| | DOI: 10.1111/mec.15204

ORIGINAL ARTICLE

A phylogenomic perspective on diversity, hybridization and evolutionary affinities in the genus

Baocheng Guo1,2 | Bohao Fang3 | Takahito Shikano3| Paolo Momigliano3 | Cui Wang3| Alexandra Kravchenko4| Juha Merilä3

1 Abstract - - 2 Pungitius - 3 - 4 Pungitius Correspondence - - Pungitius -

Funding information Pungitius - Pungitius -

KEYWORDS

4046| © 2019 John Wiley & Sons Ltd wileyonlinelibrary.com/journal/mec Molecular Ecology. 2019;28:4046–4064. GUO et al. |

| sensu Pungitius- - Pungitius- - - Pungitius - - - - - - - - Pungitius- - - - - - Gasterosteus aculeatus Gasterosteus - PungitiusP. platygasterP. hellenicus - - Pungitius | GUO et al.

60°N 130°E1140°E 150°E 60°E

PS-RU-BOL

PP-RU-BLS 50°N PS-RU-TAN PP-RU-GOR PP-JP-ROK(OM) PT-RU-VLA

PT-RU-VLA PS-RU-PRE PK-RU-NRI PS-RU-TAN PK-RU-NRI PS-RU-PRE PP-RU-GOR PS-RU-BOL 40°N PP-JP-ROK(OM)

0250 500 km 180°E 150° W E 150° 120° W

E 120° 90° E W 90°

60°W E

60°

30°W 30°E 0° 75°N

60°N

P. pungitius P. laevis

45°N P. platygaster P. sinensis PH-GR-SPE P. tymensis P. hellenicus 0 1,000 2,000 km 30°N P. kaibarae

Sample site

Pungitius

Pungitius Pungitius - Pungitius - - - GUO et al. |

fastqc | fastx | - Pungitius - Pungitius pungitiusP. lae- visP. platygasterP. hellenicusP. sinensisP. tymensis and P. kaibarae - P. bussei and P. stenurus- -

P. polyakovi bwa mem

samtools -

P. pungitius samtools and bcftools

vcftools P. occidentalis - - P. laevis Gasterosteus aculeatus- G. wheatlandiCulaea incon- in C. inconstansG. wheatlandi stans Apeltes quadracus - - - Pungitius - | r p = 4.12 × 10 preparation and sequencing Pstr - p = 4.09 × 10 - Pst de novo

- pyrad - - -

- Dpyrad

pyrad | - - rp < 2.2 × 10 | GUO et al.

(a) (b)

Gasterosteus aculeatus GA-US-ALA Gasterosteus aculeatus 100 GW-US-MAI-02 100 Gasterosteus wheatlandi GW-US-MAI-01 B AQ-US-MAI-02 100 AQ-US-MAI-01 Apeltes quadracus CI-CA-GUE-02 100 Culaea inconstans CI-CA-GUE-01 Gasterosteus wheatlandi PS-RU-TAN-01 100 PS-RU-TAN-02 100 PS-RU-BOL-02 100 100 PS-RU-BOL-01

PP-JP-NAK-02[FW] SINENSIS-CLADE 100 100 PP-JP-NAK-01[FW] Pungitius sinensis 96 PP-JP-YON-01[FW] 100 PP-JP-YON-02[FW] Culaea inconstans 95 PS-CN-LIU-01 100 100 PS-CN-LIU-02 100 PS-RU-PRE-02 100 Pungitius kaibarae 100 PS-RU-PRE-01 100 PS-RU-KIE-01 100 PS-RU-KIE-02 PK-RU-NRI-01 100 PK-RU-NRI-02 100 Pungitius tymensis Pungitius sinensis PP-JP-ROK-02[OM] 100 PP-JP-ROK-01[OM] SINENSIS-CLADE 100 PT-JP-MOT-02 A 100 PT-JP-MOT-01 100 100 PT-RU-VLA-01 100 Pungitius hellenicus PT-RU-VLA-02

100 PH-GR-SPE-02 PH-GR-SPE-01 Pungitius kaibarae 100 PR-GR-LOU-01 100 PR-GR-LOU-02 PL-FR-ANT-01[II] 100 Pungitius platygaster PL-FR-ANT-02[II] 100 100 PL-FR-NOH-02[I]

