Bull. Natl. Mus. Nat. Sci., Ser. B, 45(2), pp. 77–86, May 22, 2019

Growth Dynamics of Independent Gametophytes ofPleurosoriopsis makinoi (Polypodiaceae)
Atsushi Ebihara1,*, Joel H. Nitta1,**, Yurika Matsumoto2, Yuri Fukazawa2,
Marie Kurihara2, Hitomi Yokote2, Kaoru Sakuma2, Otowa Azakami2,
Yumiko Hirayama1 and Ryoko Imaichi2
1

Department of Botany, National Museum of Nature and Science,
4–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan2
Department of Chemical and Biological Sciences, Japan Womenʼs University,
Mejirodai 2–8–1, Bunkyo-ku, Tokyo 112–8681, Japan* E-mail: ebihara@kahaku.go.jp** Current afﬁliation: Department of Botany, National Museum of Natural History,
Smithsonian Institute, Washington, D.C., 20013, U.S.A.
(Received 12 February 2019; accepted 27 March 2019)

Abstract Despite their importance to the fern life cycle, little is known about the ecology of fern
gametophytes due to their cryptic morphology and small size. This study documents growth
dynamics over ﬁve years in a population of Pleurosoriopsis makinoi gametophytes growing independently of sporophytes in Okutama-machi, Tokyo, Japan. Cover was measured in four
10×10 cm quadrats, and the number and size of gemmae born on gametophytes measured ca.
every two months from March 2009 to March 2014. The population survived for the duration of
the study, and no seasonality was observed in the cover measurements. Gemmae gradually growthroughout the spring and summer, and mostly detach in winter. Based on our preliminary comparison between the microenvironment of the independent gametophyte site and a site supporting sporophytes of the same species, a possible cause of suppression of sporophyte formation is low light
conditions in winter due to planting of evergreen conifers.
Key words : fern, gametophyte, gemma, microenvironment, sporophyte.

Ferns possess an unusual life cycle consistingof two morphologically distinct, free-living
stages: the diploid sporophyte and the haploid
gametophyte. Unlike seed plants, in which thegametophyte is nutritionally dependent upon the
sporophyte, in ferns gametophytes may growseparately from sporophytes. The phenomenonof ʻindependent gametophytesʼ includes fern
gametophyte populations in various degrees of
independence from sporophytes (Pinson et al.,2017). Independent gametophyte species, lacking
any known counterpart sporophyte, are rather
extreme cases (Farrar, 1967, 1978, 1992). Conversely, facultatively independent species, which
may or may not grow separately from sporophytes depending on environmental conditions,have been observed in many more species (Rumsey and Shefﬁeld, 1996). Almost without exception, species capable of growing as independent
gametophytes have noncordiform morphology
with indeterminate growth (Ebihara et al., 2013).
Facultatively independent fern gametophytes
represent an understudied but useful group forunderstanding the ecological correlates of thetransition from haploid to diploid phase in land
plants.
We recently conducted a survey of the fern
gametophyte ﬂora of Japan (Ebihara et al.,2013). During the survey, we located a population of Pleurosoriopsis makinoi (Maxim. ex
Makino) Fomin (Polypodiaceae) gametophytes
in Unazawa Valley, Okutama-machi, Tokyo,

Atsushi Ebihara et al.

