38 species 204 genera (fishbase)

Phylogenetic position of the Altaic fish

genus Oreoleuciscus in Leuciscinae

based on mitogenome sequences

Junichi ImotoJunichi Imoto11, Kenji Saitoh, Kenji Saitoh22, Jun Adachi, Jun Adachi33, Takeshi Sasaki, Takeshi Sasaki44, , Yuri P. KartavtsevYuri P. Kartavtsev55, Satoru N. Chiba, Satoru N. Chiba11, Ryo O. Gotoh, Ryo O. Gotoh11, , Masaki MiyaMasaki Miya55, Mutsumi Nishida, Mutsumi Nishida66 and Naoto Hanzawa and Naoto Hanzawa11

11Grad Sch Sci Eng, Yamagata Univ, Grad Sch Sci Eng, Yamagata Univ, 22Nat Res Inst Fish SciNat Res Inst Fish Sci, , 33Inst Stat Math, Inst Stat Math, 44Inst Marine Biol, Inst Marine Biol, 55Nat Hist Mus Inst Chiba, Nat Hist Mus Inst Chiba,

66Ocean Res Inst, Univ TokyoOcean Res Inst, Univ Tokyo

Phylogenetic position of the Altaic fish

genus Oreoleuciscus in Leuciscinae

based on mitogenome sequences

38 species 204 genera (FishBase)leuciscin: Eurasian plus North American Notemigonus cryleucas

48 species 319 genera (FishBase)phoxinin: North American and Far Eastern species 　

Phylogeny based on morphology (Cavender and Coburn, 1992)

Leuciscinae

Widely distributed in Northern Hemisphere

Morphologically similar (even among different genera)

Important to consider the evolutional history of Leuciscinae

Central Asia and Far East

The genus Oreoleuciscus 　 (Warpachowskii, 1889)

Endemic to inland lakes in western Mongolia and Tuva, the upper Ob River system within the Altai Mountain region of Siberia (Berg, 1962)

Planktonic algae, insects, invertebrates and fishes

Oreoleuciscus potanini

Feeding habitat

Distribution

Far E

asternE

uropeanN

orth Am

erican

ML tree based on mt genome

Phylogenetic position of Oreoleuciscus

Oreoleucuscus potanini

Closely related to Pseudaspius and Tribolodon ? (Howes, 1984, 1991)Tribolodon

sachalinensis

Tribolodon hakonensis

Pseudaspius leptocephalus

?

Tribolodon nakamurai

Far E

asternE

uropeanN

orth Am

erican




Closely related to Phoxinus ? (Travers, 1989; Bogutskaya, 1990)

Phoxinus phoxinusPhoxinus lagowskii Phoxinus perenurus

?

No molecular phylogenetic analyses for Oreoleuciscus species

High degree of morphological variability that obscures differences among species

Taxonomy of the genus Oreoleuciscus is controversial

Paucity of specimens from key geographical areas

Determine complete mitochondrial (mt) genome sequences of Oreoleuciscus

Conduct phylogenetic analysis based on mt genome sequences

Clarify the phylogenetic position of genus Oreoleuciscus and evolutional history of the Leuciscinae

The purpose of this study

Mt genome analyses

Long DNA sequences

(Inoue et al., 2001a; Miya et al., 2003; Saitoh et al., 2006)

Correct estimation of phylogeny at various taxonomic levels

with strong statistical support

17 genera 31 species

Outgroup 9 species (Cypridae 8, Cobitidae 1)

ML RAxML v7.0.4 (Stamatakis, 2006)

Bayes MrBayes v3.1.2 (Ronquist & Huelsenbeck 2003)

Determined mt genome sequences

Genomic structure Common with vertebrates

Exclude ND6, control region and gaps (14,826 bp)

Mt genome of Oreoleuciscus potanini

0.1

Phoxinus perenurus mantschricus Vladivostok RussiaPhoxinus perenurus sachalinensis Hokkaido Japan

