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Gigantic chloroplasts, including bizonoplasts, are common in shade-adapted species of the ancient vascular plant family Selaginellaceae: Gigantic chloroplasts of Selaginellaceae

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  • Universidad de Panamá, Panama
Article

Gigantic chloroplasts, including bizonoplasts, are common in shade-adapted species of the ancient vascular plant family Selaginellaceae: Gigantic chloroplasts of Selaginellaceae

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Premise: Unique among vascular plants, some species of Selaginella have single giant chloroplasts in their epidermal or upper mesophyll cells (monoplastidy, M), varying in structure between species. Structural variants include several forms of bizonoplast with unique dimorphic ultrastructure. Better understanding of these structural variants, their prevalence, environmental correlates and phylogenetic association, has the potential to shed new light on chloroplast biology unavailable from any other plant group. Methods: The chloroplast ultrastructure of 76 Selaginella species was studied with various microscopic techniques. Environmental data for selected species and subgeneric relationships were compared against chloroplast traits. Results: We delineated five chloroplast categories: ME (monoplastidy in a dorsal epidermal cell), MM (monoplastidy in a mesophyll cell), OL (oligoplastidy), Mu (multiplastidy, present in the most basal species), and RC (reduced or vestigial chloroplasts). Of 44 ME species, 11 have bizonoplasts, cup-shaped (concave upper zone) or bilobed (basal hinge, a new discovery), with upper zones of parallel thylakoid membranes varying subtly between species. Monoplastidy, found in 49 species, is strongly shade associated. Bizonoplasts are only known in deep-shade species (<2.1% full sunlight) of subgenus Stachygynandrum but in both the Old and New Worlds. Conclusions: Multiplastidic chloroplasts are most likely basal, implying that monoplastidy and bizonoplasts are derived traits, with monoplastidy evolving at least twice, potentially as an adaptation to low light. Although there is insufficient information to understand the adaptive significance of the numerous structural variants, they are unmatched in the vascular plants, suggesting unusual evolutionary flexibility in this ancient plant genus.
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American Journal of Botany 107(4): 1–15, 2020; http://www.wileyonlinelibrary.com/journal/AJB © 2020 Botanical Society of America 1
Gigantic chloroplasts, including bizonoplasts, are common
in shade-adapted species of the ancient vascular plant
family Selaginellaceae
Jian-Wei Liu1, Shau-Fu Li1, Chin-Ting Wu2, Iván A. Valdespino3, Jia-Fang Ho1,2, Yeh-Hua Wu1,2, Ho-Ming Chang4, Te-Yu Guu1, Mei-Fang Kao5,
Clive Chesson6, Sauren Das7, Hank Oppenheimer8, Ane Bakutis9, Peter Saenger10, Noris Salazar Allen11 , Jean W. H. Yong12 ,
Bayu Adjie13, Ruth Kiew14, Nalini Nadkarni15 , Chun-Lin Huang16 , Peter Chesson1,17 , and Chiou-Rong Sheue1,17,18
RESEARCH ARTICLE
Manuscript received 11 August 2019; revision accepted 21 January 2020.
1 Department of Life Sciences & Research Center for Global Change
Biology,National Chung Hsing University, Taichung, Taiwan
2 Department of Biological Resources,National Chiayi University,
Chiayi, Taiwan
3 Departamento de Botánica,Facultad de Ciencias Naturales,
Exactas y Tecnología,Universidad de Panamá; Sistema Nacional de
Investigación (SNI),SENACYT, Panama, Panama
4 Endemic Species Research Institute, Jiji Town, Taiwan
5 TAI Herbarium,National Taiwan University, Taipei, Taiwan
6 6 Barker Way, Valley View, S.A., Australia
7 Agricultural and Ecological Research Unit,Indian Statistical
Institute, Kolkata, India
8 Maui Nui Plant Extinction Prevention Program, Maui, USA
9 Molokai Plant Extinction Prevention Program, Molokai, USA
10 Centre for Coastal Management,Southern Cross University,
Lismore, Australia
11 Smithsonian Tropical Research Institute, Panama, Panama
12 Department of Biosystems and Technology,Swedish University
of Agricultural Sciences, Alnarp, Sweden
13 Bali Botanic Garden, Indonesia
14 Forest Research Institute Malaysia, Kepong, Malaysia
15 Department of Biology,University of Utah, Salt Lake City, USA
16 National Museum of Natural Science, Taichung, Taiwan
17 Department of Ecology and Evolutionary Biology,University of
Arizona, Tucson, USA
18Author for correspondence (e-mails: crsheue@nchu.edu.tw;
crsheue@gmail.com)
Citation: Liu, J.-W., S.-F. Li, C.-T. Wu, I. A. Valdespino, J.-F. Ho, Y.-H.
Wu, H.-M. Chang, etal. 2020. Gigantic chloroplasts, including
bizonoplasts, are common in shade-adapted species of the ancient
vascular plant family Selaginellaceae. American Journal of Botany
107(4): 1–15.
doi:10.1002/ajb2.1455
PREMISE: Unique among vascular plants, some species of Selaginella have single giant
chloroplasts in their epidermal or upper mesophyll cells (monoplastidy, M), varying in
structure between species. Structural variants include several forms of bizonoplast with
unique dimorphic ultrastructure. Better understanding of these structural variants, their
prevalence, environmental correlates and phylogenetic association, has the potential to
shed new light on chloroplast biology unavailable from any other plant group.
METHODS: The chloroplast ultrastructure of 76 Selaginella species was studied with
various microscopic techniques. Environmental data for selected species and subgeneric
relationships were compared against chloroplast traits.
RESULTS: We delineated ve chloroplast categories: ME (monoplastidy in a dorsal epidermal
cell), MM (monoplastidy in a mesophyll cell), OL (oligoplastidy), Mu (multiplastidy, present
in the most basal species), and RC (reduced or vestigial chloroplasts). Of 44 ME species,
11 have bizonoplasts, cup-shaped (concave upper zone) or bilobed (basal hinge, a new
discovery), with upper zones of parallel thylakoid membranes varying subtly between
species. Monoplastidy, found in 49 species, is strongly shade associated. Bizonoplasts are
only known in deep-shade species (<2.1% full sunlight) of subgenus Stachygynandrum but
in both the Old and New Worlds.
CONCLUSIONS: Multiplastidic chloroplasts are most likely basal, implying that monoplastidy
and bizonoplasts are derived traits, with monoplastidy evolving at least twice, potentially
as an adaptation to low light. Although there is insucient information to understand
the adaptive signicance of the numerous structural variants, they are unmatched in the
vascular plants, suggesting unusual evolutionary exibility in this ancient plant genus.
KEY WORDS bilobed chloroplast; chloroplast diversity; cup-shaped chloroplast;
monoplastidy; shade-adapted Selaginella; Selaginellaceae; Stachygynandrum;
ultrastructure.
Plants carry out photosynthesis over a huge range of environmen-
tal conditions. Although the key organelle, the chloroplast, might
be expected to vary adaptively in size, number, and structure over
this range, chloroplast traits are generally highly conserved in
land plants. Exceptions to this rule have the potential to be espe-
cially instructive. e monogeneric seedless vascular plant family
2 American Journal of Botany
Selaginellaceae (sole genus Selaginella) are noted for having sin-
gle giant chloroplasts (monoplastidy) in the dorsal epidermal cells
(Banks, 2009), but as we show here, there is great diversity in chlo-
roplast type and structure in Selaginella dorsal epidermal cells.
Moreover, monoplastidy is also found in the upper mesophyll of
some species. Although algae have high chloroplast diversity, in
most groups of land plants chloroplast diversity is very low. Among
nonvascular plants, hornworts are noted for giant chloroplasts, but
liverworts and mosses typically have chloroplasts similar to those
of the majority of vascular plants (Vanderpoorten and Gonet,
2009). Among vascular plants, Selaginella stands out as an extreme
exception.
