Journal of Systematics JSE and Evolution doi: 10.1111/jse.12247 Research Article

A preliminary species-level phylogeny of the alpine ginger : Implications for speciation

Jian-Li Zhao1,2, Jinshun Zhong3, Yong-Li Fan2,4, Yong-Mei Xia2, and Qing-Jun Li1*

1Laboratory of Ecology and Evolutionary Biology, State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, Kunming 650091, 2Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla 666303, China 3Department of Biology, University of Vermont, 63 Carrigan Drive, Burlington, VT 05405, USA 4China Forest Exploration and Design Institute of Kunming, Kunming 650216, China *Author for correspondence. E-mail: [email protected]. Tel.: 86-871-65030660. Received 13 December 2016; Accepted 1 March 2017; Article first published online xx Month 2017

Abstract Speciation, the evolutionary process forming new species, is a key mode generating biodiversity on the Earth. In this study, we produced a species-level phylogeny of Roscoea using one nuclear ribosomal and two chloroplast DNA fragments based on Bayesian inference and maximum likelihood. We then explored the possible speciation processes using the species-level phylogeny and the heterozygous sites in the nuclear DNA. The incongruence between nuclear and chloroplast phylogenies, and several heterozygous sites in the nuclear DNA, suggested that R. auriculata might have a hybrid origin with R. purpurea and R. alpina being two possible parental progenitors; however, one alternative possibility through incomplete lineage sorting cannot be ruled out. In addition, R. kunmingensis likely originated from R. tibetica Batalin through the process of “budding speciation”. These results provided a valuable framework to draw testable hypotheses for future in-depth comparative studies to further our understanding of the underpinning speciation and adaptation mechanisms that contribute to ultrahigh biodiversity in the and the Hengduan Mountains. Key words: biodiversity hotspots, budding speciation, hybridization, phylogenetics, Roscoea.

1 Introduction taxonomic treatment (Wu & Kai, 2000; Cowley, 2007; Luo et al., 2007; Mao & Bhaumik, 2007). Roscoea species are Speciation, the formation of new species through splitting, unique in that many adapt to high-elevation regions (>4000 m hybridization, or polyploidization, is a key evolutionary process above sea level), for instance, R. alpina Royle in the HIM and R. that produces biodiversity on Earth (Coyne & Orr, 2004; Butlin tibetica Batalin in the Hengduan Mountains, whereas the rest et al., 2009). Exploring the patterns of speciation in a biological of the family occur mostly in pantropics and group through phylogenetic reconstruction can provide insight subtropics (Kress et al., 2002; Cowley, 2007). Field observation into the origin of biodiversity, especially in biodiversity hotspots and molecular studies have suggested that Roscoea showed (e.g., Richardson et al., 2001a, 2001b; Liu et al., 2006; Che et al., disjunct distribution between the Himalayas and the Heng- 2010; Zhao et al., 2016b). With diverse and rich endemic species, duan Mountains in northern Indochina (NIC) (Ngamriabsakul the Himalayas (HIM) and the Hengduan Mountains are et al., 2000; Cowley, 2007) (Fig. 1). A recent study also revealed considered two such hotspots (Myers et al., 2000; Mutke & that the origin and disjunction of Roscoea was associated with Barthlott, 2005). Many genus-level phylogenies have been the uplift of the Himalayan–Tibetan Plateau and the rapid inferred to assess the patterns and origins of high biodiversity extrusion of Indochina, respectively, with Roscoea in NIC likely in these two regions, such as the vicariance of warblers being derived from those in HIM (Zhao et al., 2016b). Thus, the (Johansson et al., 2007), Paini (Che et al., 2010), and Roscoea genus Roscoea is an ideal group in which to assess the Smith (Zhao et al., 2016b), and radiation of Isodon (Schrad. ex speciation patterns and processes underlying their radiation in Benth.) Spach (Yu et al., 2014) and Abies Mill. (Peng et al., 2015). these two biodiversity hotspots. However, in-depth species-level speciation study of the Previous phylogenies of Roscoea were poorly supported endemic genera is still lacking. Thus, more detailed intrageneric due to limited taxonomic sampling and insufficient phyloge- phylogenies, especially for the endemic genera, are critically netic signals using only a single molecular marker (Ngamriab- needed to uncover the general and/or unique speciation sakul et al., 2000). Although dense population-level sampling fi processes during alpine species diversi cation. strategy in a phylogeographic study has provided compelling The genus Roscoea Smith (Zingiberaceae) is endemic to the evidence for the disjunction of Roscoea between the HIM and Sino-HIM, including 22 species according to the latest NIC (Zhao et al., 2016b), reticulate evolution caused by recent

