Applying next-generation DNA sequencing to resolve the Thelypteris angustifrons species complex (Thelypteridaceae)

class: center, middle, inverse, title-slide

# Applying next-generation DNA sequencing to resolve the Thelypteris angustifrons species complex (Thelypteridaceae)
### Joel H. Nitta, Atsushi Ebihara, Narumi Nakato National Museum of Nature and Science, Japan
### 2018/10/16

---

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background-image: url(https://www.dropbox.com/s/tr5s7p2x3rk6sfd/T_angustifrons_tsukuba.jpg?raw=1)

---
# Why NGS for ferns?

__Polyploid species complexes__ are an important part of fern biodiversity, and yet...

- Most studies are based on just a __few loci__

- Can’t distinguish between incomplete lineage sorting, introgression, etc.

![](https://ce8f5f2ec.cloudimg.io/fit/800x220/ffffff/https://ksassets.timeincuk.net/wp/uploads/sites/54/2016/11/mystery-box-1-320x180.jpg)

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# Finally!

.footnote[Li, F.W. et al. 2018. "Fern genomes elucidate land plant evolution and cyanobacterial symbioses." *Nature Plants* 4:460–472]

---
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# It is time to bring next-gen methods to fern species complexes!

---
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# The _P. angustifrons_ complex
## -The Story So Far-

---
# The _P. angustifrons_ complex

.pull-left[
*P. glanduligera*
- N. India, Indonesia, Thailand, China, Taiwan, Korea, Japan

*P. angustifrons*
- China, Taiwan, Korea, Japan

*P. cystopteroides*
- S. China, Taiwan, S. Japan

*P. miyagii*
- S. Japan only

]

.pull-right[
![](https://www.dropbox.com/s/zxiv68yr995ieuj/T_angustifrons_complex.jpg?raw=1)
]

.footnote[Ebihara, A. 2016]

---
# The _P. angustifrons_ complex

.pull-left[
*P. glanduligera*
- 4x (Japan, Nepal). In Japan X=27, but X=34, 36, or 40 reported elsewhere

*P. angustifrons*
- 2x, 3x, 6x (Japan, maybe Taiwan). X=27

*P. cystopteroides*
- 2x, 4x (S. Japan). X=27

*P. miyagii*
- 2x (Okinawa). X=27

]

.pull-right[
![](https://www.dropbox.com/s/zxiv68yr995ieuj/T_angustifrons_complex.jpg?raw=1)
]

.footnote[Ebihara, A. 2016]

---
# Hybrids

.pull-left[
*P. angustifrons* × *glanduligera*
- 4x, 5x

*P. angustifrons* × *cystopteroides*
- 4x, 5x
]

.pull-right[
![](https://ce8f5f2ec.cloudimg.io/fit/344x437/ffffff/https://www.dropbox.com/s/4fip5d9hx1xu6m8/T_angustifrons_hybrids.jpg?raw=1
)
]

.footnote[Ebihara, A. 2016]

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background-image: url(https://ce8f5f2ec.cloudimg.io/fit/800x600/ffffff/https://www.dropbox.com/s/o5mgq8jt5dfw0jn/rbcl_tree.png?raw=1)
# _rbcL_

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background-image: url(https://ce8f5f2ec.cloudimg.io/fit/800x600/ffffff/https://www.dropbox.com/s/oufhjtp692wf1pn/pgic_tree.png?raw=1)
# _PGIC_

---
# The Story So Far
.pull-left[
Poor correlation between morphology and DNA

Many polytomies

*P. glanduligera* only known from 4x

Lack of sampling outside of Japan
]

.pull-right[
![](https://ce8f5f2ec.cloudimg.io/fit/350x450/ffffff/https://www.dropbox.com/s/eohks19p8cbus5j/summary_hypothesis.png?raw=1)
]

---
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# Methods

---
.pull-left[
## Sequence Capture

Short reads (300 bp PE on MiSeq)

Hundreds of loci

Works on degraded DNA

This study: 194 genes across Eupolypod II ferns

]

.pull-right[
## Pacbio + PURC

Long reads (1—10 kb on Pacbio)

< 10 genes

Requires high-quality DNA

This study: 4 genes with complex-specific primers

]

<div id="htmlwidget-02987c16c38f53252b2e" style="width:100%;height:302.4px;" class="grViz html-widget"></div>
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---
.pull-left[
## Sequence Capture