Bizonoplasts are particularly striking chloroplast variants found
in Selaginella. ey are characterized by a dimorphic ultrastruc-
ture in which the upper zone consists of multiple layers of thyla-
koid membranes, with no grana, while the lower zone has normal
grana and stroma thylakoids. Bizonoplasts have been reported rela-
tively recently (Sheue etal., 2007; Liu etal., 2012; Reshak and Sheue,
2012; Ferroni etal., 2016) and are only known from Selaginella. e
original nding (Sheue etal., 2007) was in S. erythropus (Mart.)
Spring. Studies show that the bizonoplast develops from a pro-
plastid indistinguishable from a normal vascular plant proplastid
(Sheue etal., 2015). High light conditions were shown to prevent
the development of the bizonoplast ultrastructure. Instead, several
chloroplasts, with normal chloroplast ultrastructure, developed in
each dorsal epidermal cell (Sheue etal., 2015).
In other vascular plants, there are few variants of the basic chlo-
roplast structure. Key variants are the lamelloplast (Pao etal., 2018;
originally “iridoplast”: Gould and Lee, 1996), the recently discov-
ered minichloroplasts of Begoniaceae (Pao et al., 2018), and the
bundle sheath chloroplasts of C4 plants (Solymosi and Keresztes,
2012). Other plastids, such as chromoplasts and amyloplasts ex-
ist, but our concern here is chloroplasts sensu stricto, i.e., plastids
with well-developed thylakoid membrane systems, chlorophyll, and
photosynthetic functioning. Because chloroplasts have been studied
for more than 150 years, the discovery of the bizonoplast stands
out. e aims of this study were to explore the chloroplast varia-
tion in Selaginellaceae, with an emphasis on bizonoplasts (Bps), to
further understand their prevalence, structural variation, environ-
mental, morphological and phylogenetic correlates, and adaptive
signicance. is paper presents the results of 6 years of eldwork
on four continents to obtain fresh sample collections and habitat
information on 76 species of Selaginella, which were identied and
studied by electron microscopy. e results were then correlated
with habitat features and phylogeny.
MATERIALS AND METHODS
Sources of species and environmental variables measured
In this study, plant materials from 76 species of Selaginella (ap-
proximately 10% of Selaginellaceae) from all seven subgenera
(Weststrand and Korall, 2016) were collected worldwide from
2012 to 2018 (Table 1; Appendix S1). Among them, 64 species
were collected from natural habitats, with basic environmental
data recorded (location, GPS coordinates, soil type, local vegeta-
tion, light environment), and 12 species were obtained from bo-
tanical gardens. Both morphological features and DNA sequences
(rbcL) were used for identication. Voucher specimens have been
deposited in the herbarium of National Chung Hsing University
(TCB), Taiwan.
Environmental data (light intensity, temperature, humidity)
were recorded in the habitats of 10 selected Selaginella species [S.
aristata Spring, S. arizonica Maxon, S. ciliaris (Retz.) Spring, S. deli-
catula (Desv.) Alston, S. devolii H.M.Chang, P.F.Lu & W.L.Chiou, S.
doederleinii Hieron., S. heterostachys Baker, S. moellendorffii Hieron.,
S. repanda (Desv.) Spring, and S. tamariscina (P.Beauv.) Spring],
ranging from low to high light environments. Light intensity (pho-
tosynthetically active radiation, PAR) was measured using a porta-
ble LI-COR quantum sensor model LI-190 (Lincoln, NE, USA) for
4 years (2012–2014; 2017–2018). ese data were then converted to
percentage of full sunlight to facilitate comparison. Data were tested
by ANOVA between species, followed with Scheé’s post hoc test us-
ing SPSS (version 20; SPSS, Chicago, IL, USA). Environmental fac-
tors (light intensity, temperature, and humidity) of four local species
(S. doederleinii, S. heterostachys, S. repanda, and S. tamariscina) na-
tive to Taiwan were continuously monitored and recorded over the
year 2013 with data loggers (HOBO Pro v2 Temperature/Relative
Humidity data logger and HOBO Pendant Temperature/Light 64K
Data Logger; Onset Computer Corp., Bourne, MA, USA).
Preparation of plant materials for structural study
e materials (ventral leaves only for the species with leaves lon-
ger than 3 mm; otherwise segments of shoots ca. 2–3 mm long) of
each of three individuals (up to ve individuals in abundant popu-
lations) were used for the chloroplast study. Some branch segments
were observed directly when fresh. Others were xed in 2.5% v/v
glutaraldehyde in 0.1 M sodium phosphate buer and 70% v/v eth-
anol (segments of shoots ca. 5 mm long) in the eld or botanical
gardens, then transferred to the laboratory for structural study.
Structural study of chloroplasts and phylogenetic association
Both free hand sections (transverse view) and top views of in-
tact microphylls (leaves) were used to observe chloroplast traits
in fresh materials and some samples xed in ethanol. Selected ma-
terials were observed with a confocal scanning laser microscope
(CSLM, Leica TSC-SP5, Wetzlar, Germany) (excitation 488 nm,
emission wavelength 581–756 nm) using a 63× oil immersion ob-
jective to investigate bizonoplast (Bp) morphological features. KY
jelly (Johnson and Johnson, New Brunswick, NJ, USA) was used to
temporarily embed freehand sections before observation.
A general electron microscopy protocol was followed (Sheue
etal., 2007). Semithin sections (1 μm) were cut and stained with 1%
w/v aqueous toluidine blue for observation with a light microscope
(Olympus BH-2, Tokyo, Japan). Ultrathin sections (about 70 nm)
were cut and stained with uranyl acetate (5% w/v in 50% methanol)
and lead citrate (1% w/v in water) for examination with either a
Hitachi H 600 (Tokyo, Japan) or a JEOL (JEM-2000 EXII, Tokyo,
Japan) transmission electron microscope (TEM). e results on
chloroplast types (with or without Bps) and plant morphology were
then used to annotate the subgeneric tree of Selaginella published
by Weststrand and Korall (2016).
Comparison of ultrastructural features of Bps
e ultrastructural features of Bps from nine selected species ob-
tained from TEM micrographs were measured, including thylakoid
2020, Volume 107 Liu etal.—Gigantic chloroplasts of Selaginellaceae 3
TABLE 1. Details of the 76 Selaginella species used in this study focusing on monoplastidy and chloroplast traits in dorsal epidermal cells of a microphyll.
Abbreviations: A, anisophylly; bBp, bilobed bizonoplast; bCp, bilobed chloroplast; cBp, cup-shaped bizonoplast; cCp, cup-shaped chloroplast; D, typical disk-
shaped chloroplast; gD, giant disk-shaped chloroplast; I, isophylly; ME, monoplastidy in the dorsal epidermal cell; MM, monoplastidy in a mesophyll cell; Mu,
multiplastidy; OL, oligoplastidy; RC, reduced or vestigial chloroplasts. Chloroplast types and categories are based on dorsal epidermal cells, except for MM when
monoplastidy occurs in a mesophyll cell immediately below the epidermis.
