Botany
Seed dormancy in Campanulaceae: morphological and morphophysiological dormancy in six species of Hawaiian lobelioids
Journal: Botany
Manuscript ID cjb-2020-0009.R1
Manuscript Type: Article
Date Submitted by the 03-Mar-2020 Author:
Complete List of Authors: Baskin, Carol; University of Kentucky, Biology Baskin, Jerry; University of Kentucky, Biology Yoshinaga, Alvin; Center for Conservation Research and Training Wolkis, Dustin;Draft National Tropical Botanical Garden, Department of Science & Conservation
embryo growth, endemic species, morphological dormancy, Keyword: morphophysiological dormancy, seed dormancy
Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :
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1 Seed dormancy in Campanulaceae: morphological and morphophysiological dormancy in
2 six species of Hawaiian lobelioids
3
4 Carol C. Baskin, Jerry M. Baskin, Alvin Yoshinaga and Dustin Wolkis
5
6 Carol C. Baskin [email protected]
7 Department of Biology, University of Kentucky, Lexington, KY 40506 USA
8 Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546 USA
9 10 Jerry M. Baskin [email protected] 11 Department of Biology, University of Kentucky, Lexington, KY 40506 USA
12
13 Alvin Yoshinaga alviny@hawaii.edu
14 Center for Conservation Research and Training, 3050 Maile Way, Gilmore No. 409, Honolulu, HI
15 96822 USA
16 Dustin Wolkis [email protected]
17 Department of Science and Conservation, National Tropical Botanical Garden, 3530 Papalina
18 Road, Kalāheo, HI 96741
19
20 Running title: Germination of Hawaiian lobelioids
21
22 Author for correspondence Carol C. Baskin: [email protected]
23
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24 Abstract
25 We determined the requirements for dormancy break/germination and kind of dormancy in
26 seeds of the Hawaiian lobelioids Cyanea kunthiana, Delissea rhytidoperma, Lobelia grayana, L.
27 hypoleuca, Trematolobelia grandifolia and T. singularis. Fresh seeds were incubated in
28 light/dark at 15/6, 20/10 and 25/15°C and germination monitored at 2-week intervals for 14
29 weeks. For each species, the mean embryo length (E) : seed (S) length ratio was determined for
30 freshly-matured seeds and for seeds at the time the seed coat split but before radicle emergence
31 (germination). The embryo in seeds of all six species incubated at 25/15°C grew inside the seed
32 prior to germination (42-148% increase in E:S ratio, depending on species). Seeds of L. grayana
33 and L. hypoleuca have morphological dormancy; they germinated to 82-98% at the three 34 temperature regimes in 4 weeks. Seeds ofDraft the other species have nondeep simple 35 morphophysiological dormancy and require >4 weeks for maximum germination to occur. Our
36 results add to the growing body of knowledge about the kind (class) of seed dormancy in
37 Campanulaceae, which suggests that seeds of members of this family have either MD or MPD
38 and embryos grow at warm (≥ 15°C) temperatures.
39
40 Keywords: embryo growth, endemic species, morphological dormancy, morphophysiological
41 dormancy, seed dormancy
42
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43 Introduction
44 The eudicot family Campanulaceae (APG 2016) consist of herbaceous annuals, biennials and
45 perennials and includes pachycaul rosette plants, subshrubs, shrubs, treelets, trees to 15 m tall
46 (Lammers 2007) and some aquatics (Mabberley 2008). The family is divided into five
47 subfamilies (Campanuloideae, Lobelioideae, Nemacladoideae, Cyphioideae and
48 Cyphocarpoideae) and contains 84 genera and nearly 2400 species (Antonelli 2007; Lammers
49 2007; Crowl et al. 2014). According to Lammers (2007), 25, 25, 18, 11, 11, 6 and 4 % of the
50 species of Campanulaceae occur in Africa, South America, Asia, Europe, North America,
51 Polynesia and Australia, respectively. South Africa has 18 genera and nearly 400 species, and it 52 is the only region with a large number ofDraft both Campanulioideae and Lobelioideae; the 53 Cyphioideae with 64 species is restricted to Africa. Other centers of distribution predominantly
54 have only Campanuloideae or Lobelioideae. For example, in the Hawaiian Islands, there are six
55 genera and 126 species of Lobelioideae (Givnish et al. 2009, 2013), representing the largest
56 family of Hawaiian angiosperms (Wagner 1999), i.e. the largest plant species radiation in Hawaii
57 (Givnish et al. 2009). All the native Campanulaceae in Hawaii belong to the Lobelioideae
58 (Wagner 1999).
