ALTITUDINAL VARIATION OF FERN COMMUNITY IN YAKUSHIMA ISLAND

Genki Yumoto1, Hiroya Takiyama2,3, Renata Schmitt4, Sho Murakami5, Wanyi Lee2 and Yusuke Fuke5

1Center for Ecological Research, Kyoto University, Japan 2Primate Research Institute, Kyoto University, Japan 3The Leading Graduate Program in Primatology and Wildlife Science, Japan 4Amazon National Research Institute (INPA), Brazil 5Graduate School of Science, Kyoto University, Japan

Abstract

Yakushima Island harbors a great diversity of ferns with rich micro-habitats and high

precipitation. Mountainous geology and steep altitudinal gradient allow co-existence of distinct

climatic zones from warm to cool temperate. Survey on fern community was conducted to

explore the altitudinal gradient of sporophyte and gametophyte distribution in Yakushima Island.

We identified sporophytes by morphology and gametophyte by determining rbcL sequences.

Overall, we identified 17 genera of gametophytes and 30 genera of sporophytes. Of all the

genera found, Dryopteris were present in all three altitudinal levels. In addition to fern

distribution, present study recorded gametophyte samples possibly to be the new record of

Histopteris fern on Yakushima and possibly a new species in the genus Histopteris.

Introduction

Biogeographical research concerns about the relationships between distribution patterns

of organisms and environments and their changes along with evolutionary history. Many

researches have focused on the latitudinal gradients in species richness along environmental

gradients, such as those of temperature and humidity. Similar to research of latitudinal richness,

studies on altitudinal or elevational gradients on species richness were carried out to explore the

species community transition from warm to cold habitats (Barrington, 1993; Kessler, 2010;

Kessler et al., 2011). Latitudinal and elevational gradient is different in that elevational gradients

are more spatially compressed. Compared with latitudinal gradient, environment in altitudinal

gradient, especially in temperature, changes more rapidly than that in latitude gradient.

Therefore, altitudinal studies may provide us insight on how other abiotic factors, in addition to

temperature, may have influence on biodiversity. For example, UV radiation and diurnal

fluctuations in temperature increases more rapidly with elevation, incurring negative effect on

growth of some plants (Halbritter et al., 2013; Siefert et al., 2015).

Ferns made interesting study subject of biogeographical studies with distinctive

generations, i.e., sporophytic and gametophytic generations. The distributions of ferns have been

considered to be determined primarily by abiotic environmental factors (Barrington, 1993;

Kessler, 2010). However, most biogeographical studies on ferns are done on sporophytes due to

the fact that gametophytes are typically smaller in size and less distinguishable by morphology.

Previous studies reported that distributions of gametophytes can be different from those of

sporophytes (Ebihara et al., 2008; Ebihara et al., 2013). Despite the essential role they played in

dispersal of ferns, gametophytes distributions are therefore less studied and require more

attention (Dassler & Farrar, 2001; Pinson et al., 2017; Schneider & Schuettpelz, 2006).

Lately, DNA sequencing technique provides an opportunity for accurate identification of

gametophyte species (Li et al., 2010). In plants, ribulose-bisphosphate carboxylase (rbcL) region

on the chloroplast genome have been used for DNA barcoding analyses. As an essential protein

in photosynthesis, a subunit of rubisco, the protein rbcL codes for, has relatively high

conservativeness. The use of this gene is advantageous for inference of phylogenetic relationship

at higher taxonomic levels. In the case of ferns, their nucleotide sequences are often used for

identification of genera (Hasebe et al., 1994).

Here in our study, we aimed to investigate and compare the species compositions of fern

gametophytes and sporophytes at different altitudes in Yakushima Island. By using combination

of sporophyte identification by morphology and gametophyte by molecular information, we

determined altitudinal gradient of two distinct generations of ferns in Yakushima.

Methodology

Study site

Yakushima Island is situated at the southern-most region of Kyushu, Japan (30° N, 130°

E). The island is about 500 km2 in area, lying on the biogeographic boundary of the northern

limit of the subtropical zone and southern limit of the temperate zone. Precipitation in

Yakushima Island is high, estimated to be over 4,000 mm in the low-land areas and 10,000 mm

in the high-altitudinal areas (Tokumaru, 2003). The island is mountainous, with the highest peak

Mt. Miyanoura-dake at 1,935 m above sea level (a.s.l.). As a result of steep altitudinal gradient,

climatic zones that corresponds to warm to cool temperate are present on the island. Vegetation

on the island serves as miniature representative of vegetation in Japanese Archipelago, ranging

from evergreen broadleaf forest in the warm temperate zone below 700-800 a.s.l. to

cool-temperate coniferous forests and scrub forests of dwarf bamboo above 1,200 a.s.l. (Okano

& Matsuda, 2013; Tokumaru, 2003). Diversity in micro-habitats and rich precipitation give rise

to astonishingly high variety of fern community, including about 300 species (Ministry of the

Environment, 2013; Okano & Matsuda, 2013).