No. Species
Subgenus (Weststrand
and Korall, 2016) Isophyll/Anisophyll Chloroplast type Chloroplast category
1S. anceps (C.Presl) C.Presl Stachygynandruma A cCp ME
2S. arbuscula (Kaulf.) Spring Stachygynandrum A bCp ME
3S. arenicola Underw. Rupestrae I D Mu
4S. aristata Spring Stachygynandrum A bBp ME
5S. arizonica Maxon Rupestrae Ib D Mu
6S. arthritica Alston Gymnogynum ARC RC
7S. articulata (Kunze) Spring Gymnogynum A cCp MM
8S. australiensis Baker Gymnogynuma A cCp MM
9S. bisulcata Spring Stachygynandrum A bCp ME
10 S. bombycina Spring Stachygynandrum A cCp ME
11 S. boninensis Baker Stachygynandrum A bBp ME
12 S. chrysoleuca Spring Stachygynandruma A cCp ME
13 S. ciliaris (Retz.) Spring Stachygynandrum A D, bCPc OL, MEc
14 S. cupressina (Willd.) Spring Stachygynandrum A cCp ME
15 S. deflexa Brack. Selaginella I D Mu
16 S. delicatula (Desv.) Alston Stachygynandrum A cBp ME
17 S. devolii H.M.Chang, P.F.Lu & W.L.Chiou Stachygynandrum A bBp ME
18 S. diffusa (C.Presl) Spring Gymnogynum A cCp MM
19 S. doederleinii Hieron. Stachygynandrum A cCp ME
20 S. douglasii (Hook. & Grev.) Spring Stachygynandrum A D Mu
21 S. erythropus (Mart.) Spring Stachygynandrum A cBp ME
22 S. euclimax Alston ex Crabbe & Jermy Stachygynandruma A cCp ME
23 S. eurynota A.Braun Gymnogynuma A RC RC
24 S. exaltata (Kunze) Spring Exaltatae ARC RC
25 S. flagellata Spring Stachygynandruma A bCp ME
26 S. flexuosa Spring Stachygynandruma A cCp ME
27 S. gracillima (Kunze) Spring ex Salomon Ericetorum I D Mu
28 S. haematodes (Kunze) Spring Stachygynandrum A cCp ME
29 S. heterostachys Baker Stachygynandrum A bBp ME
30 S. hieronymiana Alderw. Stachygynandrum A D Mu
31 S. horizontalis (C.Presl) Spring Gymnogynum ARC RC
32 S. huehuetenangensis Hieron. Stachygynandruma A cCp ME
33 S. intermedia (Blume) Spring Stachygynandrum A cBp ME
34 S. involvens (Sw.) Spring Stachygynandrum A D OL
35 S. kraussiana (Kunze) A.Braun Gymnogynum A bCp MM
36 S. labordei Hieron. ex Christ Stachygynandrum A bCp ME
37 S. lepidophylla (Hook. & Grev.) Spring Lepidophyllae A D Mu
38 S. leveriana Alston Stachygynandrum A cCp ME
39 S. longipinna Warb. Stachygynandrum A cCp ME
40 S. lutchuensis Koidz. Stachygynandrum A bBp ME
41 S. martensii Spring Stachygynandrum A cBp/cCpd ME
42 S. mayeri Hieron. Stachygynandrum A cCp ME
43 S. minima Spring Stachygynandruma A cCp ME
44 S. moellendorffii Hieron. Stachygynandrum A cCp ME
45 S. mollis A.Braun. Stachygynandrum A cCp ME
46 S. monospora Spring Stachygynandrum A bCp ME
47 S. nipponica Franch. & Sav. Stachygynandrum A D OL
48 S. oregana D.C.Eaton Rupestrae I D Mu
49 S. pallescens (C.Presl) Spring Stachygynandrum A D OL
50 S. picta (Griff.) A.Braun ex Baker Stachygynandruma A cBp ME
51 S. plana (Desv.) Hieron. Stachygynandrum A bCp ME
52 S. poperangensis Hieron. Stachygynandrum A cCp ME
53 S. porelloides (Lam.) Spring Stachygynandrum A gD ME
54 S. porphyrospora A.Braun Stachygynandruma A cCp ME
55 S. pseudonipponica (Tagawa) H.M.Chang,
W.L.Chiou & J.C.Wang
Stachygynandrum A D OL
56 S. pulcherrima Liebm. Stachygynandrum A bCp ME
(Continued)
4 American Journal of Botany
group number in an upper zone, thickness of a thylakoid group, num-
ber of stacked thylakoids per thylakoid group, and stroma thickness
between thylakoid groups. Only micrographs with clear thylakoid
structures (sections perpendicular to membranes and lumens) in
the upper zones of Bps were used to obtain data. Materials collected
overseas for this comparison were more limited. For each selected
species, 3–8 individuals (3 individuals of overseas species, more of
local species) were used and 3–31 Bps were selected to study their ul-
trastructural features. To determine the number of thylakoid groups
in the upper zone, micrographs at low magnication were studied
for 7 to 31 Bps. ickness of both thylakoid groups and stroma
were measured with ImageJ (ImageJ 1.51s; National Institutes of
Health, Bethesda, MD, USA). Data were tested by ANOVA between
species, followed with Scheés post hoc test using SPSS version 20.
Estimating the prevalence of chloroplast types in Selaginella
From our data, we estimated the proportion, psg,ct, of a chloroplast
type (ct) in a subgenus (sg) simply as the number of species with
that type of chloroplast that we found in that subgenus divided by
the number of species that we studied from that subgenus. To esti-
mate the overall proportion, pct, of a given ct in the genus Selaginella,
we took a weighted average of the proportions in the subgenera as
given by the formula
where nsg is the known number of species globally in the subge-
nus sg, and the sums are over all subgenera. is formula corrects
any bias that exists in the number of species that we studied in
each subgenus and gives an unbiased estimate of the proportions
of each chloroplast type in the genus, subject to the assumption
that our sampling of chloroplast types in a subgenus is unbiased.
However, as we are unsure whether chloroplast sampling is unbi-
ased within a subgenus, our nal results can only be regarded as
approximate.
p
ct =
sg
p
sg,ct
n
sg
sg
nsg
,
No. Species
Subgenus (Weststrand
and Korall, 2016) Isophyll/Anisophyll Chloroplast type Chloroplast category
57 S. rechingeri Hieron. Stachygynandrum A bCp ME
58 S. remotifolia Spring Gymnogynum A cCp, De MM, OLe
59 S. repanda (Desv.) Spring Stachygynandrum A D OL
60 S. revoluta Baker Stachygynandrum A cBp ME
61 S. rupincola Underw. Rupestrae I D Mu
62 S. salazariae Valdespino Stachygynandruma A cCp ME
63 S. sertata Spring Gymnogynuma A RC RC
64 S. schaffneri Hieron. Stachygynandruma A D Mu
65 S. simplex Baker Stachygynandruma A cCp ME
66 S. stauntoniana Spring Stachygynandrum A D Mu
67 S. tamariscina (P.Beauv.) Spring Stachygynandrum A D Mu
68 S. uliginosa (Labill.) Spring Ericetorum I D Mu
69 S. umbrosa Lem. ex Hieron. Stachygynandrum A cCp ME
70 S. uncinata (Desv.) Spring Stachygynandrum A bCp ME
71 S. underwoodii Hieron. Rupestrae I D Mu
72 S. vogelii Spring Stachygynandrum A D OL
73 S. wallacei Hieron. Rupestrae IRC RC
74 S. wallichii (Hook. & Grev.) Spring Stachygynandrum A cCp ME
75 S. willdenowii (Desv.) Baker Stachygynandrum A bCp ME
76 S. wolffii Sodiro Stachygynandruma A cCp ME
aInfrageneric classification is based on the key provided by Weststrand and Korall (2016).
bThis species is slightly anisophyllous.
cThis species may have different chloroplast types in different environments (OL, in an open grassland; ME, in a grassland shaded by trees).
dFerroni et al. (2016) reported cBps from this species, but our material obtained from a botanic garden appears as cCps.
eThis species may have different chloroplast types in different light environments (MM, in shade; OL, in partial shade).
TABLE 1. (Continued)
FIGURE 1. Habitats and chloroplast types of Selaginella. (A–C) Shade-adapted Selaginella with monoplastids in dorsal epidermal cells (ME), which
possess bizonoplast (Bp) ultrastructure. (A) S. intermedia with cup-shaped Bps (cBps), Singapore. (B) S. devolii with bBps, Taiwan. (C) S. heterostachys,
with bilobed Bps (bBps), Taiwan. (D–F) cBps of S. delicatula. Transverse view: D, E; top view: F. (G, H) Confocal scanning laser micrographs (CSLM) of cBps
of S. erythropus showing their concave tops at two dierent angels. (I–M) bBps of S. heterostachys, arrows indicating the connections between lobes.