100

Phoxinus perenurus Poland

100

Phoxinus oxycephalus jouyi Kochi JapanPhoxinus lagowskii Mongol

Phoxinus lagowskii steindachneri Mogami River Japan100

100

Oreoleuciscus potanini Altai

100

Tribolodon brandtii Ukedo River JapanTribolodon brandtii Mogami River Japan

Tribolodon nakamurai Mogami RIver Japan

100

Pseudaspius leptocephalus Lake Bolony RussiaTribolodon sachalinensis Tadami River JapanTribolodon hakonensis Mogami River Japan

100

100

Phoxinus phoxinus MongolPhoxinus phoxinus Italy100

Cyprinella lutrensisCyprinella spiloptera

100

Notropis stramineus

100

Phenacobius mirabilis

100

Campostoma anomalum

100

Mylocheilus caurinus

100

Gila robustaGila conspersa

100

Phoxinus eos

100

Alburnus alburnusLeucaspius delineatus

100

Notemigonus crysoleucasAbramis brama

Blicca bjoerkna100

Chondrostoma lemmingii

100

Pelecus cultratus

100

100

Tinca tincaPseudorasbora pumila

Sarcocheilichthys variegatus microoculus

100

Megalobrama terminalisXenocypris argentea

10

Far E

asternE

uropeanN

orth Am

erican

ML bootstrap / Bayesian posterior probabilities

0.1

Phoxinus perenurus mantschricus Vladivostok RussiaPhoxinus perenurus sachalinensis Hokkaido Japan

100

Phoxinus perenurus Poland

100

Phoxinus oxycephalus jouyi Kochi JapanPhoxinus lagowskii Mongol

Phoxinus lagowskii steindachneri Mogami River Japan100

100

Oreoleuciscus potanini Altai

100

Tribolodon brandtii Ukedo River JapanTribolodon brandtii Mogami River Japan

Tribolodon nakamurai Mogami RIver Japan

100

Pseudaspius leptocephalus Lake Bolony RussiaTribolodon sachalinensis Tadami River JapanTribolodon hakonensis Mogami River Japan

100

100

Phoxinus phoxinus MongolPhoxinus phoxinus Italy100

Cyprinella lutrensisCyprinella spiloptera

100

Notropis stramineus

100


100

Campostoma anomalum

100


100


100

Phoxinus eos

100


100

Notemigonus crysoleucasAbramis brama

Blicca bjoerkna100

Chondrostoma lemmingii

100

Pelecus cultratus

100

100

Tinca tincaPseudorasbora pumila

Sarcocheilichthys variegatus microoculus

100

Megalobrama terminalisXenocypris argentea

10

Far E

asternE

uropeanN

orth Am

erican

ML bootstrap / Bayesian posterior probabilities


Phoxinus lagowskii


0.1

Phoxinus perenurus mantschricusPhoxinus perenurus sachalinensis

99

Phoxinus perenurus

100

Phoxinus lagowskii steindachneriPhoxinus lagowskii

10031

Phoxinus oxycephalus jouyi

100


100

Tribolodon brandtii Ukedo RiverTribolodon brandtii Mogami River

68


100


74

Tribolodon sachalinensis

97

Tribolodon hakonensis100

100

Phoxinus phoxinus ItalyPhoxinus phoxinus Mongol100

97

Cyprinella lutrensisCyprinella spiloptera100

Notropis stramineus

100


100

Campostoma anomalum

100


100

86


100

Phoxinus eos

100

80


100

Notemigonus crysoleucas

66

Blicca bjoerknaAbramis brama

100

Chondrostoma lemmingii21

100

Pelecus cultratus100

100

Tinca tinca

95

Sarcocheilichthys variegatus mPseudorasbora pumila

100

85

Megalobrama terminalisCulter mongolicus

100

Xenocypris argentea

100

Zacco sieboldtii100

100

Labeo batesiiCyprinus carpio

100

100

Misgurnus nikolskyiCobitis striata

100

Lefua echigonia

100

100

Engraulis japonicusSardinops melanostictus

100

100

Astronotus ocellatusTropheus duboisi

46

Paratilapia polleni

100

Etroplus maculatusParetroplus maculatus

100100

Oryzias latipes

100

Takifugu rubripesTetraodon nigroviridis

100

43

Paralichthys oliva

L 100, U 120

L 112, U 145

L 85, U 95

U 363

Molecular Dating

(Mya)

