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DNA Barcoding of Fern Gametophytes:
Past, Present, and Future

Joel H. Nitta

University of Tokyo

XVI Conference of the
Indian Fern Society
2022.03.18

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Goal: Provide a practival overview of
DNA barcoding in fern gametophytes

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What is DNA barcoding?

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What is DNA barcoding?

Use of one* DNA locus for identifying species (Hebert, et al., 2003; Hebert, et al., 2003)




Li, et al. (2021)

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What is DNA barcoding?

Use of one* DNA locus for identifying species (Hebert, et al., 2003; Hebert, et al., 2003)

  • "Barcode" is a misnomer

    • No locus is identical across all individuals of a species and different between different species

  • Mitochondrial COI is used in animals

  • *No single marker available in plants

≠

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Why DNA barcoding of ferns?

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Why DNA barcoding of ferns?

1. Primary taxonomy

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Why DNA barcoding of ferns?

1. Primary taxonomy

DNA sequences provide objective evidence of species status

Mean infrageneric, interspecific rbcL distance across ferns: 1.50% ± 0.78%
(n = 4,711 species, 259 genera; Nitta, in prep.)

mean ± SD

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Why DNA barcoding of ferns?

1. Primary taxonomy

DNA sequences provide objective evidence of species status

Mean infrageneric, interspecific rbcL distance across ferns: 1.50% ± 0.78%
(n = 4,711 species, 259 genera; Nitta, in prep.)

Phylogenetic analysis can make new species descriptions more robust

mean ± SD

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Why DNA barcoding of ferns?

2. Identify field-collected gametophytes

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Why DNA barcoding of ferns?

Knowlege of gametophyte ecology is practically nil compared to sporophytes

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Why DNA barcoding of ferns?

"In combination these aspects of prothallial morphology serve to characterize most of the larger groups of homosporous ferns, nearly as clearly as sporophyte morphology"
Nayar and Kaur (1971)

Gametophyte morphology is important for systematics, but cannot be relied on to consistently identify species

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Why DNA barcoding of ferns?

A, B: Cordate (many terrestrial species)

C, D: Ribbon (e.g., Vittariaceae, Hymenophyllaceae)

E, F: Filamentous (e.g., Schizaeaceae, Hymenophyllaceae)

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Why DNA barcoding of ferns?

Cordate
A: Sphaeropteris medullaris (G. Forst.) Bernh.
B: Austroblechnum raiateense (J.W.Moore) Gasper & V.A.O.Dittrich

Ribbon
C: Callistopteris apiifolia (C. Presl) Copel.
D: Hymenophyllum polyanthos (Sw.) Sw.

Filamentous
E: Crepidomanes minutum (Blume) K.Iwats.
F: Abrodictyum dentatum(Bosch) Ebihara & K.Iwats.

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The beginning: ID of a single gametophyte

  • First use of DNA barcodes in ferns
  • Sequence rbcL from gametophyte in culture, query GenBank
  • Identification as Osmunda

Schneider, et al. (2006)

Soare (2008)

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Typical approach

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Typical approach

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Choosing a marker

My recommendation: rbcL + trnL-trnF

  • Nuclear markers are too difficult to obtain (multiple copies)
  • rbcL can differentiate between species in most cases, has best coverage on GenBank
  • trnL-F can be used as secondary marker for closely related taxa
Marker Type PCR success Variability
rbcL(-a) Coding High Low
matK Coding Low Moderate to high
trnH-psbA Spacer High High in some groups, low in others
trnL-F Spacer High High
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Building the library

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Building the library

Sanger sequencing of (at least) one specimen per species in study area from sporophytes

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Building the library

Sanger sequencing of (at least) one specimen per species in study area from sporophytes

What about multiple individuals per species?

  • Needed to assess "barcode gap"
  • For rbcL, almost certain to be zero variation

Meyer, et al. (2005)

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Building the library

Sanger sequencing of (at least) one specimen per species in study area from sporophytes

What about multiple individuals per species?