78

Japan growing without any sporophytes nearby
as far as we searched―its sporophyte population
has evidently been observed in the valley, but isvery small and rare (S. Fujimoto, personal communication). We therefore considered it an ʻindependent gametophyteʼ in the broad sense. Pleurosoriopsis makinoi is a small-leaved fern species
often growing with long-creeping rhizomes
among bryophyte mats on rocks distributed in
East Asia (China, Japan, Korea and Russian Far
East) (Zhang et al., 2013). It has ribbon-likegametophytes, and gemmae and sexual organs
have been observed both in the wild and in cultivation from sown spores (Masuyama, 1975).
We conducted a continuous observational
study on the wild independent gametophytes of
P. makinoi in Unazawa Valley to answer the following questions:
1) How stable is the size of independent gametophyte populations over long periods oftime?
2) Do independent gametophytes produce sexual organs (archegonia and antheridia) or
possess the ability to produce sporophytes?
3) Do the gemmae of independent gametophytes contribute to sustainment/expansionof populations?
4) How do environmental factors affect gametophyte growth?
Materials and Methods
Location and habitat
The independent gametophyte site is located in
Unazawa Valley, Okutama-machi, Tokyo, Japan,
at ca. 315 m elevation. It grows on a small northfacing bank along a river (Fig. 1). The population
is ca. 1×5 m in size.
Measurement of coverFour 10×10 cm quadrats (q1, q2, q3 and q4)
were designated on the rockface (Fig. 1) where
the independent gametophyte population grows
to measure cover (growth area). As the ribbonlike gametophytes of Hymenophyllum barbatum(Bosch) Baker (Hymenophyllaceae) also growon the rockface, we carefully situated the quadrats to include only P. makinoi. We were able todistinguish between gametophytes of the two
species based on the results of Ebihara et al.
(2013) and morphological observation—unlike P.
makinoi, gametophytes of H. barbatum do not
produce gemmae. Each quadrat was photographed using a digital camera. The image ﬁleswere printed out, and a 1 cm2 grid was overlayedon the photo with tracing paper. Cover scores foreach 1 cm2 cell in the grid were estimated in four
discrete levels ranging from zero to 1, i.e. 1.0 cm2(100% ≥ cover ＞ 50%), 0.5 cm2 (50% ≥ cover ＞
25%), 0.25 cm2 (25% ≥ cover ＞ 0%), 0 (completely absent). Cover was calculated as the sumof the scores for each cell. The measurement was
performed ca. every two months by placing thequadrats in exactly the same positions, and was
repeated 37 times from March 2009 to March2014.
Gametophyte collection, observation and measurements
Living gametophytes were collected from theperipheries of the quadrats, and were broughtback to the laboratory in small zipped plasticbags with wet papers. Collections were made 38
times from March 2009 to March 2014. The
gametophytes were washed using brushes undera stereomicroscope (Leica M420). The numberof gemmae was counted and length of eachgemma (Figs. 2a, b) was measured under a lightmicroscope (Olympus BX51) in ten individuals
per collection, and mean of the number and thelength of gemmae calculated.
We conducted a survey for reproductive structures in 2015. We collected 90 individuals, measured their length and width, and checked for thepresence of antheridia or archegonia. During the2015 survey, no reproductive structures wereobserved, so we also inspected images of all
vouchers of P. makinoi collected at this site forarchegonia or antheridia.
In this study, an individual gametophyte isdeﬁned as a single distinct thallus.

Growth dynamics of Pleurosoriopsis gametophytesFig. 1. The quadrats for measurement of gametophyte cover of Pleurosoriopsis makinoi in
Unazawa Valley, Okutama-machi, Tokyo,
Japan. a. A full view of the site with four quadrat positions (q1, q2, q3 and q4). b. A
10×10 cm quadrat frame.

Measurement of microenvironment
HOBO Micro Station Data Loggers H21-002(Onset) with sensors S-TMB-M002 (for temperature), S-THB-M002 (for temperature and humidity) and S-LIA-M003 for photosynthetic light(PAR) were used to measure microenvironment.
In Unazawa Valley, the logger was installed from
September 22, 2012 to July 22, 2014. To compare the environment of the independent gametophyte habitat with that of conspeciﬁc sporophytes, another set of the equipment was
installed at a site where sporophytes were growing. The sporophyte site was at Uratakaomachi,
Hachioji-shi, Tokyo, at 220 m elevation (fromAugust 20, 2013 to November 22, 2014). Temperature and humidity were recorded every 30
minutes. PAR was sampled every 4 minutes andthe average value was recorded every 30 min-79

Fig. 2. Gametophytes of Pleurosoriopsis makinoi
collected in Unazawa Valley, Okutama-machi,
Tokyo, Japan. a. A typical ribbon-like gametophyte with gemmae on the apical part (arrowhead), collected in August. b. Gemmae (arrowheads) on a gametophyte collected in
November. c. A gametophyte with archegonia(arrowheads), collected in October. d. Archegonia. e. A gametophyte with antheridia (arrowhead) collected in December. f. Antheridia.
Scale bars: 2 mm for a, 200 μm for b and d,
1 mm for c and e, 100 μm for f.

utes.
The data recorded by the loggers were collected by connecting the logger to a notebook
computer (Windows 7) and downloading using
BoxCar Pro 4.3 and HOBOware Pro every two
to four months. The collected data were summarized by site as the following values: 1) dailymean temperature, 2) daily minimum humidity,3) daily light integral (DLI). Daily light integral
was calculated by summing the total PAR measurments over each day.
Statistical analysis
Comparisons of microclimate between the
sporophyte site and gametophyte site were madeusing a paired t-test. To test for the inﬂuence of
microclimate on total cover (sum of all fourAtsushi Ebihara et al.

