motomi ito , hiraku yoshitake , atsushi ebihara & …asiahorcs symposium motomi ito1, hiraku...

ASIAHORCs Symposium

Motomi Ito1, Hiraku YOSHITAKE2, Atsushi Ebihara3 & Utsugi Jinbo1

1The University of Tokyo, JAPAN2National Institute for Agro-Environmental Sciences, JAPAN

3National Museum of Nature and Science, Japan

Hierarchy of Biodiversity

Ecosystem

Species

Gene

Sp. 1 Sp. 2

Gene 1

Why Genetic Diversity?1. There are so many Species Concepts, and

sometimes delimitation of species are controversial.

2. Morphological Species are not always equal to evolutionary units.

3. Cryptic Species are often reported.4. In plants, hybridization is often occurred. 5. Variation of alleles are potential genetic

resources.

Yatabe, Darnadedi and Murakami 2002 in JPR

Cryptic Species of Bird-Nest Fern

Cryptic species in Ectropis crepuscularia

Cryptomeria type ♂

Broad-leaf tree type ♂

Difference in Barcode Area (COI)

Among types：33bp(5.5%)In each type ：2bp(0.3%)

Jinbo et al. in prep.





resources.

Aster ageratoidesKalimeris pinnatifida

=Aster iinumae

Aster microcephalus

Hybridization

Doubling of chromosomes

Birth of New Species via Hybridization





resources.

Why Genetic Diversity?

Thus, we need examine and grasp genetic diversity for better understanding the biodiversity

1. Polyploid Complex of Vandenboschia radicans (Filmy fern)

2. Genetic differentriation of Wagnerinus Weevils

Case Studies of Genetic Level Biodiversity in East Asia

Vandenboschia radicans (Hymenophyllaceae)

METHODSPlastid DNA: rbcL genePloidy: FCM (fluorescence flow cyto-meter)

+ Nuclear DNA: gapc sequences(glyceraldehyde 3-phosphate dehydrogenase c)c.600bp including 2 introns

Vandenvoschia radicans complex1. Vandenvoschia is genus of filmy fern (Hymenophyllaceae)1. Huge morphological variation and consisting of polyploid complex.2. Wide distribution area3. Hybridizations evidently would occur in V. radicans complex.

(If so, which species are the parents?)

Morphological Variation・Morphological “species”

C. birmanicum（ハイホラゴケ）

C. amabile（ヒメハイホラゴケ）

C. naseanum（オオハイホラゴケ)

C. subclathratum（コケハイホラゴケ）

C. birmanicum C. naseanum

C. subclathratum

C. amabile

Chromosomes in meiosis of the Diploid sample. (a) Light micrograph. (b) Diagram of chromosomes in (a). Scale bar = 10 µm

Examples of peaks in fluorescence for three cytotypes (2x, 3x, and 4x) of Vandenboschia radicans complex from analyses with flow cytometer

V. radicans-complex A1-type

V. radicans-complex A2-type

V. radicans-complex B-type

T speciosum (France)

T davallioides (Hawaii)

V. radicans-complex C-type

V. radicans (Bolivia)

T maximum (Malaysia)

T johnstonensis (Australia)

T auriculatum (Japan)

T rupestre (Bolivia)

9864

9988

45

9023

100

99

0.005

rbcL tree (Chloroplast Genome, maternal inheritance)

Phylogenetic tree based on chloroplast rbcL sequences of the genus Vandenboschia. Numbers at the nodes show posterior probability for supporting each clade.

Primer positions on the GapCp gene of the Vandenboschia radicans complex

For examine variation in nuclear genome, we developed primers amplifying a partial GapCp gene including introns.

Because diploids with heterozygous at this gene locus have two different alleles, we employ SSCP methods with acrylamide gel for detecting and separating the bands.

After running, we cut each band and extract DNA.

