class: hide-count <br> # DNA Barcoding of Fern Gametophytes:<br>Past, Present, and Future ### Joel H. Nitta University of Tokyo XVI Conference of the<br>Indian Fern Society 2022.03.18 <img src="images/title_back.png" height="200" style="position:absolute; right:80px; top:300px;"> --- ## Preprint - #### Nitta and Chambers 2022 *Apps. in Plant Sci.* (in press)<br>https://ecoevorxiv.org/dr25p/ ## Slides - ### https://joelnitta.github.io/gameto_barcode_ifs ## More Info - ### https://joelnitta.com --- class: center, middle ## Goal: Provide a practival overview of<br>DNA barcoding in fern gametophytes --- class: inverse, center, middle # What is DNA barcoding? --- ## What is DNA barcoding? .pull-left[ Use of one\* DNA locus for identifying species (Hebert, et al., 2003; Hebert, et al., 2003) ] <!-- [@Hebert2003; @Hebert2003a] --> .pull-right[ <img src="https://media.springernature.com/full/springer-static/image/art%3A10.1038%2Fs41598-021-81087-w/MediaObjects/41598_2021_81087_Fig10_HTML.png?as=webp" height="260"> <br><br> <img src="images/orchid_barcodes.png" height="110"> <br>.small[Li, et al. (2021)] ] --- ## What is DNA barcoding? .pull-left[ 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 ] .pull-right[ <img src="https://innovativegenomics.org/wp-content/uploads/2018/04/DNA.png" height="140"> .xxl[≠] <img src="https://upload.wikimedia.org/wikipedia/commons/8/84/EAN13.svg" height="140"> ] --- ## 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.) .footnote[mean ± SD] --- ## 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__ .footnote[mean ± SD] --- ## Why DNA barcoding of ferns? ### 2. Identify field-collected gametophytes .center[<img src="images/sessa_life_cycle.jpg" height="400">] --- ## Why DNA barcoding of ferns? ### Knowlege of gametophyte ecology is practically nil compared to sporophytes .center[<img src="images/sessa_life_cycle_question.jpg" height="400">] --- ## Why DNA barcoding of ferns? .pull-left[ > "In combination these aspects of __prothallial morphology serve to characterize most of the larger groups of homosporous ferns__, nearly as clearly as sporophyte morphology" .small[Nayar and Kaur (1971)] Gametophyte morphology is important for systematics, but __cannot be relied on to consistently identify species__ ] .pull-right[ .center[ <img src="images/nayar_kaur_1971.png" height="400"> ] ] --- ## Why DNA barcoding of ferns? .pull-left[ A, B: __Cordate__ (many terrestrial species) C, D: __Ribbon__ (e.g., Vittariaceae, Hymenophyllaceae) E, F: __Filamentous__ (e.g., Schizaeaceae, Hymenophyllaceae) ] <img src="images/nitta_2022_1.png" height="500" style="position:absolute; right:80px; top:140px;"> --- ## Why DNA barcoding of ferns? .pull-left[ __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. ] <img src="images/nitta_2022_1.png" height="500" style="position:absolute; right:80px; top:140px;"> --- ## The beginning: ID of a single gametophyte .pull-left[ - First use of DNA barcodes in ferns - Sequence *rbcL* from gametophyte in culture, query GenBank - Identification as *Osmunda* .footnote[Schneider, et al. (2006)] ] .pull-right[ .center[ <img src="images/osmunda_gameto.png" height="200"> .small[Soare (2008)] ] ] --- ## Typical approach .center[ <img src="images/barcode_flow.png" height="450"> ] --- ## Typical approach .center[ <img src="images/barcode_flow_2.png" height="450"> ] --- ## 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 --- class: inverse, center, middle # Building the library --- ## Building the library Sanger sequencing of (at least) one specimen per species in study area from sporophytes --- ## Building the library Sanger sequencing of (at least) one specimen per species in study area from sporophytes .pull-left[ What about __multiple individuals per species?