Transverse view: I, J; top view: K, with inset showing widely expanded bBp. CSLM viewed from dierent angles in images L and M. Three-dimensional
reconstructions are shown in inset in L (top view) and in M (lateral view with a bBp tilted forward revealing the connection). (N) (Left) S. kraussiana
and (right) its bilobed chloroplast in the rst mesophyll cell layer. (O) S. nipponica and its oligoplastidy (OL) in dorsal epidermal cells. (P) S. deexa and
its multiplastidy (Mu) in adaxial epidermal cells. (Q) S. wallacei and its reduced or vestigial chloroplasts (RC) in adaxial epidermal cells (ruler with 1 mm
divisions). The inset shows leaf structure close to the adaxial surface (10 μm scale bar). Abbreviations: adE, adaxial epidermal cell; bBp, bilobed bizo-
noplast; bCp, bilobed chloroplast; cBp, cup-shaped bizonoplast; Cp, typical chloroplast; cCP, cup-shaped chloroplast; dE, dorsal epidermal cell; MC,
mesophyll cell; ME, monoplastidy in dorsal epidermal cells; MM, monoplastidy in mesophyll cells; Mu, multiplastidy; OL, oligoplastidy; RC, reduced or
vestigial chloroplasts; vE, ventral epidermal cell.
2020, Volume 107 Liu etal.—Gigantic chloroplasts of Selaginellaceae 5
6 American Journal of Botany
RESULTS
Chloroplast diversity
Unlike seed plants with mesophyll as the main photosynthetic
tissue, chloroplasts of Selaginella appear in both epidermal cells
and mesophyll cells (Fig.1). In most cases, Selaginella mesophyll
cells and ventral epidermal cells have multiple chloroplasts with
the typical structure of those in vascular plants generally. Among
the 76 species studied, chloroplast variants were found in dorsal
epidermal cells or in some species in the mesophyll cells directly
below the dorsal epidermal layer. Note that only in dorsiventral
species (e.g., S. kraussiana in Fig.1N) are the dorsal and ventral
epidermises dened. e corresponding terms for nondorsiventral
species (e.g., S. deflexa in Fig.1P) are adaxial and abaxial. However,
only dorsiventral species were found to have unusual chloroplasts.
Table2 gives the chloroplast categories that we identied based
on chloroplast size, number per cell, and tissue location. Briey,
four major categories are delineated: monoplastidy (M, one large
chloroplast per cell; 49 species) (Fig.1D–N), oligoplastidy [OL,
(2)3–10 chloroplasts per cell; 7 species] (Fig.1O), multiplastidy
(Mu, more than 10 chloroplasts per cell in all photosynthetic cells;
14 species) (Fig.1P), and reduced or vestigial chloroplasts (RC; few
barely visible chloroplasts, 6 species) (Fig.1Q). We estimate from
these data that the genus Selaginella is 70% M, 11% OL, 9% Mu,
and 4% RC. ese categories of chloroplast size and number are
related to microphyll morphology (anisophylly or isophylly). e
isophyllous species (generally nondorsiventral) are the Mu type,
but the anisophyllous species (generally dorsiventral) have more
diverse chloroplast types, viz. M, OL, and RC (Table1).
Monoplastids, M, are especially large, normally occupying a
substantial fraction, up to 80%, of the cell volume, with linear
dimension up to 40 μm (Fig.1E). Monoplastidy may appear in a
dorsal epidermal cell (ME type) (Fig.1D–M) or in a mesophyll cell
immediately below the epidermal layer (MM type) (Fig.1N), or
uniquely, to date, in S. plana (Desv.) Hieron. where only the ventral
epidermal cells are not monoplastidic. Among them, 44 species are
ME, and ve are MM. Moreover, three shapes of M chloroplasts
are further recognized: cup-shaped (28 ME species and 4 MM spe-
cies), bilobed (15 ME species and 1 MM species), and giant disk
(one ME species) (Table1). e monoplastids in some ME species
are classied as bizonoplasts based on the presence of two ultra-
structure zones as reported by Sheue etal. (2007) (Table2).
Occurrence and forms of bizonoplasts
is study found nine additional species with Bps beyond the two
species previously reported (Fig.1A–C; Table1). Bizonoplasts may
appear as either cup-shaped (6 species) (cBp, Fig. 1D–H) or bi-
lobed (bBp, 5 species) (Fig.1I–M). Here, S. delicatula, S. intermedia
(Blume) Spring, S. picta (Gri.) A.Braun ex Baker, and S. revoluta
Baker are newly reported to have cBps, and S. aristata, S. boninen-
sis Baker, S. devolii, S. heterostachys, and S. lutchuensis Koidz. were
found to have bBps. From a microphyll top view, a cBp appears as
a circle in a dorsal epidermal cell (Fig.1F, G), but from a lateral
view the concave top is evident (Fig.1H). Unlike cBps, bBps ap-
pear dumbbell-shaped from the top view (Fig.1K, L). e shape of
a bBp is deeply bilobed with a narrow connection at the base of each
lobe (Fig.1I–M; AppendixS2). However, the shape and number of
bBps per dorsal epidermal cell are dicult to judge directly from
free hand and semithin sections. us, confocal microscopy was ap-
plied to construct 3-dimensional images and conrm that the bBp
is a monoplastid (Fig.1L, M).
Ultrastructural variants of chloroplasts
e upper zones of cBps consist of groups of thylakoids, which hor-
izontally traverse the entire upper part of the Bp in regular parallel
arrangements (Fig.2A). Each group of thylakoids comprises 3–5
stacked thylakoids (Fig.2A), but appears as a thin line at low mag-
nication TEM. e lower zones of cBps consist of normal granal
thylakoids and stroma thylakoids similar to the typical chloroplasts
in mesophyll cells and ventral epidermal cells (Fig.2A). From a pa-
radermal TEM view of a cBp, the thylakoid groups of the upper
TABLE 2. Selaginella chloroplast categories and characteristics. Abbreviations: cBp, cup-shaped bizonoplast; bBp, bilobed bizonoplast.
Chloroplast type Feature a Location a Shape Ultrastructure Icon
M (monoplastidy)
ME (monoplastid in a dorsal
epidermal cell)
Monoplastid Dorsal epidermal
cell
Cupped, bilobed,
or giant disk
Bizonoplast or normal; giant
disks always normal
MM (monoplastid in a mesophyll
cell)
Monoplastid Mesophyll cell Cupped or bilobed Normal
OL (oligoplastidy) (2)3–10
chloroplasts
per cell
Dorsal epidermal
cell
Disk Normal
Mu (multiplastidy) More than 10
chloroplasts
per cell
All
photosynthetic
cells
Disk Normal
RC (reduced or vestigial chloroplasts) Reduced or
vestigial
chloroplasts
Dorsal or adaxial
epidermal cell
Disk Thylakoids less developed
aThe second and third columns define the categories stated in the first column. The other columns give more chloroplast features associated with these categories.
2020, Volume 107 Liu etal.—Gigantic chloroplasts of Selaginellaceae 7
FIGURE 2. TEM of cup-shaped bizonoplasts (cBps) of S. erythropus. The chloroplast drawings indicate approximate section locations. (A) Transverse
section of cBp in dorsal epidermal cell showing regularly layered upper zone (UZ) above lower zone (LZ). Inset, closeup of LZ, with groups of 3–5
stacked thylakoids. (B, C) Paradermal sections of cBps. (B) Top of cBp showing partial UZ with groups of thylakoids. These layered thylakoids form a
pattern of concentric circles expanding toward the cell wall. (C) Boundary between two zones of cBp showing parallel thylakoids groups in UZ, grana
and starch grains in LZ. Abbreviations: cBp, cup-shaped bizonoplast; Cp, chloroplast; CW, cell wall; dE, dorsal epidermal cell; g, grana; IS, intercellular
space; LZ, lower zone; M, mitochondrion; MC, mesophyll cell; N, nucleus; S, starch grain; St, stroma; T, thylakoid; UZ, upper zone; V, vacuole.
8 American Journal of Botany
zone appear as concentric circles, which expand regularly toward
the cell wall of the dorsal epidermal cell (Fig.2B). A section at the
boundary between the two zones may show part of the upper zone
(partially parallel thylakoid groups) and part of the lower zone (ran-
domly scattered grana and stromal thylakoids) (Fig.2C). ylakoid
groups oen appear thicker and blurred in paradermal sections due
to dierent cutting angles (Fig.2B, C).
e bBps are usually slightly smaller than the cBps, and each occu-
pies less than one half of a dorsal epidermal cell in a microphyll(Fig.
