Plant Systematics and Evolution

, Volume 298, Issue 8, pp 1437–1453

Variation of pollen morphology, and its implications in the phylogeny of Clematis (Ranunculaceae)

Authors

  • Lei Xie
    • College of Biological Sciences and BiotechnologyBeijing Forestry University
    • State Key Laboratory of Systematic and Evolutionary Botany, Institute of BotanyThe Chinese Academy of Sciences
Original Article

DOI: 10.1007/s00606-012-0648-y

Cite this article as:
Xie, L. & Li, L. Plant Syst Evol (2012) 298: 1437. doi:10.1007/s00606-012-0648-y

Abstract

Clematis s.l. (including Archiclematis and Naravelia) is a genus of approximately 300 species with cosmopolitan distribution. The diversity of its pollen was surveyed in 162 taxa belonging to all infrageneric groups of Clematis s.l. Pollen morphology was investigated by use of scanning electron microscopy to identify useful characters, test taxonomic and systematic hypotheses, and elucidate pollen character evolution on the basis of the molecular phylogeny. Clematis pollen is small to medium (14.8–32.1 μm × 14.2–28.7 μm), oblate to prolate (P/E = 0.9–1.4) in shape. The apertures may be tricolpate and pantoporate sometimes with 4-zonocolpate and pantocolpate pollen grains as transitional forms. The tricolpate pollen grains are predominant and occur in all the sections of the genus, whereas pantoporate pollen grains can be found in sect. Tubulosae, sect. Viorna, sect. Viticella, and Naravelia only. Phylogenetic mapping of aperture types reveals that the pantoporate pollen type may be the apomorphy in the genus and evolved several times. The surface ornamentation in all taxa studied is similar and characterized by microechinae evenly distributed on the microperforate tectum. The size and density of spinules on the tectum vary greatly but successive in the whole genus. According to the character syndromes of the ornamentation, separating sect. Brachiata from sect. Meclatis is supported. Though pollen morphology may contribute to investigation of problematic taxa, the taxonomic value of pollen morphology is limited at the species level.

Keywords

ArchiclematisClematisNaraveliaPhylogenyPollen morphologyRanunculaceaeScanning electron microscopy

Introduction

The genus Clematis L., with approximately 280–350 species, is one of the largest genera in Ranunculaceae. This cosmopolitan genus comprises climbing lianas, small shrubs, and erect sub-shrubs distributed predominantly in the temperate zone of both hemispheres but with some species distributed in tropical areas (Tamura 1995; Johnson 1997; Grey-Wilson 2000; Wang and Li 2005). The genus is extremely diverse in temperate and subtropical regions of the Northern Hemisphere, especially in eastern Asia, with 147 species reported in China, 93 of which are endemic to the country (Wang and Bartholomew 2001). Many Clematis species are of horticultural interest (e.g. C. montana, C. patens, and C. viorna), and some others are regarded as pharmaceutically important (e.g. C. chinensis, C. henryi, and C. armandii).

According to Tamura (1991), Clematis belongs to subtribe Clematidinae Lotsy (including Clematis, Naravelia, and Archiclematis) in the tribe Anemoneae DC. Molecular phylogenetic studies support the monophyly of both the tribe Anemoneae and the subtribe Clematidinae (Johansson and Jansen 1993; Johansson 1995; Hoot 1995; Ro et al. 1997; Wang et al. 2009). However, the genus Clematis had been defined differently by different authors. For example, the genus Archiclematis was recognized by Tamura (1987, 1991, 1995), whereas Johnson (1997), Grey-Wilson (2000), and Wang and Li (2005) reduced it into Clematis. Another example is Naravelia, which was often accepted as a separate genus (Tamura 1995; Wang and Li 2005), but Johnson (1997) merged it into Clematis as sect. Naravelia. Recent molecular phylogenetic studies (Miikeda et al. 2006; Xie et al. 2011) support the widest circumscription (Clematis s.l.) of the genus defined by Johnson (1997), which including Archiclematis and Naravelia in Clematis.

The taxonomy of Clematis is regarded as difficult because Clematis species are morphologically highly variable (Tamura 1995; Johnson 1997; Brandenburg 2000; Grey-Wilson 2000; Wang and Li 2005). Classifications of the genus differ from each other, mainly because of different morphological characters emphasized in each system. In addition to recent taxonomic revisions (Tamura 1995; Johnson 1997; Brandenburg 2000; Grey-Wilson 2000; Wang and Li 2005), systematic studies based on anatomy, seedling morphology, palynology, cytology, and molecular phylogeny have also been conducted (Tobe 1974, 1980a, b; Essig 1991; Zhang 1991; Yano 1993; Miikeda et al. 1999, 2006; Yang and Moore 1999; Shi and Li 2003; Xie et al. 2011). Among these, pollen morphology had potential systematic value within the genus, because of wide variation in aperture size, configuration, and number (Tobe 1974; Nowicke and Skvarla 1995).

Pollen morphology of a small number of Clematis species has been reported in some studies of the palynologic characters of Ranunculaceae (Wodehouse 1935, 1936; Kumazawa 1936; Erdtman 1952; Petrov and Ivanova 1975; Nowicke and Skvarla 1995; Santisuk 1979; Al-Eisawi 1986; Clarke et al. 1991; Blackmore et al. 1995). Several palynologic studies specifically on Clematis have also been conducted, mainly using light microscopy (LM) (Ikuse 1956; Vishnu-Mittre and Sharma 1963; Nair 1965; Tobe 1974; Petrov and Ivanova 1975; Kapoor et al. 1989; Yang and Huang 1992; Yano 1993; Yang and Moore 1999). Kumazawa (1936) reported pollen morphology of 28 taxa of Clematis by use of LM. Three pollen types were recognized, summarized, and illustrated in the study. Kapoor et al. (1989) observed the pollen grains of 32 taxa of Clematis and one species of Naravelia representing the South Asian species of the genus by use of LM. Three-zono-colpate pollen grains were observed in most species and only C. cadmia was reported to have panporate pollen grains. Nowicke and Skvarla (1995) sampled 13 Clematis species and examined pollen morphology by use of scanning electronic microscopy (SEM). They observed three aperture types within Clematis, tricolpate, pantoporate, and pantocolpate. However, until now, there has been no comprehensive, systematic survey of Clematis pollen, and there is still a lack of knowledge about pollen morphology of most species of Clematis, especially using SEM.

Recent molecular phylogenetic studies (Miikeda et al. 2006; Xie et al. 2011) showed that the previously recognized genera Archiclematis and Naravelia are nested within Clematis and most subgenera and sections defined by morphological characters (Tamura 1995; Wang and Li 2005) are not supported. The newly established phylogenetic framework enables the examination of pollen characters from an evolutionary perspective. The purpose of this study was to:
  1. 1.

    survey the diversity of pollen morphology across the genus by use of SEM;

     
  2. 2.

    place palynologic variation into an evolutionary context; and

     
  3. 3.

    assess the usefulness of pollen morphology for systematic study of Clematis.