Upper constraint ： U

Lower constraint ： L

Parametric Bayesian approach

Multidistribute (Thorn & Kishino 2002)

Cichlids

L 100, U 120

L 112, U 145

L 85, U 95

African

South American

Madagascar

Indo/Sri Lanka

Madagascar

Cichlid

Molecular Dating

(Mya)

Upper constraint ： U

Lower constraint ： L

(Azuma et al., 2008)

Phoxinus perenurus mantschricusVladivostok RussiaPhoxinus perenurus sachalinensisHokkaidao JapanPhoxinus perenurus PolandPhoxinus lagowskii MongolPhoxinus lagowskii steindachneriMogami River JapanPhoxinus oxycephalus jouyi Kochi JapanOreoleuciscus potanini AltaiTribolodon brandtii Ukedo River JapanTribolodon brandtii Mogami River JapanTribolodon nakamurai Mogami RIver JapanPseudaspius leptocephalus Lake Bolony RussiaTribolodon sachalinensis Tadami River JapanTribolodon hakonensis Mogami River JapanPhoxinus phoxinus MongolPhoxinus phoxinus ItalyCyprinella lutrensisCyprinella spilopteraNotropis stramineusPhenacobius mirabilisCampostoma anomalumMylocheilus caurinusGila robustaGila conspersaPhoxinus eosAlburnus alburnusLeucaspius delineatusNotemigonus crysoleucasAbramis bramaBlicca bjoerknaChondrostoma lemmingiiPelecus cultratusTinca tincaPseudorasbora pumilaSarcocheilichthys variegatus microoculusCulter mongolicusMegalobrama terminalisXenocypris argenteaZacco sieboldtiiCyprinus carpioLabeo batesii

Leuciscinae / other subfamily

Cyprinidae / Cobitidae

331.2

Otocephala / Euteleostei

219.2

123.6

Mesozoic CenozoicPaleozoic

Carboniferous

Triassic Jurassic Cretaceous TertiaryPermian

Phoxinus perenurus mantschricusVladivostok RussiaPhoxinus perenurus sachalinensisHokkaidao JapanPhoxinus perenurus PolandPhoxinus lagowskii MongolPhoxinus lagowskii steindachneri Mogami River JapanPhoxinus oxycephalus jouyi Kochi JapanOreoleuciscus potanini AltaiTribolodon brandtii Ukedo River JapanTribolodon brandtii Mogami River JapanTribolodon nakamurai Mogami RIver JapanPseudaspius leptocephalus Lake Bolony RussiaTribolodon sachalinensis Tadami River JapanTribolodon hakonensis Mogami River JapanPhoxinus phoxinus MongolPhoxinus phoxinus ItalyCyprinella lutrensisCyprinella spilopteraNotropis stramineusPhenacobius mirabilisCampostoma anomalumMylocheilus caurinusGila robustaGila conspersaPhoxinus eosAlburnus alburnusLeucaspius delineatusNotemigonus crysoleucasAbramis bramaBlicca bjoerkna

phoxinin / leuciscin

84.8

Far Eastern phoxinin / P. phoxinus

78.5

Orieoleuciscus / Far Eastern Phoxinus35.5

Tribolodon / Orieoleuciscus + Far Eastern Phoxinus48.6

Mesozoic Cenozoic

Cretaceous Pa. Eocene Oli. Miocene Plio

The genus Oreoleuciscus is included in phoxinin (Cavender & Coburn 1992)

rather than Tribodolon and Pseudaspius

Oreoleuciscus formed a monophyletic clade with the Far Eastern phoxinins

Conclusion

Oreoleuciscus is closely related to the Far Eastern Phoxinus

Tribolodon, Pseudaspius and the Far Eastern Phoxinus

Tribolodon + Pseudaspius and Oreoleuciscus + Phoxinus were divided 49 Mya

Oreoleuciscus and Phoxinus were divided 34 Mya

Thank you for your attention !!



The range of Oreoleuciscus demarcates a highly ranked biogeographical unit that has a very depauperate and distinct ichthyofauna.

In the review of Eurasian cyprinid biogeography, Banarescu & Coad (1991) determined that the rank of this unit is equal to that of regions such as the entire Euro-Mediterranean or Central Asian regions.


