Pollen morphology of Indigofera (Fabaceae) in China and its taxonomic implications Xue-Li Zhao, Xin-Fen Gao & Bo Xu Plant Systematics and Evolution ISSN 0378-2697 Volume 302 Number 4 Plant Syst Evol (2016) 302:469-479 DOI 10.1007/s00606-015-1275-1 1 23 Your article is protected by copyright and all rights are held exclusively by SpringerVerlag Wien. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”. 1 23 Author's personal copy Plant Syst Evol (2016) 302:469–479 DOI 10.1007/s00606-015-1275-1 ORIGINAL ARTICLE Pollen morphology of Indigofera (Fabaceae) in China and its taxonomic implications Xue-Li Zhao1,2 • Xin-Fen Gao1 • Bo Xu1 Received: 8 July 2015 / Accepted: 19 December 2015 / Published online: 12 February 2016 Ó Springer-Verlag Wien 2016 Abstract Asia, especially the temperate Sino-Himalayan region is one of the four major diversity centers of Indigofera. Pollen morphology of Indigofera species in the Sino-Himalayan region is poorly known. In this study, pollen morphology of 52 samples representing 43 Chinese Indigofera species and two varieties was examined using scanning electron microscopy to evaluate its taxonomic significance. Parasyncolpate pollen grains were described in Indigofera for the first time. Cluster and principal component analyses were conducted based on four quantitative and three qualitative pollen characters. Five groups were recognized within Chinese Indigofera in the cluster analysis, but only one can be separated by the first three principal components. The shape and size of pollen grains in Indigofera are highly variable. Consequently, both the characters lack any significant taxonomic value. The number of apertures, tectum architecture and perforation density shows some taxonomic significance. The pollen Handling editor: Xiang-Yun Zhu. Electronic supplementary material The online version of this article (doi:10.1007/s00606-015-1275-1) contains supplementary material, which is available to authorized users. & Xin-Fen Gao xfgao@cib.ac.cn Xue-Li Zhao zhaoxueli87@126.com Bo Xu xubo@cib.ac.cn 1 Chengdu Institute of Biology, Chinese Academy of Sciences, P.O. Box 416, Chengdu 610041, Sichuan, China 2 University of Chinese Academy of Sciences, Beijing 100049, China morphology exhibits obvious phylogenetic and biogeographical significance on large scales in Indigofera. Although pollen characters alone are insufficient to reconstruct the taxonomic relationships within Indigofera, palynological data can provide some useful information for the species-level revisions. Keywords Fabaceae
Indigofera
Palynology
Scanning electron microscopy
Taxonomy Introduction Indigofera L. is the third largest genus in Fabaceae, comprising approximately 750 species (Schrire et al. 2005, 2009). The genus has a pantropical distribution with four major diversity centers: Africa and Madagascar (ca. 550 spp.), Asia, especially the temperate Sino-Himalayan region (ca. 105 spp.), Australia (ca. 50 spp.), and the New World (ca. 45 spp.) (Schrire et al. 2009). Indigofera species are annuals or perennials, and habit varies from prostrate and erect herbs to undershrubs, robust shrubs, and rarely trees. Infrageneric classification of Indigofera has long been controversial. At least twelve revisions (Schrire 1995) have been proposed based on morphological characters since the establishment of the genus in 1753 (Linnaeus 1753). The most recent and comprehensive classification was proposed by Schrire (1995) based on a morphological cladistic analysis of a dataset containing 58 morphological characters of ca. 600 Indigofereae species, in which 25 sections and 14 groups were recognized within Indigofera. Subsequently, combined DNA (nrITS or plastid DNA) sequences and morphological data analyses revealed that Indigofera species clustered into four monophyletic clades, i.e., 123 Author's personal copy 470 Palaeotropical clade, Pantropical clade, Cape clade and Tethyan (Boreotropical) clade (Barker et al. 2000; Schrire et al. 2003, 2009). In China, Fang and Zheng (1989, 1994) recognized 80 species and eight varieties, and they also divided Chinese Indigofera species into three subgenera and 14 subsections chiefly based on the shape of fruits, the number of leaflets, and the types of trichomes. In a recent revision of Chinese Indigofera, Gao and Schrire (2010) recognized 79 species and nine varieties, of which 45 are endemic. The infrageneric classification of Fang and Zheng (1989, 1994) was not adopted by Gao and Schrire (2010), and no new infrageneric classification was reconstructed. Recently, I. pseudonigrescens X.F.Gao & Xue Li Zhao (2015), a new species from China, was published. Only a few Chinese Indigofera species have been involved in previous studies (Barker et al. 2000; Schrire et al. 2003, 2009), and the systematics of Chinese Indigofera species remains poorly understood. Pollen