Three new species of Paraboea (Gesneriaceae) from limestone karsts of China based on morphological and molecular evidence

Background The limestone karsts of Southeast Asia and South China are a major biodiversity hotspot of global terrestrial biomes. With more than 130 described species, Paraboea has become one of the most characteristic plant groups in the Southeast Asian limestone flora. During the course of extensive field work on the limestone formations of southern and southwestern China, three unknown species of Paraboea were collected. Results Molecular phylogenetic analyses based on nuclear ITS and chloroplast trnL-F sequences strongly confirm the placements of the three new species in Paraboea sensu Puglisi et al. (Taxon 65:277–292. 10.12705/652.5, 2016). Moreover, these three novelties can be distinguished from known Paraboea species with distinct morphological characters, further supporting their recognition as new species. Conclusions With the support of detailed morphological studies and molecular phylogenetic analyses, Paraboea dushanensis, P. sinovietnamica and P. xiangguiensis are recognized as three species new to science. Electronic supplementary material The online version of this article (10.1186/s40529-017-0207-5) contains supplementary material, which is available to authorized users.


Background
As currently circumscribed, the Asian gesneriad genus Paraboea (C.B.Clarke) Ridl. comprises ca. 130 species of rosulate or caulescent herbs characterized by the abaxially matted leaves with densely interwoven indumentum and flowers with flat-faced to shortly campanulate corolla and non-erect anthers (Middleton et al. 2010;Puglisi et al. 2016). A majority of Paraboea species are lithophytes on limestone substrates, distributed in South China, northeastern India and the eastern Himalayas, Indochina, and Malesia as far east as Sulawesi (Middleton et al. 2010). Since the last major revision by Xu et al. (2008) in which 89 species and 5 varieties were recognized, Paraboea has been expanded to include the ca. 20 species of Phylloboea Benth. and Trisepalum C.B.Clarke (Puglisi et al. 2011), with the reduction of five taxa constituting the new genus Middletonia C.Puglisi (Puglisi et al. 2016). More than 30 new species have also been described since the revision by Xu et al. (2008), almost all narrowly distributed endemic from limestone karsts (Chen et al. 2008(Chen et al. , 2012Kiew 2010;Triboun andMiddleton 2012, 2015;Xu et al. 2012a;Triboun 2013;Wen et al. 2013;Puglisi et al. 2015;Guo et al. 2016;Wen and Wei 2016). Because a great proportion of Asian limestone karsts remain unexplored or underexplored, it is fully expected that additional new species of Paraboea will be unearthed given that further field investigations and herbarium work are conducted (Puglisi et al. 2015).
During the course of extensive floristic surveys in limestone karsts of southern and southwestern China in recent years, we collected three species of Paraboea with spectacular flowers and/or fruits not known previously. After consulting the relevant literature (Burtt 1984;Wang et al. 1990Wang et al. , 1998Li and Wang 2004;Chen et al. 2008Chen et al. , 2012Xu et al. 2008Xu et al. , 2012aKiew 2010;Triboun and Middleton 2012;Wen et al. 2013;Guo et al. 2016;Wen and Wei 2016), as well as herbarium specimens of E, GXMI, HITBC, IBK, IBSC, KUN, and PE (herbarium acronyms according to Index Herbariorum; Thiers 2017), they were identified as three new species of Paraboea based on detailed examination of salient morphological and anatomical features and molecular phylogenetic analyses.

Taxon sampling and DNA sequencing
For phylogenetic analyses, a majority of species of Paraboea available in GenBank were used, with nine additional species endemic to China sampled. A total of 83 accessions representing 67 species of Paraboea were included in this study. Based on Puglisi et al. (2016), two species of Ornithoboea Parish ex C.B.Clarke and three species of Middletonia C.Puglisi were chosen as outgroups. Species, voucher information, and NCBI accession numbers are listed in Additional file 1. Two molecular markers, including the nuclear ITS (internal transcribed spacer) and the chloroplast trnL-F intronspacer region (trnL-F), were used in this study. Total genomic DNA was extracted from silica gel-dried leaf materials using the CTAB protocol (Doyle and Doyle 1987). The primers ITS-4 and ITS-5 (Möller and Cronk 1997) were used to amplify and sequence the ITS region. The primers trnL-F e and f (Taberlet et al. 1991) were used to amplify and sequence the cp DNA region based on the PCR procedures outlined in Guo et al. (2016). The PCR products were purified using the Tian quick Midi Purification Kit (TianGen Biotech, Beijing, China) and directly sequenced. Sequencing reactions were performed using the ABI Prism Bigdye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA). Sequences were analyzed using an ABI 3730 DNA Sequencer. The program Sequencher 5.0 (Gene Codes Co., Ann Arbor, Michigan, USA) was used to evaluate chromatograms for base confirmation and to edit contiguous sequences. Sequences were initially aligned using MUSCLE 3.8.31 (Edgar 2004), followed by manual adjustments in Geneious 9.1.2 (http://www.geneious. com, Kearse et al. 2012).