Short reads (300 bp PE on MiSeq)

Hundreds of loci

Works on degraded DNA

This study: 194 genes across Eupolypod II ferns

]

.pull-right[
## Pacbio + PURC

Long reads (1—10 kb on Pacbio)

< 10 genes

Requires high-quality DNA

This study: 4 genes with complex-specific primers

]

<div id="htmlwidget-7e545af01ef0d7474bd0" style="width:100%;height:302.4px;" class="grViz html-widget"></div>
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---
<img src="index_files/figure-html/purc_flow_plain-1.png" width="1365" />

.footnote[Rothfels, C. et al. 2017 *New Phyt.* 213:413—429]

---
<img src="index_files/figure-html/purc_flow_annotated-1.png" width="1365" />

.footnote[Rothfels, C. et al. 2017 *New Phyt.* 213:413—429]

---
<img src="index_files/figure-html/purc_flow_annotated_red-1.png" width="1365" />

.footnote[Rothfels, C. et al. 2017 *New Phyt.* 213:413—429]
---
# PURC Analysis Regimes

<table class="table" style="font-size: 16px; margin-left: auto; margin-right: auto;">
 <thead>
 <tr>
 <th style="text-align:left;"> Regime </th>
 <th style="text-align:right;"> clustID1 </th>
 <th style="text-align:right;"> clustID2 </th>
 <th style="text-align:right;"> clustID3 </th>
 <th style="text-align:right;"> clustID4 </th>
 <th style="text-align:left;"> Uchime settings </th>
 </tr>
 </thead>
<tbody>
 <tr>
 <td style="text-align:left;"> a </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:left;"> normal </td>
 </tr>
 <tr>
 <td style="text-align:left;"> b </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.990 </td>
 <td style="text-align:left;"> normal </td>
 </tr>
 <tr>
 <td style="text-align:left;"> c </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.990 </td>
 <td style="text-align:right;"> 0.990 </td>
 <td style="text-align:left;"> normal </td>
 </tr>
 <tr>
 <td style="text-align:left;"> d </td>
 <td style="text-align:right;"> 0.997 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:left;"> normal </td>
 </tr>
 <tr>
 <td style="text-align:left;"> e </td>
 <td style="text-align:right;"> 0.997 </td>
 <td style="text-align:right;"> 0.997 </td>
 <td style="text-align:right;"> 0.997 </td>
 <td style="text-align:right;"> 0.997 </td>
 <td style="text-align:left;"> normal </td>
 </tr>
 <tr>
 <td style="text-align:left;"> f </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:left;"> strict </td>
 </tr>
 <tr>
 <td style="text-align:left;"> g </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.990 </td>
 <td style="text-align:left;"> strict </td>
 </tr>
 <tr>
 <td style="text-align:left;"> h </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.990 </td>
 <td style="text-align:right;"> 0.990 </td>
 <td style="text-align:left;"> strict </td>
 </tr>
 <tr>
 <td style="text-align:left;"> i </td>
 <td style="text-align:right;"> 0.997 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:right;"> 0.995 </td>
 <td style="text-align:left;"> strict </td>
 </tr>
 <tr>
 <td style="text-align:left;"> j </td>
 <td style="text-align:right;"> 0.997 </td>
 <td style="text-align:right;"> 0.997 </td>
 <td style="text-align:right;"> 0.997 </td>
 <td style="text-align:right;"> 0.997 </td>
 <td style="text-align:left;"> strict </td>
 </tr>
</tbody>
</table>

.small[normal: -abskew 2.0 -minh 0.28 -xn 8.0 -dn 1.4 strict: -abskew 1.1 -minh 0.20 -xn 3.0 -dn 0.5]

---
exclude: true
# Analysis Pipeline

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---
# Pacbio + PURC Sampling

4 loci (*COP9*, *DUF1077*, *IBR3*, *PGIC*)

