was edited using Figtree v. 1.4.3 (Morariu et al. 2008). Sequences were deposited in GenBank and the alignment and phylogenetic trees in TreeBASE (ID: S24963 and S24968).

Results
For the 17 Hiptage species, we obtained 36 sequences of ITS in total. Source information and the GenBank accession numbers of the new sequences are listed in Table 1. The dataset had an aligned length of 691 base pairs (bp), containing 128 parsimonyinformative characters.

Discussion
We provide here the first well-sampled phylogenetic study for the Asian endemic Hiptage, although this phylogeny is based on a single marker and most clades are not highly supported. Hiptage is one of the largest Old-World genera of Malpighiaceae, being adapted to various habitats such as forest edges, river valleys and limestone hills in Asia (Sirirugsa 1991;Ren 2015;Yang et al. 2018). Our phylogenetic tree shows that two species from South Vietnam and Thailand (i.e. H. stellulifera and H. detergens) were recovered as basal groups, suggesting the genus might have evolved at the southern part of Indo-China Peninsula (Fig. 1). Based on the phylogeny tree, the most widespread species in the genus, H. benghalensis, might have appeared late in the evolution of the genus, although we are not providing divergence time estimates. H. benghalensis is well-known for its extremely reflexed petals and single oversized calyx gland secreting nectar, attracting both pollinators and herbivory-defending ants (Ren et al. 2013). Such floral syndromes indicate generalized pollination by pollen-collecting bees (Ren et al. 2013;, which can explain the widespread distribution of H. benghalensis. Moreover, both our data and the results of Davis and Anderson (2010) demonstrated the polyphyly in H. benghalensis (Fig. 1), suggesting this most widespread species might be treated as two taxa. Further studies are still needed to properly address this question with more extensive sampling.The petal shape and calyx glands are diagnostic traits of the family Malpighiaceae, being used for species identification and taxonomic study (Niedenzu 1924;Anderson et al. 2006;Chen and Funston 2008;Ren 2015). The phylogeny indicates that reflexed petals may be common in both basal and nested clades, and flat petals probably evolved only once (Fig. 1). Normally there are ten oil secreting calyx glands in Neotropical Malpighiaceae, with two glands on each sepal (Anderson et al. 2006). In Hiptage, however, most species show a single calyx gland, but secreting nectar instead of oil (Ren et al. 2013). In the basal H. stellulifera, five calyx glands were found (i.e., each sepal shows a single gland) (Hô 1992). Therefore, one of the evolutionary trends in Hiptage appears to be the numeric reduction of calyx glands (Anderson et al. 2006;Ren et al. 2013;Ren 2015).
The multiple accessions of the proposed new species were recovered as a strongly supported clade (Fig. 1), separated from closely related taxa by weak molecular, but several morphological traits (Fig. 2). Specifically, the new species is distinctive in having two large glands on the dorsal sepals and two small glands on the remaining sepals (Figs 2E-F, 3C). Interestingly, the lower parts of the two large glands are fused (Figs 2F, 3C), indicating a possible explanation for the evolution of the single oversized calyx gland in H. benghalensis (Anderson et al. 2006;Ren et al. 2013). The similar evolutionary trend was also found in the Paleotropical genus Acridocarpus (Malpighiaceae) (Guesdon et al. 2019), in which the adjacent sepal glands show different degrees of fusion in several species and the single sepal gland in some species shows sagittate-acute shape, shared secretory tissues and vascular bundles, providing strong evidence of the fusion of two glands on adjacent anterior sepals (Vogel 1990;Guesdon et al. 2019). Molecular data showed that H. incurvatum is closely related to H. tianyangensis, H. multiflora and H. benghalensis. These species, however, differ significantly in habitat type, and in calyx gland and mericarp morphology (see Key). The new species grows along a river valley at very high latitudes (>1300 m) in North Yunnan, while H. multiflora and H. tianyangensis normally grow at the top of limestone mountains in Guangxi and H. benghalensis is widespread in Asia in forest margins and riversides (Anderson et al. 2006;Ren 2015).
The most distinctive trait in the new species is the arcuate anterior lateral wings of the three-winged mericarp (Figs 2J-K, 3D). Winged mericarps are an adaption for wind dispersal of fruits ) and the striking diversity of winged mericarps types in Malpighiaceae indicates that this morphology played a role in long-distance dispersals and speciation (Davis et al. 2001(Davis et al. , 2002Tan et al. 2018). Pingpo Town is located at the northern edge of the distribution range of the genus Hiptage. The surrounding mountains and gorges form a unique isolated habitat, which might be the main reason for the evolution and maintenance of the new species.

Conclusions
We presented the first well-sampled phylogeny of Hiptage, based on the ITS region, suggesting that the southern part of Indo-China Peninsula may be the area of origin of the genus. It also indicates that the erect petals have probably evolved only once in the genus. The number of calyx glands in Hiptage seems to have decreased during the genus evolutionary history. And specimens from Mt. Cangshan in North Yunnan were treated as a new species due to forming a highly supported clade in our phylogenetic study and being morphologically distinct from all accepted species in Hiptage.