     

For Clematis s.str., the classification by Tamura (1995) was followed in this study.

Materials and methods

In total, 162 taxa, including 145 species and 13 varieties from Clematis, one species from Archiclematis, and three from Naravelia, were examined (Appendix). Dried herbarium material was used for all the sampled species. The sampling covered all the currently recognized subgenera and sections (sensu Tamura 1995) throughout all the distribution areas of Clematis. Efforts were made to sample multiple accessions, particularly for those taxa spanning large biogeographical ranges or having a diverse morphology, to observe potential infraspecific variation.

Pollen samples of some Clematis species, e.g. C. akebioides, C. pinnata, and C. henryi, are fragile and the acetolysis method for pollen preparation (Erdtman 1960) is too drastic and damages the pollen grains for SEM observation (Hesse et al. 2009). Thus, in this study mature anthers were fragmented and mounted directly on the stubs with double-sided adhesive tape without acetolysis to preserve the exine and intine. This treatment for SEM observation was applied by Liu et al. (2010, 2011), Polevova et al. (2010), Welsh et al. (2010), among others. The samples were gold-coated and examined by use of an Hitachi S-800 SEM.

Morphological features of pollen grains were observed for each species and were analyzed to describe and categorize pollen types. Measurements were based on 20 pollen grains, the values of P (polar axis length), E (equatorial diameter), spinule height, and density were measured and the P/E ratio was calculated. Statistics of the palynologic characters were calculated by use of PAST 2.14 (Hammer et al. 2001). Descriptive terminology follows Clarke et al. (1991), Hesse et al. (2009), and Punt et al. (2007).

To evaluate character evolution, aperture types were mapped on to a simplified strict consensus tree of Clematis built in Mesquite 2.7 (Maddison and Maddison 2011), resulting from the phylogenetic analyses based on three plastid (atpB-rbcL spacer, psbA-trnH-trnQ spacer, and rpoB-trnC spacer) and nuclear ITS sequences (Xie et al. 2011). The strict consensus tree from Xie et al. (2011) was simplified by removing the duplicate samples from the same species. Clades with significant statistical support values (Bayesian posterior probability ≥0.95) were considered and are indicated in the tree. Aperture types were coded as tricolpate pollen (or, rarely, with pantocolpate pollen) = 0, and pantoporate pollen = 1. Other characters, for example polar axis length, equatorial diameter, and spinule feature, are quantitative characters and only analyzed statistically.

Results

Descriptions of Clematis s.l. pollen

Pollen grains of Clematis s.l. are monad, radially symmetrical, isopolar, spheroidal, oblate or suboblate to oblate spheroidal (P/E range: 0.9–1.0) with broad poles, to prolate (or subprolate) (P/E range: 1.1–1.4) with broad or relatively narrow poles. Pollen grains are small to medium (Hesse et al. 2009) in size, with P × E (polar length × equatorial diameter) of 14.8–32.1 μm × 14.2–28.7 μm (min–max) for tricolpate pollen, diameter from 14.8 to 36.6 μm for pantoporate pollen. Equatorial outline elliptical, circular (Table 1).
Table 1

Pollen characters in Archiclematis, Clematis, and Naravelia

 

Taxon

Shape

Length (μm)

Width (μm)

P/E

Type of aperture

Spinule height

No. of spinules per 3 × 3 μm2

Archiclematis (1/1) Sect. Campanella Tamura (24/40)

Archiclematis alternata

SP

23.3 (22.2–25.4)

21.9 (20.2–23.0)

1.06

Tricolpate

0.16 (0.13–0.18)

15–16

Clematis urophylla

OS

22.5 (20.9–23.7)

23.0 (20.8–24.9)

0.98

Tricolpate

0.12 (0.08–0.16)

15–16

C. qingchengshanica

OS

22.9 (20.7–24.1)

23.9 (21.0–24.8)

0.96

Tricolpate

0.11 (0.08–0.14)

13–14

C. otophora

SP

24.3 (22.2–25.1)

21.1 (19.7–22.4)

1.15

Tricolpate

0.10 (0.08–0.12)

11–13

C. kockiana

SP

26.6 (23.7–27.9)

25.6 (23.6–26.8)

1.04

Tricolpate

0.14 (0.12–0.17)

7–8

C. pseudootophora

OS

22.1 (19.6–23.1)

23.0 (21.2–23.8)

0.96

Tricolpate

0.12 (0.10–0.15)

13–14

C. pogonandra

SP

24.8 (22.7–25.6)

20.6 (18.9–22.1)

1.20

Tricolpate

0.09 (0.07–0.11)

6–7

C. leschenaultiana

SP

23.8 (22.1–24.2)

22.2 (20.4–23.1)

1.08

Tricolpate

0.09 (0.08–0.11)

9–10

C. leschenaultiana var. rubifolia

S

24.5 (22.6–26.0)

24.5 (22.7–25.9)

1.00

Tricolpate

0.17 (0.10–0.26)

18–20

C. lasiandra

SP

24.8 (22.1–26.0)

21.8 (19.8–23.7)

1.14

Tricolpate

0.16 (0.13–0.18)

17–20

C. dasyandra

SP

24.1 (22.2–26.0)

22.5 (21.1–23.8)

1.07

Tricolpate

0.17 (0.14–0.19)

28–30

C. aethusifolia

S

24.5 (22.8–25.4)

24.5 (22.1–25.6)

1.00

Tricolpate

0.16 (0.13–0.19)

18–19

C.ranunculoides

SP

26.3 (23.3–27.1)

21.3 (19.9–22.2)

1.23

Tricolpate

0.14 (0.12–0.17)

15–17

C. yuanjiangensis

SP

24.0 (22.2–26.1)

22.5 (20.1–23.6)

1.07

Tricolpate

0.10 (0.08–0.12)

18–19

C. rehderiana

SP

23.8 (21.5–24.7)

22.5 (20.4–24.1)

1.06

Tricolpate

0.16 (0.14–0.18)

13–15

C. grewiiflora

S

24.7 (21.2–26.0)

24.4 (20.9–26.0)

1.01

Tricolpate

0.18 (0.12–0.28)

13–14

C. siamensis

OS

23.3 (20.9–25.0)

23.7 (22.1–24.3)

0.98

Tricolpate

0.11 (0.08–0.14)

15–17

C. yunnanensis

SP

24.7 (22.0–25.8)

20.7 (18.4–22.2)

1.19

Tricolpate

0.08 (0.06–0.10)

17–18

C. henryi

S

23.6 (21.5–25.3)

23.1 (21.1–25.2)

1.02

Tricolpate

0.08 (0.07–0.10)

16–17

C. repens

S

24.4 (22.7–25.8)