Phylogenetic analyses
The phylogenetic analyses were conducted based on maximum likelihood (ML) and Bayesian inference (BI) methods for the individual locus datasets (ITS/trnL-F) and combined dataset (ITS-trnL-F), using RAxML v7.0.4 (Stamatakis et al. 2008) and MrBayes v3.3.5 (Ronquist et al. 2012), respectively. The model GTR + Ґ was selected as the optimal model for both DNA regions based on the Akaike Information Criterion via jModeltest v2.1.4 (Posada 2008). For ML analyses, node support was estimated with nonparametric bootstrap (1000 replicates) following a thorough search for the best ML tree. For BI analyses, four runs of Metropolis-coupled Markov chain Monte Carlo (MCMCMC) analyses were conducted with one tree sampled for every 2000 generations over 20 million generations, starting with a random tree. Analyses were run until the average standard deviation of the split frequencies approached 0.01, indicating that two runs converged to a stationary distribution. The first 25% of sampled trees corresponding to the burn-in period was discarded, and the remaining trees were used to construct a majority-rule consensus tree. We used bootstrap support (BS) ≥ 70% and posterior probability (PP) ≥ 0.95 as the thresholds for strongly supported clades (Wang et al. 2014). To investigate congruence between the nuclear and chloroplast genomes, topologies of the ITS and trnL-F datasets of both ML and BI analyses were compared. Because a majority of clades with BS ≥ 70% and PP ≥ 0.95 were congruent without significant conflicts, the concatenated dataset was presented for further discussion.
Distribution, habitat and ecology: Paraboea dushanensis is only found at the type locality on limestone substrate (Fig. 8), and only one population has so far been identified by us during field investigations in 2015. Paraboea dushanensis grows on rock faces of the limestone karst, at an elevation between 900 and 960 m.
Phenology: This new species had been observed in flower from May to June, and fruit from July to August.
Etymology: The specific epithet is derived from the type locality, Dushan County, Southern Guizhou Province.
Notes: Paraboea dushanensis is most similar to P. velutina (Fig. 9a, b) in the habit and the leaf blade shape, but it can be distinguished from the latter by many leaf characters (see Diagnosis). Paraboea dushanensis is also similar to Paraboea crassifolia (Hemsl.) B.L.Burtt, but differs in the obvious subterete rhizomes, the branching at the apex of rhizome (vs. rhizomes very unobvious, rosulate), the leaf blade oblanceolate, rarely oblong, 4-8 × 0.7-1.5 cm, 3-6 times as long as wide, (vs. obovate, 3-16 × 1.5-5 cm, 2-3 times as long as wide), tertiary venation conspicuously reticulate on the upper leaf surface (vs. smooth on the upper leaf surface), calyx 3.5-5 mm long (vs. 2 mm long), capsule not twisted (vs. spirally twisted). Phylogenetic analyses revealed that these three species are closely related and yet considerably different from each other, supporting the recognition of P. dushanensis as a new species.
Distribution, habitat and ecology: Paraboea sinovietnamica is found near the border between China and Vietnam on limestone substrate, and only three populations have been identified so far by us during field investigations from 2006 to 2015 (Fig. 8). Paraboea sinovietnamica grows on rock faces of the limestone karst top, at an elevation between 400 and 1000 m.
Phenology: This new species had been observed in flower from May to June, and fruit from July to August.
Etymology: The specific epithet is derived from the type locality, the Sino-Vietnamese border.
Notes: Paraboea sinovietnamica is most similar to P. sinensis (Fig. 9c, d), but it is easily distinguished from the latter by the habit, leaves, inflorescences, and flowers (see Diagnosis). Phylogenetic analyses revealed that these two species are closely related and yet considerably different from one another, supporting the recognition of P. sinovietnamica as a new species.
Phenology: This new species had been observed in flower from March to April.
Etymology: The specific epithet is derived from the type locality, the border between Hunan province (Abbr. Xiang) and Guangxi Zhuangzu Autonomous Region (Abbr. Gui).
Notes: Paraboea xiangguiensis is most similar to P. guilinensis (Fig. 9e, f ) in its corolla shape, but it is easily distinguished from the latter by habit, leaves, and inflorescences (see Diagnosis). Paraboea xiangguiensis is also similar to P. dictyoneura (Fig. 9g, h), differing in leaf shape (blade spathulate, narrowly obovate to obovate-elliptic vs. narrowly obovate-elliptic), size (9-20 × 2.5-4.6 vs. 7-15 × 2-4.5 cm), margins (entire to shallowly repand vs. more or less serrullate), apex (obtuse to subround vs. acute to obtuse), and hairiness of peduncle and pedicel (glabrous vs. grayish matted indumentum) and bract and calyx (glabrous outside vs. grayish matted indumentum). Phylogenetic analyses revealed that these three species are closely related and yet considerably differentiated from one another, supporting the recognition of P. xiangguiensis as a new species.