Sample Collection

Study sites were categorized into three altitudinal levels, 1) low altitude, less than 300 m,

2) mid altitude, between 300–1,000 m and 3) high altitude, higher than 1,000 m. We sampled

two sites for each altitudinal level. To equalize sampling efforts across altitudes, each sampling

session lasted for about 30 minutes. For sporophytes, we collected one sample for one species to

get the distribution information at each altitude level. For gametophytes, samples were collected

using tweezers. Sporophyte samples were preserved as pressed specimens using newspapers

immediately after returning to the field station. We also examined the proportion of sporophyte

samples with spores. For species that we were able to get more than two samples in one altitude

level, we include it as “with spore” when at least one sample had spores. Gametophyte samples

were carefully washed using a stereomicroscope, were cut into half for molecular analysis and

for preservation as voucher specimens, and both of them were preserved in ethanol 100%.

Sporophytes were identified by examining morphological characteristics (e.g. leaf

arrangement, spore appearance and root morphology). Gametophyte were identified by DNA

sequencing of rbcL region.

DNA Isolation and Amplification by PCR (Polymerase Chain Reaction)

We performed tissue-direct PCR for 192 out of 349 gametophyte samples using ca. 1

mm2 of gametophytes tissue preserved in 100% Ethanol. The tissue samples were cut with

razors, placed in 0.2 ml tubes and mixed with 10 µl of PCR mix for a nested PCR technique that

combines two distinct reactions. For the first PCR, we used a 10 µL reaction mixture containing

1X PCR buffer (Takara Bio), 0.2 mM dNTPs (Takara Bio), 5 pmol of both forward and reverse

primers, and 0.5 units of Taq DNA polymerase (Takara Bio) using TaKaRa PCR Thermal Cycler

Dice TP650 (Takara Bio) or 2720 Thermal Cycler (Applied Biosystems). The cycling parameters

for the first PCR were; 94°C for 5 min (heat shock), 35 cycles each of 30 sec at 94°C

(denaturing), 30 sec at 50°C (annealing), and 72°C for 60 sec (extension), 7 min at 72°C (final

extension), followed by 10°C (storage). For the second PCR reaction, we used 0.5 µl of the first

PCR as template and the same conditions as the first one. In both PCR reactions, the chloroplast

fragment of the large-subunit of rbcL region was amplified using the following pair of primers

aF (5’- ATG TCA CCA CAA ACA GAG ACT AAA GC – 3’ ) and bR (5’ – CGT TCG CCT

TCC AAT TTG CCC ACT ACA GT – 3’) (Hasebe et al., 1994).

PCR products were electrophoresed in a 1% agarose gel in 1X TAE buffer with 1 µl of

loading solution (BFB) and 0.6 µl of Safety-dye (Natural Immunity) (diluted to 20 times) to

confirm PCR amplification for 10 min at 100V using Mupid-exU (Mupid). The PCR products

were purified using ExoSAP-IT (Affymetrix) according to manufacturer's instructions before the

DNA sequencing by Sanger method using an automated sequencer (ABI 3130) with only a single

strand, primer aF.

Molecular Analyses

The sequences obtained were confirmed and edited in MEGA 7.0.20 (Kumar et al., 2016)

and then “blasted” in BLAST nucleotide (Basic Local Assignment Search Tool -

https://blast.ncbi.nlm.nih.gov/Blast.cgi) to infer the gametophytes genera, based on the hit with

the highest similarity percentage. After, the rbcL sequences were aligned using the multiple

sequence alignment program Clustal W (Thompson et al., 1994). The resulting alignment was

subject to the Neighbour-Joining (NJ) method using MEGA 7. The confidence of the internal

branches from the resulting tree was tested by bootstrap analysis with 500 replications.

We also performed Maximum likelihood analyses for the genus Histiopteris. The

alignments of nucleotide sequences were done by Clustal W implemented in MEGA 7. To find

the best model that fits our data set we also performed a search in MEGA 7. The branch support

was assessed using 500 bootstrap replicates.