80

quadrats), mean gemmae length, and mean gemmae number, linear models were ﬁt with each ofthese as the dependent variable and the microclimatic variables as the independent variable. All
statistical analysis was done using R 3.5.1 (RCore Team, 2018). Plots were made with the“ggplot2” package (Wickham, 2016). Raw dataand code to replicate all analyses and ﬁgures areavailable at https://github.com/joelnitta/pleurosoriopsis.

Results
Measurement of cover
The cover of Pleurosoriopsis gametophytes
measured in four quadrats over ﬁve years isshown in Fig. 3a. Cover irregularly ﬂuctuatedlower than 40% of original size, but neverreached zero. Cover in three of the four quadrats(q1, q2, and q4) decreased during the last two
years of the survey (2012–2014).
Measurement of number and size of gemmae
The mean number and length of gemmae perindividual are summarized in Figs. 3b and 3c.

Fig. 3. Change in growth of Pleurosoriopsis makinoi gametophytes observed from May 2009 to May 2014. a.
Cover (cm2) of gametophytes in four 10×10 cm quadrats. b. Mean number of gemmae per individual. c. Mean
gemma length. For b and c, n＝10 individuals per observation; grey bars show SD. Shaded bars show alternating years.

Growth dynamics of Pleurosoriopsis gametophytes

81Fig. 4. Change in microclimate at site with Pleurosoriopsis makinoi independent gametophytes (Okutama) andsporophytes (Uratakao) from 2013 to 2014. a, b. Daily light integral (DLI) (mol m-2 day-1). c, d. Daily minimum relative humidity (%). e, f. Daily mean temperature (°C). Shaded bars show alternating years. Data collected at Okutama from September 22, 2012 to July 22, 2014 and at Uratakao from August 20, 2013 toNovember 22, 2014. Some data missing due to equipment malfunction (see Results).

The highest number of gemmae tend to beobserved in late summer to autumn and the lowest number tend to be observed in spring (Fig.
3b). Longer gemmae are frequently observed
during November to January (Fig. 3c).
Measurement of microenvironment
The measured temperature, humidity and DLI
are summarized in Fig. 4. At the gametophyte
site, data collection was interrupted by technicalmalfunction during the following periods: March
8–22, 2014, May 4–6, 2014, May 11–July 13,
2014.
The sporophyte site and gametophyte site differed signiﬁcantly in nearly all measured micro-

climatic variables (Fig. 5, Table 1). The gametophyte site had lower light levels, especially infall (0.21±0.06 mmol per day, vs. 0.33±0.12 at
the sporophyte site; p＜0.001, paired t-test) andwinter (0.36±0.27 mmol per day, vs. 1.51±
0.74; p＜0.001), and tended to be drier, particularly in spring (50±22% min. rel. humidity, vs.
69±21%; p＜0.001) and winter (78±18%, vs.
91±10%; p＜0.001). Differences in temperature
were signiﬁcant but small (all estimateddifferences＜1°C; Table 1).
Relationship between growth and climate
No signiﬁcant relationship was observed
between gametophyte growth and microclimate

Atsushi Ebihara et al.

82

Fig. 5. Density plots showing difference between microclimate at Pleurosoriopsis makinoi independent gametophyte site (Okutama) and sporophyte site (Uratakao). Signiﬁcant differences (paired t-test) shown by asterisks:***, p＜0.001; **, p＜0.01; * p＜0.05. Y-axis is relative probability density; curves scaled so that total probability of each scales to 1. Vertical lines shown medians.

except for length of gemmae, which was negatively related to temperature (linear model; r2＝
0.51, p＝0.013) (Fig. 6, Table 2).
Reproductive structures
None of the gametophytes surveyed in 2015

possessed any sexual organs (n＝90). When we
searched all other images of specimens collectedfrom the Okutama site, archegonia (Figs. 2c, d)were observed in 32 individuals (Fig. 7). Antheridia (Figs. 2e, f) were much less common thanarchegonia—we only found them in four individ-

Growth dynamics of Pleurosoriopsis gametophytes

83

Table 1. Results of paired t-test for difference in means between microclimatic variables (daily light integral[DLI], daily minimum relative humidity, and daily mean temperature) at two sites in Japan hosting
independent gametophytes or sporophytes of Pleurosoriopsis makinoi (Okutama and Uratakao, respectively)by season
EstimatetpOkutamadf