24.0 (22.9–26.0)

1.02

Tricolpate

0.09 (0.08–0.11)

23–25

C. acuminata var. longicaudata

OS

23.0 (21.2–25.5)

24.0 (22.3–26.7)

0.96

Tricolpate

0.08 (0.06–0.10)

14–15

C. jingdungensis

OS

24.0 (22.0–26.1)

24.5 (21.7–26.2)

0.98

Tricolpate

0.11 (0.08–0.14)

19–22

C. kweichowensis

SP

24.8 (22.2–25.9)

22.0 (20.4–24.0)

1.13

Tricolpate

0.09 (0.08–0.11)

13–15

C. buchananiana

S

24.3 (21.7–26.7)

24.3 (22.2–26.5)

1.00

Tricolpate

0.23 (0.20–0.25)

7–9

C. connata

SP

23.3 (21.0–25.5)

22.2 (20.1–24.4)

1.05

Tricolpate

0.25 (0.20–0.31)

10–14

C. pinchuanensis

SP

23.8 (21.1–25.9)

22.1 (20.1–24.2)

1.07

Tricolpate

0.14 (0.12–0.17)

15–17

Sect. Tubulosae Decne. (3/8)

C. heracleifolia

P

23.6 (20.1–24.5)

17.3 (15.4–19.1)

1.37

Tricolpate

0.21 (0.16–0.26)

6–7

C. pinnata

SP

22.9 (21.3–24.8)

21.6 (20.2–22.8)

1.09

Tricolpate

0.26 (0.20–0.31)

5–6

C. stans

S

21.1 (20.5–21.8)

21.1 (20.5–21.8)

1.00

Pantoporate*

0.27 (0.20–0.34)

8–10

Sect. Bebaeanthera Edgew. (3/4)

C. japonica

SP

23.4 (21.1–24.6)

21.1 (19.9–22.3)

1.11

Tricolpate

0.14 (0.12–0.17)

21–23

C. tosaensis

SP

23.0 (20.8–24.1)

21.5 (20.3–22.4)

1.07

Tricolpate

0.15 (0.13–0.18)

18–20

C. pseudopogonandra

OS

22.5 (20.8–23.6)

23.0 (21.1–24.8)

0.98

Tricolpate

0.16 (0.13–0.18)

16–19

Sect. Atragene (L.) DC. (2/9)

C. macropetala

S

24.6 (23.1–25.4)

24.6 (22.9–25.7)

1.00

Tricolpate

0.15 (0.12–0.18)

16–19

C. sibirica

S

23.8 (22.1–24.6)

23.6 (21.9–24.7)

1.01

Tricolpate

0.16 (0.12–0.19)

17–19

Sect. Meclatis (Spach) Tamura (10/20)

C. wightiana

S

23.3 (22.1–24.7)

23.3 (22.2–24.9)

1.00

Tricolpate

0.30 (0.28–0.33)

8–9

C. simensis

SP

27.2 (25.4–29.1)

23.1 (20.9–25.6)

1.18

Tricolpate

0.35 (0.29–0.42)

6–7

C. hirsuta

SP

28.8 (26.6–30.0)

26.7 (24.3–28.7)

1.08

Tricolpate

0.26 (0.22–0.30)

9–11

C. orientalis

SP

26.1 (24.6–27.1)

23.6 (22.1–24.1)

1.10

Tricolpate

0.14 (0.10–0.16)

21–24

C. tangutica

SP

24.8 (22.1–26.2)

22.9 (20.3–24.2)

1.08

Tricolpate

0.13 (0.12–0.15)

19–20

C. tangutica var. obtusiuscula

SP

24.3 (22.2–25.1)

23.3 (22.1–24.8)

1.04

Tricolpate

0.18 (0.09–0.25)

16–19

C. akebioides

SP

23.1 (22.2–24.5)

22.4 (20.8–23.6)

1.03

Tricolpate

0.13 (0.10–0.15)

22–24

C. serratifolia

OS

24.1 (22.8–25.3)

24.6 (22.6–25.4)

0.98

Tricolpate

0.14 (0.12–0.16)

20–21

C. graveolens

SP

25.5 (23.7–26.4)

22.4 (20.8–24.1)

1.14

Tricolpate

0.16 (0.13–0.18)

20–21

C. intricata

S

23.0 (21.8–24.3)

22.8 (21.2–24.1)

1.01

Tricolpate

0.17 (0.14–0.19)

17–19

C. viridis

S

23.5 (22.2–24.6)

23.5 (22.5–24.5)

1.00

Tricolpate

0.17 (0.15–0.19)

28–29

Sect. Pseudanemone Prant (6/25)

C. caleoides

S

24.6 (22.8–25.7)

24.6 (22.7–25.6)

1.00

Tricolpate

0.30 (0.25–0.36)

14–16

C. corniculata

S

25.0 (24.2–25.9)

25.0 (24.2–26.0)

1.00

Tricolpate

0.26 (0.20–0.31)

11–14

C. trifida

S

24.8 (23.9–26.4)

24.8 (23.9–26.4)

1.00

Tricolpate or pantocolpate

0.32 (0.29–0.36)

5–6

C. villosa

S

25.0 (24.4–25.8)

25.0 (24.6–25.7)

1.00

Tricolpate

0.33 (0.24–0.41)

8–10

C. stanleyi

SP

30.5 (26.4–32.1)

23.3 (20.8–26.5)

1.31

Tricolpate

0.26 (0.20–0.32)

7–9

C. chrysocarpa

S

25.5 (24.2–26.7)

25.5 (24.1–26.5)

1.00

Tricolpate

0.25 (0.20–0.31)

7–9

Sect. Viorna (Reichb.) Prantl (9/21)

C. albicoma

S

32.2 (31.1–33.4)

32.2 (31.1–33.4)

1.00

Pantoporate

0.16 (0.13–0.18)

17–18

C. bigelovii

 

32.3 (30.0–34.5)

32.3 (30.0–34.5)

1.00

Pantoporate

0.18 (0.11–0.34)

13–14

C. reticulata

S

31.0 (28.9–34.6)

31.0 (28.9–34.6)

1.00

Pantoporate

0.22 (0.11–0.39)

12–14

C. pitcheri

S

34.2 (30.2–36.5)

34.2 (30.2–36.5)

1.00

Pantoporate

0.24 (0.10–0.38)

14–15

C. glaucophylla

S

35.2 (34.5–36.5)

35.2 (34.5–36.5)

1.00

Pantoporate

0.23 (0.14–0.31)

14–16

C. versicolor

S

31.6 (29.8–33.2)

31.6 (29.8–33.2)

1.00

Pantoporate

0.24 (0.12–0.36)

15–17

C. crispa

S

31.7 (30.2–32.5)

31.7 (30.2–32.5)

1.00

Pantoporate

0.28 (0.14–0.42)