59 The Afrotropics are the inferred ancestral place of origin of the Campanulaceae,
60 including Lobelioideae (Crowl et al. 2016). Antonelli (2009) found that the giant lobelias of
61 eastern Africa, South America, Hawaiian Islands, French Polynesia and southeast Asia form a
62 monophyletic group and suggested that they are derived from a woody ancestor that grew in
63 Africa. Using molecular phylogeny data, Givnish et al. (2009) showed that the Lobelioideae in
64 Hawaii are from one immigration event. The lobelioids have diversified in Hawaii and can be
65 found in a wide range of habitats and exhibit diversity in growth form, pollination biology
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66 (Givnish et al. 2009) and seed coat morphology (Buss et al. 2001), but except for Clermontia
67 fauriei, C. hawaiiensis, C. kakeana, C. pyrularia, Cyanea angustifolia and Trematolobelia
68 macrostachys (Baskin et al. 2005), we have limited knowledge about seeds of the Hawaiian
69 lobelioids.
70 For the Campanulaceae, a relatively short linear embryo and copious endosperm occur in
71 the seeds of Campanula, Clermontia, Cyanea, Delissea, Lobelia,, Trematolobelia, Triodanis and
72 Wahlenbergia (Baskin and Baskin unpublished embryo data base). Seeds of L. dentata and
73 perhaps other species in the Australian endemic section Holopogon of Lobelia (sensu Lammers,
74 2011) have an undifferentiated embryo (Fraser 1931; Warcup 1988). In the seeds of temperate- 75 zone Campanula americana, Lobelia appendiculataDraft and L. spicata (Baskin and Baskin 2005) 76 and the Hawaiian lobelioid shrubs Clermontia fauriei, C. hawaiiensis, C. kakeana, C. pyrularia,
77 Cyanea angustifolia and Trematolobelia macrostachys (Baskin et al. 2005), the embryo grows
78 inside the seed prior to germination (radicle emergence). If embryo growth and germination in
79 freshly-matured seeds occur over a wide range of temperatures during incubation for about 4
80 weeks, seeds have morphological dormancy (MD), i.e. no physiological dormancy (PD) and
81 germination is delayed until the embryo grows to its full length (Nikolaeva 1977; Baskin and
82 Baskin 2014). On the other hand, freshly matured seeds have morphophysiological dormancy
83 (MPD) if dormancy-breaking treatments are required to break the PD component of dormancy
84 and embryo growth and germination take longer than 4 weeks. If seeds with MPD germinate at
85 some temperatures in about 4 weeks but over a wider temperature range after 4 weeks, MPD is
86 conditional, i.e. conditional MPD. Seeds of C. americana have MD, while those of C. fauriei, C.
87 hawaiiensis, C. kakeana, C. pyrularia, C. angustifolia, L. appendiculata, L. spicata and T.
88 macrostachys have nondeep simple MPD (Baskin and Baskin 2005; Baskin et al. 2005).