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Fig. 1. Sampling of Roscoea species along the Himalayas (HIM) and Hengduan Mountains of northern Indochina (NIC). gene flow and introgression after speciation may be too Sang et al., 1995; Techaprasan et al., 2006). Polymerase chain noisy to infer species-level phylogeny, despite diverse distinct reaction (PCR) was carried out in a total volume of 50 mL morphologies among species (Fig. 2). Therefore, extended reaction including 1 PCR buffer (50 mmol/L KCl, 10 mmol/L phylogenetic analyses for Roscoea with broader species-level Tris, pH 8.0), 2 mmol/L MgCl2, 0.2 mmol/L dNTP, 1 mmol/L sampling and additional molecular markers are needed. In this primer, 0.25 U Taq DNA polymerase, and approximately 10 ng study, we inferred phylogenies of Roscoea using nuclear genomic DNA template. We used the following PCR internal transcribed spacers (nrITS) and chloroplast DNA conditions: 94 °C for 5 min, followed by 35 cycles of (cpDNA). We then used these results to assess species 94 °C for 30 s, annealing temperatures for 30 s and 72 °C for relationships and proposed hypotheses of likely speciation 1.5 min, with a final extension at 72 °C for 15 min. The annealing modes for future in-depth speciation analyses. temperatures were 56 °C, 53 °C, and 56 °C for nrITS, psbA-trnH, and trnL-F, respectively. The PCR products were bidirectionally sequenced using the amplification primers on an ABI 3730 2 Material and Methods DNA Analyzer (Applied Biosystems, Foster City, CA, USA). If unreadable peak signals or conflict were found between 2.1 Taxon sampling and DNA sequencing bidirectional sequences at the same locus for one individual, Morphological species concept of Roscoea (sensus Wu & Kai, sequences were re-amplified and resequenced to ensure 2000; Cowley, 2007) was used for field sampling. Forty-eight that heterozygous sites (double peaks) were not the artifacts accessions representing 18 of 22 total delimited Roscoea of sequencing error. DNA sequences of H. gardnerianum species were collected from the HIM and NIC (Fig. 1). The were obtained from GenBank (nrITS, AY424759; psbA-trnH, remaining four species are distributed in a very small, KC597931; trnL-F, AY424787). The newly generated sequences restricted area and were not sampled in this study, including of Roscoea and Cautleya have been submitted to the GenBank R. ganeshensis Cowley & W.J. Baker, R. brandisii (King ex database with accession numbers KY210313–KY210363 (nrITS), Baker) K. Schum., R. ngainoi A.A. Mao & Bhaumik, and R. KY210364–KY210414 (psbA-trnH), and KY210415–KY210465 cangshanensis M.H. Luo, X.F. Gao & H.H. Lin (Table 1). We used (trnL-F). Cautleya J.D. Hooker (C. gracilis (Sm.) Dandy and C. spicata (Sm.) Baker) and Hedychium gardnerianum Roscoe as out- groups according to previous studies (Kress et al., 2002; Zhao 2.2 Phylogenetic reconstruction et al., 2016b). Sequences were aligned using MAFFT (Katoh et al., 2002) Total genomic DNA from silica-dried leaves for each followed by manual adjustment in MEGA version 5 (Tamura accession was extracted using the CTAB protocol (Doyle & et al., 2011). Chloroplast psbA-trnH and trnL-F were Doyle, 1987). We used ITS1-5.8S–ITS2 nrITS and cpDNA psbA- concatenated for phylogenetic inference. Gaps were coded trnH, including the partial trnH gene, and trnL-F, including the using the sample-coding method (Simmons & Ochoterena, partial trnF gene using previous primers (Taberlet et al., 1991; 2000) in SeqState version 1.4.1 (Muller,€ 2005). We ran the

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Fig. 2. Diverse species of Roscoea (A–L) and phylogram of the 50% major consensus tree (M,N). A, R. tibetica Batalin. B, R. kunmingensis. C, R. debilis. D, R. cautleoides. E, R. humeana. F, R. schneideriana. G, R. alpina. H, R. bhutanica. I, R. auriculata. J, R. purpurea. K, R. tumjensis. L, R. capitata. Bayesian inference and maximum likelihood analyses of nuclear internal transcribed spacer (M) and chloroplast DNA (N). The topology of the phylogram is a best Bayesian inference tree. Bayesian posterior probability and maximum likelihood bootstrap support values are listed above and below the corresponding branches, respectively. Asterisks show that support values are higher than 90%; oblique lines show that support values are lower than 50%. Thick lines indicate species with heterozygous sites. Numbers following the species are the sample identifiers. Double slash indicates branch is shortened. Cautleya (C. gracilis and C. spicata)andHedychium gardnerianum are the outgroups. HIM, Himalayan; NIC, northern Indochina.

