38 species 204 genera (FishBase)

1. Phylogenetic position of the Altaic fish genus Oreoleuciscus in Leuciscinae based on mitogenome sequences Phylogenetic position of the Altaic fish genus Oreoleuciscus in Leuciscinae based on mitogenome sequences Junichi Imoto1, Kenji Saitoh2, Jun Adachi3, Takeshi Sasaki4, Yuri P. Kartavtsev5, Satoru N. Chiba1, Ryo O. Gotoh1, Masaki Miya5, Mutsumi Nishida6 and Naoto Hanzawa1 1Grad Sch Sci Eng, Yamagata Univ, 2Nat Res Inst Fish Sci, 3Inst Stat Math, 4Inst Marine Biol, 5Nat Hist Mus Inst Chiba, 6Ocean Res Inst, Univ Tokyo

2. Phylogeny based on morphology (Cavender and Coburn, 1992) leuciscin: Eurasian plus North American Notemigonus cryleucas 38 species 204 genera (FishBase) phoxinin: North American and Far Eastern species 48 species 319 genera (FishBase) Leuciscinae Widely distributed in Northern Hemisphere Morphologically similar (even among different genera) Central Asia and Far East Importantto consider the evolutional history of Leuciscinae

3. The genus Oreoleuciscus(Warpachowskii, 1889) Oreoleuciscus potanini Distribution Endemic to inland lakes in western Mongolia and Tuva, the upper Ob River system within the Altai Mountain region of Siberia (Berg, 1962) Feeding habitat Planktonic algae,insects, invertebrates and fishes

4. ? Closely related to Pseudaspius and Tribolodon ? (Howes, 1984, 1991) Tribolodon sachalinensis Pseudaspius leptocephalus Tribolodon hakonensis Phylogenetic position of Oreoleuciscus Far Eastern Oreoleucuscus potanini North American Tribolodon nakamurai European ML tree based on mt genome

5. Phylogenetic position of Oreoleuciscus Far Eastern Oreoleucuscus potanini ? North American Closely related to Phoxinus ? (Travers, 1989; Bogutskaya, 1990) European Phoxinus lagowskii Phoxinus perenurus Phoxinus phoxinus ML tree based on mt genome

6. The purpose of this study 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 Taxonomy of the genus Oreoleuciscus is controversial Paucity of specimens from key geographical areas High degree of morphological variability that obscures differences among species No molecular phylogenetic analyses for Oreoleuciscus species

7. Mt genome of Oreoleuciscus potanini Determined mt genome sequences Genomic structure Common with vertebrates Exclude ND6, control region and gaps (14,826 bp) 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) Mt genome analyses Long DNA sequences Correct estimation of phylogeny at various taxonomic levels with strong statistical support (Inoue et al., 2001a; Miya et al., 2003; Saitoh et al., 2006)

8. Far Eastern North American European ML bootstrap / Bayesian posterior probabilities

9. Far Eastern North American European ML bootstrap / Bayesian posterior probabilities

10. Far Eastern North American Phoxinus lagowskii European Tribolodon nakamurai Oreoleuciscus potanini ML bootstrap / Bayesian posterior probabilities

11. Molecular Dating Parametric Bayesian approach Multidistribute (Thorn & Kishino 2002) Upper constraint ： U Lower constraint ： L (Mya) L 100, U 120 L 112, U 145 U 363 Cichlids L 85, U 95

12. Molecular Dating Upper constraint ： U Lower constraint ： L (Mya) Cichlid L 100, U 120 African L 112, U 145 South American Madagascar Indo/Sri Lanka Madagascar L 85, U 95 (Azuma et al., 2008)

13. Leuciscinae / other subfamily 123.6 Cyprinidae / Cobitidae 219.2 Otocephala / Euteleostei 331.2 Paleozoic Mesozoic Cenozoic Permian Triassic Jurassic Cretaceous Tertiary Carboniferous