__ - Needed to assess __"barcode gap"__ - For __*rbcL*__, almost certain to be __zero variation__ ] .pull-right[ .center[<img src="images/paulay_2005_1.png" height="350">] .small[Meyer, et al. (2005)] ] --- ## Building the library Sanger sequencing of (at least) one specimen per species in study area from sporophytes .pull-left[ 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) ] .pull-right[ .center[<img src="images/paulay_2005_1.png" height="350">] .small[Meyer, et al. (2005)] ] --- ## Case study 1:<br>Pteridophytes of Japan .pull-left[ - *rbcL* + *trnH-psbA* - 733 taxa, 1 individual per species - High success in sexual diploids, lower in polyploid or apogamous taxa .footnote[Ebihara, et al. (2010)] ] <img src="images/ebihara_2010_1.png" height="600px" style="position:absolute; right:30px; top:30px;"> --- ## Case study 2: Ferns of Moorea and Tahiti, French Polynesia .pull-left[ - *rbcL* + *trnH-psbA* - 145 spp., 1 individual per species for most - High success rate overall (better than Japan) .footnote[Nitta, et al. (2017)] ] .pull-right[ .center[<img src="images/nitta_2017_1.png" height="300">] ] --- class: inverse, center, middle # Using the barcode --- ## Case study 1: Independent gametophytes in Japan <br> .middle[.center[<img src="images/indep_gameto.png" height="450">]] --- ## Case study 1: Independent gametophytes in Japan __Focus on gametophyte mats__ - *Hymenophyllum mikawanum* (Seriz.) Seriz.:<br> sporophyte endangered - *Haplopteris mediosora* (Hayata) X.C.Zhang:<br> sporophyte extremely rare - *Antrophyum plantagineum* (Cav.) Kaulf:<br> sporophyte unknown in Japan .footnote[Murakami, et al. (2021)] <img src="images/murakami_2021_1.png" height="230" style="position:absolute; right:130px; top:160px;"> <img src="images/murakami_2021_2.png" height="220" style="position:absolute; right:130px; top:430px;"> --- ## Case study 1: Independent gametophytes in Japan Focus on gametophyte mats - *Hymenophyllum mikawanum* (Seriz.) Seriz.:<br> __sporophyte endangered__ - *Haplopteris mediosora* (Hayata) X.C.Zhang:<br> __sporophyte extremely rare__ - *Antrophyum plantagineum* (Cav.) Kaulf:<br> __sporophyte unknown in Japan__ .footnote[Murakami, et al. (2021)] <img src="images/murakami_2021_1.png" height="230" style="position:absolute; right:130px; top:160px;"> <img src="images/murakami_2021_2.png" height="220" style="position:absolute; right:130px; top:430px;"> --- ## Case study 1: Independent gametophytes in Japan Focus on gametophyte mats - *Hymenophyllum mikawanum* (Seriz.) Seriz.:<br> sporophyte endangered - __*Haplopteris mediosora*__ (Hayata) X.C.Zhang:<br> sporophyte extremely rare - *Antrophyum plantagineum* (Cav.) Kaulf:<br> sporophyte unknown in Japan .footnote[Murakami, et al. (2021)] <img src="images/murakami_2021_3.png" height="400" style="position:absolute; right:30px; top:160px;"> --- ## Case study 2: Gametophyte community surveys in Japan .pull-left[ - First use of __garden net__ for sampling - Non-cordate gametophytes tend to occur separate from sporophytes - Identify several new independent gametophytes .footnote[Ebihara, et al. (2013)] ] .pull-right[ .center[<img src="images/ebihara_2013_1.png" height="350">] ] --- ## Case study 2: Gametophyte community surveys in Japan .pull-left[ - First use of __garden net__ for sampling - Non-cordate gametophytes tend to occur separate from sporophytes - Identify several new independent gametophytes .footnote[Ebihara, et al. (2013)] ] .pull-right[ .center[ <img src="images/ebihara_2013_2.png" height="150"> <img src="images/ebihara_2013_3.png" height="150"> ] ] --- ## Case study 3: Community structure of ferns in Tahiti .pull-left[ - __96-well plates for DNA extraction, PCR__ - Sporophytes are more affected by environment - Gametophytes are widely distributed, but observed fewer than expected .footnote[Nitta, et