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Table 1 Sampling and vouchers of Roscoea Sm. and Cautleya Hook. f † Species Sample ID Location Specimen code R. alpina Royle 64301 Thyangsyapu, Nepal XYM643 70006 Yadong, Xizang, China HITBC150257 R. auriculata K. Schum. 75101 Nagahot, Nepal XYM751 75102 Nagahot, Nepal XYM751 R. bhutanica Ngamr. 69901 Yadong, Xizang, China HITBC150256 R. capitata Sm. 63701 Bamboo, Nepal XYM637 76002 Ricemphet, Nepal XYM760 77101 Tumje, Nepal XYM771 77601 Charikot, Nepal XYM776 R. nepalensis Cowley 4104 Lete, Nepal R41 4105 Lete, Nepal R41 R. purpurea Sm. 64705 Ulleri, Nepal XYM647 64902 Ghandruk, Nepal XYM649 R. tumjensis Cowley 76901 Tumje, Nepal XYM769 77001 Tumje, Nepal XYM770 R. debilis Gagnep. 60103 Lincang, Yunnan, HITBC150237 71404 Shidian, Yunnan, China XYM714 R. cautleoides Gagnep. 27801 Lijiang, Yunnan, China XYM278 28204 Lijiang, Yunnan, China XYM282 R. forrestii Cowley 73201 Binchuan, Yunnan, China XYM732 73203 Binchuan, Yunnan, China XYM732 R. humeana Balf. f. & W.W. Sm. 20707 Lijiang, Yunnan, China XYM207 73906 Ninglang,Yunnan, China HITBC150278 R. scillifolia (Gagnep.) Cowley 23403 Lijiang, Yunnan, China HITBC150227 26501 Yanyuan, Sichuang, China XYM265 27706 Yanbian, Sichuang, China XYM277 62509 Zhongdian, Yunnan, China XYM625 62907 Zhongdian, Yunnan, China XYM629 78503 Zhongdian, Yunnan, China XYM785 R. kunmingensis S.Q. Tong 4404 Kunming, Yunnan, China R44 4405 Kunming, Yunnan, China R45 R. praecox K. Schum. 4804 Kunming, Yunnan, China R48 R. schneideriana (Loes.) Cowley 24801 Lijiang, Yunnan, China XYM248 25001 Zhongdian, Yunnan, China XYM250 26201 Yanyuan, Sichuang, China XYM262 27401 Lijiang, Yunnan, China XYM274 Roscoea sp. 71101 Fugong, Yunnan, China HITBC150267 Roscoea sp. 71102 Fugong, Yunnan, China HITBC150267 Roscoea sp. 26120 Xichang, Sichuang, China XYM261 Roscoea sp. 70502 Dali, Yunnan, China HITBC150264 R. tibetica Batalin 22901 Lijiang, Yunnan, China HITBC150221 25201 Zhongdian, Yunnan, China XYM252 29312 Menglian, Yunnan, China XYM293 60210 Jingdong, Yunnan, China XYM602 70610 Dali, Yunnan, China XYM706 R. tibetica var. emarginata S.Q. Tong 74401 Wenshan, Yunnan, China HITBC150280 R. wardii Cowley 26603 Yanbian, Sichuang, China XYM266 26604 Yanbian, Sichuang, China XYM266 Cautleya gracilis (Sm.) Dandy 29901 Jingdong, Simao, China XYM299 60401 Jingdong, Simao, China XYM604 C. spicata (Sm.) Baker 65001 Langtang, Nepal XYM650 † XYM and R refer to the herbarium and DNA samples that are deposited in the Plant Evolutionary Ecology Group of Xishuangbanna Tropical Botanical Garden (XTBG). HITBC, Herbarium of XTBG. incongruence length difference (ILD) to test the compatibility congruence between datasets from different genomes; between cpDNA and nrDNA datasets (Farris et al., 1994). The however, this test is too conservative as it examines whether null hypothesis (congruence) of ILD is rejected if the P-value the combined dataset would produce one parsimonious tree, is less than 0.05. The ILD test is routinely used to test but does not compare the topologies between trees of