9–11

C. fusca

S

24.5 (22.7–25.8)

24.5 (23.0–25.7)

1.00

Tricolpate

0.16 (0.10–0.28)

18–20

C. integrifolia

SP

25.3 (23.7–26.9)

23.5 (22.1–24.7)

1.08

Tricolpate

0.15 (0.13–0.17)

20–22

Sect. Clematis Eichler (12/25)

C. apiifolia

S

20.1 (19.1–21.8)

18.5 (17.2–20.0)

1.00

Tricolpate

0.25 (0.20–0.31)

9–12

C. grandidentata

SP

20.5 (18.9–21.8)

19.8 (17.7–21.4)

1.04

Tricolpate

0.28 (0.24–0.32)

7–10

C. peterae

SP

21.5 (19.7–22.8)

17.7 (15.9–19.2)

1.21

Tricolpate

0.23 (0.16–0.30)

8–9

C. peterae var. trichocarpa

SP

21.6 (19.5–22.9)

17.4 (16.0–19.4)

1.24

Tricolpate

0.24 (0.12–0.32)

8–9

C. subumbellata

SP

19.1 (17.8–21.1)

16.2 (15.2–17.4)

1.18

Tricolpate

0.24 (0.22–0.28)

8–9

C. ganpiniana

SP

19.6 (17.7–20.9)

18.6 (16.8–20.1)

1.05

Tricolpate

0.30 (0.20–0.46)

8–9

C. ganpiniana var. tenuisepala

SP

19.5 (17.6–20.7)

18.7 (17.0–20.3)

1.04

Tricolpate

0.28 (0.16–0.45)

8–9

C. gouriana

SP

17.6 (15.8–19.5)

15.3 (14.2–17.7)

1.15

Tricolpate

0.24 (0.20–0.32)

8–9

C. parviloba

SP

21.6 (18.9–23.0)

20.0 (18.9–21.7)

1.08

Tricolpate

0.25 (0.22–0.31)

8–9

C. parviloba var. longianthera

SP

21.5 (18.7–22.8)

19.2 (18.4–21.3)

1.12

Tricolpate

0.25 (0.21–0.32)

7–8

C. mashanensis

SP

21.5 (18.9–23.0)

18.2 (17.1–19.7)

1.18

Tricolpate

0.28 (0.25–0.32)

8–9

C. tsaii

S

18.7 (17.8–19.6)

18.7 (17.8–19.6)

1.00

Tricolpate

0.33 (0.29–0.46)

11–12

C. puberula

OS

18.7 (16.8–19.9)

19.1 (17.8–21.0)

0.97

Tricolpate

0.30 (0.26–0.45)

8–9

C. chingii

SP

19.6 (17.8–20.9)

19.1 (17.4–20.6)

1.03

Tricolpate

0.21 (0.16–0.32)

14–16

C. tenuipes

S

20.1 (18.4–21.7)

20.1 (19.2–21.3)

1.00

Tricolpate

0.16 (0.14–0.18)

8–9

Sect. Cheiropsis DC. (13/13)

C. laxistrigosa

S

25.0 (23.6–26.5)

25.0 (23.5–26.5)

1.00

Tricolpate

0.16 (0.11–0.22)

12–14

C. wenshanensis

OS

24.8 (23.6–25.7)

24.8 (23.5–26.0)

0.97

Tricolpate

0.24 (0.12–0.32)

10–13

C. venusta

S

18.2 (16.8–19.7)

18.2 (16.5–19.9)

1.00

Tricolpate

0.25 (0.21–0.32)

15–17

C. chrysocoma

S

22.4 (20.5–24.1)

22.1 (20.8–23.9)

1.01

Tricolpate

0.17 (0.13–0.22)

16–19

C. gracilifolia

SP

22.5 (20.7–24.3)

19.4 (17.9–21.6)

1.16

Tricolpate

0.20 (0.16–0.32)

17–19

C. gracilifolia var. dissectifolia

SP

22.7 (20.5–23.9)

19.6 (17.7–21.4)

1.16

Tricolpate

0.21 (0.14–0.30)

16–18

C. montana

SP

21.6 (20.1–23.0)

19.4 (17.7–21.2)

1.11

Tricolpate

0.25 (0.17–0.32)

17–18

C. acerifolia

SP

23.8 (21.4–25.1)

18.6 (16.6–20.0)

1.28

Tricolpate

0.14 (0.10–0.17)

15–17

C. glabrifolia

S

24.1 (22.8–26.1)

24.0 (22.5–25.8)

1.00

Tricolpate

0.25 (0.19–0.30)

19–21

C. fasciculiflora

SP

26.4 (24.1–27.9)

22.7 (20.7–24.1)

1.16

Tricolpate

0.16 (0.13–0.20)

11–12

C. napaulensis

OS

27.6 (25.3–29.0)

28.2 (26.6–29.5)

0.98

Tricolpate

0.17 (0.13–0.22)

9–10

C. cirrhosa

S

25.1 (24.3–26.2)

25.1 (24.2–26.2)

1.00

Tricolpate

0.25 (0.17–0.32)

18–20

C. potaninii

SP

25.8 (23.9–27.1)

19.7 (18.2–21.8)

1.31

Tricolpate

0.28 (0.25–0.33)

12–13

C. williamsii

SP

25.7 (23.8–27.2)

24.1 (22.5–26.2)

1.07

Tricolpate

0.14 (0.10–0.17)

17–18

Sect. Naraveliopsis Hand–Mazz. (7/13)

C. metouensis

S

16.7 (15.2–18.1)

16.7 (15.2–18.1)

1.00

Tricolpate or pantocolpate

0.32 (0.19–0.47)

12–13

C. menglaensis

S

16.6 (14.8–17.2)

16.6 (14.8–17.2)

1.00

Tricolpate or pantocolpate

0.30 (0.25–0.36)

10–12

C. smilacifolia

S

16.5 (14.9–17.6)

16.5 (14.9–17.6)

1.00

Tricolpate

0.32 (0.20–0.45)

12–14

C. tashiroi

S

16.6 (15.2–17.1)

16.6 (15.3–17.3)

1.00

Tricolpate

0.25 (0.17–0.32)

19–22

C. loureiriana

S

16.8 (15.4–17.5)

16.6 (15.4–17.2)

1.01

Tricolpate

0.16 (0.13–0.20)

16–17

C. pianmaensis

S

16.0 (14.8–17.2)

16.0 (14.8–17.2)

1.00

Tricolpate or pantocolpate

0.30 (0.27–0.33)

15–18

C. crassipes

S

17.3 (15.9–18.7)

17.3 (15.9–18.7)

1.00

Tricolpate or pantocolpate

0.16 (0.11–0.24)

12–14

Sect. Aspidanthera Spach (24/72)

C. aristata

SP

21.0 (18.9–22.6)

18.7 (16.7–20.0)