34 samples
<table class="table" style="font-size: 16px; margin-left: auto; margin-right: auto;">
 <thead>
 <tr>
 <th style="text-align:left;"> Taxon </th>
 <th style="text-align:left;"> Ploidy (no. samples) </th>
 </tr>
 </thead>
<tbody>
 <tr>
 <td style="text-align:left;font-style: italic;"> P. angustifrons </td>
 <td style="text-align:left;"> 3x (1), 6x (9), unknown (2) </td>
 </tr>
 <tr>
 <td style="text-align:left;font-style: italic;"> P. cystopteroides </td>
 <td style="text-align:left;"> 4x (1), unknown (2) </td>
 </tr>
 <tr>
 <td style="text-align:left;font-style: italic;"> P. glanduligera </td>
 <td style="text-align:left;"> 4x (8) </td>
 </tr>
 <tr>
 <td style="text-align:left;font-style: italic;"> P. miyagii </td>
 <td style="text-align:left;"> 2x (3) </td>
 </tr>
 <tr>
 <td style="text-align:left;font-style: italic;"> P. sp. </td>
 <td style="text-align:left;"> 3x (1) </td>
 </tr>
 <tr>
 <td style="text-align:left;font-style: italic;"> P. angustifrons × glanduligera </td>
 <td style="text-align:left;"> 4x (1), 5x (2) </td>
 </tr>
 <tr>
 <td style="text-align:left;font-style: italic;"> P. cystopteroides × T. angustifrons </td>
 <td style="text-align:left;"> 4x (1), 5x (3) </td>
 </tr>
</tbody>
</table>

Distribution: Japan (30), South Korea (2), Taiwan (2)
---
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# Results

---
## Dataset
<table class="table" style="font-size: 16px; margin-left: auto; margin-right: auto;">
 <thead>
 <tr>
 <th style="text-align:left;"> Run </th>
 <th style="text-align:left;"> Platform </th>
 <th style="text-align:right;"> CCS Reads </th>
 <th style="text-align:right;"> Filtered Reads* </th>
 </tr>
 </thead>
<tbody>
 <tr>
 <td style="text-align:left;"> This Study </td>
 <td style="text-align:left;"> RSII </td>
 <td style="text-align:right;"> 25879 </td>
 <td style="text-align:right;"> 18751 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> Rothfels et al. R2 </td>
 <td style="text-align:left;"> RSI </td>
 <td style="text-align:right;"> 30732 </td>
 <td style="text-align:right;"> 18189 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> Rothfels et al. R3 </td>
 <td style="text-align:left;"> RSI </td>
 <td style="text-align:right;"> 44721 </td>
 <td style="text-align:right;"> 27994 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> Rothfels et al. R4 </td>
 <td style="text-align:left;"> RSI </td>
 <td style="text-align:right;"> 38982 </td>
 <td style="text-align:right;"> 28550 </td>
 </tr>
</tbody>
</table>

.small[*length > 600bp and expected errors < 5]

---
## Dataset
<img src="index_files/figure-html/ccs_phred_scores-1.svg" width="120%" />

---
## Dataset
<img src="index_files/figure-html/ccs_length-1.svg" width="120%" />

---
<img src="index_files/figure-html/plot_allele_coverage-1.svg" width="120%" />

---
exclude: true
<img src="index_files/figure-html/plot_allele_coverage_scales_free-1.svg" width="120%" />

---
<img src="index_files/figure-html/plot_pacbio_allele_count-1.svg" width="120%" />

---
class: small-margins

.left-tree[
![](index_files/figure-html/COP9_tree-1.svg)