Cut a band and extract DNA

The result of SSCP (Single Strand Conformation Polymorphisms)

3ｘA2birm.Mie

2ｘA1subc.Is. Iriomote

2ｘ?Cnase.Taiwan

2ｘA1birm.Mie

3ｘBamab.Niigata

3ｘA1birm.Is. Iriomote

?A1birm.Is. Chichi

3ｘ?Cbirm.Is. Haha

?Cnase.Kagosima

3ｘBbirm.Chiba

4ｘCnase.Is. Iriomote

4ｘA2amab.Shimane

PlioidyChloroplast

Morph. sp.Locality

The band patterns strongly suggest occurring of hybridizations between several parental species.

H13-4 6H46-4 5H46-1

H53-1H13-1 3H46-3

T.radicans (Bolivia)W3-2

H51-2W3-1H26-2

H6H53-2H13-2 5H2-3H36H20

H51-3H26-3

H2-2T. auriculatum (Outgroup)

87

79

6497

6894

93

8362

100

56

0.02

C-type(Southern type)

A-type(widespread)

B-type(Northern type)

H13(3X・Niigata) = ABBH53(4X・Shimane) = AB**

H26(3X・Iriomote) = AC*H51(4X・Iriomote) = AC**

: maternal lineage

gapc tree (Nuclear Genome, biparental inheritance)

Examples of hybridization in JAPAN

A consensus phylogram resulting from Bayesian analysis of nuclear GapCp sequences identified from the Vandenboschia radicanscomplex of Japan and adjacent areas and V. liukiuensis. Numbers at the nodes are the posterior probabilities for the support of each clade.

Group A seqs

Group B seqsGroup D seqs+ V. liukiuensis

Group C seqs

C

C

○A1○C A1△

△B

A1

C△

A2A1△○

A1△BB

A2C

△△△△

△

△

△

□

○

○

○

△A2A2△

A2A1

Distribution of Vandenboschia radicans -complex in Japan

・Morphological “species”●： C. birmanicum（ハイホラゴケ）

●： C. amabile（ヒメハイホラゴケ）

●： C. naseanum（オオハイホラゴケ)

●： C. subclathratum（コケハイホラゴケ）

・Ploidy-level○： 2X△： 3X□： 4X (sexual)

： 4X (meiosis irregular)： 4X (not investigated)

・Chloroplast DNA typeA1 / A2 / B / C

△△△C△B

△C

○

△

△

A2

A hypothesized process of reticulate evolution in the Japanese Vandenboschia radicans complex. All the fronds are fertile. The bold line shows chromosome doubling, the broken line shows polyploidization that concerns unreduced spores, and the dotted line shows alternative hybridization process of the genotype αßγ.

● α genome● β genome● γ genome

Distribution of Diploids

α genome β genome γ genome

Estimation of Potential Distribution Area using Ecological Niche Modeling

Based on climate data, and for ENM, we use Maxent.

Example 2:

An example of genetically and ecologically diversified species

Evolution of Obligatory Cecidophagy in the Genus Wagnerinus Korotyaev(Coleoptera: Curculionidae)

Wagnerinus Weevil Weigela hortensis

This result is by-product of the study:

Cecidophage --- An organism that exhibits cecidophagy

Cecidophagy & Cecidophages

Cecidophagy --- Gall-eating habit

W. costatus larvaWagnerinus costatus

Weigela hortensis

Asphondylia baca

Gall induced byA. baca

Wagnerinus costatus

Cecidophagous Habit by the Weigela weevil

Oviposition

W. costatus larva

OvipositionGall formation

1. W. carinulatus Russian Far East2. W. sp. n. 1 Japan: Hokkaido3. W. sp. n. 2 Japan: Honshu4. W. sp. n. 3 Japan: Honshu5. W. sp. n. 4 Japan: Sado Is.6. W. sp. n. 5 Japan: Honshu7. W. costatus Japan: Honshu8. W. sp. n. 6 Japan: Honshu9. W. sp. n. 7 Japan: Honshu

10. W. sp. n. 8 Japan: Shikoku11. W. sp. n. 9 South Korea12. W. shikotanus Japan: S Kuriles13. W. harmandi Japan: Honshu14. W. sp. n. 10 Japan: Honshu15. W. sp. n. 11 Japan: Honshu16. W. sp. n. 12 Japan: Shikoku

Species Distribution Wagnerinus is composedof 16 species in 2 groups(Yoshitake et al., unpubl.)