Far E

asternE

uropeanN

orth Am

erican

1.81 ~ 4.37 Mya Tribolodon speciation

The glacial period

12.90 ~ 15.41 Mya Radiation of Leuciscinae

Rapid global cooling of Middle Miocene

The sea level dropped

The land area expansion, freshwater and brackish water area increased

Environment habitable for Leuciscinae extended

Enlarge their distribution and occurred speciation

Far E

asternE

uropeanN

orth Am

erican


Ancestral area of the Far Eastern Leuciscinae



The range of Oreoleuciscus demarcates a highly ranked biogeographical unit that has a very depauperate and distinct ichthyofauna.

In the review of Eurasian cyprinid biogeography, Banarescu & Coad (1991) determined that the rank of this unit is equal to that of regions such as the entire Euro-Mediterranean or Central Asian regions.

10

Tribolodon brandtii UKETribolodon brandtii MGMTribolodon nakamuraiPseudaspius leptocephalusTribolodon sachalinensisTribolodon hakonensisPhoxonus perenurus sachalinensisPhoxonus perenurus mantschricusPhoxonus perenurusPhoxinus oxycephalus jouyiPhoxinus lagowskii steindachneriPhoxinus lagowskiiPhoxinus phoxinus MONPhoxinus phoxinus ITACyprinella lutrensisNotropis stramineusMylocheilus caurinusGila conspersaPhoxinus eosAlburnus alburnusLeucaspius delineatusNotemigonus crysoleucasAbramis bramaPelecus cultratusTinca tincaSarcocheilichthys variegatus microoculusMegalobrama treminalisXenocypris argenteaCyprinus carpioLabeo batesiiCobitis striataLefua echigoniaEngraulis japonicusSardinops melanostictusAstronotus ocellatusTropheus duboisiParatilapia polleniEtroplus maculatusParetroplus maculatusOryzias latipesParalichthys olivaceusTakifugu rubripesTetraodon nigroviridisGasterosteus aculeatus

100 0200

中世代

第三紀

第四紀古世代

白亜紀ジュラ紀三畳紀ペルム紀

(Mya)

ウグイ亜科と他の亜科

コイ科とドジョウ科

271.0

ニシン・骨鰾下区と正新骨下区

189.7

100.3

化石記録よりも古い年代

コイ科魚類　最古の化石Parabarbus sp. ca. 50

Mya

24

ユーラシアおよび北米に広く分布するコイ科ウグイ亜科魚類は、主にユーラシアに分布する leuciscin と、主に北米および極

東に分布する phoxinin の 2 つの系統に大別される。

ウグイ亜科魚類の系統地理学的考察を行うためには、 2 つの系統群が接する極東から中央ユーラシアにかけて分布するタクソ

ンのサンプリングが必要と考えられ、発表者らは、アルタイ共和国においてサンプリングを行い、ウグイ亜科 Oreoleuciscus

属魚類を採集した ( 千葉ら , 2008) 。

Oreoleuciscus 属魚類は中央アジア・オビ川上流のアルタイ山地、モンゴルの一部にのみ分布する非常に希少な魚類である。

Howes (1984, 1991) は、形態的特徴から Oreoleuciscus 属は Pseudaspius 属やウグイ属と共に aspinin の系統に含まれる

とした。

Howes (1984, 1991) 　ウグイ亜科を Abramin 、 Aspinin 、 Phoninin の 3 つに分類

しかし、 Oreoleuciscus 属は形態的に Phoxinus 属に近いという考えもあり (Travers, 1989; Bogutskaya, 1990) 、未だ

に系統的位置は不明だが、これまで分子系統解析は全く行われていない。

そこで、我々は Oreoleuciscus potanini のミトコンドリアゲノムを決定して、他のウグイ亜科魚類とともに分子系統解析を

行った。

その結果、 Oreoleuciscus 属は極東産 Phoxinus 属の姉妹群となり、これらにウグイ属を加えたクレードは単系統群を形成し、

他の欧米産ウグイ亜科魚類各クレードからは大きく離れていた。

本研究の結果は、ウグイ亜科魚類の進化史を考察する上で重要なデータと考えられる。

10


100 0200

中世代

第三紀

第四紀古世代

白亜紀ジュラ紀三畳紀ペルム紀

(Mya)