Uratakao

Lower

Upper

Mean

SD

Mean

SD

Winter
DLI−1.14 −19.17 2.34e-33
Min. Rel. Hum. −12.72 −8.38 7.29e-13
Mean Temp.
0.38
5.25 1.03e-06

89
89
89

−1.26
−15.74
0.24

−1.02
−9.70
0.52

0.36
78.26
1.53

0.27
18.15
1.75

1.51
90.98
1.15

0.74
9.59
2.01

SpringDLI−1.52 −10.17 3.71e-14
Min. Rel. Hum. −19.27 −6.20 8.03e-08
Mean Temp.
−0.40 −2.10 4.03e-02

54
54
54

−1.82 −1.22−25.50 −13.04−0.78 −0.02

1.03
49.58
9.49

0.78
21.90
4.17

2.56
68.85
9.89

1.06
20.82
4.20

SummerDLI
Min. Rel. Hum.
Mean Temp.

0.28
1.32
−0.41

1.99 5.93e-02
0.50 6.23e-01−2.30 3.14e-02

21
21
21

−0.01
−4.19
−0.77

0.58
6.84
−0.04

0.76
96.22
22.51

0.77
5.82
1.65

0.48
94.90
22.92

0.21
10.85
1.70

Fall
DLI
Min. Rel. Hum.
Mean Temp.

−0.12 −10.04 9.35e-16−4.06 −3.42 1.00e-03
0.68
7.43 1.10e-10

79
79
79

−0.15
−6.43
0.49

−0.10
−1.70
0.86

0.21
93.52
14.74

0.06
11.94
5.43

0.33
97.58
14.07

0.12
4.61
5.92

Table 2. Results of linear models testing for relationship between growth traits (total cover, count of gemmae,
and length of gemmae) as dependent variable on microclimate (monthly averages of daily light integral [DLI],
daily minimum relative humidity, and daily mean temperature) as independent variable in gametophytes ofPleurosoriopsis makinoi at Unazawa Valley, Okutama-machi, Tokyo, Japan. Results presented by independent
variabler2

adj. r2

Sigma

t

p

df

df
residual

DLI
Total coverGemmae countGemmae length

0.34
0.26
0.06

0.25
0.18
−0.05

25.20
2.72
46.18

3.68
3.14
0.53

0.096
0.110
0.486

2
2
2

Min. Rel. Hum.
Total coverGemmae countGemmae length

0.38
0.18
0.10

0.29
0.09
0.00

24.49
2.86
44.99

4.31
2.00
1.04

0.077
0.191
0.335

Mean Temp.
Total coverGemmae countGemmae length

0.00
0.03
0.51

−0.14
−0.07
0.46

31.12
3.11
33.23

0.01
0.32
9.40

0.942
0.588
0.013

uals. Females tended to be larger than asexuals(it was not possible to conduct statistical analysis
of size differences between sexes due to the low
number of individuals with antheridia and inconsistency in sampling season and effort). No
bisexual individuals were observed. We did notobserve any juvenile sporophytes on the gametophytes.

logLikAIC

BIC

Deviance

799−40.68 87.36 87.96−25.52 57.03 58.23−56.66 119.33 120.52

4,446
67
19,196

2
2
2

7
9
9

−40.43 86.85 87.44−26.06 58.12 59.31−56.38 118.75 119.95

4,200
74
18,220

2
2
2

7
9
9

−42.58 91.16 91.75−26.98 59.95 61.14−53.04 112.09 113.28

6,780
87
9,939

Discussion
The gametophyte patches in each of the four
quadrats survived through all ﬁve years of ourstudy. Even though we observed change in cover
over time in each quadrat, there seems to be noseasonality in this ﬂuctuation (Fig. 3a). In contrast, clear seasonal patterns were observed inAtsushi Ebihara et al.