14. Orieoleuciscus/ Far Eastern Phoxinus 35.5 Tribolodon/ Orieoleuciscus + Far Eastern Phoxinus 48.6 Far Eastern phoxinin / P. phoxinus 78.5 phoxinin / leuciscin 84.8 Mesozoic Cenozoic Pa. Eocene Oli. Miocene Plio Cretaceous

15. Conclusion The genus Oreoleuciscus is included in phoxinin (Cavender & Coburn 1992) Oreoleuciscus formed a monophyletic clade with the Far Eastern phoxinins Tribolodon, Pseudaspius and the Far Eastern Phoxinus Oreoleuciscusisclosely related to the Far Eastern Phoxinus rather thanTribodolonandPseudaspius Tribolodon + Pseudaspius and Oreoleuciscus + Phoxinus were divided 49 Mya Oreoleuciscus and Phoxinus were divided 34 Mya

16. Thank you for your attention !!

18. The genus Oreoleuciscus(Warpachowskii, 1889) Oreoleucuscus potanini Endemic to inland lakes in western Mongolia and Tuva, the upper Ob River system within the Altai Mountain region of Siberia (Berg, 1962) 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.

19. The sea level dropped Environment habitable for Leuciscinae extended 12.90 ~ 15.41 MyaRadiation of Leuciscinae Enlarge their distribution and occurred speciation The land area expansion, freshwater and brackish water area increased Rapid global cooling of Middle Miocene 1.81 ~ 4.37 MyaTribolodon speciation The glacial period Far Eastern North American European

20. Far Eastern North American European ML tree based on mt genome

21. Ancestral area of the Far Eastern Leuciscinae

22. 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) 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.

23. 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 ウグイ亜科と他の亜科 100.3 コイ科とドジョウ科 189.7 ニシン・骨鰾下区と 正新骨下区 271.0 200 100 0 (Mya) 古世代 中世代 第四紀 ペルム紀 三畳紀 ジュラ紀 白亜紀 第三紀

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。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。

25. 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) コイ科とドジョウ科 189.7 ニシン・骨鰾下区と 正新骨下区 271.0 200 100 0 (Mya) 古世代 中世代 第四紀 ペルム紀 三畳紀 ジュラ紀 白亜紀 第三紀

26. Orieoleuciscus/ Far Eastern Phoxinus 21.2 Tribolodon/ Orieoleuciscus + Far Eastern Phoxinus 請戸川 最上川 39.2 Far Eastern phoxinin / P. phoxinus 61.7 モンゴル phoxinin / leuciscin イタリア 74.7 100 0 (Mya) 中世代 新生代 ジュラ紀 白亜紀 始新生 中新生 暁新生 漸新世 鮮新世

27. 7.5 7.5 ～ 21.2 MyaTribolodon 属各種が種分化 請戸川 21.2 最上川 寒冷化・乾燥化 モンゴル イタリア 海水面の低下 陸地が拡大、淡水域や汽水域などが増える 100 0 (Mya) ウグイ亜科魚類が生息できる環境が広がる 中世代 新生代 ジュラ紀 白亜紀 始新生 中新生 暁新生 漸新世 鮮新世 新しい生息場所に分布を広げながら種分化

28. Phylogenetic position of Oreoleuciscus Closely related to Pseudaspius and Tribolodon ? (Howes, 1984, 1991) Tribolodon sachalinensis Tribolodon brandtii Pseudaspius leptocephalus Tribolodon hakonensis Tribolodon nakamurai Closely related to Phoxinus ? (Travers, 1989; Bogutskaya, 1990) Phoxinus lagowskii Phoxinus perenurus Phoxinus phoxinus Taxonomy of the genus Oreoleuciscus is controversial Paucity of specimens from key geographical areas High degree of morphological variability that obscures differences among species

29. 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.