1.12

Tricolpate

0.32 (0.22–0.46)

12–14

C. ibarensis

SP

28.0 (26.7–29.2)

21.3 (20.1–23.0)

1.31

Tricolpate

0.32 (0.24–0.39)

8–9

C. virginiana

SP

23.3 (21.7–25.2)

21.1 (20.0–23.2)

1.11

Tricolpate

0.40 (0.32–0.48)

6–8

C. ligusticifolia

OS

23.6 (21.8–25.0)

24.0 (22.3–26.4)

0.98

Tricolpate

0.30 (0.26–0.36)

7–8

C. drummondii

SP

24.0 (22.2–26.1)

23.0 (21.3–24.5)

1.04

Tricolpate

0.25 (0.21–0.30)

6–7

C. dioica

SP

24.7 (22.3–26.2)

24.0 (22.2–27.1)

1.03

Tricolpate

0.32 (0.16–0.48)

6–7

C. acapulcensis

SP

24.8 (22.1–26.2)

19.6 (17.4–21.3)

1.27

Tricolpate

0.35 (0.26–0.45)

5–6

C. bonariensis

OS

23.1 (21.5–24.9)

23.5 (21.4–25.2)

0.98

Tricolpate

0.30 (0.27–0.32)

6–7

C. campestris

SP

28.2 (26.4–30.3)

22.1 (20.5–24.2)

1.28

Tricolpate

0.25 (0.17–0.32)

7–8

C. alborosea

SP

28.5 (26.4–30.2)

22.8 (20.2–24.9)

1.25

Tricolpate

0.29 (0.17–0.39)

9–11

C. populifolia

S

26.5 (24.7–28.1)

26.5 (25.3–28.0)

1.00

Tricolpate

0.28 (0.26–0.32)

9–11

C. grossa

SP

26.2 (22.3–28.1)

21.2 (19.8–23.4)

1.24

Tricolpate

0.32 (0.30–0.36)

8–9

C. grahami

SP

27.2 (25.5–28.8)

24.6 (22.7–26.3)

1.11

Tricolpate

0.33 (0.30–0.35)

7–8

C. affinis

OS

25.4 (23.3–27.1)

25.8 (22.9–27.6)

0.98

Tricolpate

0.26 (0.19–0.31)

5–6

C. peruviana

S

26.4 (24.2–28.5)

26.4 (24.5–28.7)

1.00

Tricolpate

0.27 (0.24–0.31)

8–9

C. seemanni

S

24.5 (22.8–26.2)

24.5 (22.8–26.2)

1.00

Tricolpate

0.29 (0.25–0.34)

6–7

C. glycinoides

SP

27.7 (24.6–29.1)

21.6 (19.8–23.4)

1.28

Tricolpate

0.36 (0.30–0.46)

9–10

C. haenkeana

S

21.3 (19.6–23.4)

21.3 (19.7–23.5)

1.00

Tricolpate

0.32 (0.16–0.45)

9–10

C. millefoliata

SP

31.1 (28.9–33.0)

26.6 (24.7–27.5)

1.17

Tricolpate

0.28 (0.25–0.30)

9–10

C. viridiflora

SP

25.3 (23.1–27.6)

19.6 (17.6–22.1)

1.29

Tricolpate

0.21 (0.12–0.39)

9–11

C. dissecta

S

25.1 (22.3–27.4)

25.1 (23.1–27.2)

1.00

Tricolpate

0.31 (0.27–0.33)

14–15

C. foetida

SP

22.6 (19.8–24.3)

18.3 (16.8–21.1)

1.23

Tricolpate

0.25 (0.19–0.29)

8–9

C. microphylla

S

25.0 (24.1–26.3)

25.0 (24.0– 26.1)

1.00

Tricolpate

0.28 (0.20–0.40)

9–11

C. paniculata

S

26.3 (23.2–28.1)

26.3 (23.2–28.1)

1.00

Tricolpate or pantocolpate

0.35 (0.29–0.49)

5–6

Sect. Lasiantha Tamura (2/2)

C. lasiantha

SP

25.2 (23.1–27.6)

22.5 (20.7–24.8)

1.12

Tricolpate

0.30 (0.24–0.36)

8–10

C. pauciflora

S

26.0 (24.8–28.5)

26.1 (24.9–28.3)

1.00

Tricolpate or pantocolpate

0.31 (0.27–0.33)

6–7

Sect. Flammula DC. (17/25)

C. flammula

SP

23.2 (20.9–25.2)

20.6 (18.7–22.1)

1.13

Tricolpate

0.09 (0.08–0.10)

13–16

C. dilatata

SP

22.5 (20.1–24.3)

19.1 (17.3–21.4)

1.18

Tricolpate

0.22 (0.18–0.28)

12–15

C. recta

SP

24.4 (22.2–26.1)

21.7 (19.6–23.4)

1.12

Tricolpate

0.21 (0.14–0.29)

15–18

C. chinensis

SP

23.1 (21.1–25.3)

20.6 (18.4–22.5)

1.12

Tricolpate

0.18 (0.14–0.21)

13–15

C. obscura

S

22.7 (20.2–24.3)

22.5 (20.4–24.1)

1.01

Tricolpate

0.16 (0.13–0.19)

23–24

C. meyeniana

SP

23.1 (21.2–25.4)

22.2 (20.8–24.5)

1.04

Tricolpate

0.17 (0.11–0.26)

19–20

C. terniflora

S

25.0 (23.8–26.4)

25.0 (24.0–26.1)

1.00

Tricolpate

0.16 (0.13–0.19)

20–21

C. finetiana

S

24.6 (22.1–25.7)

24.6 (22.0–26.0)

1.00

Tricolpate

0.17 (0.14–0.20)

17–19

C. mandshurica

S

25.9 (23.6–27.1)

25.9 (23.7–27.3)

1.00

Tricolpate

0.17 (0.13–0.22)

22–24

C. jialasaensis

S

25.1 (23.3–27.4)

25.1 (23.3–27.2)

1.00

Tricolpate

0.21 (0.14–0.29)

10–12

C. uncinata

P

27.3 (25.5–28.6)

19.7 (17.4–21.3)

1.39

Tricolpate

0.29 (0.25–0.34)

9–10

C. akoensis

SP

23.0 (21.8–25.2)

22.6 (20.7–24.4)

1.02

Tricolpate

0.24 (0.19–0.28)

9–11

C. armandii

SP

22.6 (20.2–24.5)

21.7 (19.8–23.0)

1.04

Tricolpate

0.21 (0.14–0.29)

9–11

C. armandii var. hefengensis

SP

22.2 (20.1–24.2)

21.9 (19.9–23.5)

1.01

Tricolpate

0.21 (0.15–0.28)

9–12

C. quinquefoliolata

S

22.7 (20.2–24.9)

22.5 (20.4–24.8)