.small[RAxML, 1000 bootstrap replicates]
]
.right-table[<table class="table table-striped table-condensed" style="font-size: 9px; margin-left: auto; margin-right: auto;">
 <thead>
 <tr>
 <th style="text-align:left;"> Taxon </th>
 <th style="text-align:left;"> Specimen </th>
 <th style="text-align:left;"> Formula </th>
 <th style="text-align:right;"> Ploidy </th>
 </tr>
 </thead>
<tbody>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Ebihara_3199_f </td>
 <td style="text-align:left;"> B2/C14/C8 </td>
 <td style="text-align:right;"> 3 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Ebihara_3313 </td>
 <td style="text-align:left;"> B1/C8 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_2998 </td>
 <td style="text-align:left;"> B1/C8 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_2952 </td>
 <td style="text-align:left;"> B1/C15/C8/C9 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_3028 </td>
 <td style="text-align:left;"> B2/C10/C8 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_3036 </td>
 <td style="text-align:left;"> B1/C8 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_3041 </td>
 <td style="text-align:left;"> B1/C2/C8 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_3068 </td>
 <td style="text-align:left;"> B1/C2/C8 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_3069 </td>
 <td style="text-align:left;"> B1/C1/C2/C8 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_3072 </td>
 <td style="text-align:left;"> B2/C2/C8 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Ebihara_2823 </td>
 <td style="text-align:left;"> B2/C2/C8 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Ebihara_2866 </td>
 <td style="text-align:left;"> B2/C2/C8 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons × glanduligera </td>
 <td style="text-align:left;"> Nakato_2855 </td>
 <td style="text-align:left;"> B2/C10/C8 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons × glanduligera </td>
 <td style="text-align:left;"> Nakato_3024 </td>
 <td style="text-align:left;"> B2/C2/C7/C8 </td>
 <td style="text-align:right;"> 5 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons × glanduligera </td>
 <td style="text-align:left;"> Nakato_3025 </td>
 <td style="text-align:left;"> B1/B2/C4/C8 </td>
 <td style="text-align:right;"> 5 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides </td>
 <td style="text-align:left;"> Ebihara_3250_3 </td>
 <td style="text-align:left;"> C10/C13/C2 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides </td>
 <td style="text-align:left;"> Ebihara_2007_251 </td>
 <td style="text-align:left;"> C10/C13 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Nakato_2953 </td>
 <td style="text-align:left;"> B2/C11/C13/C8 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Hyodo_2837 </td>
 <td style="text-align:left;"> B1/C3/C8 </td>
 <td style="text-align:right;"> 5 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Ebihara_3249_3 </td>
 <td style="text-align:left;"> B2/C10/C8 </td>
 <td style="text-align:right;"> 5 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Nakato_3083 </td>
 <td style="text-align:left;"> B1/C13/C16/C8 </td>
 <td style="text-align:right;"> 5 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. glanduligera </td>
 <td style="text-align:left;"> Nakato_3046 </td>
 <td style="text-align:left;"> B1/C6 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. glanduligera </td>
 <td style="text-align:left;"> Nakato_3050 </td>
 <td style="text-align:left;"> B1/B2/C5/C8 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. miyagii </td>
 <td style="text-align:left;"> Nakato_2861 </td>
 <td style="text-align:left;"> C2 </td>
 <td style="text-align:right;"> 2 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. miyagii </td>
 <td style="text-align:left;"> Ebihara_3197_b </td>
 <td style="text-align:left;"> C11/C2 </td>
 <td style="text-align:right;"> 2 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. sp0 </td>
 <td style="text-align:left;"> NA </td>
 <td style="text-align:left;"> C10/C12/C8 </td>
 <td style="text-align:right;"> 3 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> Thelypteris_kunthii </td>
 <td style="text-align:left;"> NA </td>
 <td style="text-align:left;"> A1 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
</tbody>
</table>

]

---
class: small-margins

.left-tree[
![](index_files/figure-html/DUF1077_tree-1.svg)

]

---
class: small-margins

.left-tree[
![](index_files/figure-html/IBR3SHORT_tree-1.svg)