J. Syst. Evol. 9999 (9999): 1–10, 2017 www.jse.ac.cn Species-level phylogeny of Roscoea 5 different genes (Cunningham, 1997; Darlu & Lecointre, 2002). maximum agreement subtree test suggested the phyloge- Therefore, we also tested the topological similarity between netic topologies of cpDNA and nrITS were more congruent ¼ < trees using the maximum agreement subtree with congru- than expected by chance (Icong 2.01, P 0.001). Thus, our ence index (de Vienne et al., 2007). The null hypothesis discussions focus more on the discordance between cpDNA (incongruence) is rejected if the P-value is less than 0.05. and nrITS, and we are cautious in interpreting the findings of Indeed, regardless of discordance, combined datasets would the combined dataset. provide better phylogenetic inference (Poe & Swofford, 1999; Phylogenetic positions of several Roscoea species were Rosenberg & Kumar, 2001). Thus, phylogenetic trees of the inconsistent between nrITS and cpDNA phylogenies. Most combined dataset of the two genomes were also recon- notably, R. tumjensis Cowley is sister to R. capitata Sm. in structed using parameters as those in individual datasets. the nrITS tree (BPP > 0.90; MBS > 90) (Fig. 2M), but to The optimal nucleotide substitution model was selected R. auriculata, R. alpina, and R. nepalensis Cowley in the cpDNA using jModelTest version 0.1 with the Akaike information phylogeny (BPP > 0.90; MBS ¼ 83) (Fig. 2N). Likewise, R. criterion (Posada, 2008). The best-fit models of nrITS and auriculata formed a well-supported monophyletic clade with cpDNA were GTR (general time reversal) þ G (gamma) and R. purpurea and R. bhutanica Ngamr. in the nrITS phylogeny GTR þ I (invariant) þ G, respectively. The model of gap-coded (BPP > 0.90; MBS ¼ 86), but it formed a strong monophyletic dataset was F81 þ G. Bayesian inference was implemented in clade with R. alpina, R. tumjensis, and R. nepalensis in MrBayes version 3.2 (Ronquist et al., 2012). Specifically, two the cpDNA phylogeny (BPP > 0.90; MBS > 90). Although independent Bayesian runs were carried out using 5 million the species of the R. humeana Balf. f. & W. W. Sm. and generations with four Markov chains and sampling every R. cautleoides Gagnep. clade in the nrITS phylogeny were 500 replicates. A consensus tree with Bayesian posterior ambiguous due to low support values, potential relatives of probability (BPP) was generated after discarding the first 25% these two species in the nrITS phylogeny were distinct from trees as burn-in. Maximum likelihood (ML) tree was inferred those in the cpDNA tree (R. schneideriana (Loes.) Cowley was using RAxML version 8.1 (Stamatakis, 2006; Stamatakis et al., closely related to R. humeana and R. cautleoides; BPP > 0.90; 2008). The support values (MBS) of the ML tree were MBS > 90). Roscoea wardii and R. scillifolia Gagnep. formed a estimated using 1000 bootstrap replicates with the model monophyletic clade in the nrITS tree (BPP > 0.90; MBS ¼ 82) GTRGAMMA. whereas R. wardii is embedded within the R. tibetica/R. kunmingensis S.Q. Tong clade in the cpDNA tree (BPP > 0.90). Some species were strongly supported as closely related 3 Results to each other in the combined dataset (Fig. 3). For instance, R. humeana and R. cautleoides formed a well-supported 3.1 Sequence characteristics monophyletic group (BPP > 0.90; MBS > 90). The narrowly The lengths of aligned sequences of nrITS, psbA-trnH, and endemic species R. kunmingensis, R. bhutanica,andR. nepalensis trnL-F were 685 bp, 847 bp, and 1003 bp, respectively. The each clustered with the widespread species R. tibetica, fi combined cpDNA was 1850 bp. There were ve insertions/ R. purpurea,andR. alpina, respectively. deletions (ranging from 1 bp to 54 bp) in the nrITS dataset, seven insertions/deletions (ranging from 1 bp to 438 bp) and a 92-bp high variation region in the psbA-trnH dataset, and 12 insertions/deletions (ranging from 1 bp to 15 bp) in the trnL-F 4 Discussion dataset. The 438-bp deletion of psbA-trnH is found in the Roscoea species are largely endemic to the Himalayas and the outgroup Cautleya. Hengduan Mountains, and exhibit extremely diverse floral In total, 75 polymorphic sites were found in the nrITS traits that may link to local adaptations and/or distinct dataset of Roscoea excluding gaps. Among these variations, pollination syndromes (Zhang & Li, 2008; Zhang et al., 2011; 32 sites contained heterozygous sites (Tables 2, S1). Twenty- Fan & Li, 2012; Paudel et al., 2015, 2016). Compared to previous seven heterozygous sites were present in R. auriculata K. phylogenetic results (Ngamriabsakul et al., 2000), this study Schum., R. purpurea Sm., and R. debilis Gagnep. The remaining produced a more informative phylogenetic backbone using five heterozygous sites were found in one individual each of nrDNA and cpDNA from which testable hypotheses can be R. alpina, R. tibetica, and R. wardii Cowley, respectively. drawn for future in-depth speciation studies for this group. Species that might involve hybridization are listed in Table 2 with supporting polymorphic sites (Fig. 2). 4.1 Hybridization and incomplete lineage sorting in Roscoea Nuclear ribosomal ITS and cpDNA are biparentally inherited 3.2 Phylogenetic relationships among Roscoea species and maternally inherited in most angiosperms, respectively Two distinct monophyletic clades of Roscoea were strongly (Corriveau & Coleman, 1988). Hybridization/introgression and/ supported in the nrITS phylogeny, the HIM clade (BPP > 0.90; or incomplete lineage sorting (ILS) are the key factors leading MBS ¼ 75) and the NIC clade (BPP > 0.90; MBS ¼ 80) to the phylogenetic incongruence between nrITS and cpDNA (Fig. 2M); however, the monophyly of each of these two trees (Degnan & Rosenberg, 2009; Joly et al., 2009; Petit & clades were not well supported in the cpDNA (Fig. 2N). Excoffier, 2009). Hybridization integrates the genetic materi- Likewise, the two Roscoea clades were strongly supported in als from two distinct parental species while incomplete the concatenated matrix of nrITS and cpDNA (HIM clade, lineage sorting preserves common ancestral polymorphism BPP > 0.90 and MBS ¼ 87; NIC clade, BPP > 0.90 and MBS (e.g., Jakob & Blattner, 2006; Carstens & Knowles, 2007). In ¼ 81) (Fig. 3). Although the ILD test suggested that cpDNA the case for hybridization/introgression, concerted evolution and nrITS were not suitable for combination (P < 0.01), the of nrITS could occur in the descendants (e.g., Sang et al., 1995; www.jse.ac.cn J. Syst. Evol. 9999 (9999): 1–10, 2017 6 Zhao et al.