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89 Actually, fresh seeds of C. fauriei, C. hawaiiensis, C. kakeana and C. angustifolia have
90 conditional MPD because the level of PD in the seeds is nondeep and seeds exhibit an increase in
91 range of temperatures for germination as PD is broken. In these species, the embryo grew and
92 seeds germinated to a high percentage at 25/15 in 4 weeks, but after 8-12 weeks seeds gained the
93 ability to germinate to high percentages at 20/10 and 15/6°C. It is well known that seeds with
94 the nondeep level of PD may exhibit an increase in the range of temperatures (or other
95 conditions) at which they can germinate to a high percentage. Thus, dormancy break in most
96 seeds with PD occurs along a dormancy continuum that starts at ≤ 1.0 (seeds dormant = 1.0 and
97 seeds conditionally dormant <1.0 but >0) and ends with 0.0 (seeds nondormant). Seeds along the
98 continuum between <1.0 and >0.0 are in conditional dormancy (Soltani et al. 2017). Draft 99 Seeds of various species of Campanulaceae, including 131 taxa [of Campanulaceae] from
100 around the world (Koutsovoulou et al. 2014), nine species of Campanula from Mt Olympos in
101 Greece (Blionis and Vokou 2005), Campanula fragilis subsp. cavolinii (Frattaroli et al. 2013), C.
102 glomerata (Gülbağ and Özzambak 2017), C. uniflora (Aegisdóttir and Thórhallsdóttir 2006),
103 Jasione crispa (Giménez-Benavides et al. 2005), Lobelia inflata (Muenscher 1936; Baskin and
104 Baskin 1992), Lobelia spp. from Australia (Fraser 1931; Warcup 1988) and Physoplexis comosa
105 (Cerabolinin et al. 2004), have been investigated with regard to the effect of dormancy-breaking
106 treatments and/or effect of incubation temperatures and/or light-dark on germination. Nikolaeva
107 et al. (1985) included 22 species of Campanulaceae (16 Campanula, 1 Codonopsis and 5
108 Phyteuma) in their reference book on seed dormancy and germination, and seeds of all of them
109 were assigned to PD. However, none of these studies gave any attention to pregermination size
110 of growth of the embryo; thus, it is not possible to classify the kind of dormancy in seeds of these
111 species with confidence.
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112 The broad objective of the present study was to further classify the kind of dormancy and
113 the dormancy-breaking and germination requirements of the Campanulaceae in general and of
114 the Hawaiian lobelioids specifically. Studies were conducted on seeds of Cyanea kunthiana,
115 Delissea rhytidoperma, Lobelia grayana, L. hypoleuca, Trematolobelia grandifolia and T.
116 singularis (Table 1). We hypothesized that the seeds of these species have either MD or MPD.
117 Methods
118 Germination
119 Seeds of the six species were collected in Hawaii (Table 1) and immediately sent to the
120 University of Kentucky where studies were conducted. Freshly-matured seeds were tested for 121 germination in light (14-hour daily exposureDraft of about 40 µmol m-2 s-1, 400-700nm, of cool white 122 fluorescent light; hereafter light) at 15/6, 20/10 and 25/15°C (12/12 hour). At each temperature
123 regime, the light period in the incubator extended from 1 hour before the daily high-temperature
124 period began to 1 hour after it ended. Seeds were sown in 5.5-cm-diameter Petri dishes on two
125 sheets of Whatman No. 1 filter paper moistened with distilled water, and three replicates of 50
126 seeds each were used for each test condition. Each dish was wrapped with clear plastic film to
127 reduce rate of water loss. Seed germination (radicle emergence) was monitored at 2-week
128 intervals for 14 weeks, and each time germinated seeds were counted and removed from the
129 dishes water was added to the dishes as needed to keep the filter paper moist. The temperatures
130 regimes used in this study simulate those at high, mid- and low elevations in the Hawaiian
131 Islands (see Baskin et al. 2005), thus broadly covering the range of temperature regimes over
132 which the species occur in nature (see Table 1).
133 Embryo growth
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134 For each species, the embryo length (E) : seed (S) length ratio was determined for each
135 of 15 freshly matured seeds. To do this, seeds were placed on moist filter paper and incubated in
136 the dark at room temperature for 24 hours. Then, each seed was cut open lengthwise with a razor
137 blade, and length of the seed and of the embryo was measured using a dissecting microscope
138 equipped with a micrometer.