morphology of Indigofera was examined between 1980 and 2000 (Ferguson and Strachan 1982; Wu and Huang 1995; Schrire and Sims 1997), mainly for African species. Using a sampling of 77 Indigofera species, Ferguson and Strachan (1982) defined four types and eight subtypes of pollen morphology based on the pollen size, shape, tectal sculpturing and exine stratification. Wu and Huang (1995) proposed a different classification of pollen for Taiwan Indigofera species, and these authors recognized five types of pollen morphology characterized by exine structure, perforations, shape of tectum channels, granular or columellate interstitium, and prominent or reduced foot layer. Schrire and Sims (1997) examined 79 Indigofera species including two species of Vaughania S.Moore that has been reduced to synonymy under Indigofera (Schrire 2008). In the study (Schrire and Sims 1997), perforation density on pollen grain surfaces was newly added, and it was also revealed to be of significant taxonomic value. Cluster analysis recognized three major pollen groups in Indigofera. Previous studies have provided extensive palynological data in Indigofera (Ferguson and Strachan 1982; Wu and Huang 1995; Schrire and Sims 1997), but pollen morphology of Chinese endemisms is still largely unknown. Thus far, only two Chinese endemisms (I. dumetorum Craib and I. taiwaniana T.C.Huang & M.J.Wu) have been studied (Ferguson and Strachan 1982; Wu and Huang 1995). In the present study, we aim to (1) investigate the pollen morphology of Chinese Indigofera species using scanning electron microscopy (SEM), and (2) assess the potential significance of pollen characters in the taxonomy of Indigofera. 123 X.-L. Zhao et al. Materials and methods Sampling and scanning electron microscopy Pollen grains examined in this study were obtained from the opened flowers, in which the anthers were still wrapped by the keel. Most of the flowers were removed from herbarium specimens deposited in Herbarium of Chengdu Institute of Biology (CDBI), Chinese Academy of Sciences (CAS), and some were obtained from the transplanted plants. In total, 52 samples representing 43 species and two varieties were examined, of which 29 species and one variety are endemic to China. For species with a large geographic distribution, at least two samples from different geographic areas were examined. A list of taxa sampled with voucher information is shown in ‘‘Appendix’’ section. Pollen grains were directly taped on stubs and sputter coated with gold–palladium. Observations were conducted using scanning electron microscope (Phenom proX, Phenom-world, The Netherlands) at 10 kV at Chengdu Institute of Biology, CAS. SEM photographs of the polar and equatorial views were taken at 7 or 8 K, and the exine surfaces of the apocolpium and mesocolpium areas were both taken at 25 K. Pollen character Pollen measurements were performed on digital SEM images using software ImageJ (Abràmoff et al. 2004). For each sample, the polar length (P), equatorial length (E), the length of colpi (C), and the diameter of perforations were measured for at least 20 pollen grains. The ratio of polar length to equatorial length (P/E) and that of colpus length to polar length (C/P) were calculated. Descriptive terminology follows that of Erdtman (1952), Ferguson and Strachan (1982), and Punt et al. (2007). Perforation density on pollen grain surfaces was calculated according to the method described by Schrire and Sims (1997). The number of perforations in 1 cm2 area in the same apocolpium or mesocolpium areas of each pollen grain was counted and converted to number per lm2, and then used in the analyses. Phenetic analysis Cluster analysis (CA) and principal component analysis (PCA) were performed using the program SPSS version 21 (IBM SPSS Statistics). Prior to doing the CA and PCA, the data were standardized. The characters used in the CA and PCA included four quantitative and three qualitative characters: P, P/E, C/P, the number of apertures, tectum architecture in the apocolpium and mesocolpium areas, and Author's personal copy Pollen morphology of Indigofera perforation density in the apocolpium area. The three qualitative characters were coded. Tectum architecture in the apocolpium area was treated as five types: (1) imperforate, (2) perforate, (3) microperforate, (4) finely microperforate, and (5) pitted perforate. Tectum architecture in the mesocolpium area was classified into six types: (1) perforate, (2) microperforate, (3) finely microperforate, (4) rugulate and perforate, (5) pitted microperforate, and (6) pitted perforate. The number of apertures was treated as two types: (1) three apertures, and (2) three and more than three apertures. Hierarchical cluster analysis was conducted using the shortest Euclidean distances according to the Ward’s (1963) method. For principal