  • Needed to assess "barcode gap"
  • For rbcL, almost certain to be zero variation

My recommendation:

  • One specimen/species for most taxa
  • Multiple specimens in case of "difficult" taxa (species complexes)

Meyer, et al. (2005)

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Case study 1:
Pteridophytes of Japan

  • rbcL + trnH-psbA

  • 733 taxa, 1 individual per species

  • High success in sexual diploids, lower in polyploid or apogamous taxa

Ebihara, et al. (2010)

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Case study 2: Ferns of Moorea and Tahiti, French Polynesia

  • rbcL + trnH-psbA

  • 145 spp., 1 individual per species for most

  • High success rate overall (better than Japan)

Nitta, et al. (2017)

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Using the barcode

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Case study 1: Independent gametophytes in Japan


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Case study 1: Independent gametophytes in Japan

Focus on gametophyte mats

  • Hymenophyllum mikawanum (Seriz.) Seriz.:
    sporophyte endangered
  • Haplopteris mediosora (Hayata) X.C.Zhang:
    sporophyte extremely rare
  • Antrophyum plantagineum (Cav.) Kaulf:
    sporophyte unknown in Japan

Murakami, et al. (2021)

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Case study 1: Independent gametophytes in Japan

Focus on gametophyte mats

  • Hymenophyllum mikawanum (Seriz.) Seriz.:
    sporophyte endangered
  • Haplopteris mediosora (Hayata) X.C.Zhang:
    sporophyte extremely rare
  • Antrophyum plantagineum (Cav.) Kaulf:
    sporophyte unknown in Japan

Murakami, et al. (2021)

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Case study 1: Independent gametophytes in Japan

Focus on gametophyte mats

  • Hymenophyllum mikawanum (Seriz.) Seriz.:
    sporophyte endangered
  • Haplopteris mediosora (Hayata) X.C.Zhang:
    sporophyte extremely rare
  • Antrophyum plantagineum (Cav.) Kaulf:
    sporophyte unknown in Japan

Murakami, et al. (2021)

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Case study 2: Gametophyte community surveys in Japan

  • First use of garden net for sampling
  • Non-cordate gametophytes tend to occur separate from sporophytes
  • Identify several new independent gametophytes

Ebihara, et al. (2013)

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Case study 2: Gametophyte community surveys in Japan

  • First use of garden net for sampling
  • Non-cordate gametophytes tend to occur separate from sporophytes
  • Identify several new independent gametophytes

Ebihara, et al. (2013)

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Case study 3: Community structure of ferns in Tahiti

  • 96-well plates for DNA extraction, PCR
  • Sporophytes are more affected by environment
  • Gametophytes are widely distributed, but observed fewer than expected

Nitta, et al. (2017)

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Case study 3: Community structure of ferns in Tahiti

  • 96-well plates for DNA extraction, PCR
  • Sporophytes are more affected by environment
  • Gametophytes are widely distributed, but observed fewer than expected

Nitta, et al. (2017)

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Case study 4: Hemi-epiphytism in Vandenboschia

  • Unclear if V. collariata was primary or secondary hemi-epiphyte

Nitta, et al. (2009)

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Case study 4: Hemi-epiphytism in Vandenboschia

  • Unclear if V. collariata was primary or secondary hemi-epiphyte
  • Find gametophytes at base of tree
  • Sequence DNA to confirm identity
  • ➡︎ Shows V. collariata is primary hemiepiphyte

Nitta, et al. (2009)

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Case study 5: Desiccation tolerance of filmy ferns

  • DT is important trait in transition of plants to life on land
  • DT is known from both sporophytes and gametophytes of filmy ferns
  • Compare DT between sporophytes and gametophytes

Nitta, et al. (2021)

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Case study 5: Desiccation tolerance of filmy ferns

  • Gametophytes have less DT than sporophytes

Nitta, et al. (2021)

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Case study 5: Desiccation tolerance of filmy ferns

  • Perhaps filmy ferns rely on gemmae and microhabitats, not DT

Nitta, et al. (2021)

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The future: Next-generation DNA sequencing

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Disadvantages of Sanger sequencing

  • Slow
  • Limited number of samples
  • Limited number of loci

High-throughput methods could allow for continuous monitoring over time and space of gametophyte populations

https://www.pacb.com/

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Next-Gen DNA sequencing: Microfluidic PCR

  • Allows to massively scale-up sequencing
  • Expensive

Gostel, et al. (2020)

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Next-Gen DNA sequencing: MinION

  • Portable DNA sequencer
  • Long reads (ca. 1,000 bp)
  • Enables identification of species
    in the field

Pomerantz, et al. (2018)

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Tissue-direct PCR

  • Skips DNA extraction step
  • Used to survey Lomariopsis in Taiwan
  • Possible to combine with next-gen sequencing?

Li, et al. (2009); Wu, et al. (2022)

Photo: L.-Y. Kuo

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Tissue-direct PCR

  • Skips DNA extraction step
  • Used to survey Lomariopsis in Taiwan
  • Possible to combine with next-gen sequencing?