W. carinulatus

W. costatus W. harmandi

W. sp. n. 3

Morphological Classification

carinulatus-groupharm

andi-group

1. W. carinulatus Unknown Unknown2. W. sp. n. 1 Weigela Seed capsule3. W. sp. n. 2 Midge gallWeigela4. W. sp. n. 3 Weigela Midge gall5. W. sp. n. 4 Unknown Unknown6. W. sp. n. 5 Midge gallWeigela7. W. costatus Midge gallWeigela8. W. sp. n. 6 Unknown Unknown9. W. sp. n. 7 Weigela Unknown

10. W. sp. n. 8 Unknown Unknown11. W. sp. n. 9 Unknown Unknown12. W. shikotanus Unknown Unknown13. W. harmandi Abelia Flower bud14. W. sp. n. 10 Weigela Unknown15. W. sp. n. 11 Abelia Unknown16. W. sp. n. 12 Abelia Unknown

- Host plants of 10 spp.and larval foods of 6 spp. have been confirmed(Yoshitake et al., unpubl.)

Available Ecological InformationSpecies Host plant Larval food resource


andi-group

- They exhibit different waysof host plant utilization,feeding on flower buds, midge galls & seed capsules during their larval stage

- Wagnerinus weevils are associated at least with 2 plant genera: Weigela & Abelia (Caprifoliaceae)

Objectives

1. To gather additional ecological data, collectingflesh samples for molecular studies

2. To investigate phylogenetic relationships among Wagnerinus weevils based on molecular data

To understand the evolution of host plant utilization patterns within Wagnerinus containing obligatory cecidophages …

- The evolutionary histories in host plant utilization among Wagnerinus weevilswere reconstructed on phylogenies under parsimony, using the character tracing tools of MESQUITE 1.11 (Maddison & Maddison, 2006)

- A 1,425bp fragment of mitochondrial cytochrome oxidase subunit I (COI) gene was sequenced for the molecular phylogenetic analysis

- An unweighted parsimony analysis of a molecular dataset (OTU: 48) was conducted with PAUP* 4.0b10 (Swofford, 2003)

- From 2005 to 2006, fundamental ecological data and flesh samples for molecular studies were collected through field surveys at 32 sites in Japan, Korea & the Russian Far East

Molecular Phylogenetic Analysis

Field surveys

2. Materials & Methods

1. W. carinulatus Weigela Flower bud2. W. sp. n. 1 Weigela Seed capsule3. W. sp. n. 2 Midge gallWeigela4. W. sp. n. 3 Weigela Midge gall5. W. sp. n. 4 Weigela Midge gall6. W. sp. n. 5 Midge gallWeigela7. W. costatus Midge gallWeigela8. W. sp. n. 6 Weigela Midge gall9. W. sp. n. 7 Weigela Flower bud & Midge gall

10. W. sp. n. 8 Weigela Flower bud & Midge gall11. W. sp. n. 9 Weigela Flower bud12. W. shikotanus Unknown Unknown13. W. harmandi Abelia Flower bud14. W. sp. n. 10 Weigela Flower bud15. W. sp. n. 11 Abelia Flower bud16. W. sp. n. 12 Abelia Flower bud

Fundamental Ecological InformationSpecies Host plant genus Larval food resources

Host plants & larvalfood resources of 15spp. were confirmed


andi-group

- The host range of Wagnerinus is limited in Caprifoliaceae

Type 1: Weigela Type 2: Abelia

11 spp. of the carinulatus-group 3 spp. of the harmandi-group1 sp. of the harmandi-group

Host Plant Association

- Each species is mono- or stenophagous- Host plant associations can be grouped into two types

3: Seed Capsule

1 sp.

capsuleLarva

2: Midge Gall

6 spp.