component analysis, the standardized data were computed to obtain the correlation matrix. The first three principal components representing the maximum variance were extracted and plotted as a scatter diagram (Fig. 4). Results Pollen morphology Pollen characters of all examined samples are summarized in Table 1. Representative pollen grains are illustrated in Figs. 1 and 2, while the others are illustrated as in the electronic supplementary material. Pollen grains are shed as monads, and their sizes range from small to medium. The average polar length is 18.86 lm (I. pseudonigrescens; Fig. 2i) -28.63 lm (I. hirsuta L., Fig. 2h), and the average equatorial length is 23.41 lm (I. suffruticosa Mill.; VI: f) -33.49 lm (I. hirsuta L., Fig. 2h) lm. Indigofera hirsuta possesses the largest (P 9 E = 28.83 lm 9 33.49 lm) pollen grains, while I. pseudonigrescens has the smallest (P 9 E = 18.86 lm 9 24.08 lm). Three-colporate and four-colporate pollen grains were observed in all and eight species (i.e., I. carlesii Craib, I. chaetodonta Franch., I. dumetorum, I. henryi Craib, I. heterantha Wall., I. jikongensis Y.Y.Fang & C.Z.Zheng, I. pampaniniana Craib and I. pseudonigrescens), respectively. We also identified parasyncolpate pollen grains in three species and one variety (i.e., I. carlesii, I. chaetodonta, I. decora var. ichangensis (Craib) Y.Y.Fang & C.Z.Zheng and I. suffruticosa) (Figs. 1, 2 and I–VII). The shapes of the 3-colporate pollen grains are spherical to oblate, with the ratio of polar axis to equatorial axis varying from 0.71 (I. bungeana Walp. 4; II: b and I. silvestrii Pamp. 1; VI: b) to 0.92 (I. megaphylla X.F.Gao; V: a). The average length of colpi ranges from 13.29 lm (I. bungeana 3; II: a) to 21.28 lm (I. pendula Franch.; V: d). The ratio of the colpus length to polar length is from 0.52 (I. hirsuta; Fig. 2h) to 0.91 (I. franchetii X.F.Gao & Schrire; III: d). 471 Results of CA and PCA Five groups (Fig. 3a–e) were resolved in cluster analysis at the Euclidean distance of 7.5. Pollen of group A can be distinguished by a combination of 3-colporate pollen grains and microperforate tectum. Group B is unique in having pollen with more than three apertures. Group C pollen is mainly distinguished by the low perforate density in the apocolpium area, while group D differs mainly by the pitted perforate/microperforate. Pollen of group E is characterized by the finely microperforate tectum. The scatter diagram of the first three principal components from the PCA is shown in Fig. 4. The first three principal components accounted for the total variance of 70.27 % in differentiating the taxa. The first principal component explained 32.63 % of the total variance, the second principal component accounted for 21.99 %, while the third principal component accounted for 15.65 %. Two quantitative characters (P and C/P) and one qualitative character (tectum architecture in the mesocolpium area) in the first axis, tectum architecture and perforation density in the apocolpium area in the second axis, and the number of apertures in the third axis showed the strongest loadings. The three-dimensional scatter plot resulting from PCA indicated that all the samples analyzed can be divided into two groups. Group E can be recognized as an independent group, while overlap occurs among groups A, B, C and D. Discussion Pollen shape and size Pollen shape and size have been considered as having a significant taxonomic value in Indigofera (Ferguson and Strachan 1982; Schrire and Sims 1997). In this study, the stability of pollen shape and size at infraspecific level was tested among the multiple samples of I. amblyantha Craib, I. bungeana and I. silvestrii. The results indicated that pollen shape and size are relatively stable at the infraspecific level (Table 1). However, pollen shapes and sizes showed dramatic differences when comparing our results with those from previous studies (Ferguson and Strachan 1982; Wu and Huang 1995; Schrire and Sims 1997). For example, the ratios of P/E for I. colutea (Burm.f.) Merr., I. decora Lindl., I. hirsuta, I. dumetorum, I. linnaei Ali, I. pendula, I. stachyodes Lindl. and I. suffruticosa were reported to be 0.91, 1.14, 1.13, 1.11, 1.03, 1.12, 1.19 and 0.94 in previous studies as opposed to 0.77, 0.79, 0.85, 0.79, 0.76, 0.89, 0.87 and 0.86 reported in this study, respectively. According to the results of our preliminary 123 472 123 Table 1 Summary of pollen details in Indigofera examined in this study Avg. perforation (lm2) Apocolpium Mesocolpium Apocolpium Mesocolpium 0.8 m. perf. m. perf. 19.3 22.7 0.82 m. perf. m. perf. 16.7 21.3 17.25 (16.77–18.69) 0.74 m. perf. m. perf. 6.7 21.3 3 17.46 (16.76–19.23) 0.76 m. perf. m. perf. 11.3 42.7 3 18.24 (16.38–19.06) 0.82 perf. perf. 5.3 13.3 0.78 3 17.68 (17.45–17.89) 0.84 m. perf. m. perf. 13.3 17.3 29.82 (28.53–31.14) 26.92 (25.48–28.43) 0.93 0.82 3 3 17.27 (16.13–18.03) 16.62 (15.09–17.69) 0.62 0.76 m. perf. m. perf. perf. m. perf. 0 12.7 30.7 18.7 21.25 (19.28–22.19) 25.57 (23.63–26.29) 0.83 3 17.49 (15.61–19.71) 0.82 m. perf. m. perf. 18.7 30.7 18.93 (16.94–19.70) 24.20 (20.97–25.84) 0.77 3 13.29 (11.83–15.99) 0.71 m. perf. m. perf. 20.7 29.3 I. bungeana 4 20.13 (17.94–21.56) 26.39 (25.60–28.12) 0.71 3 14.21 (13.02–16.36) 0.71 m. perf. m. perf. 10.7 14.7 I. calcicola 25.22 (23.30–26.52) 27.95 (26.25–29.29) 0.9 3 19.89 (19.36–20.88) 0.79 perf. perf. 22.7 21.3 I. carlesii 24.21 (21.13–28.25) 29.11 (25.70–30.63) 0.83 3,4,5/6 17.41 (15.53–20.73) 0.72 m. perf. p. m. perf. 14.7 18.7 I. cassioides 24.17 (22.83–24.65) 28.45 (27.27–29.42) 0.85 3 18.98 (18.53–19.74) 0.79 m. perf. m. perf. 13.3 18.7 I. chaetodonta 1 21.30 (18.73–23.41) 25.10 (22.21–27.12) 0.85 3 16.09 (14.67–17.98) 0.76 perf. perf. 21.3 17.3 – 3, 4,4–12 – perf. perf. 24 28 Colpus length (C) lm C/P 0.78 3 16.13 (15.31–16.73) 0.87 3 17.61 (16.97–18.65) 27.41 (25.04–28.70) 0.85 3 22.81 (20.61–24.14) 26.17 (24.99–29.39) 0.87 22.28 (20.98–23.42) 26.48 (24.22–28.28) 0.84 I. balfouriana 21.17 (19.66–24.06) 27.14 (25.60–28.85) I. bracteata I. bungeana 1 27.82 (26.97–28.91) 22.00 (20.17–22.95) I. bungeana 2 I. bungeana 3 Polar length (P) lm Equatorial length (E) lm P/E I. amblyantha 1 20.17 (18.92–21.33) 25.86 (23.73–27.87) I. amblyantha 2 21.42 (19.64–22.76) 24.50 (21.68–25.64) I. amblyantha 3 23.30 (21.69–24.24) I. argutidens I. atropurpurea I. chaetodonta 2 – – – I. colutea 21.46 (19.91–23.74) 27.80 (24.77–30.84) 0.77 3 15.86 (14.95–17.73) 0.65 f. m. perf. f. m. perf. 69.3 73.3 I. decora 25.38 (22.15–29.07) 29.47 (27.76–30.63) 0.86 3 17.34 (15.71–19.46) 0.68 m. perf. p. m. perf. 2.7 12.7 I. decora var. ichangensis 24.16 (20.93–25.62) 30.13 (28.87–32.03) 0.80 3, 5/6 19.54 (18.75–20.11) 0.78 m. perf. p. m. perf. 5.3 30.7 I. delavayi 25.14 (24.64–25.50) 32.53 (31.75–34.33) 0.77 3 17.10 (15.23–18.41) 0.71 perf. p. perf. 0 10.7 I. dolichochaete 21.02 (19.18–22.85) 24.38 (23.37–25.36) 0.86 3 18.67 (18.00–19.60) 0.89 m. perf. m. perf. 13.3 21.3 I. dumetorum I. esquirolii 24.37 (22.84–26.17) 24.53 (22.25–25.99) 30.76 (29.18–33.43) 26.93 (25.66–28.62) 0.79 0.91 3, 4 3 18.63 (18.51–18.77) 17.05 (15.45–17.99) 0.76 0.7 p. perf. p. perf. p. perf. p. perf. 14.7 0 11.3 10.7 I. franchetii 22.00 (20.50–24.45) 26.17 (25.56–27.12) 0.84 3 19.96 (17.89–22.30) 0.91 m. perf. m. perf. 10.7 25.3 21.56 (20.64–22.13) 27.81 (26.41–28.70) 0.78 3 18.35 (16.94–19.37) 0.85 perf. perf. 21.3 25.3 I. hebepetala var. glabra 28.45 (27.27–30.36) 31.40 (30.43–32.67) 0.91 3 15.67 (14.23–16.70) 0.55 perf. perf. 0 14.7 I. hendecaphylla 21.22 (20.49–22.86) 28.59 (27.19–29.81) 0.74 3 16.69 (16.32–17.10) 0.79 f. m. perf. f. m. perf. 70.7 72 I. henryi 23.23 (21.99–25.61) 25.03 (23.63–26.48) 0.93 3, 4 16.63 (15.04–18.10) 0.72 m. perf. m. perf. 22.7 21.3 I. heterantha 22.21 (21.72–23.24) 29.85 (28.76–31.50) 0.74 3, 4 17.18 (15.81–18.86) 0.77 perf. perf. 10.7 48 I. hirsuta 28.63 (25.41–30.52) 33.49 (29.73–36.11) 0.85 3 14.86 (11.37–17.52) 0.52 perf. r. and perf. 6.7 9.3 I. jikongensis 25.10 (27.23–23.84) 31.65 (28.62–34.90) 0.79 3, 4 17.61 (15.76–19.97) 0.7 m. perf. p. m. perf. 0 21.3 I. lenticellata 24.43 (22.08–25.57) 27.16 (25.36–28.79) 0.9 3 17.92 (15.86–18.85) 0.73 perf. perf. 10.7 14.7 I. linnaei 22.04 (19.30–24.92) 29.03 (26.25–31.28) 0.76 3 18.20 (17.14–19.12) 0.83 m. perf. f. m. perf. 17.3 17.3 X.-L. Zhao et al. I. hancockii Author's personal copy Tectum architecture No. of apertures Species Avg. perforation (lm2) Apocolpium Mesocolpium Apocolpium Mesocolpium 0.78 m. perf. m. perf. 18.7 18.7 0.74 m. perf. m. perf. 13.3 21.3 m. perf. m. perf. 6.7 22.7 m. perf. m. perf. p. m. perf. m. perf. 22.7 13.3 20 22.7 m. perf. m. perf. 14.7 24 m. perf. r. and perf. 6.7 12 0.72 m. perf. m. perf. 29.3 33.3 14.45 (13.80–14.91) 0.72 m. perf. m. perf. 18.7 16 16.16 (15.82–16.64) 0.74 m. perf. p. m. perf. 0 21.3 3 15.01 (14.45–15.32) 0.73 perf. perf. 26.7 18.7 0.71 3 15.77 (14.37–16.75) 0.82 perf. perf. 25.3 24 0.86 3 15.40 (15.11–15.07) 0.73 m. perf. perf. 30.7 30.7 26.41 (24.62–29.76) 0.87 3 16.49 (15.24–18.75) 0.72 m. perf. p. perf. 1.3 10.7 23.55 (23.16–24.10) 0.9 3 15.53 (14.56–17.06) 0.73 m. perf. m. perf. 33.3 30.7 20.20 (18.40–21.68) 23.41 (21.42–25.30) 0.86 3, 5–12 16.03 (14.63–17.17) 0.79 m. perf. m. perf. 21.3 28 23.60 (22.23–25.06) 30.50 (29.48–32.87) 0.77 3 19.19 (18.47–19.98) 0.81 m. perf. m. perf. 26.7 16 I. wightii 24.63 (23.07–25.45) 30.21 (28.63–32.63) 0.82 3 16.47 (14.26–17.83) 0.67 f. m. perf. f. m. perf. 86.7 96 I. wilsonii 25.19 (21.82–28.28) 29.41 (27.87–31.58) 0.86 3 17.67 (15.97–20.97) 0.7 p. perf. p. m. perf. 10.7 17.3 Colpus length (C) lm C/P 0.8 3 17.12 (16.38–18.64) 0.92 3 16.76 (16.24–17.52) 28.96 (25.33–31.50) 0.84 3, 4 18.00 (16.89–20.09) 0.74 30.44 (27.77–34.23) 31.11 (28.10–34.20) 0.73 0.89 3 3 18.88 (18.24–19.21) 21.28 (20.79–21.70) 0.85 0.77 23.49 (21.82–25.16) 30.90 (29.33–33.09) 0.76 3 17.02 (14.47–19.80) 0.72 25.26 (21.99–27.64) 33.29 (31.74–34.66) 0.76 3 14.66 (13.79–15.45) 0.58 I. pseudonigrescens 18.86 (18.16–19.92) 24.08 (23.48–25.12) 0.78 3, 4 13.51 (13.04–14.01) I. rigioclada 20.18 (19.02–20.98) 26.10 (24.58–27.20) 0.77 3 I. scabrida 21.86 (20.88–23.54) 26.49 (23.51–28.91) 0.83 3 I. sensitiva 20.61 (19.45–21.56) 24.76 (23.93–25.51) 0.83 I. silvestrii 1 19.24 (17.70–20.38) 27.24 (26.22–28.02) I. silvestrii 2 20.98 (20.04–22.64) 24.51 (23.68–25.29) I. stachyodes 22.97 (21.26–24.55) I. sticta 21.17 (20.44–21.77) I. suffruticosa I. szechuensis Polar length (P) lm Equatorial length (E) lm P/E I. mairei 21.83 (20.02–23.31) 27.13 (26.39–28.12) I. megaphylla 22.60 (21.78–23.35) 24.52 (21.31–26.16) I. pampaniniana 24.29 (22.71–25.99) I. parkesii I. pendula 22.24 (20.53–23.62) 27.58 (26.21–29.00) I. penduloides I. pseudoheterantha Author's personal copy Tectum architecture No. of apertures Species Pollen morphology of Indigofera Table 1 continued A dash (–) indicates missing data perf. perforate, m. perf. microperforate, f. m. perf. finely microperforate, p. perf. pitted perforate, r. rugulate 473 123 Author's personal copy 474 Fig. 1 SEM micrographs of pollen grains in Indigofera. a-1–c-1, e1–g-1 Pollen grains in polar view, scale bar 5 lm; a-2–c-2, e-2–g-2 Exine surfaces in polar view, scale bar 0.5 lm; a-3–c-3, e-3–g-3 Apertures of pollen grains in equatorial view, scale bar 5 lm; a-4–c4, e-4–g-4 Pollen grains in equatorial view, scale bar 5 lm; a-5–c-5, 123 X.-L. Zhao et al. e-5–g-5 Exine surfaces in equatorial view, scale bar 0.5 lm; d:1–5 Parasyncolpate pollen grains, scale bar 5 lm. a I. bracteata. b I. bungeana 2. c I. chaetodonta 1. d I. chaetodonta 2. e I. colutea. f I. delavayi. g I. dumetorum Author's personal copy Pollen morphology of Indigofera Fig. 2 SEM micrographs of pollen grains in Indigofera. g-6, h-1, i-1, i-6, j-1–l-1. Pollen grains in polar view, scale bar 5 lm; g-7, h-2, i-2, i-7, j-2–l-2. Pollen grains in equatorial view, scale bar 0.5 lm; g-8, h3, i-3, i-8, j-3–l-3 Apertures of pollen grains in equatorial view, scale 475 bar 5 lm; g-9, h-4, i-4, i-9, j-4–l-4 Pollen grains in equatorial view, scale bar 5 lm; g-10, h-5, i-5, i-10, j-5–l-5 Exine surfaces in equatorial view, scale bar 0.5 lm. g I. dumetorum. h I. hirsuta. i I. pseudonigrescens. j I. sensitiva. k I. wightii. l I. wilsonii 123 Author's personal copy 476 X.-L. Zhao et al. Fig. 3 Phenogram of the cluster analysis (CA) investigations (see Online Resource 2), these differences may result from the differences in material selections. As a consequence, material selection is critical for palynological study of Indigofera. The pollen shape and size are not suitable to be treated as valuable taxonomic characters in Indigofera. 123 Number of apertures The number of apertures was generally reported to be three in previous studies (Ferguson and Strachan 1982; Wu and Huang 1995; Schrire and Sims 1997). A 4-colporate pollen grain of I. demissa Taub. was reported by Ferguson and Author's personal copy Pollen morphology of Indigofera 477 the sample might be triggered by the environmental factors, such as drought, poor and rocky soil in the region. Exine Fig. 4 Scatter plot of the first three components from principal component analysis (PCA); members of the different groups generated from the CA were marked as different colors Strachan (1982), and 4-colporate pollen grains were reported in Taiwan Indigofera species (Wu and Huang 1995), but no illustrations or descriptions were available. In this study, pollen grains with four colpi were examined in eight species. Additionally, parasyncolpate pollen grains were examined in four species for the first time. Indigofera chaetodonta has the pollen grains with the number of apertures ranging from three to twelve (Fig. 1d). Variations in the number and arrangement of apertures have been reported as the most common type of variation within the eudicots (Ressayre et al. 2002; Fukuda et al. 2008; Golshan et al. 2014), which can occur at the population level (polymorphism) or at the individual level (heteromorphism) (Walker and Doyle 1975). In this study, aperture variations were observed to be restricted to the individual level. Aperture variations can be caused by genetic factors and environmental conditions, such as the ploidy level, temperature, humidity, latitude, altitude, and pollinator activity (Thanikaimoni 1986; Bredenkamp and Van Wyk 1996; Till-Bottraud et al. 1995; do Pico and Dematteis 2010). For aperture variations in Indigofera, we speculated that polyploidization might be one possible reason, especially in I. decora complex. In this study, four species of I. decora complex (I. carlesii, I. decora, I. decora