139 To determine if embryo growth occurred prior to germination, three replicates of 50 seeds
140 each for each species were sown on moist filter paper in Petri dishes and incubated in light at
141 25/15 °C. At weekly intervals (for up to 14 weeks), the seeds were checked using the dissecting
142 microscope to find those with a split seed coat but with no emergence of the radicle. When 143 appropriate seeds were found, they wereDraft cut lengthwise and the embryo and seed lengths 144 measured. Eventually, 15 seeds of each species with a split seed coat were found and the embryo
145 length and seed length measured. A split seed coat indicates that a seed is starting to germinate
146 (“incipient germination”) and that the embryo has reached the critical length for germination to
147 occur (Baskin and Baskin 2005; Baskin et al. 2005).
148 Statistical analysis
149 Germination proportions at weeks four and 14 (final germination) were square root arcsine
150 transformed and compared against temperature treatments using analysis of variance (ANOVA)
151 and post-hoc Tukey tests when appropriate (α = 0.05) with the software environment R (R Core
152 Team, 2019) and RStudio (RStudio Team, 2018). When either no germination occurred (e.g.
153 Trematolobelia grandifolia at week four), or all seeds germinated (e.g. final germination for
154 Lobelia grayana and L. hypoleuca) data were not analyzed.
155 Results
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156 Germination
157 Except for seeds of C. kunthiana incubated at 15/6°C, final germination of all six species
158 incubated at 15/6, 20/10 and 25/15°C, ranged from 74 - 100% (Fig. 1) with significant
159 differences between temperature regimes observed for C. kunthiana, D. rhytidosperma, L.
160 hypoleuca, and T. singularis (P values = <0.001 – 0.015; Table 1). At 25/15°C, ≥ 50% of the
161 seeds of L. grayana and L. hypoleuca had germinated at 2 weeks, and ≥ 50 % of the seeds of C.
162 kunthiana and D. rhytidosperma had germinated at 4 weeks. The 20/10°C temperature regime
163 was the most favorable for germination of T. grandifolia and T. singularis seeds, with
164 germination beginning between 2 and 4 and 4 and 6 weeks, respectively, and reaching and 87 165 and 77%, respectively, at 8 weeks. For allDraft species, final germination at 20/10°C ranged from 80 - 166 100%. Only 3% of C. kunthiana seeds germinated at 15/6°C and was significantly different from
167 20/10 and 25/15°C (both P values <0.001; Table 1), but seeds the other five species germinated
168 to 74 - 100% at this temperature regime.
169 Embryo growth
170 The mean E : S ratio for imbibed freshly-matured seeds ranged from 0.267 in T.
171 grandifolia to 0.598 in T. singularis (Table 2). The embryo in seeds of the six species incubated
172 at 25/15°C grew inside the seed prior to germination, and the increase in E:S ratio ranged from
173 .42 fold in T. singularis to 1.48 fold in D. rhytidopserma.
174 Discussion
175 Seeds of the six lobelioid species have small (underdeveloped) embryos that grow inside the
176 seeds prior to germination; thus, the seeds have either MD or MPD, as hypothesized. Seeds of L.
177 grayana and L. hypoleuca had germinated to ≥ 80% at 15/6, 20/10 and 25/15°C at 4 weeks.
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178 Seeds of C. kunthiana incubated at 25/15°C germinated rapidly, but those incubated at 20/10°C
179 were slow to germinate. Only a few seeds of this species germinated at 15/6°C. Seeds of D.
180 rhytidosperma germinated rapidly at 25/15 and 20/10°C, but germination at 15/6°C was delayed,
181 reaching ≥ 80% at 8 weeks. Seeds of T. grandifolia and T. singularis did not reach ≥50% at the
182 three temperatures regimes until ≥ 8 weeks. Thus, we conclude that seeds of L. grayana and L.
183 hypoleuca have MD, those of C. kunthiana and D. rhytidosperma conditional MPD and those
184 of T. grandifolia and T. singularis MPD. Like seeds of Cyanea kunthiana and D. rhytidosperma
185 those of Clermontia. fauriei, Clermontia. hawaiiensis, Clermontia. kakeana and Cyanea.
186 angustifolia have conditional MPD (Baskin et al. 2005). 187 Nine levels of class MPD have beenDraft distinguished, and they are divided into two 188 subclasses: simple and complex (sensu Baskin and Baskin 2014). In the simple subclass of
189 MPD, embryo growth occurs at temperatures suitable for warm stratification (≥ 15°C), whereas
190 in the complex subclass embryo growth occurs at temperatures suitable for cold stratification (ca.