Results

Species identification of Sporophyte

In total, 93 samples of sporophytes were collected; 40 from low, 50 from mid and 3 from

high altitudes. The species were from 30 different genera. Dryopteris was found distributing in

all three altitude levels (Table 1, Figure 1). Different altitudinal levels generally have limited

sharing genus, although some genera were found in both low and high altitudes. There were 20

genera of sporophyte that exclusively present in only one altitude (Table 1, Figure 1 and 2). In

every altitude level, the proportion of sporophyte species with spores was less than 60% (Table

2).

Molecular Data of Gametophyte

We obtained part of rbcL gene sequences from 62 out of 192 samples, that resulted in a

final alignment with 700 bp. Using nucleotide BLAST, 57 samples were identified as

gametophyte ferns (used as ingroup in our NJ analysis) and five as moss (used as outgroup in the

NJ analysis) (Figure 1). For the ferns, we found seventeen genera belonging to 11 families. The

most abundant genus was Dryopteris, occurring in all three altitudes (Table 1, Figure 1). There

were thirteen gametophyte genera exclusively presented in only one altitude (Table 1, Figure 1

and 2). Between the altitude levels, there were few genera overlapping for gametophytes (Figure

2).

For the Neighbor Joining (NJ) analysis, we included 25 sequences downloaded from

GenBank for comparisons with our own sequences (Figure 1). The analysis recovered 17 genera

identified in our BLAST search, and for some of the genera (the colored boxes in the

cladogram), it might be possible that we have more than one species (e.g. Dryopteris,

Arachniodes).

Furthermore, we found a new record for distribution to Japan for the genus Histiopteris,

or even a new species not yet described for the genus. The genus Histiopteris has seven nominal

species. BLAST results for Histiopteris showed that our samples are more closely related with a

sequence of H. incisa (a worldwide distributed species) from New Zealand (KT983822.1) than

those ones found in the mainland of Japan. These result is corroborated by ML analysis,

recovering Histiopteris as a monophyletic group (Figure 3).

Table 1. Genus list of fern sporophytes and gametophyte

Altitude level high, middle, low represented by “H”, “M”, “L” respectively. “o” means the genera is present.

Table 2. Percentage of sporophyte with spore

Figure 1. Neighbor-Joining (NJ) tree for gametophytes. The

optimal tree with the sum of branch length = 1.24 is shown.

The percentage of replicate trees in which the associated

taxa clustered together in the bootstrap test (500 replicates)

are shown next to the branches – only values over 70%. The

tree is drawn to scale, with branch lengths in the same units

as those of the evolutionary distances used to infer the

phylogenetic tree. The evolutionary distances were

computed using the p-distance method and are in the units

of the number of base differences per site. The analysis

involved 87 nucleotide sequences. All ambiguous positions

were removed for each sequence pair. There were a total of

700 positions in the final dataset. Evolutionary analyses

were conducted in MEGA7. The color boxes represents the

genera and the dots besides each sample represent the

altitude (yellow = high, blue = middle, red = low).

Figure 2. Genus distribution of gametophyte (left) and sporophyte (right) across altitude. Color

presents the altitude level (yellow = high, blue = middle, red = low)

Figure 3. Phylogeny of Histiopteris by Maximum Likelihood method (ML) based on partial rbcL

sequence data (689 bp). Number on each nodes is bootstrap values in 500 replicates. Red dots

represent the samples collected in this study.

Discussion

Phenology of ferns

Two possibilities are assumed from the fact that only about 50% of the sporophyte

samples collected in this study germinate spores. Firstly, we can confirm that seasonality exists

in fern sporophyte and gametophyte reproduction. In our sampling, which was conducted within

a relatively short span of time, we could still found about half of the sporophyte samples with

spores. The ratio of the sporophyte samples that have spores in the low and middle altitude level

were 40% and 59%, respectively. Previous study revealed that fern spores can maintain

dormancy until the environment becomes more suitable for germination (Inoue, 2003). And

humidity, precipitation, temperature and sunlight hour, which change seasonally, could influence

the length of dormancy. Accordingly, there may be difference between season of spore dispersal

and that of germination.

Secondly, the result may be affected by our sample size. In high altitude, we got only

three sporophyte samples, which may not reveal the actual reproductive state of the fern

community. Since the sampling of present study only lasts for few days, it is highly possible that

we underestimate the diversity of fern gametophytes. We recommend conducting study in

different seasons so as to resolve the problem of underestimation. To further confirm the

seasonality in fern reproduction, close observation in phenology of sporophyte individuals and

its relationship with environmental factors like humidity, precipitation is also recommended.