var. ichangensis and I. jikongensis) exhibit some variations in the number of apertures. Indigofera decora was reported to be hexaploid which is rare in Indigofera (Fedorov 1969), and polyploidization played an important role in the speciation of I. decora complex (Choi and Kim 1997). In this study, parasyncolpate pollen grains with 4–12 apertures were observed in one sample of I. chaetodonta. Indigofera chaetodonta is a subshrub species distributed in Yunnan Province, and the sample was collected from a dried limestone slope in Yulong County, Yunnan Province. Variations in the number of aperture observed in Congruent with all previous palynological studies of Indigofera (Ferguson and Strachan 1982; Wu and Huang 1995; Schrire and Sims 1997), we found that exine characteristics are highly stable at the infraspecific level. Combining with the phylogeny of Indigofereae (Schrire et al. 2009), we found that pollen grains of species in the Palaeotropical clade (I. wightii Graham ex Wight & Arn.; Fig. 2k and I. colutea; Fig. 1e) and Tethyan clade (I. linnaei; IV: f) have the finely microperforate tectum. Pollen grains of species in Pantropical clade (Sino-Himalayan region) show high diversity level of tectum architecture, which is consistent with the high level of morphological diversity of Indigofera in the Sino-Himalayan region. Perforation density and perforation type (especially pitted perforate and pitted microperforate) provide useful information in distinguishing species in Sino-Himalayan region (Table 1). Exine is a stable character in Indigofera, and it also showed some phylogenetic and biogeographical significance in Indigofera. Exine characteristics including tectum architecture, perforation type and density can be treated as valuable taxonomic characters in Indigofera. Taxonomic significance of pollen characters in Indigofera Indigofera is a large and complex genus taxonomically. Sino-Himalayan region has the highest diversity level of Indigofera species in China. The infrageneric classification of Chinese Indigofera (Fang and Zheng 1989, 1994) is controversial. The pollen morphology is partly consistent with the infrageneric classification of Fang and Zheng (1989, 1994). For example, species of subsect. Decorae (I. carlesii, I. decora, I. decora var. ichangensis, I. jikongensis and I. parkesii Craib) share the similar microperforate tectum in the apocolpium area and pitted microperforate tectum in the mesocolpium. Additionally, pollen grains with more than three apertures were observed in I. carlesii (II: d), I. decora var. ichangensis (III: a) and I. jikongensis (IV: d). However, most of the subsections are not supported by pollen characters. Several sister species did not show similar pollen morphology [e.g., I. szechuensis Craib (VII: a) and I. calcicola Craib (II: c)], while several species that do not have close relationships show similar pollen morphology [e.g., I. hirsuta (Fig. 2h) and I. pseudoheterantha X.F.Gao & Schrire (V: f)]. Those results indicated that macromorphology and pollen morphology might evolve at different diversification patterns and rates in Indigofera. Palynological data also provide further significant evidence 123 Author's personal copy 478 X.-L. Zhao et al. for the newly published species (I. pseudonigrescens; Fig. 2i) (Zhao and Gao 2015). Pollen morphology of I. pseudonigrescens differs from both its morphologically similar species (I. nigrescens Kurz ex King & Prain) (Wu and Huang 1995) and phylogenetically related species (I. delavayi Franch.; Fig. 1f) in pollen size, the number of apertures, tectum architecture and perforation density. Given the above results, it is clear that pollen characters alone are insufficient to reconstruct taxonomic relationships within Indigofera, but the variations of pollen are useful for the further taxonomic revisions at the species level. Conclusions Our study focused on pollen morphology of Chinese Indigofera, especially on Sino-Himalayan endemisms. Pollen morphology shows relatively high homogeneity in the examined Chinese Indigofera species. The number of apertures varied from 3-colporate type, to 4-colporate type, and the apomorphic (parasyncolpate) types that were observed for the first time. The number of apertures, tectum architecture and perforation density can provide palynological evidence in the taxonomy of Indigofera, and these characters also exhibit relatively obvious phylogenetic and biogeographical significance on large scales in Indigofera. But pollen shape and size show strong variations and, therefore, are not suitable to be used for taxonomic purposes. Acknowledgments This research was supported by the National Natural Science Foundation of China (Grant Nos. 31370230 and 31070173), and project of platform construction for plant resources of Sichuan Province to Xin-Fen Gao. We are grateful to