1J, K). At the apex of each lobe of the bBp is an upper zone, which is
similar in structure to the upper zone of the cBps and lines the in-
terior side of the lobe, becoming thinner farther from the apex, and
eventually disappearing (Fig.3A–E). Although bBps appear as various
shapes when viewed with TEM, depending on the section location
and angle, their ultrastructural features are similar to cBps (Fig.3B–
D). In longitudinal TEM views, bBps occasionally appear as two sepa-
rate lobes even though connected at the base (Fig.3A).
e cBps of four selected Selaginella species with sucient ma-
terials for study (S. delicatula, S. erythropus, S. intermedia, and S.
revoluta) show morphological similarity. ere are no signicant
ultrastructural dierences in the upper chloroplast zones of these
four species (Fig.4A–D, le group). e average number of thyla-
koid groups in the upper zone of a bBp is close to that of a cBP
(Fig.4B). Although there is evidence of variation between species
in the thickness of the stroma between two thylakoid groups, the
species dierences are not well resolved (Fig.4C, D).
Bilobed chloroplasts need not be bizonoplasts, and then they have
normal ultrastructure (Fig.3F). ey are either ME (monoplastidy in
dorsal epidermal cells) or MM (monoplastidy in mesophyll cells). In
general, bilobed chloroplasts (bizonoplast or not) are located at the
narrow base of a funnel-shaped cell, with a nucleus between its lobes
and a large vacuole above (Fig.3A, F). Normal chloroplast structure
is found in OL, Mu(Fig. 3G), and RC chloroplasts(Fig. 3H, I), but
an RC is relatively small (3–4 μm or smaller), with less-elaborated
thylakoid membranes and rarely contains starch grains. Reduced or
vestigial chloroplasts (RC) were found in epidermal cells of some
isophyllous (e.g., S. wallacei, adaxial epidermal cells) (Fig.1Q) and
some anisophyllous species (e.g., S. exaltata, dorsal epidermal cells)
(Fig.3H, I). Mesophyll tissues with larger chloroplasts are the main
photosynthetic tissues in the RC species studied.
The association between chloroplast traits, habitats, and
phylogeny
e light habitats of 10 selected species range from extremely low-
light montane forests (0.4–2.1% full sunlight), partial shade (11.2–
25.5% full sunlight) to a high-light desert (40.5–53.8% full sunlight)
(Fig.5A). A strong association between the chloroplast number in
a dorsal epidermal cell (M, OL, and Mu) and light environment was
found. e species with M (ME, Bp or not) are found in low-light
environments (Fig.1A–C). For the species with Bps, light intensity
ranges from 0.4% to 2.1% full sunlight in their natural habitats. By
contrast, the species with OL are found in partial-shade environ-
ments, while the species with Mu are found in high-light environ-
ments (Fig.5A).
e annual environmental data (light, temperature, and hu-
midity; near 24°N) of four native Taiwanese species of Selaginella
recorded in their natural habitats provide more detailed infor-
mation (Fig.5B–D; AppendixS1). e two species with MEs and
bBps are shade-adapted (S. doederleinii with cCps, S. heterostachys
with bBps), growing in the forest understory in relatively low light.
Selaginella doederleinii lives in a closed forest with evergreen trees
and deep shade all year, but S. heterostachys (in a forest with de-
ciduous trees) gradually receives more light when it sporulates in
late summer and autumn. e species with OL (S. repanda, on a
partially shaded slope) was found in partial shade. In contrast, the
species with Mu (S. tamariscina, on a rocky slope) is oen sun-
exposed and receives large uctuations in light due to changes in
canopy openness in the deciduous forest (Fig.5B).
e variations in mean monthly temperature of these four spe-
cies from Taiwan represent three types of northern hemisphere,
subtropical, montane habitats: lowland (S. repanda), middle el-
evation (S. doederleinii and S. heterostachys), and relatively high
elevation (S. tamariscina) (Fig. 5C). In these habitats, June to
September are the warmest months, but humidity generally uc-
tuates over the year. Only the habitat of S. doederleinii (with cCps)
has relatively high humidity throughout the year. Selaginella het-
erostachys, a species with bBps, encounters relatively high humidity
in the spring and summer growing season, but conditions are drier
during sporulation and senescence in autumn and winter (Fig.5D).
Unfortunately, humidity data for S. heterostachys are missing for
January and November to December.
Our annotation of the Selaginella phylogeny tree published by
Weststrand and Korall (2016) (Fig. 6; Tables1,3) shows that the
basal clade of Selaginella, subg. Selaginella, features isophyllous,
nondorsiventral shoots and Mu chloroplasts. Similar results were
also found for subg. Ericetorum and Rupestrae, but the drought-
tolerant species have apparent thick cell walls in their dorsal epi-
dermis. Although the other four subgenera share the same features
of anisophyllus and dorsiventral shoots, they have dierent chlo-
roplast types in their dorsal epidermal cells. Subgenus Exaltatae
has reduced or vestigial chloroplasts in its dorsal epidermal cells
(RC) and subg. Lepidophyllae is Mu. Only subg. Gymnogynum and
subg. Stachygynandrum possess high chloroplast diversity in their
FIGURE 3. TEM of dierent forms of chloroplasts in the dorsal or adaxial epidermal cells of Selaginella. (A–F) Monoplastidy in a dorsal epidermal
cell (ME), including bilobed bizonoplasts (bBps) in A–E and a bilobed chloroplast (bCp) with normal ultrastructure in F; (G) Multiplastidy (Mu); (H, I)
Reduced or vestigial chloroplasts (RCs). (A) bBp of S. heterostachys in funnel-shaped dorsal epidermal cell. At the apex, each lobe has an upper zone,
that runs along the interior side of the lobe, narrowing farther from the apex until it disappears. The open arrow (bottom left) at the base of the two
lobes indicates the location of the connection, which is just out of view. Inset shows part of upper zone. (B) Close-up of upper zone of S. aristata. Each
group consists of 4–6 thylakoids. Note that some terminal thylakoids can be seen at connections. (C–E) bBps from dierent section angles: S. aristata
in (C, D) and S. devolii in (E). (F) bCp in dorsal epidermal cell of S. willdenowii. (G) Disk-like chloroplast in adaxial epidermal cell of S. deexa. (H, I) RC in S.
exaltata dorsal epidermal cells. Mesophyll cells have larger chloroplasts (Cp). Abbreviations: adE, adaxial epidermal cell; bBp, bilobed bizonoplast; bCp,
bilobed chloroplast with normal ultrastructure; Cp, chloroplast; g, granum; IS, intercellular space; L, lumen (1–3); LZ, lower zone; M, mitochondrion;
MC, mesophyll cell; ME, monoplastidy in a dorsal epidermal cell; Mu, multiplastidy; N, nucleus; RC, reduced or vestigial chloroplasts; S, starch grain; St,
stroma; T, thylakoid; UZ, upper zone; V, vacuole.
2020, Volume 107 Liu etal.—Gigantic chloroplasts of Selaginellaceae 9
10 American Journal of Botany
dorsal epidermal cells (Bp, ME, MM, Mu, OL, and RC), includ-
ing monoplastidy. It is noteworthy that the ME chloroplast appears
in subg. Stachygynandrum and the MM chloroplast appears in subg.
Gymnogynum, respectively. However, Bps were only found in subg.
Stachygynandrum.
DISCUSSION
Long before modern microscopy, Haberlandt (1888, 1914) re-
ported monoplastidy, describing the chloroplasts as bowl-shaped,
in several species of Selaginella including S. martensii Spring
and S. grandis T.Moore. Later, monoplastidy was reported in
a few more species of Selaginella (Ma, 1930; Jagels, 1970a, b).
Webster (1992) highlighted monoplastidy as a major pecu-
liarity of Selaginella. e results reported here, with 49 of 76
species monoplastidic, mean that monoplastidy is not an uncom-
mon phenomenon in Selaginellaceae, but also that it is not univer-
sally found, contrary to previous reports (Wesbter, 1992; Banks,
2009). We estimate that approximately 70% of Selaginella species
are monoplastidic. However, the most basal species in our study,
viz., S. deflexa Brack., is Mu; i.e., it has multiple typical chloroplasts
per cell. Based on the types of monoplastids (M) recognized in this
study, previously reported monoplastids are (1) the ME type (M in
the dorsal epidermal cell), e.g., S. apus [current name S. apoda (L.)