Table 2 Roscoea species linked to hybridization inference and polymorphic sites of nuclear internal transcribed spacers, grouped according to clade Species Polymorphic sites in HIM clade R. alpina_70006 GGCGTGTCATG G CCCGG R. alpina_64301 GGCGTGTCATG G CCCGG R. nepalensis_4104 GGCGTGTCATG G CCCGG R. nepalensis_4105 GGCGTGTCATG G CCCGG C A A R. auriculata_75101 ACCGCGTCA CTCGG T G G C A A R. auriculata_75102 ACCGCGTCA CTCGG T G G C A R. purpurea_64705 ACCGCGT ACA A TCGG T C C A R. purpurea_64902 ACCGCGT A CA A TCGG T C R. bhutanica_69901 ACCGCGTCACA A CTCGG R. capitata_63701 GGTACAGCGCA A CTTAA R. capitata_76002 GGTACAGCGCA A CTTAA R. capitata_77101 GGTACAGCGCA A CTTAA R. capitata_77601 GGTACAGCACA A CTTAA R. tumjensis_76901 AGTGCAGTACG A ATTAA R. tumjensis_77001 AGTGCAGTACG A ATTAA Polymorphic sites in NIC clade R. cautleoides_27801 ATGGAGAGAGG C ATCCCGC R. cautleoides_28204 ATGGAGAGAGG C ATCCCGC R. humeana_20707 ATGGAGAGAGG C ATTACGC R. humeana_73906 ATGGAGAGAGG C ATTACGC R. scillifolia_23403 ATGGAGAGAGG C AATACAC R. scillifolia_26501 ATGGAAAGAGG C AATACAC R. scillifolia_27706 ATGGAGAGAGG C AATACAC R. scillifolia_62509 ATGGAGAGAGG C AATACAC R. scillifolia_62907 ATGGAGAGAGG C AATACAC R. scillifolia_78503 ATGGAGAGAGG C AATACAC R. wardii_26603 ATGGAGAGAGG C AATACAC R. wardii_26604 ATGGAGAGAGG C AATACAC G A G A A A C R. debilis_60103 ATA AA GG A CCGC C G A C C T T A A A A A C R. debilis_71404 ATAGA G GCA CCGC G G C G T T R. tibetica_22901 TTACGAGGCAA A GTCCGAA A R. tibetica_25201 TTAC AGGCAA C GTCCGAA G R. tibetica_29312 ATGCGATGCAA C ATCCGAA R. tibetica_60210 AAACAAGACAA C GTCCGAA R. tibetica_70610 AAACAAGACAA C GTCCGAA Heterozygous sites are in bold. Species showing phylogenetic incongruence are shaded gray. HIM, Himalayas; NIC, northern Indochina.