GallLarva

- There are three kinds of larval food resources

1: Flower Bud

Larva

Flower bud

6 spp. 2 spp.

Host Plant Utilization

- Each species utilizes a certain organ of its host plant during larval stage

- Two species can utilize both of flower bud & midge gall

How the obligatory cecidophagy has evolved in this genus?

Host Utilization of Wagnerinus Weevils

- Host utilization of Wagnerinus weevils can be divided into four types:

Type 1. Flower bud feeding

Type 2. Flower bud & Gall feeding (= Facultative cecidophagy)

Type 3. Gall feeding (= Obligatory cecidophagy)

Type 4. Seed capsule feeding

Cecidophages can be seen only in the carinulatus-group

Wagnerinus carinulatus (Kedrovaya Pad, Russian Far East)Wagnerinus carinulatus (Arimkovka River, Russian Far East)

Wagnerinus carinulatus (Kedrovaya Pad, Russian Far East)

Wagnerinus sp. n. 9 (Mt. Wollaksan, Korea)Wagnerinus sp. n. 9 (Mt. Sudosan, Korea)

Wagnerinus sp. n. 9 (Mt. Jirisan, Korea)

Wagnerinus sp. n. 5 (Nasu, Tochigi)

Wagnerinus sp. n. 8 (Mt. Tsurugisan, Tokushima)Wagnerinus sp. n. 8 (Mt. Tsurugisan, Tokushima)Wagnerinus sp. n. 8 (Mt. Tsurugisan, Tokushima)Wagnerinus sp. n. 8 (Mt. Takashiroyama, Tokushima)

Wagnerinus sp. n. 4 (Sado Is., Niigata)

Wagnerinus sp. n. 7 (Mt. Andosan, Wakayama)Wagnerinus sp. n. 7 (Mt. Andosan, Wakayama)Wagnerinus sp. n. 7 (Mt. Gomadanzan, Wakayama)Wagnerinus sp. n. 7 (Mt. Gomadanzan, Wakayama)

Wagnerinus sp. n. 7 (Mt. Gomadanzan, Wakayama)

Wagnerinus sp. n. 6 (Otarionsen, Nagano)Wagnerinus sp. n. 6 (Otarionsen, Nagano)Wagnerinus costatus (Mt. Nougou-Hakusan, Gifu)

Wagnerinus sp. n. 1 (Aizankei, Hokkaido)Wagnerinus sp. n. 1（Yukomanbetsu, Hokkaido)

Wagnerinus sp. n. 1 (Horoshikatouge, Hokkaido)Wagnerinus sp. n. 2 (Mt. Iwakisan, Aomori)

Wagnerinus sp. n. A (Fuji-subaruline, Yamanashi)Wagnerinus sp. n. A (Fuji-subaruline, Yamanashi)

Wagnerinus sp. n. 12 (Mt. Tsurugisan, Tokushima)Wagnerinus sp. n. 12 (Mt. Tsurugisan, Tokushima)

Wagnerinus sp. n. 10 (Mt. Daigatake, Hakone, Kanagawa)Wagnerinus sp. n. 10 (Mt. Daigatake, Hakone, Kanagawa)Wagnerinus sp. n. 10 (Mt. Chausuyama, Toyone, Aichi)Wagnerinus sp. n. 10 (Taroubou, Gotenba, Shizuoka)Wagnerinus sp. n. 10 (Taroubou, Gotenba, Shizuoka)Wagnerinus harmandi (Mt. Komagatake, Hakone, Kanagawa)Wagnerinus harmandi (Nasu-kougen, Tochigi)

Wagnerinus sp. n. D (Mt. Ishizuchi, Ehime)Wagnerinus sp. n. D (Mt. Ishizuchi, Ehime)

Wagnerinus sp. n. 2 (Mt. Hakkoudasan, Aomori)Wagnerinus sp. n. 3 (Kotamagawa, Oguni, Yamagata)

Cyphosenus grouvellei (outgroup)