Spring], S. serpens (Desv.) Spring, and S. uncinata (Desv.) Spring
(Ma, 1930; Jagels, 1970a, b), and (2) the MM type (M in the meso-
phyll cell), e.g., S. kraussiana (Kunze) A.Braun (Haberlandt, 1914;
Jagels, 1970a). Monoplastidy in Selaginella truly stands out among
vascular plants. All other vascular plants with chloroplasts have a
population of small chloroplasts in each photosynthetic cell (usu-
ally mesophyll cells).
FIGURE 4. Comparative ultrastructural features of upper zones of bizonoplasts (Bps) of nine Selaginella species. Four species have cup-shaped bizo-
noplasts (cBps, left icon): S. delicatula (S. del), S. erythropus (S. ery), S. intermedia (S. int), and S. revoluta (S. rev); ve species have bilobed Bp (bBps, right
icon): S. aristata (S. ari), S. boninensis (S. bon), S. devolii (S. dev), S. heterostachys (S. het), and S. lutchuensis (S. lut). (A) Number of stacked thylakoids per
thylakoid group (ANOVA F8, 24 = 7.857, p < 0.001). (B) Number of thylakoid groups in upper zone (ANOVA F8, 24 = 1.198, p = 0.341). (C) Thickness of a thyla-
koid group (nm) (ANOVA F8, 32 = 1.707, p = 0.135). (D) Stroma spacing between adjacent thylakoid groups (nm) (ANOVA F8, 24 = 4.410, p < 0.01). Values
are means ± SE. Dierent letters indicate a signicant dierence between species within a group as determined by Scheé’s post hoc test (p < 0.05).
S. del
S. ery
S. int
S. rev
S. ari
S. bon
S. dev
S. het
S. lut
0
2
4
sdiokalyhtdekcatsfo.oN
puorgdiokalyhtrep
S. del
S. ery
S. int
S. rev
S. ari
S. bon
S. dev
S. het
S. lut
0
5
10
15
enozreppunanispuorgdiokalyhtfo.oN
S.
del
S. er
y
S. int
S. re
v
S.
ari
S.
bon
S
. de
v
S. he
t
S. lut
0
50
100
150
neeewtebgnicapsamortS
)mn(spuorgdiokalyht2
S. de
l
S. er
y
S. in
t
S. re
v
S.
ari
S. bo
n
S. de
v
S. he
t
S. lu
t
0
50
100
)mn(puorgdiokalyhtafossenkcihT
a
A
C
B
D
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
aa
ab
a
ac ac
bc
ac ab ab
ab
ab
a
ab
b
ab
b
ab ab
species
species
species species
2020, Volume 107 Liu etal.—Gigantic chloroplasts of Selaginellaceae 11
Monoplastid chloroplasts vary not just in leaf tissue location
(ME versus MM), but also in form (cup-shaped, bilobed, and giant
disk) and in ultrastructure. A bizonoplast (Bp) uniquely has a di-
morphic ultrastructure with distinctive and regularly layered thyla-
koids at the top of the single giant chloroplast. Before this study,
Bps had only been reported in two Selaginella species, S. erythropus
(Sheue etal., 2007, 2015) and S. martensii (Ferroni etal., 2016), al-
though the material of S. martensii that we obtained from a botanic
garden showed cup-shaped chloroplasts with typical ultrastructure
(no upper zone) in the present study. Unexpectedly, here an addi-
tional nine species of Selaginella were newly found to have Bps in
their dorsal epidermal cells, bringing the total to 11 of 76 species
with Bps. Seven of these species are from Taiwan where we studied
all known native species of Selaginella. Because most of these spe-
cies with Bps are small and cryptic, the potential exists that species
with Bps are under-sampled elsewhere.
Although the chloroplast traits in the dorsal epidermal cells
of Selaginella are species-specic and persistent, rare variations
FIGURE 5. Environmental data recorded from natural habitats for selected Selaginella. (A) Light intensity (photosynthetically active radiation) inter-
preted as percentage of full sunlight for 10 species with chloroplast traits noted for comparison. Average values are based on 3–10 measurements at
dierent locations (ANOVA F9, 54 = 31.035, p < 0.001). The values are means ± SE. Dierent letters indicate a signicant dierence between species as
determined by Scheé’s post hoc test. (B–D) Annual environmental data for four selected Selaginella in Taiwan. The data for the dormant period for
S. repanda (September–March) are indicated by lighter symbols. (B) Mean monthly light intensity. (C) Mean monthly temperature. (D) Mean monthly
relative humidity. Icons show chloroplast types in the epidermal cells, which are the same as in Fig.6.
AB
CD
12 American Journal of Botany
on chloroplast number per cell and ultrastructure were observed.
For example, most of the studied material of S. remotifolia has
bilobed giant chloroplasts in its mesophyll cells beneath dorsal
epidermal cells (MM trait, bCp), but the OL trait (2–10 chloro-
plasts per cell) is rare in these mesophyll cells. In S. erythropus,
the ME trait with Bp ultrastructure, occurs in dorsal epidermal
cells of microphylls, but two Bps in a cell adjacent to a stoma were
observed once (C. R. Sheue, unpublished data). Ma (1930) also
pointed out that in S. apoda (syn. S. apus in Ma, 1930), the dorsal
epidermal cells near the tip of a microphyll usually contain one
chloroplast, but the cells near the base of a microphyll may have
two or more chloroplasts.
e species of Selaginella with monoplastidy (both ME and
MM) in this study all live in deep shade, receiving only on aver-
age 0.4~2.1% of full sunlight (Fig.5A). In strong contrast, species
with multiple chloroplasts live in open places, with on average more
than 40.5% of full sunlight. Selaginella doederleinii with ME cup-
shaped chloroplasts was recorded with the lowest light intensity.
Among the four species with Bps, the species with cBp (S. delicat-
ula) was found at higher light intensities than the three species with
bBps (bilobed Bp). More in-depth studies are needed to understand
the eects of Bps on plant physiology and how species with cBps
and bBps dier ecophysiologically.
Despite the shape dierences between cBp and bBp, the ultra-
structure of their upper zones of parallel thylakoid groups is similar.
A bBp has bivalve, shell-like lobes with an upper zone at the apex.
is upper zone runs along the interior side of the lobe, becoming
thinner farther from the apex until it disappears. Our observations
show that the angle between the two lobes can change in response
to light, potentially optimizing its shape in a given light environ-
ment. In contrast, the concave top of a cBp is similar to a basin,
yet still has some ability to change shape. e comparative data for
the nine Selaginella species (Fig.4) with Bps show that the upper
zones typically contain about 11 parallel thylakoid groups, with
each group comprising 3–5 stacked thylakoids. eir ultrastructural
features are very similar even in dierent species. Some variations
are found in the thicknesses of thylakoid groups and in the stroma
thickness between two thylakoid groups, but these variations are
subtle. Convergent evolution is suggested by the occurrence of these
structures in distantly related species from distant parts of the Earth
(from the New World to the Old World) in shaded environments.
Both bBps and cBps are located at the bases of funnel-shaped
dorsal epidermal cells surrounded by intercellular space. e
refractive index inside a dorsal epidermal cell (ncell = 1.425,
Gausman etal., 1974) is higher than air (nair = 1), which means
that some portion of the light striking base of the cell, depending
FIGURE 6. Chloroplast diversity in the dorsal epidermal cells of Selaginellaceae associated with subgenera and morphological traits. The phyloge-
netic tree is modied from that of Weststrand and Korall (2016). Abbreviations: AD, anisophyllous, dorsiventral; IN, isophyllous, nondorsiventral; Bp,
bizonoplast (b, bilobed; c, cup-shaped); ME, monoplastidy in the dorsal epidermal cell; MM, monoplastidy in mesophyll cell; Mu, multiplastidy, OL,
oligoplastidy; RC, reduced or vestigial chloroplasts.