30. Purpose Determine complete mitochondrial (mt) genome sequences of Leuciscinae species Conduct phylogenetic analysis based on mt genome sequences Clarify the phylogenetic position of genus Oreoleuciscus and evolutional history of the Leuciscinae

31. Purpose Determine complete mitochondrial (mt) genome sequences of Leuciscinae species Conduct phylogenetic analysis based on mt genome sequences Clarify the phylogenetic position of genus Oreoleuciscus and evolutional history of the Leuciscinae

32. Leuciscinae Phylogeny based on morphology (Cavender and Coburn, 1992) leuciscin: Eurasian plus North American Notemigonus cryleucas 38 species 204 genera (FishBase) phoxinin: North American and Far Eastern species 48 species 319 genera (FishBase)

34. 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?

35. T. sachalinensis T. nakamurai Far Eastern phoxinin Tribolodon T. brandtii T. hakonensis Endemic in the Far East Inhabit the upper reaches of rivers, lakes and coastal regions of the sea Low level of morphological differentiation Their taxonomy had been contentious (1 ~ 5 species). (Ikeda, 1937; Nakamura, 1963,1969; Onodera & Honma, 1976; Kurawaka, 1977)

36. 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. The pharyngeal teeth of Pseudaspius and Tribolodon species are similar. (Tomoda, 1998) P. leptocephalus T. nakamurai T. sachalinensis Pseudaspius leptocephalus

37. Far Eastern phoxinin Phoxinus Degree of morphological differentiation is lower than the other genera P. lagowskii P. perenurus P. phoxinus Widely distributed in Northern Hemisphere (Nelson, 2006) Body size small Classified into a single genus “Phoxinus” 7 species live in the Far East Previous molecular phylogenetic studies (Sasaki et al., 2007) Far Eastern Phoxinus species and P. phoxinus are polyphyletic

38. Phylogenetic analysis based on cyt b gene

39. cyt bgene sequences 1140 bp Useful to estimate phylogenetic relationships in closely related species Large number of sequence are registered in databases. North American 27 species European 36 species Far Eastern 30 species 45 genera 93 species Outgroup 9 species (Cypridae 8, Cobitidae 1) Neighbor Joining (NJ) Analysis

40. NJ tree based on cyt b gene

41. Relationship among clades Unclear European leuciscin & North American Notemigonus Far Eastern phoxinin North American phoxinin North American phoxinin Phoxinus phoxinus North American phoxinin NJ tree based on cyt b gene

42. Phylogenetic analysis based on mt genome

43. mt genome analysis Long DNA sequences Correct estimation of phylogeny at various taxonomic levels with strong statistical support (Inoue et al., 2001a; Miya et al., 2003; Saitoh et al., 2006) mt genome of Leuciscinae species Determined mt genome sequences ca. 16,600 bp (circular) Genomic structure common with vertebrates Exclude ND6, control region and gaps (14,969 bp) Selected species from major clades in the cyt b-based tree Far Eastern 13 species European 7 species North American 10 species 16 genera 30 species Outgroup 9 species (Cypridae 8, Cobitidae 1) Maximum likelihood (ML) Analysis (RAxML)

44. Far Eastern North American European ML tree based on mt genome

45. Far Eastern North American European ML tree based on mt genome gene ML tree based on mt genome

46. Far Eastern phoxinin North American leuciscin European This analysis supports the classification by Cavender and Coburn (1992) Divided into 2 clades; phoxinin & leuciscin

47. Far Eastern North American European The Far Eastern phoxinins Tribolodon, Pseudaspius, Phoxinus Monophyletic and greatly diverged from the other Leuciscinae clades

48. Pseudaspius leptocephalus Far Eastern Tribolodon brandtii North American European Tribolodon species and Pseudaspius leptocephalus Subclade with 100% statistical supports A single genus ?

49. Notemigonus cryleucasEuropean leuciscin clade Far Eastern North American European North American species Divided into some independent clades

50. Far Eastern Phoxinus species, European P. phoxinus, North American P. eos P. phoxinus P. eos P. lagowskii Some different genera ? Polyphyletic Far Eastern North American European