al. (2017)] ] .pull-right[ .center[<img src="images/plates.jpg" height="380">] ] --- ## Case study 3: Community structure of ferns in Tahiti .pull-left[ - 96-well plates for DNA extraction, PCR - __Sporophytes are more affected by environment__ - Gametophytes are widely distributed, but observed fewer than expected .footnote[Nitta, et al. (2017)] ] <img src="images/nitta_2017_2.png" height="500" style="position:absolute; right:80px; top:140px;"> --- ## Case study 4: Hemi-epiphytism in *Vandenboschia* .pull-left[ - Unclear if *V. collariata* was __primary__ or __secondary__ hemi-epiphyte .footnote[Nitta, et al. (2009)] ] .pull-right[ .center[<img src="images/nitta_2009_1.png" height="450">] ] --- ## Case study 4: Hemi-epiphytism in *Vandenboschia* .pull-left[ - 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__ .footnote[Nitta, et al. (2009)] ] .pull-right[ .center[<img src="images/nitta_2009_2.png" height="230">] .center[<img src="images/nitta_2009_3.png" height="230">] ] --- ## Case study 5: Desiccation tolerance of filmy ferns .pull-left[ - 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 .footnote[Nitta, et al. (2021)] ] <img src="images/nitta_2021_1.png" height="500" style="position:absolute; right:80px; top:140px;"> --- ## Case study 5: Desiccation tolerance of filmy ferns <img src="images/nitta_2021_2_trans.png" height="520" style="position:absolute; right:80px; top:140px;"> - Gametophytes have __less DT than sporophytes__ .footnote[Nitta, et al. (2021)] --- ## Case study 5: Desiccation tolerance of filmy ferns <img src="images/nitta_2021_3.png" height="350" style="position:absolute; right:140px; top:240px;"> - Perhaps filmy ferns rely on __gemmae and microhabitats__, not DT .footnote[Nitta, et al. (2021)] --- class: inverse, center, middle # The future: Next-generation DNA sequencing --- ## 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 .center[<img src="https://www.pacb.com/wp-content/uploads/Evolution-of-sequencing-technology.jpg" height="260">] .footnote[https://www.pacb.com/] --- ## Next-Gen DNA sequencing: Microfluidic PCR .pull-left[ - Allows to __massively scale-up sequencing__ - Expensive .footnote[Gostel, et al. (2020)] ] <img src="images/gostel_2020_1.png" height="500" style="position:absolute; right:80px; top:140px;"> --- ## Next-Gen DNA sequencing: MinION .pull-left[ - Portable DNA sequencer - Long reads (ca. 1,000 bp) - Enables __identification of species<br>in the field__ .footnote[Pomerantz, et al. (2018)] ] <img src="images/pomerantz_2018_1.png" height="500" style="position:absolute; right:80px; top:140px;"> --- ## Tissue-direct PCR .pull-left[ - __Skips DNA extraction step__ - Used to survey *Lomariopsis* in Taiwan - Possible to combine with next-gen sequencing? .footnote[Li, et al. (2009); Wu, et al. (2022)] ] .pull-right[ .center[<img src="images/kuo_2022_3.png" height="350">] .small[Photo: L.-Y. Kuo] ] --- ## Tissue-direct PCR .pull-left[ - Skips DNA extraction step - __Used to survey *Lomariopsis* in Taiwan__ - Possible to combine with next-gen sequencing? .footnote[Li, et al. (2009); Wu, et al. (2022)] ] <img src="images/kuo_2022_2.png" height="360" style="position:absolute; right:30px; top:140px;"> --- ## 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) .center[<img src="images/nitta_2022_2.png" height="380">] --- ## Acknowledgements - Co-author Sally Chambers - Marie Selby Botanical Garden - Li-Yaung Kuo - Organizing Committee of the XVI Conference of the Indian Fern Society --- ## References .small[ 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](https://doi.org/10.1371%2Fjournal.pone.0015136). Ebihara, A. et al. (2013). "A survey of the fern gametophyte flora of Japan: Frequent independent occurrences of noncordiform gametophytes". 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