.small[RAxML, 1000 bootstrap replicates]
]
.right-table[<table class="table table-striped table-condensed" style="font-size: 8px; margin-left: auto; margin-right: auto;">
 <thead>
 <tr>
 <th style="text-align:left;"> Taxon </th>
 <th style="text-align:left;"> Specimen </th>
 <th style="text-align:left;"> Formula </th>
 <th style="text-align:right;"> Ploidy </th>
 </tr>
 </thead>
<tbody>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Ebihara_3199_f </td>
 <td style="text-align:left;"> C11/C12/C3 </td>
 <td style="text-align:right;"> 3 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Ebihara_3313 </td>
 <td style="text-align:left;"> C11/C12/C17/C18 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_2998 </td>
 <td style="text-align:left;"> C12/C18/C2 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_2952 </td>
 <td style="text-align:left;"> C1/C12/C7 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_3028 </td>
 <td style="text-align:left;"> C1/C12 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_3036 </td>
 <td style="text-align:left;"> C1/C12/C16/C23 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_3041 </td>
 <td style="text-align:left;"> C11/C12/C18 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_3068 </td>
 <td style="text-align:left;"> C12/C18 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_3069 </td>
 <td style="text-align:left;"> C1/C12 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_3072 </td>
 <td style="text-align:left;"> C1/C12/C9 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Ebihara_2866 </td>
 <td style="text-align:left;"> C12/C20 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons × glanduligera </td>
 <td style="text-align:left;"> Nakato_2855 </td>
 <td style="text-align:left;"> C1/C5/C6 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons × glanduligera </td>
 <td style="text-align:left;"> Nakato_3024 </td>
 <td style="text-align:left;"> C12/C18/C7 </td>
 <td style="text-align:right;"> 5 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons × glanduligera </td>
 <td style="text-align:left;"> Nakato_3025 </td>
 <td style="text-align:left;"> C1/C12/C13/C16 </td>
 <td style="text-align:right;"> 5 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides </td>
 <td style="text-align:left;"> Ebihara_2007_251 </td>
 <td style="text-align:left;"> C1/C18 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides </td>
 <td style="text-align:left;"> Fujimoto_070106_2 </td>
 <td style="text-align:left;"> C1/C18 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Nakato_2953 </td>
 <td style="text-align:left;"> C10/C18 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Hyodo_2837 </td>
 <td style="text-align:left;"> C1/C12/C18 </td>
 <td style="text-align:right;"> 5 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Ebihara_3249_3 </td>
 <td style="text-align:left;"> C11/C12/C18/C4 </td>
 <td style="text-align:right;"> 5 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Nakato_3083 </td>
 <td style="text-align:left;"> C1/C12/C18 </td>
 <td style="text-align:right;"> 5 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. glanduligera </td>
 <td style="text-align:left;"> Ebihara_3312 </td>
 <td style="text-align:left;"> C12 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. glanduligera </td>
 <td style="text-align:left;"> Ebihara_3266 </td>
 <td style="text-align:left;"> C12/C7 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. glanduligera </td>
 <td style="text-align:left;"> Nakato_2859 </td>
 <td style="text-align:left;"> C12/C15/C16 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. glanduligera </td>
 <td style="text-align:left;"> Nakato_3029 </td>
 <td style="text-align:left;"> C12/C16 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. glanduligera </td>
 <td style="text-align:left;"> Nakato_3030 </td>
 <td style="text-align:left;"> C12/C14/C16 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. glanduligera </td>
 <td style="text-align:left;"> Nakato_3040 </td>
 <td style="text-align:left;"> C12/C13/C8 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. glanduligera </td>
 <td style="text-align:left;"> Nakato_3046 </td>
 <td style="text-align:left;"> B1/C12 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. miyagii </td>
 <td style="text-align:left;"> Nakato_2861 </td>
 <td style="text-align:left;"> C18 </td>
 <td style="text-align:right;"> 2 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. miyagii </td>
 <td style="text-align:left;"> Ebihara_3197_b </td>
 <td style="text-align:left;"> C18/C19/C21/C22 </td>
 <td style="text-align:right;"> 2 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> Thelypteris_kunthii </td>
 <td style="text-align:left;"> NA </td>
 <td style="text-align:left;"> A1 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
</tbody>
</table>

]

---
class: small-margins

.left-tree[
![](index_files/figure-html/PGIC_tree-1.svg)