2020, Volume 107 Liu etal.—Gigantic chloroplasts of Selaginellaceae 13
on its angle, will be reected back into the Bp (Liu etal., 2012)
increasing the amount of light absorbed (Fig.7). is particular
feature of a Bp, however, is shared with other monoplastids that
do not have a layered structure in the upper zone. e layered
structure of the upper zone of a Bp may additionally interfere
with the light waves, with the potential to enhance absorption
TABLE 3. Chloroplast categories of subgenera of the 76 studied Selaginella. Abbreviations: A, anisophylly; bBp, bilobed bizonoplast; bCp, bilobed chloroplast;
cBp, cup-shaped bizonoplast; cCp, cup-shaped chloroplast; D, typical disk-shaped chloroplast; gD, giant disk-shaped chloroplast; I, isophylly; ME, monoplastidy
in the dorsal epidermal cell; MM, monoplastidy in the mesophyll cell; Mu, multiplastidy; OL, oligoplastidy; RC, reduced or vestigial chloroplasts.
Subgenus
Number of species
(studied no./ total no.) Microphyll (sp. no.) Chloroplast category (sp. no.) Chloroplast type (sp. no.)
Ericetorum 2/8 I (2) Mu (2) D (2)
Exaltatae 1/3 A (1) RC (1) RC (1)
Gymnogynum 9/40 A (9) MM (5), RC (4) bCp (1), cCp (4), RC (4)
Lepidophyllae 1/2 A (1) Mu (1) D (1)
Rupestrae 6/50 I (6) Mu (5), RC (1) D (5), RC (1)
Selaginella 1/2 I (1) Mu (1) D (1)
Stachygynandrum 56/600 A (56) ME (44), Mu (5), OL (7) bBp (5), bCp (10), cBp (6),
cCp (22), D (12), gD (1)
FIGURE 7. Diagrams of bizonoplasts (Bps) in dorsal epidermal cells, with a proposed 3-dimensional structure for their upper zones. Potential light
paths are marked to show optical properties. The cup-shaped Bps (cBp) and bilobed Bps (bBp) have regularly arranged groups of thylakoids in their
upper zones, which are located above lower zones that have typical chloroplast ultrastructures. The concave top of a cBp is similar to a basin, while a
bBp is similar to a bivalve shell with a narrow connection at the base. Both types of Bp have the potential to open and close as light conditions change,
and both are located at the base of funnel-shaped dorsal epidermal cells surrounded by intercellular space. The refractive index inside cells (ncell =
1.425) is higher than air (nair = 1) which may cause multiple reections when light paths hit the cell–air boundaries obliquely, potentially increasing the
opportunity for light absorption as the light passes through the Bp multiple times. Abbreviations: Bp, bizonoplast (b, bilobed; c, cup-shaped); CW, cell
wall; IS, intercellular space; L, lumen; LZ, lower zone; N, nucleus; S, starch grain; Si, silica body; T, thylakoid; UZ, upper zone; V, vacuole.
14 American Journal of Botany
and reection depending on the wavelengths and light angles
(Jacobs et al., 2016). e presence of light interference is evi-
dent from blue iridescent features found on microphylls of some
species (S. erythropus, S. heterostachys, and S. delicatula) during
development (C. R. Sheue, unpublished data). Recently, Masters
etal. (2018, pp. 1, 6) successfully characterized iridescence in S.
erythropus and determined that it is from “one-dimensional pho-
tonic multilayers” (the upper zone of the Bp). ese light interfer-
ence eects of the upper zone, together with internal reections
at the cell boundary potentially make the Bp a unique photonic
system deserving further study. is iridescence from a Bp is
in contrast to the iridescence caused by layered lamellae on the
outer cell wall of dorsal epidermal cells (Hébant and Lee, 1984)
in S. willdenowii (Desv.) Baker and S. uncinata (Desv.) Spring.
Among the seven subgenera, subg. Selaginella (erect with heli-
cally arranged isophylls [equal-sized leaves] and stems lacking rhi-
zophores) is the most basal group of Selaginellaceae (Zhou etal.,
2015; Weststrand and Korall, 2016). Because Mu chloroplasts are
found in subgenus Selaginella, we may infer that the ancestors of ge-
nus Selaginella were Mu. e Mu trait (7 species) and the RC trait (1
species) are characteristic of the other two subgenera (Ericetorum
and Rupestrae) that share similar morphological traits with subg.
Selaginella (but with rhizophores, and a few species of Ericetorum
with anisophylls) (Weststrand and Korall, 2016). However, the ma-
jority of Selaginellaceae with dorsiventral shoots and anisophylls
(usually with smaller dorsal leaves and larger ventral leaves)
(ca. 91%) belong to four subgenera (Exaltatae, Gymnogynum,
Lepidophyllae, and Stachygynandrum) (Table 3). ese subgenera
have the highest chloroplast diversity in this family, exhibiting all
chloroplast types (Mu, OL, ME, MM, and RC). However, M oc-
curs only in the two subgenera with the highest species diversity,
Gymnogynum (40 spp., 5 of 10 studied spp. have MM chloroplasts)
and Stachygynandrum (600 spp., 44 of 56 studied spp. with ME
type). About 79% and 55% of studied species of Stachygynandrum
and Gymnogynum, respectively, have the M trait. Notably, the 11
species with Bps all belong to subg. Stachygynandrum, the largest
subgenus of Selaginella, comprising about 600 species and occur-
ring in both the Old World and the New World (Weststrand and
Korall, 2016). Eight species with Bps are from the Old World, and
three species are from the New World (S. erythropus, S. martensii,
and S. revoluta). Although monoplastidy occurs in two subgenera,
Stachygynandrum (ME type, 44 species) and Gymnogynum (MM
type, 5 species), all Bps (cBp and bBp) are the ME type. us, Bp ap-
pears to be an apomorphy of subg. Stachygynandrum in Selaginella
because the members of other subgenera have only typical chloro-
plast ultrastructures regardless of chloroplast size and number.
In contrast with most plants with multiple chloroplasts per pho-
tosynthetic cell, algae oen contain only a few or a single giant chlo-
roplast in a cell (Solymosi, 2012). In algae, monoplastidy is common
in green algae and some species of Rhodophyta (unicellular or mu-
ticellular), but multiplastidy also occurs in both groups (Solymosi,
2012; de Vries and Gould, 2018). Chloroplasts of algae have di-
verse shapes, including cup-shaped, reticulate, ring-shaped, heli-
cal, cuboidal, star-shaped, and bilobed (Solymosi, 2012). However,
in land plants multiplastidy and small chloroplasts, appearing as
disk-shaped, spherical, ellipsoidal, or lens-shaped, are prevalent,
with few exceptions. Monoplastidy in mature photosynthetic cells
of land plants is restricted to hornworts and Selaginella. However,
hornwort monoplastids vary in shape from spherical to ellipsoidal,
to lens-shaped, spindle-shaped, and star-shaped (Renzaglia etal.,
2007), unlike Selaginella monoplastids. Moreover, many species
of hornwort have pyrenoids in their chloroplasts and grana con-
sisting of stacks of short thylakoids, lacking end membranes, and
surrounded by channel thylakoids (Vaughn et al., 1992; Villarreal
and Renner, 2012). ese major dierences in monoplastidy be-
tween hornworts and Selaginella are suggestive of independent
evolution of this trait in these distantly related plant groups. is
inference is further supported by the absence of monoplastidy in
basal Selaginella species.
e high diversity of chloroplast types in Selaginella revealed
here greatly expands the known chloroplast diversity in vascular
plants. Selaginellaceae are a widely distributed species-rich family
(Jermy, 1990), which arose more than 370 million years ago (Banks,
2009) and successfully adapted to environments ranging from trop-
ical forests to hot deserts. eir strikingly dierent chloroplasts,
especially the bizonoplast with its special internal structure and po-
tential optical integration with its containing cell, imply that much
is to be learned from these otherwise unprepossessing plants, espe-
cially from multidisciplinary approaches combining physics, physi-
ology, systematics, and ecology.
ACKNOWLEDGMENTS
is article is dedicated to the memories of the late Professor V.