Drs. Yu-Lan Peng, Zhang-Ming Zhu, Ms. Xiao-Mei Wei, Xia Zheng, Chun-Qiao Peng, Li–Na Guo, and Mr. Wen-Bin Ju, Meng Li for their help in the fieldwork. Special thanks to Drs. Timothée Le Péchon, Yun-Dong Gao, Liang Zhang, Zhang-ming Zhu and Mr. Abubakar Bello for their constructive comments, and Ms. Tian-Fang Huang for her technical assistance in SEM. We also thank two anonymous reviewers for their constructive comments. Compliance with ethical standards Conflict of interest of interest. The authors declare that they have no conflict Appendix Voucher information of Indigofera species included in this study. Indigofera amblyantha Craib 1, China, Sichuan: Maoxian, X. F. Gao and X. L. Zhao 15878-9 (CDBI). I. amblyantha 2, China, Shannxi: Shiquan, X. L. Zhao and C. Q. Peng 208-9 (CDBI). I. amblyantha 3, China, Zhejiang: Linan, X. L. Zhao and C. Q. Peng 95-13 (CDBI). I. argutidens Craib, China, 123 Yunnan: Yulong, X. F. Gao et al. 9618 (CDBI). I. atropurpurea Buch.-Ham. ex Hornem., China, Guizhou: Wangmo, Z. M. Zhu and W. B. Ju 397-5 (CDBI). I. balfouriana Craib, China, Yunnan: Yulong, X. L. Zhao et al. 2014-64-4 (CDBI). I. bracteata Graham ex Baker, China, Xizang: Jilong, X. F. Gao et al. 14709 (CDBI). I. bungeana Walp. 1, China, Sichuan: Maoxian, X. F. Gao and X. L. Zhao 15868-1 (CDBI). I. bungeana 2, China, Guizhou: Dafang, Z. M. Zhu and W. B. Ju 312-12 (CDBI). I. bungeana 3, China, Zhejiang: Linan, X. L. Zhao and C. Q. Peng 94-1 (CDBI). I. bungeana 4, China, Jiangsu: Jurong, X. L. Zhao and C. Q. Peng 63-4 (CDBI). I. calcicola Craib, China, Sichuan: Derong, X. F. Gao et al. 9577-26 (CDBI). I. carlesii Craib, China, Hubei: Xiangyang, X. L. Zhao and C. Q. Peng 33 (CDBI). I. cassioides Rottler ex DC., China, Yunnan: Yuanjiang, X. F. Gao and B. Xu 10077 (CDBI). I. chaetodonta Franch. 1, China, Yunnan: Yulong, X. F. Gao et al. 9616-4 (CDBI). I. chaetodonta 2, China, Yunnan: Yulong, X. L. Zhao et al. 2014-43-8 (CDBI). I. colutea (Burm.f.) Merr., China, Hainan: Sanya, X. L. Zhao 215-4 (CDBI). I. decora Lindl., China, Anhui: Jinzhai, X. L. Zhao and C. Q. Peng 56 (CDBI). I. decora var. ichangensis (Craib) Y.Y.Fang & C.Z.Zheng, China, Henan: Xinyang, X. L. Zhao and C. Q. Peng 35 (CDBI). I. delavayi Franch., China, Sichuan: Muli, X. F. Gao et al. 9689-19 (CDBI). I. dolichochaete Craib, China, Yunnan: Eryuan, X. L. Zhao et al. 201427 (CDBI). I. dumetorum Craib, China, Yunnan: Yongsheng, X. L. Zhao et al. 2014-20 (CDBI). I. esquirolii H. Lév., China, Guizhou: Wangmo, Z. M. Zhu and W. B. Ju 396-2 (CDBI). I. franchetii X.F.Gao & Schrire, China, Sichuan: Shimian, X. F. Gao et al. 11616-5 (CDBI). I. hancockii Craib, China, Sichuan: Dechang, X. L. Zhao et al. 2014-10-6 (CDBI). I. hebepetala var. glabra Ali, China, Xizang: Jilong, X. F. Gao et al. 14753 (CDBI). I. hendecaphylla Jacq., China, Yunnan: Baoshan, X. F. Gao et al. 11494 (CDBI). I. henryi Craib, China, Yunnan: Yulong, X. F. Gao et al. 9628-10 (CDBI). I. heterantha Wall., China, Yunnan: Xianggelila, X. F. Gao et al. 9579-2 (CDBI). I. hirsuta L., China, Hainan: Sanya, X. L. Zhao 220-1 (CDBI). I. jikongensis Y.Y.Fang & C.Z.Zheng, China, Henan: Xinyang, X. L. Zhao and C. Q. Peng 53 (CDBI). I. lenticellata Craib, China, Sichuan: Luding, X. F. Gao et al. 9738-12 (CDBI). I. linnaei Ali, China, Hainan: Sanya, X. L. Zhao 216-8 (CDBI). I. mairei Pamp., China, Sichuan, Luding, X. F. Gao et al. 9367-22 (CDBI). I. megaphylla X.F.Gao, China, Yunnan: Yuanjiang, X. F. Gao and B. Xu 10057-1 (CDBI). I. pampaniniana Craib, China, Yunnan: Shiping, X. F. Gao et al. 10086-29 (CDBI). I. parkesii Craib, China, Zhejiang: Tiantai, X. L. Zhao and C. Q. Peng 81 (CDBI). I. pendula Franch., China, Yunnan: Yongsheng, X. L. Zhao et al. 2014-14 (CDBI). I. penduloides Y.Y.Fang & C.Z.Zheng, China, Yunnan: Yulong, B. Xu and A. N. Egan 459 (CDBI). I. pseudoheterantha X.F.Gao & Schrire, China, Sichuan: Miyi, X. F. Gao et al. 9748 (CDBI). I. pseudonigrescens X.F.Gao & Xue Li Zhao, China, Sichuan: Mianning, Author's personal copy Pollen morphology of Indigofera X. L. Zhao et al. 2014-1-1 (CDBI). I. rigioclada Craib, China, Yunnan: Yulong, X. F. Gao et al. 9610-26 (CDBI). I. scabrida Dunn, China, Yunnan: Yulong, X. F. Gao et al. 9609-6 (CDBI). I. sensitiva Franch., China, Sichuan: Muli, X. L. Zhao et al. 2014-123-2 (CDBI). I. silvestrii Pamp. 1, China, Sichuan: Maoxian, X. F. Gao and X. L. Zhao 15893-9 (CDBI). I. silvestrii Pamp. 2, China, Yunnan: Weixi, X. L. Zhao et al. 2014-71-2 (CDBI). I. stachyodes Lindl., China, Yunnan: Yuanjiang, X. F. Gao and B. Xu 10079-11 (CDBI). I. sticta Craib, China, Yunnan: Yuanjiang, X. F. Gao and B. Xu 1007325 (CDBI). I. suffruticosa Mill., China, Hainan: Ledong, X. L. Zhao 235-2 (CDBI). I. szechuensis Craib, China, Sichuan: Maoxian, X. F. Gao and X. L. Zhao 15912-1 (CDBI). I. wightii Graham ex Wight & Arn., China, Hainan: Ledong, X. L. Zhao 236-1 (CDBI). I. wilsonii Craib, China, Sichuan: Wenchuan, X. F. Gao and X. M. Wei 6-1 (CDBI). Information on Electronic Supplementary Material Online Resource 1. SEM micrographs of pollen grains in Indigofera. Online Resource 2. Comparisons of pollen shape of pollen grains from the flowers with different maturity. References Abràmoff MD, Magelhães PJ, Ram SJ (2004) Image processing with image. J Biophotonics Int 11:36–42 Barker NP, Schrire BD, Kim JH (2000) Generic relationships in the tribe Indigofereae (Leguminosae: Papilionoideae) based on sequence data and morphology. In: Herendeen PS, Bruneau A (eds) Advances in legume syst, part 9. Royal Botanic Gardens, Kew, Melbourne, pp 311–337 Bredenkamp CL, Van Wyk AE (1996) Palynology of the genus Passerina (Thymelaeaceae): relationships form and functions. Grana 35:335–346. doi:10.1080/00173139609429092 Choi BH, Kim JH (1997) ITS sequences and speciation on far eastern Indigofera (Leguminosae). J Pl Res 110:339–346. doi:10.1007/ BF02524932 do Pico GMV, Dematteis M (2010) Meiotic behavior and pollen morphology variation in Centaurium pulchellum (Gentianaceae). Pl Syst Evol 290:99–108. doi:10.1007/s00606-010-0352-8 Erdtman G (1952) Pollen morphology and plant taxonomy. Angiosperms. Almqvist & Wiksell, Stockholm Fang YY, Zheng CZ (1989) A study on the genus Indigofera Linnaeus from China. Acta Phytotax Sin 27:161–177 Fang YY, Zheng CZ (1994) Indigofera Linnaeus. In: Wei Z (ed) Fl Reipubl Popularis Sin, vol 40. Science Press, Beijing, pp 239–325 Fedorov AA (1969) Chromosome numbers of flowering plants. Academy of Sciences of the USSR, Leningrad Ferguson IK, Strachan R (1982) Pollen morphology and taxonomy of the tribe Indigofereae (Leguminosae: Papilionoideae). Pollen & Spores 24:171–210 479 Fukuda T, Naiki A, Nagamasu H (2008) Pollen morphology of the genus Skimmia (Rutaceae) and its taxonomic implications. J Pl Res 121:463–471. doi:10.1007/s10265-008-0174-8 Gao XF, Schrire BD (2010) Indigofera Linnaeus. In: Wu ZY, Raven PH (eds) Flora of China. Science Press, Botanical Garden Press, Beijing, St. Louis, pp 137–164 Golshan Z, Ali AD, Emel OD (2014) Pollen morphology and evolution in the genus Orobanche L. s.l. and its allied genera (Orobancheae/Orobanchaceae) in Turkey. Pl Syst Evol 300:783–802. doi:10.1007/s00606-013-0919-2 Linnaeus C (1753) Species plantarum. Laurentius Salvius, Stockholm Punt W, Hoen PP, Blackmore S, Nilsson S, Le Thomas A (2007) Glossary of pollen and spore terminology. Rev Paleobot Palynol 143:1–81. doi:10.1016/j.revpalbo.2006.06.008 Ressayre A, Raquin C, Mignot A, Godelle B, Gouyon PH (2002) Correlated variation in microtubule distribution, callose deposition during male, post-meiotic cytokinesis, and pollen aperture number across Nicotiana species (Solanaceae). Amer J Bot 89:393–400. doi:10.3732/ajb.89.3.393 Schrire BD (1995) Evolution of the tribe Indigofereae (LeguminosaePapilionoideae). In: Crisp M, Doyle JJ (eds) Advances in legume syst, part 7. Royal Botanic Gardens, Kew, Melbourne, pp 161–244 Schrire BD, Lavin M, Barker NP, Cortes-Burns H, von Senger I, Kim JH (2003) Towards a phylogeny ofIndigofera (LeguminosaePapilionoideae): identification of major clades and relative ages. In: Klitgaard BB, Bruneau A (eds) Advances in legume syst, part 10. Royal Botanic Gardens, Kew, Melbourne, pp 269–302 Schrire BD (2008) The Madagascan genus Vaughania is reduced to synonymy under Indigofera (Leguminosae–Papilionoideae– Indigofereae). Kew Bull 63:477–479 Schrire BD, Sims JR (1997) A re-evaluation of pollen morphology and taxonomy in the tribe Indigofereae (Leguminosae–Papilionoideae). Kew Bull 52:841–878 Schrire BD, Lavin M, Lewis GP (2005) Global distribution patterns of the Leguminosae: insights from recent phylogenies. Biol Skr 55:375–422 Schrire BD, Lavin M, Barker NP, Forest F (2009) Phylogeny of the tribe Indigofereae (Leguminosae–Papilionoideae): geographically structured more in succulent-rich and temperate settings than in grass-rich environments. Amer J Bot 96:816–852. doi:10. 3732/ajb.0800185 Thanikaimoni G (1986) Pollen apertures: form and function. In: Blackmore S, Ferguson IK (eds) Pollen and spores: form and function. Academic Press, London, pp 119–136 Till-Bottraud I, De Paepe R, Mignot A, Dajoz I (1995) Pollen heteromorphism in Nicotiana tabacum (Solanaceae). Amer J Bot 82:1040–1048 Walker JW, Doyle JA (1975) The bases of angiosperm phylogeny: palynology. Ann Missouri Bot Gard 62:664–723 Ward JH (1963) Hierarchical grouping to optimize an objective function. J Amer Statist Assoc 58:236–244. doi:10.1080/ 01621459.1963.10500845 Wu MJ, Huang TC (1995) A palynological study of the genus Indigofera (Leguminosae) in Taiwan. Grana 34:160–181. doi:10. 1080/00173139509429040 Zhao XL, Gao XF (2015) Indigofera pseudonigrescens (Fabaceae: Papilionoideae): A new species from Sichuan, China. Phytotaxa 222:251–258 123