.small[RAxML, 1000 bootstrap replicates]
]
.right-table[<table class="table table-striped table-condensed" style="font-size: 9px; margin-left: auto; margin-right: auto;">
 <thead>
 <tr>
 <th style="text-align:left;"> Taxon </th>
 <th style="text-align:left;"> Specimen </th>
 <th style="text-align:left;"> Formula </th>
 <th style="text-align:right;"> Ploidy </th>
 </tr>
 </thead>
<tbody>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Ebihara_3199_f </td>
 <td style="text-align:left;"> C3/C4/C5/C6 </td>
 <td style="text-align:right;"> 3 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_3069 </td>
 <td style="text-align:left;"> C2 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_2952 </td>
 <td style="text-align:left;"> C2/C8 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_3028 </td>
 <td style="text-align:left;"> B1/B2/C3 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_3036 </td>
 <td style="text-align:left;"> C2/C5/C6 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_3072 </td>
 <td style="text-align:left;"> C2/C3 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Ebihara_2866 </td>
 <td style="text-align:left;"> C2 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Ebihara_2823 </td>
 <td style="text-align:left;"> C3/C4 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons × glanduligera </td>
 <td style="text-align:left;"> Nakato_2855 </td>
 <td style="text-align:left;"> C2/C3/C5/C6 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons × glanduligera </td>
 <td style="text-align:left;"> Nakato_3024 </td>
 <td style="text-align:left;"> C3/C5/C6 </td>
 <td style="text-align:right;"> 5 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons × glanduligera </td>
 <td style="text-align:left;"> Nakato_3025 </td>
 <td style="text-align:left;"> C2/C3/C5/C6 </td>
 <td style="text-align:right;"> 5 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides </td>
 <td style="text-align:left;"> Ebihara_3250_3 </td>
 <td style="text-align:left;"> B1/B4/C1/C9 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides </td>
 <td style="text-align:left;"> Ebihara_2007_251 </td>
 <td style="text-align:left;"> B1/C2/C3 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides </td>
 <td style="text-align:left;"> Fujimoto_070106_2 </td>
 <td style="text-align:left;"> B1/C2 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Nakato_2953 </td>
 <td style="text-align:left;"> B1/C3 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Hyodo_2837 </td>
 <td style="text-align:left;"> B1/B2/C3 </td>
 <td style="text-align:right;"> 5 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Ebihara_3249_3 </td>
 <td style="text-align:left;"> B1/C3 </td>
 <td style="text-align:right;"> 5 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Nakato_3083 </td>
 <td style="text-align:left;"> B1/B3/C7/C8 </td>
 <td style="text-align:right;"> 5 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. glanduligera </td>
 <td style="text-align:left;"> Nakato_2859 </td>
 <td style="text-align:left;"> C5 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. glanduligera </td>
 <td style="text-align:left;"> Nakato_3030 </td>
 <td style="text-align:left;"> C3 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. glanduligera </td>
 <td style="text-align:left;"> Nakato_3040 </td>
 <td style="text-align:left;"> C5 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. glanduligera </td>
 <td style="text-align:left;"> Ebihara_3266 </td>
 <td style="text-align:left;"> C4/C5 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. glanduligera </td>
 <td style="text-align:left;"> Nakato_3050 </td>
 <td style="text-align:left;"> C4/C5/C6 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. miyagii </td>
 <td style="text-align:left;"> Ebihara_3194 </td>
 <td style="text-align:left;"> C3 </td>
 <td style="text-align:right;"> 2 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. miyagii </td>
 <td style="text-align:left;"> Nakato_2861 </td>
 <td style="text-align:left;"> C3 </td>
 <td style="text-align:right;"> 2 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. sp0 </td>
 <td style="text-align:left;"> NA </td>
 <td style="text-align:left;"> B1 </td>
 <td style="text-align:right;"> 3 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> Thelypteris_kunthii </td>
 <td style="text-align:left;"> NA </td>
 <td style="text-align:left;"> A1 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
</tbody>
</table>

]

---
exclude: true
class: small-margins

.left-tree[
![](index_files/figure-html/ebihara_PGIC_tree-1.svg)