Overlapping genus

In gametophyte, only one genus was found to be distributed in all three altitude levels.

In sporophyte, overlapping genus between high and low altitude was similar result with

gametophyte. However, overlapping between mid and low altitude was different, which had 8

genera overlapping between mid and low altitude. Regarding this, we propose two possible

reasons, firstly the sampling method and secondly the effect of climate.

During sampling, different strategies were implemented when sporophytes and

gametophytes were collected. For sporophytes, we collected samples on the way to sampling site

for gametophytes. Whereas for gametophyte, we collected samples in smaller areas at the

sampling sites. Therefore, sampling range for sporophytes was larger than that of gametophytes.

There is thus a possibility of underestimate the overlapping gametophyte genus since we may not

be able to cover all ecotones in elevation. Other than that, there were differences in sampling

efforts that may underestimate the distribution of sporophytes in high altitude and overestimate

the overlapping genera found in both mid and low altitude.

Scientists have consensus on the importance of climatic condition on distribution and

species richness (Kessler, 2010). In Yakushima, 750 m a.s.l. is temperature transition zone, and

above this altitude, diurnal and annual ranges of temperature fluctuation tend to increase

considerably (Kawarai, 2009). Above 1,000 m a.s.l., snow accumulates during winter (Osawa,

2006). The ferns in high altitude may be specific to be able to survive under low temperature and

snow accumulation conditions, so there may be few genera found overlapping between high and

mid altitude, or between high and low altitude. However, it is not the case for gametophytes. We

think it is due to the aforementioned bias in sampling and the possibility of underestimation for

overlapping of gametophyte genera.

The genus Dryopteris

In this study, Dryopteris was collected in all altitude levels in Yakushima. So far in

Yakushima, species from Dryopteris genus is most abundant around our research sites (Nature

Conservation Bureau of the Environment Agency, 1993). However, if we identify the samples to

lower taxonomic level i.e. to species level, it is possible that the degree of overlapping will

decrease.

Putative new Histopteris spp.

Two of the gametophyte samples we collected (Middle-23 and Middle-40) were from the

genus Histopteris. By BLAST, they are genetically different from H. incisa, a species widely

distributed in temperate and tropical areas including Japan. Almost all H. incisa including that

reported in Wakayama (Ebihara et al., 2010) made one clade, but our samples and H. incisa

reported in New Zealand (Perrie et al., 2015) made different clade (Figure 3). Hence, we suggest

that our samples are not H. incisa.

In total, there are seven species in the genus Histopteris. However, we can only obtain

rbcL DNA sequence of two species i.e. H. incisa and H. sinuate from GenBank. Therefore, our

samples could either be undescribed species or described species whose DNA sequence of rbcL

are not available in GenBank. In any case, these two samples are the first report of Histopteris

spp. other than H. incisa in Japan, because. H. incisa was the only one Histopteris reported in

Japan.

Sporophyte samples from Histopteris were also collected in this study. With reference to

morphological characteristics and distribution information, it was identified as H. incisa.

However, no molecular analysis was done on that sporophyte sample. Therefore, by the limited

information, we could not eliminate the possibility that our sporophyte sample is from a species

that is reported in Japan. For further study, we recommend molecular analysis on the sporophyte

sample and compare it with our gametophyte sample. Moreover, comparison of genetic and

morphological information with other species from Histopteris including the five lacking from

GenBank is highly recommended.

Acknowledgement

This course was supported by PWS. Our sincere thanks go to Ms. Sakai and Ms.

Akiyama, staff of the PWS office. We would like to express our deep gratitude to Prof. Wataru

Shinohara, Prof. Hiroshi Kudoh and the other lecturers for precious knowledge they shared. We

would also like to acknowledge Prof. Shizuka Fuse and staffs of laboratory of Plant Physiology,

who advised on DNA analysis.

Bibliography

Barrington, D. S. (1993). Ecological and historical factors in fern biogeography. Journal of

Biogeography, 275-279.

Dassler, C. L. & Farrar, D. R. (2001). Significance of gametophyte form in long-distance

colonization by tropical, epiphytic ferns. Brittonia, 53(2), 352-369.

Ebihara, A., Farrar, D. R. & Ito, M. (2008). The sporophyte-less filmy fern of eastern North

America Trichomanes intricatum (Hymenophyllaceae) has the chloroplast genome of an

Asian species. American journal of botany, 95(12), 1645-1651.

