Three New Species of Primulina
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Guo et al. Bot Stud DOI 10.1186/s40529-015-0115-5 ORIGINAL ARTICLE Open Access Three new species of Primulina (Gesneriaceae) from limestone karsts of China based on morphological and molecular evidence Jing Guo 1,2 , Bo Pan2 , Jing Liu2 , Wei‑Bin Xu 2* and Kuo‑Fang Chung 3* Abstract Background: With more than 160 described species, Primulina is one of the most characteristic plant groups of the Sino‑Vietnamese limestone fora. In our continous botanical inventory of the limestone fora of South China, we col‑ lected three new Primulina species not identifable to known species. Results: Molecular phylogenetic analyses based on nuclear ITS and chloroplast trnL‑F and trnH‑psbA sequences strongly support the placement the three new species in Primulina. In addition to morphological diferences, DNA sequences of all these three new species show substantial divergencies, sustaining the recognition of these three new species. Conclusions: Based on morphological and molecular data, we describe and illustrate three new Primulina species: P. maculata, P. pengii, and P. yangshanensis. Keywords: Flora of Guangdong, Molecular taxonomy, Primulina maculata, Primulina pengii, Primulina yangshanensis, Sino‑Vietnamese limestone karst Background et al. 2015). Currently, Primulina comprises ca. 160 As a consequence of recent molecular recircumscription pre dominately calciphilous species distributed almost (Wang et al. 2011; Weber et al. 2011), Primulina Hance exclusively to limestone karsts of China south of Yangtze has been expanded greatly from a monotypic genus River and northern Vietnam (Xu et al. 2012a, b; (Wang et al. 1990; Ying et al. 1993; Wang et al. 1998; L et al. 2013 ; Xu et al. 2013 ; Li et al. 2015 ). Among them, and Wang 2004; Zheng and Xia 2005; W 2010) to one ca. 130 species (85 %) are described from southern and of the largest genera of the Old World Gesneriaceae (Xu southwestern China (i.e., Guangxi, Guangdong, and Yun- et al. 2013). nan), an area recognized as a global biodiversity hotspot new Primulina species are being described (e.g., Huang (Xu 1995; Hou et al. 2010; López-Pujol et al. 2011), with et al. 2012; W et al. 2012; X et al. 2012a; Chung et al. a majority of the species known only from a single or a 2013; Pan et al. 2013; Xu et al. 2013; Zhao et al. 2013; Li handful of localities (Xu et al. 2012a; Ch2013; et al. 2014; Zheng and Deng 2014; Li et al. 2015; Ning Xu e 2013; Ch 2014). However, because Primulina was circumscribed based on molecular evidence and lacks apparent synapomor- [email protected]; kuofa 2 Guangxi Key Laboratory of Plant Conservation and Restoration phies (Weber et al. 2011), ome species might have been Ecology in Karst Terrain, Guangxi Institute of Botany, Guangxi Zhuang misplaced (e.g., Christie et al. 2012; Xu 2014). M Autonomous Region and Chinese Academy of Sciences, Guilin 541006, overnewly described species are China difered only by trivial morphological diferences, Weber 3 School of Forestry and Resource Conservation, National Taiwan University, Taipei 106, Taiwan et al. (2011) suspects that the diversity in Primulina is Full list of author information is available at the end of the article © 2015 Guo et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Guo et al. Bot Stud Page 2 of 13 likely overestimated. Terefore, it is essential to com- (1000 replicates) and a search for the best-scoring tree in bine molecular data with morphological observation for a single program run. Bayesian analyses were executed in generic placement and species description, taking into MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003), account of both morphological and genetic divergences the + Γ sian analyses (e.g., Xu et al. 2012a; Chung et al. 2013; Pan et al. 2013; were started from random trees, sampling one tree every Xu et al. 2013). 1000th generation, with four incrementally heated chains. During our botanical explorations in limestone karsts Te Markov chain Monte Carlo (MCMC) algorithm was of South China, we discovered three distinct species of run with two replicates for 400,000,000 generations for Primulina not identifable to any known species. Tis each data set. Analyses were run until the average stand- article provides molecular and morphological evidence ard deviation of the split frequencies approached 0.01, for the description of these three new species. indicating that two runs converged to a stationary distri- bution. Te frst 5000 trees corresponding to the ‘burn- Methods in’ period were discarded, and the remaining trees were Phylogenetic analyses used to construct a