Saras and the late Dr. C. C. Tsai, both of whom greatly assisted
with this study. e authors thank Prof. M. S. B. Ku and two anon-
ymous reviewers for providing help and valuable comments; the
University of Bristol Botanic Garden (UK), Cairns Botanic Gardens
(Australia), Sun Yat-sen University (China),South China Botanic
Garden (China),e Dr. Cecilia Koo Botanic Conservation Center
(KBCC, Taiwan), Smithsonian Tropical Research Institute,and Mr.
Z. X. Chang for providing materialsor eld trip help; and Dr. W.
N. Jane (Academia Sinica, Taiwan) and Miss P. C. Chao (Precision
Instruments Center, National Chung Hsing University) for assisting
with TEM. is study was supported by the Ministry of Science and
Technology, Taiwan (awards MOST-101-2621-B-005-002-MY3;
MOST 104-2621-B-005-002-MY3; MOST 107-2621-B-005-001).
AUTHOR CONTRIBUTIONS
J.W.L. participated in structural studies, plant collection and identi-
cation, collection of environmental data, preparation of gures, and
draing the manuscript. S.F.L. participated in structural and mo-
lecular studies, plant collection and identication, and collection
of environmental data. C.T.W participated in structural studies,
plant collection, and identication. I.A.V. participated in plant
collection and identication, preparation of a table, and editing of
the manuscript. J.F.H. and Y.H.W. participated in structural studies.
H.M.C. participated in plant collection, identication, and molecu-
lar studies. T.Y.G. participated in plant collection and identication
and collection of environmental data. W.H.Y. participated in plant
collection and identication and editing of the manuscript. N.S.A.
participated in planning expeditions, preparing specimens, and ed-
iting the manuscript. M.F.K., C.C., S.D., H.O., A.B., P.S., B.A., R.K.,
and N.N. participated in plant collection and identication. C.L.H.
supervised the molecular identications and preparing data les.
P.C. organized expeditions, participated in plant collection and
statistical analyses, and edited the manuscript. C.R.S. coordinated
2020, Volume 107 Liu etal.—Gigantic chloroplasts of Selaginellaceae 15
the entire project, supervised the structural studies, participated in
plant collection and identication, writing the manuscript, and pre-
paring data les and gures.
DATA AVAILABILITY
All data underlying the study are included in the manuscript, its
supplementary materials, or access information is provided in the
supplementary tables.
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the
supporting information tab for this article.
APPENDIX S1. Materials of Selaginella used in this study, with in-
formation on distribution, voucher specimens, and habitat.
APPENDIX S2. Video of bilobed bizonoplast of Selaginella het-
erostachys viewed with a confocal laser scanning microscope.
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The largest genus of seed-free vascular plants Selaginella alone constitutes the family Selaginellaceae, the largest of the lycophyte families. The genus is estimated to contain ca. 800 species distributed on all continents except Antarctica, with the highest species diversity in tropical and subtropical regions. The monophyly of Selaginella has rarely been doubted, in contrast its infrageneric classification has been contentious. In the present study, based on chloroplast and nuclear DNA evidence, macromorphology, spore features, and/or distribution information, Selaginella is classified into six subgenera: S. subg. Selaginella, S. subg. Boreoselaginella, S. subg. Pulviniella, S. subg. Ericetorum, S. subg. Heterostachys, and S. subg. Stachygynandrum. The latter three subgenera are further classified into six, five, and seven sections, respectively. All of these infrageneric divisions, identified with molecular data, are supported by non-molecular features. A key to infrageneric taxa is given. Thirty-seven infrageneric taxa published in earlier literature are lectotypified and classified into those infrageneric taxa here recognized. A nomenclatural account of each infrageneric taxon is given.
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Iridescence in shade-dwelling plants has previously been described in only a few plant groups, and even fewer where the structural colour is produced by intracellular structures. In contrast with other Selaginella species, this work reports the first example in the genus of structural colour originating from modified chloroplasts. Characterization of these structures determines that they form one-dimensional photonic multilayers. The Selaginella bizonoplasts present an analogous structure to recently reported Begonia iridoplasts; however, unlike Begonia species that produce iridoplasts, this Selaginella species was not previously described as iridescent. This therefore raises the possibility of widespread but unobserved and uncharacterized photonic structures in plants.
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Iridescent blue leaf coloration in four Malaysian rain forest understory plants, Diplazium tomentosum Bl. (Athyriaceae), Lindsaea lucida Bl. (Lindsaeaceae), Begonia pavonina Ridl. (Begoniaceae), and Phyllagathis rotundifolia Bl. (Melastomataceae) is caused by a physical effect, constructive interference of reflected blue light. The ultrastructural basis for this in D. tomentosum and L. lucida is multiple layers of cellulose microfibrils in the uppermost cell walls of the adaxial epidermis. The helicoidal arrangement of these fibrils is analogous to that which produces a similar color in arthropods. In B. pavonina and P. rotundifolia the blue-green coloration is caused by parallel lamellae in specialized plastids adjacent to the abaxial wall of the adaxial epidermis. The selective advantage of this color production, if any, is unknown.
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The iridescent blue color of several Selaginella species is caused by a physical effect, thin-film interference. Predictions for a model film have been confirmed by electron microscopy of S. willdenowii and S. uncinata. For the latter species iridescence contributes to leaf absorption at wavelengths above 450 nm and develops in environments enriched with far-red (730 nm) light. This evidence supports the involvement of phytochrome in the developmental control of iridescence.
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Enhanced light harvesting is an area of interest for optimizing both natural photosynthesis and artificial solar energy capture1,2. Iridescence has been shown to exist widely and in diverse forms in plants and other photosynthetic organisms and symbioses3,4, but there has yet to be any direct link demonstrated between iridescence and photosynthesis. Here we show that epidermal chloroplasts, also known as iridoplasts, in shade-dwelling species of Begonia⁵, notable for their brilliant blue iridescence, have a photonic crystal structure formed from a periodic arrangement of the light-absorbing thylakoid tissue itself. This structure enhances photosynthesis in two ways: by increasing light capture at the predominantly green wavelengths available in shade conditions, and by directly enhancing quantum yield by 5-10% under low-light conditions. These findings together imply that the iridoplast is a highly modified chloroplast structure adapted to make best use of the extremely low-light conditions in the tropical forest understorey in which it is found5,6. A phylogenetically diverse range of shade-dwelling plant species has been found to produce similarly structured chloroplasts⁷⁻⁹, suggesting that the ability to produce chloroplasts whose membranes are organized as a multilayer with photonic properties may be widespread. In fact, given the well-established diversity and plasticity of chloroplasts10,11, our results imply that photonic effects may be important even in plants that do not show any obvious signs of iridescence to the naked eye but where a highly ordered chloroplast structure may present a clear blue reflectance at the microscale. Chloroplasts are generally thought of as purely photochemical; we suggest that one should also think of them as a photonic structure with a complex interplay between control of light propagation, light capture and photochemistry.
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Vascular plants have evolved a long-term light acclimation strategy primarily relying on the regulation of the relative amounts of light-harvesting complex II (LHCII) and of the two photosystems, photosystem I (PSI) and photosystem II (PSII). We investigated whether such a model is also valid in Selaginella martensii, a species belonging to the early diverging group of lycophytes. Selaginella martensii plants were acclimated to three natural light regimes (extremely low light (L), medium light (M) and full sunlight (H)) and thylakoid organization was characterized combining ultrastructural, biochemical and functional methods. From L to H plants, thylakoid architecture was rearranged from (pseudo)lamellar to predominantly granal, the PSII : PSI ratio changed in favour of PSI, and the photochemical capacity increased. However, regulation of light harvesting did not occur through variations in the amount of free LHCII, but rather resulted from the flexibility of the association of free LHCII with PSII and PSI. In lycophytes, the free interspersed LHCII serves a fixed proportion of reaction centres, either PSII or PSI, and the regulation of PSI-LHCII(-PSII) megacomplexes is an integral part of long-term acclimation. Free LHCII ensures photoprotection of PSII, allows regulated use of PSI as an energy quencher, and can also quench endangered PSI.