Li, et al. (2009); Wu, et al. (2022)

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Conclusions

  • DNA barcoding can provide unprecedented insights into fern biology
  • Sanger sequencing is (still) useful, but limited
  • Next-generation DNA barcoding has the potential to revolutionize fern biology (again)

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Acknowledgements

  • Co-author Sally Chambers
  • Marie Selby Botanical Garden
  • Li-Yaung Kuo
  • Organizing Committee of the XVI Conference of the Indian Fern Society
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References

Ebihara, A. et al. (2010). "Molecular species identification with rich floristic sampling: DNA barcoding the pteridophyte flora of Japan". In: PLoS ONE 5.12, p. e15136. DOI: 10.1371/journal.pone.0015136.

Ebihara, A. et al. (2013). "A survey of the fern gametophyte flora of Japan: Frequent independent occurrences of noncordiform gametophytes". In: American Journal of Botany 100.4, pp. 735-743. DOI: 10.3732/ajb.1200555.

Gostel, M. R. et al. (2020). "Microfluidic Enrichment Barcoding (MEBarcoding): A New Method for High Throughput Plant DNA Barcoding". In: Scientific Reports 10.1 (1), p. 8701. DOI: 10/gg539d.

Hebert, P. et al. (2003). "Ten Species in One: DNA Barcoding Reveals Cryptic Species in the Neotropical Skipper Butterfly Astraptes Fulgerator". In: Proceedings of the National Academy of Sciences of the United States of America 101, pp. 114812-14817.

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References (cont.)

Hebert, P. et al. (2003). "Barcoding Animal Life: Cytochrome c Oxidase Subunit 1 Divergences among Closely Related Species". In: Proceedings of the Royal Society of London, Series B: Biological Sciences 270, pp. S96-S99.

Li, F. et al. (2009). "Tissue Direct PCR, a Rapid and Extraction Free Method for Barcoding of Ferns". In: Molecular Ecology Resources 10.1, pp. 92-95.

Li, H. et al. (2021). "The Specific DNA Barcodes Based on Chloroplast Genes for Species Identification of Orchidaceae Plants". In: Sci Rep 11.1, p. 1424. DOI: 10.1038/s41598-021-81087-w.

Meyer, C. P. et al. (2005). "DNA Barcoding: Error Rates Based on Comprehensive Sampling". In: PLoS Biology 3.12, p. e422.

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References (cont.)

Murakami, N. et al. (2021). "Inventories of fern independent gametophytes in Japan using DNA barcoding based on nucleotide sequences of rbcL gene (in Japanese)". In: BSJ-Review 12, pp. 159-168. DOI: 10.24480/bsj-review.12c3.00211.

Nayar, B. K. et al. (1971). "Gametophytes of Homosporous Ferns". In: The Botanical Review 37.3, pp. 295-396.

Nitta, J. H. et al. (2009). "Hemi-epiphytism in Vandenboschia collariata (Hymenophyllaceae)". In: Brittonia 61.4, pp. 392-397. DOI: 10.1007/s12228-009-9097-5.

Nitta, J. H. et al. (2017). "Life cycle matters: DNA barcoding reveals contrasting community structure between fern sporophytes and gametophytes". In: Ecological Monographs 87.2, pp. 278-296. DOI: 10.1002/ecm.1246.

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References (cont.)

Nitta, J. H. et al. (2021). "Ecophysiological differentiation between life stages in filmy ferns (Hymenophyllaceae)". In: J Plant Res 134, pp. 971-988. DOI: 10.1007/s10265-021-01318-z.

Pomerantz, A. et al. (2018). "Real-Time DNA Barcoding in a Rainforest Using Nanopore Sequencing: Opportunities for Rapid Biodiversity Assessments and Local Capacity Building". In: GigaScience 7.4, p. giy033. DOI: 10/gc84n7.

Schneider, H. et al. (2006). "Identifying Fern Gametophytes Using DNA Sequences". In: Molecular Ecology Notes 6, pp. 989-991. DOI: 10.1111/j.1471-8286.2006.01424.x.

Soare, L. C. (2008). "In Vitro Development of Gametophyte and Sporophyte in Several Fern Species". In: Notulae Botanicae Horti Agrobotanici Cluj-Napoca 36. DOI: 10.15835/nbha36183.

Wu, Y. et al. (2022). "Integrating Tissue-Direct PCR into Genetic Identification: An Upgraded Molecular Ecology Approach to Survey Fern Gametophytes in the Field". In: Applications in Plant Sciences 10.2, p. e11462.

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