Wendel et al., 1995; Fuertes Aguilar et al., 1999; Koch et al., species may form a distinct clade in phylogenetic trees where 2003). There are three possibilities of concerted evolution for it is difficult to identify its parental donors. In the third, two different copies in an individual (Alvarez & Wendel, 2003; heterozygous sites can be used to infer the potential parental Koch et al., 2003): fixation of one copy and loss of the other progenitors. Therefore, incongruence between markers from one, mixture of the two copies forming a new genotype, or different genomes, integrating with the observed heterozy- maintenance of two copies with heterozygous sites. Under gous sites, offers an opportunity to detect speciation through the first scenario, if concerted evolution of nrITS directs to the hybridization (e.g., Sang & Zhong, 2000; Kim & Donoghue, paternal parent, the phylogenetic trees derived from nrITS 2008; Russell et al., 2010; Yu et al., 2014; Xiang et al., 2015), and cpDNA may exhibit conflicts that can help identify the although we cannot completely rule out the possibility of ILS parental donors of the hybrid. In the second situation, a hybrid in this study. Phylogenetic analyses and heterozygous sites

J. Syst. Evol. 9999 (9999): 1–10, 2017 www.jse.ac.cn Species-level phylogeny of Roscoea 7

Fig. 3. The 50% consensus Bayesian tree of the combined nuclear internal transcribed spacer and chloroplast DNA of Roscoea. Values next to the branch are Bayesian posterior probability (above) and maximum likelihood bootstrap support values (below). Support values greater than 90% and lower than 50% were marked with asterisks and oblique lines, respectively. Double slash indicates branch is shortened for better presentation. Cautleya (C. gracilis and C. spicata) and Hedychium gardnerianum are outgroups. alone are insufficient to identify the exact underlying R. purpurea/R. bhutanica clade being parental progenitor and evolutionary processes of species divergence; however, our the R. alpina/R. nepalensis/R. tumjensis group being maternal phylogenies with multiple accessions for each species in donor (Fig. 2M, 2N). It remains unclear which species could particular allow us to draw some reasonable preliminary be the direct parental donors for this hybrid species. hypotheses on speciation/population divergence for future However, morphological characters and geographical distri- in-depth population-level analyses. bution seem to suggest that R. purpurea and R. alpina are the In this study, phylogenetic positions of R. auriculata were most likely parental progenitors. Roscoea purpurea morpho- inconsistent between the nrITS and cpDNA trees (Fig. 2), logically resembles R. auriculata by having purple flower, indicating possible hybridization or ILS in Roscoea. Three narrowly elliptic dorsal , and broadly obovate labellum heterozygous sites of nrITS in R. auriculata (Table 2) suggest (Fig. 2) and geographically overlaps with R. auriculata and that this species is the result of hybridization with the R. alpina from west to east in the Himalayas. Furthermore, www.jse.ac.cn J. Syst. Evol. 9999 (9999): 1–10, 2017 8 Zhao et al.