1.01

Tricolpate

0.29 (0.25–0.34)

14–15

C. shensiensis

SP

22.9 (20.8–25.2)

22.3 (20.6–25.1)

1.03

Tricolpate

0.24 (0.19–0.28)

8–11

C. kirilowii

SP

24.5 (22.1–26.8)

18.3 (16.4–20.5)

1.34

Tricolpate

0.22 (0.16–0.29)

15–16

C. crassifolia

SP

18.8 (16.4–20.2)

18.4 (16.1–20.0)

1.02

Tricolpate

0.12 (0.08–0.14)

13–15

Sect. Angustifolia (Tamura) Serov (1/1)

C. hexapetala

SP

22.9 (20.2–24.4)

21.8 (19.1–23.3)

1.05

Tricolpate

0.16 (0.10–0.23)

18–19

C. hexapetala var. tchefouensis

SP

21.5 (20.2–23.4)

20.5 (19.2–22.1)

1.20

Tricolpate

0.19 (0.12–0.28)

20–21

Sect. Fruticella Tamura (6/7)

C. songarica

S

18.7 (16.9–20.0)

18.7 (17.0–20.0)

1.00

Tricolpate

0.16 (0.13–0.18)

19–20

C. songarica var. aspleniifolia

SP

22.9 (20.1–24.4)

20.6 (18.2–22.1)

1.11

Tricolpate

0.09 (0.08–0.10)

17–18

C. lancifolia

SP

23.8 (21.1–25.4)

18.2 (16.6–19.7)

1.31

Tricolpate

0.19 (0.14–0.25)

10–12

C. lancifolia var. ternata

SP

26.6 (24.4–28.6)

19.7 (17.7–21.6)

1.32

Tricolpate

0.18 (0.13–0.23)

10–11

C. delavayi

SP

25.5 (22.1–28.2)

23.2 (21.2–25.3)

1.10

Tricolpate

0.16 (0.08–0.23)

12–13

C. delavayi var. calvescens

SP

22.9 (20.2–25.0)

19.7 (17.1–22.2)

1.16

Tricolpate

0.17 (0.11–0.22)

11–12

C. nanophylla

SP

23.0 (20.9–25.5)

20.6 (17.8–22.9)

1.12

Tricolpate

0.13 (0.09–0.17)

35–37

C. fruticosa

OS

23.1 (21.7–25.3)

23.6 (20.8–25.9)

0.98

Tricolpate

0.20 (0.14–0.25)

14–15

C. tomentella

OS

20.0 (18.8–22.3)

21.0 (18.9–22.8)

0.95

Tricolpate

0.17 (0.13–0.22)

17–19

Sect. Viticella (Moench) DC. (7/10)

C. cadmia

S

26.5 (23.4–28.7)

26.5 (23.4–28.7)

1.00

Pantoporate

0.24 (0.19–0.28)

19–20

C. lanuginosa

S

26.0 (24.2–28.4)

26.0 (24.2–28.4)

1.00

Pantoporate

0.38 (0.16–0.48)

10–12

C. hancockiana

S

27.3 (25.1–29.0)

27.3 (25.1–29.0)

1.00

Pantoporate

0.17 (0.13–0.20)

17–18

C. patens var. tientaiensis

S

25.5 (24.1–27.2)

25.5 (24.1–27.2)

1.00

Pantoporate

0.28 (0.20–0.34)

11–12

C. courtoisii

S

26.2 (24.8–28.1)

26.2 (24.8–28.1)

1.00

Pantoporate

0.25 (0.19–0.29)

18–20

C. huchouensis

SP

26.6 (24.5–28.6)

22.7 (20.2–25.1)

1.17

Tricolpate

0.16 (0.14–0.18)

8–10

C. viticella

SP

25.5 (23.1–28.0)

21.4 (19.8–23.3)

1.19

Tricolpate

0.16 (0.12–0.19)

9–12

Sect. Pterocarpa Tamura (1/1)

C. brachyura

SP

18.2 (16.7–20.2)

17.5 (15.4–19.7)

1.04

Tricolpate

0.19 (0.11–0.27)

15–16

Naravelia DC. (3/7)

N. zylanica

S

16.6 (15.2–18.0)

16.6 (15.2–18.0)

1.00

Pantoporate

0.19 (0.12–0.26)

18–20

N. pilurifera

S

17.7 (16.4–18.8)

17.7 (16.4–18.8)

1.00

Pantoporate

0.21 (0.12–0.30)

22–23

N. laurifolia

S

16.2 (14.9–18.7)

16.2 (14.9–18.7)

1.00

Pantoporate

0.20 (0.11–0.30)

23–25

S spheroidal, SP subprolate, OS oblate spheroidal, P prolate

Aperture is tricolpate, occasionally 4-zonocolpate and pantocolpate, and pantoporate (Fig. 1a–d). Colpi sunken or open, wider in the middle, gradually acute at the ends. Colpus membrane is often covered with granular or microechinate elements usually the same size as or larger than those on the tectum. For pantoporate grains, pores are irregular in shape and distribution, and in pantocolpate pollen, the colpi are also arranged sporadically.
https://static-content.springer.com/image/art%3A10.1007%2Fs00606-012-0648-y/MediaObjects/606_2012_648_Fig1_HTML.jpg
Fig. 1

Variation of aperture types (ad). aClematis grahami, scale bar 6 μm, b, cC. trifida, scale bars 10 μm, dC. patens, scale bar 10 μm. Tectum variation (ef). eC. nanophyllascale bar 8.6 μm, fC. gourianascale bar 6 μm. Variation of spinule density (gi). gC. gouriana, hC. finetiana, iC. dasyandra. Variation of spinule height (jl). jC. stans, kC. villosa, lC. japonica. Scale bars (gl) 3 μm

Aperture in Clematis s.l. can be divided into two major types: tricolpate and pantoporate. One-hundred and forty-five sampled taxa have the former pollen type, whereas all sampled species of Naravelia and three sections of Clematis s.str., sect. Tubulosae, sect. Viorna, and sect. Viticella, have the pantoporate pollen grains (Table 1). Pantocolpate pollen is rare and always accompanied by tricolpate or pantoporate grains.