コイ科とドジョウ科

271.0

ニシン・骨鰾下区と正新骨下区

189.7

化石記録よりも古い年代

コイ科魚類　最古の化石Parabarbus sp. ca. 50

Mya分子を用いた年代推定とほぼ一致

(Inoue et al., 2005; Yamanoue et al., 2006; Azuma et al., 2008)

10


中世代新生代

白亜紀ジュラ紀

暁新生

始新生

漸新世

中新生

鮮新世

100 0 (Mya)

phoxinin / leuciscin

74.7

Far Eastern phoxinin / P. phoxinus

61.7

Orieoleuciscus / Far Eastern Phoxinus

21.2Tribolodon / Orieoleuciscus + Far Eastern Phoxinus

39.2

イタリアモンゴル

最上川請戸川

10


中世代新生代

白亜紀ジュラ紀

暁新生

始新生

漸新世

中新生

鮮新世

100 0 (Mya)

7.5 ～ 21.2 Mya Tribolodon 属各種が種分化

寒冷化・乾燥化

陸地が拡大、淡水域や汽水域などが増える

ウグイ亜科魚類が生息できる環境が広がる

新しい生息場所に分布を広げながら種分化

海水面の低下

7.5

21.2

イタリアモンゴル

最上川請戸川

Closely related to Pseudaspius and Tribolodon ? (Howes, 1984, 1991)

Closely related to Phoxinus ? (Travers, 1989; Bogutskaya, 1990)

Tribolodon sachalinensis

Tribolodon hakonensis

Tribolodon brandtii


Phoxinus phoxinusPhoxinus lagowskii Phoxinus perenurus


High degree of morphological variability that obscures differences among species

Taxonomy of the genus Oreoleuciscus is controversial

Paucity of specimens from key geographical areas


Oreoleuciscus is thought to be allied to the Eurasian cyprinid genera Pseudaspius and Tribolodon which are within the aspinin lineage of the subfamily Leuciscinae (Howes, 1984, 1991).

On the other hand, there is also the idea that Oreoleuciscus is rather closely related to the phoxinin lineage (Travers, 1989; Bogutskaya, 1990).

Thus, taxonomy of the genus Oreoleuciscus is controversial, in part because of the paucity of specimens from key geographical areas, and in part because there is high degree of morphological variability that obscures differences among species.

Determine complete mitochondrial (mt) genome sequences of Leuciscinae species

Purpose

Conduct phylogenetic analysis based on mt genome sequences

Clarify the phylogenetic position of genus Oreoleuciscus and evolutional history of the Leuciscinae

Determine complete mitochondrial (mt) genome

sequences of Leuciscinae species

Purpose

Conduct phylogenetic analysis based on

mt genome sequences

Clarify the phylogenetic position of genus Oreoleuciscus

and evolutional history of the Leuciscinae

38 species 204 genera (FishBase)leuciscin: Eurasian plus North American Notemigonus cryleucas

48 species 319 genera (FishBase)phoxinin: North American and Far Eastern species 　

Phylogeny based on morphology (Cavender and Coburn, 1992)

Leuciscinae

ウグイ亜科の分類　 phoxinin と leuciscin

中央アジアが Point だけどサンプルが少ない

ウグイ亜科の進化史を考えると重要

今回アルタイで Oreoleuciscus を採集

Oreoleuciscus はどこに入るか ?

T. sachalinensis

T. hakonensis

T. brandtii

T. nakamurai

Low level of morphological differentiation

Their taxonomy had been contentious (1 ~ 5 species).

Tribolodon

(Ikeda, 1937; Nakamura, 1963,1969; Onodera & Honma, 1976; Kurawaka, 1977)

Endemic in the Far East

Far Eastern phoxinin

Inhabit the upper reaches of rivers, lakes and coastal regions of the sea

The pharyngeal teeth of Pseudaspius and Tribolodon species are similar.