191 0 - 10°C) (Nikolaeva 1977; Baskin and Baskin 2014). With the combination of underdeveloped
192 embryos that grow at 25/15°C and the presence of nondeep PD, we conclude that seeds of C.
193 kunthiana, D. rhytidosperma, T. grandifolia and T. singularis have nondeep simple MPD.
194 There is much to be learned about seed dormancy-break and germination in the
195 Campanulaceae, and questions remain. For example, do all members of this family have seeds
196 with MD or MPD, and after dormancy is broken do seeds have similar germination
197 requirements? Data for presence/absence of MD and MPD in the family are limited, but
198 available information indicates that small linear embryos occur in many genera (Baskin and
199 Baskin unpublished embryo data base; see Introduction). On the other hand, many germination
200 tests have been conducted to determine the temperature requirements for germination of seeds of
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201 Campanulaceae. Koutsovoulou et al. (2014) tested germination of 131 taxa in 27 genera of
202 Campanulaceae from five continents at constant and alternating temperature regimes. For taxa
203 that germinated to ≥ 80%, the mean (± s.e.) constant and alternating temperatures for best
204 germination were 16.4 ± 0.6°C and 16.9 ± 0.5°C, respectively. In the book on seeds by Baskin
205 and Baskin (2014), the mean temperature reported for or a high germination percentage for 51
206 species in 14 genera of Campanulaceae was 17.7 ± 0.6°C. For the six lobelioids in the present
207 study, the mean of the best temperature for germination of each species [based on speed (slope of
208 germination curve) and percentage] is 17.5 ± 1.6°C.
209 Conclusions 210 Dormancy and germination amongDraft the Hawaiian lobelioids is predictable: MD or MPD 211 and a high germination percentage at 25/15°C. An exception to this statement is the Hawaiian
212 Clermontia. pyrularia whose seeds have MPD but germinate to a high percentage only at 15/6°C
213 and with little or no germination at 25/15 or 20/10°C (Baskin et al. 2005). Given that the
214 Campanulaceae, especially the Lobelioideae, may have originated in the same place, i.e. the
215 Afrotropics (Antonelli 2009; Crowl et al. 2016), two further research questions come to mind.
216 (1) Do seeds of all Campanulaceae have underdeveloped embryos and thus MD or MPD? (2)
217 Does embryo growth occur only at warm-stratifying temperatures? Although the Campanulaceae
218 now occurs in Africa, South America, Asia, Europe, North America, Polynesia and Australia, the
219 available data seem to hint that the species in this family have similar seed dormancy (MD or
220 MPD) and that embryos require warm-stratifying temperature to grow even if cold stratification
221 is required to break the PD part of MPD. For example, seeds of Lobelia spicata and L.
222 appendiculata require cold stratification before they will germinate, but embryo growth does not
223 occur until the cold-stratified seeds are transferred to high (spring) temperatures (Baskin and
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224 Baskin 2005). With regard to the first question, although the “dust seeds” (0.31 to 0.39 mm long
225 x 0.21 to 0.29 mm wide) of L. dentata, in section Holopogon of Lobelia have an organless
226 (undifferentiated) embryo when they mature (Fraser 1931), germination of the seeds is bipolar
227 (Warcup 1988) and thus not unipolar as occurs in dust seeds of holoparasites and
228 mycoheterotrophs with an undifferentiated embryo (Baskin and Baskin 2014). We suggest that
229 the organless embryo in L. dentata differentiates into an underdeveloped embryo that grows
230 before the seed germinates, as occurs in several other species (see page 71 in Baskin and Baskin
231 2014).
232 Acknowledgment
233 We thank the Hawaii Conservation AllianceDraft and HATCH Project Accession No. 0210780 for 234 financial support.
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318 RStudio Team. 2018. RStudio: Integrated Development for R. RStudio, Inc., Boston, MA. URL:
319 http://www.rstudio.com/.