Ebihara, A., Nitta, J. H. & Ito, M. (2010). Molecular species identification with rich floristic

sampling: DNA barcoding the pteridophyte flora of Japan. PLoS one, 5(12), e15136.

Ebihara, A., Yamaoka, A., Mizukami, N., Sakoda, A., Nitta, J. H. & Imaichi, R. (2013). A survey

of the fern gametophyte flora of Japan: frequent independent occurrences of noncordiform

gametophytes. American journal of botany, 100(4), 735-743.

Eguchi, T. (2004). Formation and Maintenance of Yakushima Ecology Vol. 1 : Forest as Center.

Journal of Industry and Management of Industrial Management Institute 36, 91-101.

[Japanese]

Halbritter, A. H., Alexander, J. M., Edwards, P. J. & Billeter, R. (2013). How comparable are

species distributions along elevational and latitudinal climate gradients?. Global Ecology

and Biogeography, 22(11), 1228-1237.

Hasebe, M., Omori, T., Nakazawa, M., Sano, T., Kato, M. & Iwatsuki, K. (1994). rbcL gene

sequences provide evidence for the evolutionary lineages of leptosporangiate ferns.

Proceedings of the National Academy of Sciences, 91(12), 5730-5734.

Inoue, H. (2003). Research frontier: study about fern moisture responding gene. The Hokuriku

Journal of Radioisotope Research 5, 46-48. [Japanese]

Kawarai, H. (2009). Vegetation distribution along temperature gradient with special reference to

altitude and landforms in Yakushima Island, SW Japan. Graduate School of Frontier

Sciences, The University of Tokyo, Master's thesis. (summary)

Kessler, M. (2010). Biogeography of ferns. Fern ecology, 22-60.

Kessler, M., Kluge, J., Hemp, A. & Ohlemüller, R. (2011). A global comparative analysis of

elevational species richness patterns of ferns. Global Ecology and Biogeography, 20(6),

868-880.

Li, F. W., Kuo, L. Y., Huang, Y. M., Chiou, W. L., & Wang, C. N. (2010). Tissue-direct PCR, a

rapid and extraction-free method for barcoding of ferns. Molecular ecology resources, 10(1),

92-95.

Ministry of the Environment. (2013). World Natural Heritage in Japan. Retrieved from

http://www.env.go.jp/nature/isan/worldheritage/en/yakushima/uiversal/index.html

Nature Conservation Bureau of the Environment Agency. (1993). Nature of Yakushima: Survey

Report for Conservation of Natural Environment in Yakushima Island. Tokyo: The

Nature Conservation Society of Japan.

Ohsawa, M., Tagawa, H. & Yamagiwa, J. (2006). World Heritage Yakushima/Nature and

Ecosystem at Subtropical, Tokyo, Asakura Publishing Co., Ltd., pp. 288. [Japanese]

Okano, T. & Matsuda, H. (2013). Biocultural diversity of Yakushima Island: mountains, beaches,

and sea. Journal of Marine and Island Cultures, 2(2), 69-77.

Perrie, L. R., Shepherd, L. D. & Brownsey, P. J. (2015). An expanded phylogeny of the

Dennstaedtiaceae ferns: Oenotrichia falls within a non-monophyletic Dennstaedtia, and

Saccoloma is polyphyletic. Australian Systematic Botany, 28(4), 256-264.

Pinson, J. B., Chambers, S. M., Nitta, J. H., Kuo, L.-Y., Sessa, E. B. & Edwards, E. (2017). The

Separation of Generations: Biology and Biogeography of Long-Lived Sporophyteless Fern

Gametophytes. International Journal of Plant Sciences, 178(1), 1-18.

Schneider, H. & Schuettpelz, E. (2006). Identifying fern gametophytes using DNA sequences.

Molecular Ecology Notes, 6(4), 989-991.

Siefert, A., Lesser, M. R. & Fridley, J. D. (2015). How do climate and dispersal traits limit

ranges of tree species along latitudinal and elevational gradients? Global Ecology and

Biogeography, 24(5), 581-593.

Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity

of progressive multiple sequence alignment through sequence weighting, position-specific

gap penalties and weight matrix choice Thompson, Julie D.; Higgins, Desmond G.; Gibson,

Toby J. Nucleic Acids Research, 22(22), 4673-4680.

Tokumaru, H. (2003). Nature Conservation on Yakushima Island: Kagoshima Prefecture's

Efforts. GLOBAL ENVIRONMENTAL RESEARCH-ENGLISH EDITION-, 7(1), 103-112.