P. leptocephalus T. nakamurai T. sachalinensis

(Tomoda, 1998)

Previous molecular phylogenetic studies(Kartavtsev et al., 2002; Sakai et al., 2002, 2006; Sasaki et al., 2007)

Pseudaspius and Tribolodon species are closely related

in the Far Eastern phoxinin branch.

Widely distributed in Northern Hemisphere (Nelson, 2006)

Body size small

Classified into a single genus “Phoxinus”

7 species live in the Far East

Degree of morphological differentiation is lower than the other genera

P. phoxinusP. lagowskii P. perenurus

Phoxinus

Previous molecular phylogenetic studies (Sasaki et al., 2007)

Far Eastern Phoxinus species and P. phoxinus are polyphyletic


Phylogenetic analysis based on cyt b gene

cyt b gene sequences 1140 bp

Useful to estimate phylogenetic relationships in closely related species

Large number of sequence are registered in databases.

North American27 species

Far Eastern30 species

European36 species


Neighbor Joining (NJ) Analysis


NJ tree based on cyt b gene


North American phoxinin


European leuciscin

&North American Notemigonus


Phoxinus phoxinus

Relationship among clades

Unclear

NJ tree based on cyt b gene

Phylogenetic analysis based on mt genome

mt genome analysis

Long DNA sequences

(Inoue et al., 2001a; Miya et al., 2003; Saitoh et al., 2006)

Determined mt genome sequences ca. 16,600 bp (circular)

Genomic structure common with vertebrates

Exclude ND6, control region and gaps (14,969 bp)

mt genome of Leuciscinae species

North American10 species

Far Eastern13 species

European7 species

Selected species from major clades in the cyt b-based tree



Maximum likelihood (ML) Analysis (RAxML)

Correct estimation of phylogeny at various taxonomic levels

with strong statistical support

Far E

asternE

uropeanN

orth Am

erican


ML tree based on mt genome gene

Far E

asternE

uropeanN

orth Am

erican


phoxinin

leuciscin

Far E

asternE

uropeanN

orth Am

erican

This analysis supports the classification by Cavender and Coburn (1992)

Divided into 2 clades; phoxinin & leuciscin

Far E

asternE

uropeanN

orth Am

erican

The Far Eastern phoxinins Tribolodon, Pseudaspius, Phoxinus

Monophyletic and greatly diverged from the other Leuciscinae clades

Far E

asternE

uropeanN

orth Am

erican


Tribolodon brandtii

Tribolodon species and Pseudaspius leptocephalus

A single genus ?

Subclade with 100% statistical supports

Far E

asternE

uropeanN

orth Am

erican

North American species

Divided into some independent clades

Notemigonus cryleucas European leuciscin clade

Far E

asternE

uropeanN

orth Am

erican

Far Eastern Phoxinus species, European P. phoxinus, North American P. eos

P. eos

P. lagowskii

P. phoxinus

Polyphyletic Some different genera ?

Far E

asternE

uropeanN

orth Am

erican

mt genome tree

Divergence time estimation

L 0.014 Mya

L 0.014 Mya

North America isolation from Eurasia

The oldest and reliable fossils of Cyprinidae (Cavender, 1991)

Fossil record of Pseudorasbora (Liu and Su, 1962)

U 54.8 L 49.0 Mya

L 3.0 Mya

Lower constraint : L

Upper constraint : U

Bayesian method; mcmctree (PAML 4; Yang and Rannala, 2006)

15.14 Mya

12.90 Mya

8.82 Mya

4.37 Mya

19.74 Mya

Far E

asternE

uropeanN

orth Am

erican

Far E

asternE

uropeanN

orth Am

erican

1.81 ~ 4.37 Mya Tribolodon speciation

The glacial period

12.90 ~ 15.41 Mya Radiation of Leuciscinae

Rapid global cooling of Middle Miocene

The sea level dropped

The land area expansion, freshwater and brackish water area increased

Environment habitable for Leuciscinae extended

Enlarge their distribution and occurred speciation

(Miya and Nishida, 1999)

Long PCR

Cycle sequencing

Same primers as for the nested PCR

Nested PCR

162 Fish-versatile primers55 cyprinid-specific primers

Determination of whole mt genome sequences

4 combinations of Fish-versatile primers

38 species 204 genera (fishbase)

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