84

Fig. 7. Sizes of gametophytes with antheridia(male), archegonia (female), or no reproductive
structures (asexual). a. Length (mm). b. Width(mm). Boxplots show medians and extend tothe ﬁrst and third quartile; whiskers extend to
1.5× the interquartile range. A single archegoniate (female) outlier with length of 46.6 mm
was removed prior to plotting. Points randomly
scattered within each x-axis category to reduceoverlap.
Fig. 6. Relationships between growth of Pleurosoriopsis makinoi gametophytes (total cover,mean gemma number, and mean gemma length)
and microclimate at the Okutama site. Microclimatic variables are monthly means of dailylight integral (DLI) (mol m−2 day−1), daily
minimum relative humidity (%), and dailymean temperature (°C). Grey bars show SD(except for total cover, which is a sum, not amean). Signiﬁcant relationships (linear model)
shown with a line.

gemmae formation and growth (Figs. 3b, c).
Gemmae gradually grow and mostly detach inwinter, the coldest and driest season. Considering
the fact that no obvious new patches of gametophytes appeared during our study, the function ofgemmae as asexual propagules was not demonstrated by our results.
Although our measurement data of microenvironment are too short-termed to discuss the factors causing the independent gametophyte phenomenon, an interesting trend was observed in
the DLI values—they are higher in the sporophyte site than the gametophyte site, particularlyin the fall and winter (Fig. 5, Table 1). The difference is undoubtedly caused by the different tree
species covering the P. makinoi habitats—summer-green broad-leaved trees at the sporophyte
site (Figs. 8a, b) versus evergreen conifer

Fig. 8. A comparison of vegetation of the independent gametophyte site with that of a sporophyte
site of Pleurosoriopsis. a–b. Typical vegetationof sporophyte habitat covered with summergreen broad-leaved trees (Chichibu-shi, Saitama
Pref., Japan). c–d. Vegetation of the independent gametophyte site in Unazawa Valley covered with evergreen cypress trees. a and c inAugust, b and d in December.

[Chamaecyparis obtusa (Siebold et Zucc.) Endl.]
plantation in Unazawa Valley, Okutama, theindependent gametophyte site (Figs. 8c, d). It ishighly likely that sporophytes of P. makinoi,
which have winter-green life history, are probably adapted to the summer-green forest ﬂoor
environment and are not able to grow and sur-

Growth dynamics of Pleurosoriopsis gametophytes

vive on the evergreen forest ﬂoor due to a shortage of light. The stable presence of the gametophyte patches in Okutama suggests that theenvironment is suitable for gametophytes, but
not sporophytes. Since we only observed a verysmall number of individuals with gametangia andno juvenile sporophytes, it seems likely that suppression of sporophyte production occurs prior to
fertilization, or shortly thereafter. Archegoniate(female) individuals tended to be larger thanasexual individuals (Fig. 7), suggesting thatgametophytes may need to reach a size threshold
before they are able to sexually reproduce. The
low winter light conditions at Okutama may prevent gametophytes from reaching this size consistently and thereby inhibit fertilization.
“Inter-generational ecological niche separation”, or different ecological preferences between
sporophytes and gametophytes of the same species, has been reported in several ferns (Sato andSakai, 1981; Rumsey and Shefﬁeld 1996; Pinson
et al., 2017). In most cases, the gametophyte
tends to tolerate a wider range of environments
than the conspeciﬁc sporophyte (Watkins et al.,2007). This seems also to be the case for P.
makinoi with regards to light condition. A possible scenario for formation of the independent
gametophyte population in Okutama is as follows: 1) natural vegetation of the area was summer-green forest preferred by both sporophytes
and gametophytes of P. makinoi; 2) after evergreen conifer plantations were established in the
mid-20th century, sporophyte growth in the plantation areas was hampered by light shortage inwinter and eventually ceased; 3) gametophytes
still survive by indeterminate growth and vegetative reproduction by gemmae.
We could not obtain any evidence for pastpresence of sporophytes at the site, but we founda similar case in Kyoto City, Kyoto Prefecture,
Japan. At that site as well, evergreen conifer
plantations dominated the forests starting fromalmost the same era. At least six herbarium specimens of Pleurosoriopsis sporophytes collectedin the mountains surrounding Kyoto from 1928
to 1958 deposited in KYO, TNS and TOFO

85

imply that it was not a very rare species at thattime. Nowadays it is quite difﬁcult to ﬁnd Pleurosoriopsis sporophytes in the same area (Y.
Kazumi, personal communication), but independent gametophytes are occasionally found (A.
Ebihara, unpublished data). Such gametophytes
whose sporophyte formation has been suppressedby recent environmental change during the past
100 years may resume their sexual life cycles
when or if the environment returns to its priorcondition. Further studies are necessary to determine the conditions needed for sporophyte formation, in particular frequencies of sexual organ
formation.
Acknowledgments
This study was supported by a Grant-in-Aid
for Scientiﬁc Research (KAKENHI 16370046 to
Ryoko Imaichi) from the Japan Society of thePromotion of Science.
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