320 Soltani, E., Baskin, C.C., and Baskin, J.M. 2017. A graphical method for identifying
321 the six types of nondeep physiological dormancy in seeds. Plant Biol. 19(5): 673-682.
322 doi: 10.1111/plb.12590.
323 Wagner, W.l., Herbst, D.R., and Sohmer, S.H. (Editors). 1999. Manual of the flowering plants of
324 Hawaii. Revised edition. Vol. 1. University of Hawaii Press and Bishop Museum Press,
325 Honolulu.
326 Warcup, J.H. 1988. Mycorrhizal associationsDraft and seedling development in Australian
327 Lobelioideae (Campanulaceae). Aust. J. Bot. 36(4): 461-472. DOI:10.1071/bt9880461
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331 Table 1. Island of seed collection for the six study species, island(s) of occurrence in Hawaii and 332 elevation range and embryo length (E) : seed length (S) ratio (mean ± s.e.) of freshly-matured 333 seeds and at time of “incipient” germination. Percent increase in E:S ratio is shown in 334 parenthesis.
E:S ratio Island of Elevation (m) seed and island(s) in Fresh Incipient germination Species collection Hawaiia Cyanea kunthiana Maui 1000-1600 0.563±0.04 0.881±0.02 (56.5)b (Gaudich.) Hillebr. Maui Delissea rhytidosperma Hawaii 300-1000 0.387±0.01 0.961±0.02 (148.3) H. Mann. Kauai Lobelia grayana E. Wimm. Maui 1700-2300 0.428±0.04 0.671±0.01 (56.8) Maui Lobelia hypoleuca Hillebr. OahuaDraft600-1500 0.359±0.04 0.613±0.04 (70.8) all except Niihau and Kahoolawe Trematolobelia grandifolia Hawaii 625-1200 0.267±0.04 0.488±1.04 (82.1) (Rock) O. Deg. Hawaii Trematolobelia singularis Oahua 900-915 0.598±0.07 0.851±0.02 (42.3) H. St. John Oahu
335
336 ainformation from Wagner et al.,1999
337 b(Final E:S ratio minus original E:S ratio) divided by original E:S ratio x 100 338
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344 Table 2: Comparison of square root arcsin transformed germination proportions between
345 temperature treatments at weeks 4 and 14 (final) with of analysis of variance (ANOVA) and
346 post-hoc Tukey test if ANOVA P value < 0.05 (α = 0.05). There was no germination at week
347 four in Trematolobelia grandifolia and was therefore not analyzed.
Germination at week 4 Final Germination Temperature Comparison ANOVA Tukey ANOVA Tukey Species (°C) P value P value P value P value 20/10 - 15/6 0.002 <0.001 Cyanea kunthiana 25/15 - 15/6 <0.0001 <0.001 <0.0001 <0.001 25/15 - 20/10 <0.001 1.000 20/10 - 15/6 <0.001 0.001 Delissea <0.0001 <0.001 <0.0001 rhytidosperma 25/15 - 15/6 0.001 25/15 - 20/10 0.699 1.000 20/10 - 15/6 Draft Lobelia grayana 25/15 - 15/6 0.133 NA 0.422 NA 25/15 - 20/10 20/10 - 15/6 0.010 Lobelia hypoleuca 25/15 - 15/6 0.003 0.003 0.422 NA 25/15 - 20/10 0.546 20/10 - 15/6 Trematolobelia No germination 25/15 - 15/6 NA 0.752 NA grandifolia occurred 25/15 - 20/10 20/10 - 15/6 0.016 Tramatolobelia 25/15 - 15/6 0.015 0.725 0.263 NA singularis 25/15 - 20/10 0.041 348
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350 Figure Legend
351 Fig. 1. Germination (mean % ± s.e., if ≥ 5%) of seeds of Cyanea kunthiana (A), Delissea
352 rhytidoperma (B), Lobelia grayana (C), L. hypoleuca (D), Trematolobelia grandifolia (E) and T.
353 singularis (F) incubated in light/dark at 15/6, 20/10 and 25/15°C.
354
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Draft
194x162mm (300 x 300 DPI)
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