In most cases, pollen surface is perforate, with microechinate ornamentation evenly distributed. Spinules vary in size (ranging from 0.08 to 0.40 μm) and density (ranging from 5 to 37 per 9 μm2) in the genus (Figs. 2, 3, 4).
https://static-content.springer.com/image/art%3A10.1007%2Fs00606-012-0648-y/MediaObjects/606_2012_648_Fig2_HTML.gif
Fig. 2

Box plot showing variation of spinule height (a) and spinule density (b) of Clematis s.l. Rectangles define 25–75 %; horizontal lines show median; whiskers show 5–95 %; circles indicate extreme values

https://static-content.springer.com/image/art%3A10.1007%2Fs00606-012-0648-y/MediaObjects/606_2012_648_Fig3_HTML.gif
Fig. 3

Scatter diagram illustrating the relationship between the height and density of the sculpture element. Squares: sect. Campanella, sect. Bebaeanthera, sect. Atragene, sect. Fruticella, and Archiclematis; red crosses: sect. Tubulosae, sect Aspidanthera, sect. Lasiantha, sect. Clematis, and sect. Pseudanemone; points: samples from sect. Viticella, sect. Flammula, sect. Naraveliopsis, sect. Viorna, sect. Pterocarpa, sect. Cheiropsis, sect. Angustifolia, and Naravelia; green crosses: sect. Meclatis

https://static-content.springer.com/image/art%3A10.1007%2Fs00606-012-0648-y/MediaObjects/606_2012_648_Fig4_HTML.gif
Fig. 4

Scatter diagram illustrating the size of pantoporate pollen. Crosses: sect. Viorna; squares: sect. Viticella; diamond: sect. Tubulosae; rectangles: Naravelia

Evolution of aperture types

Although a molecular phylogenetic study by Xie et al. (2011) did not generate a robust phylogenetic framework for Clematis, especially in deep branches, analysis of ancestral pollen character reconstruction still provided some systematic information. Phylogenetic mapping of aperture types (Fig. 5) demonstrates that the tricolpate pollen type may be the ancestral type and the pantoporate pollen may be the apomorphy in the genus and evolved several times.
https://static-content.springer.com/image/art%3A10.1007%2Fs00606-012-0648-y/MediaObjects/606_2012_648_Fig5_HTML.gif
Fig. 5

Aperture types optimized on to a tree resulting from molecular phylogenetic study of Clematis based on the combined data of nrITS, atpB-rbcL, psbA-trnH-trnQ, and rpoB-trnC sequences (Xie et al. 2011). Vertical bars indicate the clades supported by ≥0.95 Bayesian posterior probability values

Discussion

Aperture types

Pollen of Clematis s.l. is heteromorphic (Fig. 1). This has also been reported after previous studies, for example those of Kumazawa (1936), Nowicke and Skvarla (1995), and Kapoor et al. (1989). The prevalent aperture type in Clematis s.l. is tricolpate; 89.5 % of the taxa examined can be characterized as this type. It can be found in all the sections of Clematis s.str. and Archiclematis. However, this apertural type may be accompanied in the same anther by a small proportion of 4-zonocolpate or pantocolpate grains (Fig. 1b, c). Pantoporate pollen is also occasionally accompanied by pantocolpate grains and are confined to sect. Tubulosae, sect. Viorna, sect. Viticella, and Naravelia. All the sampled Naravelia species have pantoporate pollen, but this pollen type is not section-specific in Clematis s.str. In sect. Tubulosae, two sampled Chinese species, C. heracleifolia and C. pinnata (Wang and Xie 2007), have tricolpate pollen, whereas the Japanese C. stans has pantoporate pollen. Another interesting distribution pattern was found in sect. Viorna. The American species of the section has pantoporate pollen whereas the Eurasian species (C. integrifolia and C. fusca) have tricolpate pollen. Both aperture types were also found in sect. Viticella but no distribution patterns were recognized.

Apertural heteromorphism is not rare in Ranunculaceae (Kumazawa 1936; Xi 1985; Nowicke and Skvarla 1995). An extreme example is Anemone, which has the most diverse aperture types including tricolpate, 5–8-zonocolpate, spiral, pantocolpate, and pantoporate grains (Huynh 1970; Nowicke and Skvarla 1995; Ehrendorfer et al. 2009). Ranunculus and Pulsatilla were also reported to have at least three aperture types (Xi 1985; Nowicke and Skvarla 1995). In eudicots, tricolpate pollen has been regarded as Plesiomorphic, whereas pantocolpate and pantoporate grains are regarded as progressively derived from tricolpate pollen (Wodehouse 1936; Xi 1985; Tellería and Daners 2003). If this evolutionary trend is applied to Clematis s.l., Japanese species in sect. Tubulosae, North American species of sect. Viorna, most species of sect. Viticella, and Naravelia can be inferred as derived groups, although the most primitive groups of the genus cannot be inferred from aperture types.

Tectal variation

Like most genera of Ranunculaceae, for example Caltha, Cimicifuga, Thalictrum, Anemone, Pulsatilla, and Ranunculus, pollen grains of Clematis s.l. have spinulose tecta that are usually punctate or microperforate (Kumazawa 1936; Xi 1985; Nowicke and Skvarla 1995). Although it has uniformity in tectal ornamentation in Clematis, the size and density of sculpture elements vary substantially (Fig. 1e–l). The spinules are very small and indistinct (<20 μm) in some sections, for example sect. Campanella and sect. Atragene, whereas they are larger (up to 40 μm) in sect. Clematis, sect. Tubulosae, and sect. Aspidanthera (Fig. 2a).

The density of the spinules also varied substantially (Figs. 1g–i, 2b). It is noteworthy that smaller spinules are often more densely distributed than larger ones. Although exceptional pollen grains may be observed, pollen of sect. Aspidanthera, sect. Tubulosae, sect. Lasiantha, sect. Clematis, and sect. Pseudanemone tends to have larger sculpture elements that are more sparsely distributed than in sect. Campanella, sect. Bebaeanthera, sect. Atragene, sect. Fruticella, and Archiclematis (Figs. 2, 3).

Combining these two characters, pollen grains of sect. Campanella, sect. Bebaeanthera, sect. Atragene, and sect. Fruticella can be distinguished from those of sect. Aspidanthera, sect. Lasiantha, sect. Tubulosae, and sect. Clematis (Fig. 1f, i). Thus, the combined characters provide some systematic information. For example, two groups of pollen grains in sect. Meclatis (sensu Tamura 1995) can be recognized (Fig. 3, green cross). Clematis wightiana, C. hirsuta, and C. simensis have pollen with large and sparse spinules which is very similar to the pollen in sect. Clematis and sect. Aspidanthera, whereas other species in sect. Meclatis (C. tangutica, C. akebioides, etc.) have pollen grains similar to those of sect. Campanella. This result supports Snoeijer’s (1992) treatment separating the former species as a section Brachiata. These species have often been placed in sect. Meclatis (e.g. Tamura 1991, 1995) mainly because they all have hairy filaments. They differ from those of sect. Meclatis s.str. (sensu Snoeijer 1992) in their spreading, white sepals and narrowly linear filaments. In species of sect. Meclatis s.str., the sepals are usually ascending and yellow in color, and the filaments are widened below. Wang (2004) hypothesized that sect. Brachiata may be closely related to sect. Clematis on the basis of gross resemblance. Our palynologic data support this point. Both sections have pollen grains with sparse and large spinules. However, in the whole genus these two quantitative characters of ornamentation are continuous and cannot be used to delimitate sectional groups.