Wagnerinus sp. n. 2 (Mt. Iwakisan, Aomori)

Wagnerinus sp. n. 10 (Mt. Daigatake, Hakone, Kanagawa)

Wagnerinus sp. n. C (Otarionsen, Nagano)Wagnerinus sp. n. C (Otarionsen, Nagano)Wagnerinus sp. n. B (Toyosato, Koumi, Nagano)Wagnerinus sp. n. B (Itabashi, Minamimaki, Nagano)Wagnerinus sp. n. B (Itabashi, Minamimaki, Nagano)Wagnerinus sp. n. B (Hikawarindou, Yamanashi)Wagnerinus sp. n. B (Mt. Takahachiyama, Shizuoka)

98

73

100

99

100

56

100

9885

63

99

67

71

97

8467

92

100

92

10099

85

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100

85

77

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6495

98

64

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64

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82

100

Molecular Phylogeny of WagnerinusStrict consensus of 4 MP trees inferred from COI sequence data with bootstrap values(TL: 1,105; CI: 0.618; RI: 0.841)

The topology agrees well with classification based onmorphological features

W. sp. n. A

W. sp. n. B

W. sp. n. C

W. sp. n. D

carinulatus-groupharmandi-group

The topology agrees well with classification based onmorphological features


















Cyphosenus grouvellei (outgroup)




98

73

100

99

100

56

100

98

85

63

99

67

71

97

8467

92

100

92

10099

85

92

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100

85

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6495

98

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82

100

W. sp. n. A

W. sp. n. B

W. sp. n. C

W. sp. n. D

Potential New Species were found by Genetic Analysis

Evolution of Host Association in Wagnerinus





















Weigela-feeding

Abelia-feeding

- The ancestral state for Wagnerinuswas Abelia-feeding

Abelia→Weigela

Abelia→Weigela

- Weigela-feeding occurredtwice within this genus

harmandi-group

carinulatus-group

Evolution of Host Utilization in Wagnerinus





















Flower bud feeding

Obligatory cecidophagy

Facultative cecidophagy

Seed capsule feeding

Cecidophagy

- The ancestral state reconstruction on the molecular phylogenysuggests that the plesiotypicstate for Wagnerinus wasflower bud feeding

harmandi-group

carinulatus-group





















- After the evolution of cecidophagy, a specialization to flower budoccurred


Flower bud feeding

Cecidophagy→Flower bud feeding

Cecidophagy

harmandi-group

carinulatus-group

Summary• Analysis of genetic variation is important to

understanding biodiversity both in local and global scale.• It is not enough to monitoring and/or to conserve a

species, because genetic differentiation has often occurred or sometimes reproductive isolation had already occurred among local populations.

• Thus, understanding genetic level of biodiversity is necessary both from interest of basic science and conservation of biodiversity.

• Now, accessing to genetic diversity is not difficult, owing to rapid development of Bio-Technology and Information Technology

AckonwkedgementVandenboschia filmy fern• Atsushi Ebihara• Hirosi Ishikawa • Sadamu Matsumoto• Su-Juan Lin• Kunio Iwatsuki• Masayuki Takamiya• Yasuyuki Watano

Wagnerinus weevils • Hiraku Yoshitake• Toshihide Kato• Naoko Nakahara

In General (for Financial Support)

• JSPS• MEXT• GBIF• JST





















- Seed capsule feeding occurred once after the evolution of obligatory cecidophagy

obligatory cecidophagy→seed capsule feeding


Seed capsule feeding

harmandi-group

carinulatus-group





















Evolution of cecidophagy(obligatory or facultative)

- Cecidophagy occurred oncewithin the carinulatus-groupObligatory cecidophagy: 1-3 timesFacultative cecidophagy: 1-3 times


Obligatory cecidophagy

Facultative cecidophagy

Cecidophagy

harmandi-group

carinulatus-group

motomi ito , hiraku yoshitake , atsushi ebihara & …asiahorcs symposium motomi ito1, hiraku...

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