.small[RAxML, 1000 bootstrap replicates]
]
.right-table[<table class="table table-striped table-condensed" style="font-size: 8px; margin-left: auto; margin-right: auto;">
 <thead>
 <tr>
 <th style="text-align:left;"> Taxon </th>
 <th style="text-align:left;"> Specimen </th>
 <th style="text-align:left;"> Formula </th>
 <th style="text-align:right;"> Ploidy </th>
 </tr>
 </thead>
<tbody>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Ebihara_3199_f </td>
 <td style="text-align:left;"> B5/C15 </td>
 <td style="text-align:right;"> 3 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_2952 </td>
 <td style="text-align:left;"> C17/C18/C5 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Ebihara_3313 </td>
 <td style="text-align:left;"> C15/C17 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Nakato_2998 </td>
 <td style="text-align:left;"> B3/B5/C15/C17 </td>
 <td style="text-align:right;"> 6 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Ebihara_2823 </td>
 <td style="text-align:left;"> B1/B5/C13/C17 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Ebihara_3199_a </td>
 <td style="text-align:left;"> B5/C13 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. angustifrons </td>
 <td style="text-align:left;"> Ebihara_2008_1714 </td>
 <td style="text-align:left;"> B2/B5/C3 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides </td>
 <td style="text-align:left;"> Ebihara_2007_251 </td>
 <td style="text-align:left;"> C17/C3 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides </td>
 <td style="text-align:left;"> Fujimoto_070106_2 </td>
 <td style="text-align:left;"> C17/C3 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides </td>
 <td style="text-align:left;"> Ebihara_3199_d </td>
 <td style="text-align:left;"> C10/C14 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides </td>
 <td style="text-align:left;"> Ebihara_3198_b </td>
 <td style="text-align:left;"> C14/C9 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides </td>
 <td style="text-align:left;"> Ebihara_3250_4 </td>
 <td style="text-align:left;"> C10/C12/C13/C3 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides </td>
 <td style="text-align:left;"> Ebihara_3251 </td>
 <td style="text-align:left;"> C13/C3 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Nakato_2953 </td>
 <td style="text-align:left;"> C15/C3 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Hyodo_2837 </td>
 <td style="text-align:left;"> C13/C3/C9 </td>
 <td style="text-align:right;"> 5 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Ebihara_3249_3 </td>
 <td style="text-align:left;"> C13/C3 </td>
 <td style="text-align:right;"> 5 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Ebihara_3198_a </td>
 <td style="text-align:left;"> C11/C13/C17 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Ebihara_3250_1 </td>
 <td style="text-align:left;"> C13/C2/C3/C5 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Ebihara_3250_2 </td>
 <td style="text-align:left;"> C1/C3/C6 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Ebihara_3250_7 </td>
 <td style="text-align:left;"> B4/B5/C15/C4 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. cystopteroides × angustifrons </td>
 <td style="text-align:left;"> Hyodo_2839 </td>
 <td style="text-align:left;"> C15/C3 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. glanduligera </td>
 <td style="text-align:left;"> Ebihara_3312 </td>
 <td style="text-align:left;"> C15/C3/C7/C8 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. glanduligera </td>
 <td style="text-align:left;"> Ebihara_3266 </td>
 <td style="text-align:left;"> B5/C15/C3 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. glanduligera </td>
 <td style="text-align:left;"> Nakato_2859 </td>
 <td style="text-align:left;"> C15 </td>
 <td style="text-align:right;"> 4 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. miyagii </td>
 <td style="text-align:left;"> Ebihara_3197_b </td>
 <td style="text-align:left;"> C13/C16 </td>
 <td style="text-align:right;"> 2 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. miyagii </td>
 <td style="text-align:left;"> Ebihara_3194 </td>
 <td style="text-align:left;"> C13 </td>
 <td style="text-align:right;"> 2 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> P. miyagii </td>
 <td style="text-align:left;"> Nakato_2861 </td>
 <td style="text-align:left;"> C13/C3 </td>
 <td style="text-align:right;"> 2 </td>
 </tr>
 <tr>
 <td style="text-align:left;"> Thelypteris_kunthii </td>
 <td style="text-align:left;"> NA </td>
 <td style="text-align:left;"> A1 </td>
 <td style="text-align:right;"> NA </td>
 </tr>
</tbody>
</table>

]

---
# Sequence Capture Sampling
<img src="index_files/figure-html/specimen_map-1.svg" width="120%" style="display: block; margin: auto;" />

---
# Spore Sizes
<img src="index_files/figure-html/spore_size_plot-1.svg" width="120%" />

---
# DNA yield from herbarium specimens
<img src="index_files/figure-html/dna_conc-1.svg" width="120%" />

---
# Conclusions

Pacbio + PURC success varies by gene

- _COP9_  ✓ 
- _PGIC_ ✓ 
- _DUF1077_ ▵
- _IBR3_ ×

Different genes have different evolutionary histories

Diploid *P. glanduligera* quite rare

Sampling herbarium specimens should provide greater insight

---
class: center
# Acknowledgements

.pull-left-narrow[
![](https://ce8f5f2ec.cloudimg.io/width/100/n/http://demo.joyhsu.com/tfri/wp-content/uploads/2018/04/AFS2018-LOGO.jpg)
]

.pull-right-wide[
.asia-ferns[ASIAN SYMPOSIUM OF FERNS & LYCOPHYTES]
]

![](https://ce8f5f2ec.cloudimg.io/width/700/n/http://web.cla.kobe-u.ac.jp/group/Promis/_src/30890/jsps_logo.jpg)

![](https://ce8f5f2ec.cloudimg.io/width/700/n/http://pe.ibcas.ac.cn/images/title.gif)

Motomi Ito, Li-Yaung Kuo, Fay-Wei Li, Zhongyang Li, Carl Rothfels, Xinping Qi, Haruyo Yamaguchi