majority-rule consensus tree. Pos- Total genomic DNA of the three new species was terior probability (PP) was used to estimate robustness. extracted from silica-gel dried leaves collected from the We used the parsimony-based incongruence length dif- type locality using the CTAB protocol (Xu et al. 2012a). ference (ILD) test (Farris et al. 1994) to test for phyloge- DNA sequences of the nuclear internal transcribed spac- netic incongruence between cpDNA and nrDNA data ers (ITS) and the chloroplast intergenic spacer trnL-F, sets. Although the ILD test indicated that the two data- which were used for the recircumscription of Primulina sets are incongruent (P = 0.01), in the combined analy- (Wang et al. 2011; Weber et al. 2011), were amplifed and ses framework, the interaction of characters from the sequenced based on the PCR procedures outlined in Xu two incongruent partitions actually increase phylogenetic et al. (2012a). A resolution and support than that observed in separate plasttrnH-psbA sequences that are amply available in the analyses of individual data sets (DeSalle et al. 2002). C GenBank based on PCR conditions detailed in Kang et al. sequions were com- (2014). bined into a concatenated matrix and analyzed using ML In our initial BLAST search through GenBank using and BI methods with same parameters stated above. DNA sequences of the three new species, the closest relatives with the highest hit scores were species in the Results recently recircumscribed Primulina as expected based Phylogenetic analyses on morphological observations. To further elucidate Te aligned positions of ITS, trnL-F and trnH-psbA data- phylogenetic afnities of these new taxa, we included sets were 722, 852 and 643 base pairs, respectively. Te sequences of 92 species for ITS, 58 species for trnL- combined matrix of the three markers consisted of 2219 F, and 53 species for trnH-psbA of Primulina available characters. Te best ML phylogram, with bootstrap (BS) in GenBank. Te species and the GenBank accession supports and PP values of Bayesian analyses, are depicted numbers are listed in Appendix 1. Petrocodon dealbatus in Fig. 1. Hance, Petrocodon scopulorus (Chun) Yin Z. Wang, and Pylogenetic relationships of the concatenated matrix Petrocodon guangxiensis (Yan Liu & W.B. Xu) W.B. Xu are congruent with those reported in Kang et al. (2014) & K.F. Chung were chosen as outgroups based on recent and species phylogenetic analyses (Xu et al. 2014). (Primulina maculata, P. pengii, and P. yangshanensis) in inclof the three DNA regions Primulina (BS = 100 %; PP = 1.00). Additionally DNA in 101 species with 15 newly obtained. Sequences were sequences of all these three species show substantial dif- aligned using MUSCLE 3.8.31 (Edgar 2004) and adjusted ferences from other species of Primulina (Fig. 1) manually in Bioedit 5.0.9 (Hall 1990). ts. Within Te e frst analyzed separately Primulina, P. maculata was placed in Clade B, allied with using maximum likelihood (ML) and Bayesian inference P. gueilinensis, P. subulata, and P. lungzhouensis with no (BI) approaches. Optimal substitution models for each support (Fig. 1). Interestingly, none of these latter three data set were determined using the Akaike information species have the white maculation along the veins. Prim- criterion (AIC) using jModeltest 0.1.1 (Posada 2008) wi ulina pengii, together with P. orthandra and P. hunanen- GTR + Γ selected as the best model. ML analyses of each sis, formed a well-supported clade (BS = 77 %, PP = 0.99) datasets were performed using RAxML 7.0.3 (Stamatakis closely allied with P. lutea, P. eburnean, and P. xiziae that et al. 2008), + Γ all possess big bracts with strong support (BS = 99 %, tion selected and the option of a rapid bootstrap analyses PP = 1.00). Primulina yangshanensis is sister to the group Guo et al. Bot Stud Page 3 of 13 73 97 P. andengensis 0.88 1.00 P. minu maculata 0.99 59 59 P. bullata P. brachytricha 1.00 67 P. mollifolia 0.93 P. rongshuiensis 68 0.92 96 99 P. spinulosa P. hedyo dea 1.00 P. wentsaii P. longzhouensis 100 1.00 P. pungen sepala 91 P. renifolia P. jingxiensis 861.00 100 100 P. longgangensis P. linearis P. purpurea P. ophiopogoides 100 P. weii 611.00 P. macrodonta 1.00 P. orthandraP. ningmingensis 81 77 1.00 P. hunanensis 99 1.00 P. lutea 99 P. xiziae P. eburnea 100 P. chizhouensis 94 0.88 P. tenuituba 0.97 Primulina pengii sp. nov. 0.99 0.96 0.99 P. mabaensis P. depressa 50 P. danxiaensis 72 1.00 P. juliae P. lobulata 1.00 80 1.00 0.99 P. tabacum P. la P. nervis xiuningensis 73 66 P. roseoalba 1.00 95 0.99 P. langshanica P. tribracteata 95 P. sclerophylla 82 P. varicolor 0.99 97 0.99 P. liboensis 80 P. leiophylla 1.00 P.