Pollen size

Pollen of Clematis is generally small to medium, on the basis of the standard of Hesse et al. (2009). Our measurements largely agree with the size range described by Kapoor et al. (1989) but are slightly smaller in most of the species sampled. Kumazawa (1936) considered that different wet and dry conditions or different preparations for pollen samples may substantially affect pollen size. We agree with Kumazawa (1936) and attribute the difference between our study and that of Kapoor et al. (1989) to the preparation and observation methods. Kapoor et al. (1989) acetolyzed the pollen and observed them under LM, whereas this study mounted grains without treatment and observed them under SEM.

In this study, there was little regular variation of the size of pollen grains, especially the tricolpate type, in Clematis s.l. However, the size of pantoporate pollen provides some information. Pantoporate pollen grains of Naravelia, sect. Tubulosae, sect. Viticella, and sect. Viorna can be clearly separated by their size (Fig. 4). Sect. Viorna has the largest grains. Pollen of sect. Viticella is smaller than that of Sect. Viorna and larger than that of sect. Tubulosae. Naravelia has the smallest pantoporate pollen. This indicates that although only a small proportion is of the pantoporate type, it may have evolved several times independently.

Pollen and phylogeny of Clematis

Clematis is one of the most difficult groups taxonomically. There are major conflicts among currently published classifications (Tamura 1995; Johnson 1997; Grey-Wilson 2000; Wang and Li 2005; reviewed by Xie et al. 2011). Furthermore, recent molecular phylogenetic studies (Miikeda et al. 1999, 2006; Xie et al. 2011) are largely inconsistent with the above mentioned classifications. These studies did not generate a robust phylogenetic framework for Clematis, especially in deep branches, which indicated that the crown group of Clematis diverged recently, and a recent species radiation may have happened in the genus.

Similar to other characters, for example seedling morphology (Essig 1991) and leaf epidermis (Shi and Li 2003), pollen morphology alone is insufficient to reconstruct phylogenetic relationships within the genus. In Clematis s.l., palynologic characters are rather conservative, and only two major aperture types are recognized in the genus. We mapped this character on the simplified phylogeny (Fig. 5) resulting from Xie et al. (2011) and confirmed that the pantoporate type is derived and has evolved several times in the genus. At least three significantly supported clades have derived this character.

Naravelia has been recognized as a distinctive genus by most taxonomists (Eichler 1963; Tamura 1995; Grey-Wilson 2000; Wang and Li 2005). This genus was found nested within Clematis and closely related with sect. Naraveliopsis by recent molecular phylogenetic analyses (Miikeda et al. 2006; Xie et al. 2011). The aperture type of sect. Naraveliopsis (C. loureiriana in Fig. 5) is tricolpate, whereas Naravelia has pantoporate pollen. The result indicates that Naravelia may be derived from sect. Naraveliopsis and extends its distribution to tropical areas in South and South East Asia.

Pantoporate pollen also occurs in sect. Tubulosae. The molecular phylogenetic study by Xie et al. (2011) revealed that C. pinnata from northern China is more closely related to sect. Clematis than to sect. Tubulosae, and C. heracleifolia from China and C. stans from Japan are sibling species (Fig. 5). The Chinese species was found to have tricolpate pollen and its Japanese ally has pantoporate pollen. The dispersal route of sect. Tubulosae from China to Japan may be inferred from our palynologic observation.

In previous classifications, sect. Viorna and sect. Viticella are thought to be distantly related groups in Clematis, because of their different floral characters (Tamura 1995; Johnson 1997; Grey-Wilson 2000; Wang and Li 2005). However, these two sections were clustered together and their relationships were not clearly supported in the molecular phylogenetic study by Xie et al. (2011). It is interesting that both sections have the two aperture types. Sect. Viorna is disjunctly distributed in North America and North Eurasia. All the North American species have pantoporate pollen and Eurasian species have tricolpate species. Sect. Viticella is distributed in Eurasia and most of the sampled species from East Asia have pantoporate pollen except European C. viticella and East Asian C. huchouensis. Because the relationships of these two sections are not resolved, the evolution of the pantoporate pollen of this clade cannot be inferred. The size of the pantoporate pollen in sect. Viorna is larger than that in sect. Viticella (Fig. 4) indicating that this aperture type may have evolved more than once in this clade.

Previous molecular phylogenetic studies (Miikeda et al. 2006; Xie et al. 2011) suggested recent species radiation in the genus. Molecular dating analysis indicated that the current species of Clematis may have diverged in the late Miocene (Xie et al. 2011). Approximately 300 species with highly diverse morphological characters evolved within a relatively short period of time. However, it is noteworthy that there is no evidence for the adaptive evolution of pollen morphology in Clematis, because the aperture types in the genus are rather simple. Tricolpate pollen grains are predominant in the genus and accompany all kinds of diverse morphological characters, for example floral structure (Jiang et al. 2010), inflorescence type, seedling phyllotaxy, sexuality of flowers, etc. (Xie et al. 2011).

Conclusions

Different morphotypes of pollen maximize the chances of successful fertilization under different conditions (Welsh et al. 2010). Tricolpate, 4-6-zonocolpate, pantocolpate, and pantoporate grains are regarded as progressively derived in Angiosperms (Wodehouse 1936; Muller 1970; Tellería and Daners 2003; Welsh et al. 2010), and this “successiformy” (Van Campo 1976) also can be observed in Ranunculaceae (Nowicke and Skvarla 1995). Pollen of Clematis is heteromorphic. Tricolpate pollen is predominant and may be the primitive type and pantoporate pollen may be derived but is not synapomorphic among the sections which have this type of pollen. Size and ornamentation characters are variable and can provide some systematic information about the genus; however, the variation of these characters is quantitative, continuous within and among sections. Although evolutionary trends can be inferred from palynologic characters in some groups of Clematis s.l., the taxonomic value of pollen morphology is limited in Clematis at species level.

Acknowledgments

The authors thank the herbaria of the Institute of Botany, Chinese Academy of Sciences (PE), Harvard University (GH), the Smithsonian Institute (US), South China Botanical Garden (IBSC), Missouri Botanical Garden (MO), Muséum National d’Histoire Naturelle (P), and the Herbarium of Uppsala University (UPS) for the loans of specimens for examination. We are indebted to Yin-Hou Xiao and Xue-Jian Yang for their assistance with SEM experiments and film development. This study was supported by the Fundamental Research Funds for the Central Universities (grant no. YX2010-31), the State Key Laboratory Program from the State Key Laboratory of Systematic and Evolutionary Botany (grant no. LSEB2011-07), the Main Direction Program of Knowledge Innovation of the Chinese Academy of Sciences (grant no. KSCX2-EW-Z-1), and the National Natural Science Foundation of China (grant nos. 31170201, 81072317, 31110103911).

Copyright information

© Springer-Verlag 2012