R. nepalensis is morphologically different from R. auriculata by speciation of Picea morrisonicola Hayata from P. wilsonii Mast. having white flower, circular , and undulate edges (Zou et al., 2013). Thus, R. kunmingensis likely originated from of labellum, and is endemic to a restricted area where it is very a budding speciation process derived from its progenitor unlikely that it could hybridize with other potential parental R. tibetica. donors. Alternatively, if heterozygous sites represent ances- tral polymorphisms, the phylogenetic inconsistence of R. auriculata between the cpDNA and nrDNA trees may result from ILS. However, further population genetic analyses and/ 5 Conclusions or additional nuclear DNA are needed to better determine the Our work presented a preliminary phylogenetic framework of underlying evolutionary processes for the origin and species/ an endemic alpine ginger genus Roscoea in the Himalayas and population differences of R. auriculata and its allies. Hengduan Mountains. This study found that diversification of Some other putative hybrid species (R. cautleoides/R. Roscoea may experience various speciation processes, includ- humeana, R. debilis, R. tumjensis,andR. wardii)maybeinferred ing hybridization and/or ILS, and budding speciation. Limited from the comparative tree analyses and heterozygous sites; evolutionary information restricted us to make more conclu- however, the possible underpinning evolutionary processes sive inferences; however, the phylogenetic framework of remain less clear in this work. Although R. cautleoides/R. Roscoea is the cornerstone for extended research (e.g., Zhao humeana and R.schneideriana formed a sister clade in the cpDNA et al., 2016a). Further in-depth work on populations or closely tree, it was very unlikely that R. schneideriana is a potential related species using next-generation sequencing (Zimmer & progenitor of the hybrid species because this species is strictly Wen, 2015) and common garden experiments will provide autonomously selfing (Zhang & Li, 2008). Thus, monophyly of R. novel insights into patterns and underlying processes of alpine cautleoides/R. humeana and R. schneideriana in the cpDNA tree diversification and adaptations. may be due to incomplete lineage sorting. Other species showed either heterozygous sites with low resolution of the cpDNA tree (R. debilis) or phylogenetic inconsistencies without Acknowledgements heterozygous sites (R. tumjensis and R. wardii), making it This study was financially supported by the National Natural challenging to determine the exact processes (hybridization or Science Foundation of China (Project No. U1202261, 41601061). incomplete lineage sorting) from our results. We thank Babu Ram Paudel for sampling assistance in Nepal. 4.2 Budding speciation gave rise to R. kunmingensis Roscoea kunmingensis and R. tibetica are allopatrically isolated and formed a strongly supported clade in the nrITS References and combined phylogenies (Figs. 2M, 3). Morphologies and Alvarez I, Wendel JF. 2003. Ribosomal ITS sequences and plant phenology between these two species are different. The phylogenetic inference. Molecular Phylogenetics and Evolution 29: – lateral staminodes and dorsal petal are almost equal in length 417 434. in R. kunmingensis, whereas the lateral staminodes are about Butlin R, Bridle J, Schluter D. 2009. Speciation and patterns of half the length of, and concealed within, the dorsal petal in diversity. Cambridge: Cambridge University Press. R. tibetica. Blooming time of R. kunmingensis in mid-May is Carstens BC, Knowles LL. 2007. Estimating species phylogeny from earlier than that of R. tibetica, from June to July. Another gene-tree probabilities despite incomplete lineage sorting: An major difference between R. kunmingensis and R. tibetica is example from Melanoplus grasshoppers. Systematic Biology 56: – the relative timing of leafing and flowering, being flowering 400 411. first in the former and leafing first in the latter. Additionally, Che J, Zhou WW, Hu JS, Yan F, Papenfuss TJ, Wake DB, Zhang YP. 2010. R. kunmingensis and R. tibetica are allopatrically isolated Spiny frogs (Paini) illuminate the history of the Himalayan region and largely selfing, producing fruits without pollinators and Southeast Asia. Proceedings of the National Academy of – (J. L. Zhao, personal observation, 2013). Finally, phylogeneti- Sciences USA 107: 13765 13770. cally, R. kunmingensis has a longer branch than R. tibetica Corriveau JL, Coleman AW. 1988. Rapid screening method to detect (Fig. 3). These observations collectively indicate a phenome- potential biparental inheritance of plastid DNA and results for non named “budding speciation” (also called progenitor- over 200 angiosperm species. American Journal of Botany 75: 1443–1458. derivative speciation) (Crawford, 2010). Likewise, phyloge- netic results alone seem to suggest that R. nepalensis also Cowley EJ. 2007. The genus Roscoea. 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