100 PUNGITIUS-CLADE PL-FR-NOH-01[I] 100 PL-FR-YON-01[III] Pungitius tymensis 100 PL-FR-YON-02[III] Pungitius laevis 100 PP-CA-TEM-02 100 PP-CA-TEM-01

PP-CA-FLO-01 NA 100 C PP-CA-FLO-02 100 PP-RU-BLS-01 100 PP-RU-BLS-02 100 PP-JP-BIW-02[BW] Pungitius platygaster 100 100 PP-JP-BIW-01[BW]

100 FE PP-RU-GOR-02 100 PP-RU-GOR-01

PP-RU-LEN-01 PUNGITIUS-CLADE 100 100 PP-RU-LEN-02 PP-RU-USH-01 100 PP-RU-UNG-01 91 Pungitius hellenicus PP-FI-HEL-01 100 PP-FI-HEL-02 78 53 PP-SE-BYN-02

100 EE PP-SE-BYN-01 Pungitius pungitius 100 PP-FI-RYT-02 DISTRIBUTION 100 PP-FI-RYT-01 100 100 PP-RU-LEV-02 100 PP-RU-LEV-01 Pungitius laevis PP-SE-FIS-02 100 PP-SE-FIS-01 100 PP-NO-STO-01 PP-NO-UGE-01 95 100 PP-NO-UGE-02

81 100 PP-FR-MAR-01 WE PP-FR-MAR-02 Pungitius pungitius PP-UK-HAR-01 100 DISTRIBUTION 100 PP-UK-HAR-02 56 PP-BE-MAL-02 0.03 97 20 15 10 50 PP-BE-MAL-01 MIOCENE PLIOCENE Mya PLEISTOCENE

P. pungitius P. pungitius- | | delimitation - -

- snapp

beast2 -

raxml- - Pungitius- - | P. pungitius - GUO et al. |

treeannotator (Drummond

- figtree

snappbeast2; |

snapp- P. pungitius and G. acu-

snapp leatus-

vcftools in P. pungitius G. aculeatus and G. aculeatus G. wheatlandi - Pungitius Pungitius

phylippgdspider SD

snapp G. aculeatus and G. wheatlandi-

snapp SD- - Pungitius P. pungitius- - A. quadracus

snapp A. quadracus | phylogenetic inference snapp A. quadracus Pst- A. quadracus - - Pungitius A. quadracus- P. kaibaraeP. sinensis

snapp P. hellenicus P. platygaster - - b

ruby - Pungitius

beast2

snapp b beast2

snapp

tracer

snapp

densitree | GUO et al.

P. tymensis | D- P. pungitius PungitiusD- |

snapp- Pungitius

snapp

snapp

snapp-

Dpyradpyrad - Pungitius D (psinensis - P. tymensis, P. sinensis and P. kaibarae pungitius- - P. platygaster, P. hellenicus, P. laevis and P. pun- gitius The sinensis - p included P. sinensis D P. tymensisP. kai-

D- barae

D pyrad The pungitiusp P. platygaster and P. hellenicus - - clade included P. laevis - P. pungitius

DD

DILD and DOL - - P. pungitius

DD P. pungitius p -

snapp | sinensispungitius | P. tymensis, P. sinensis and P. kaibarae P. hellenicus, - P. platygaster, P. laevis and P. pungitius P. laevis and P. pungitius were Pungitius pun- - gitiusP. sinensis P. pungitius P. sinensis - P. pungitiusP. kaibarae GUO et al. |

Nuclear phylogeny Mitochondrial phylogeny

GA-USA-ALA GA-FI-HEL

PT-JP-MOT PT-JP-MOT 58 100 PK-RU-NRI PK-RU-NRI 100

100 PP-JP-ROK[OM] PH-GR-SPE 100 PS-CN-LIU PR-GR-LOU 100 PP-JP-NAK[FW] PL-FR-ANT[II] 100

PH-GR-SPE 100 PP-FI-HEL

99 100 PR-GR-LOU PL-FR-YON[III]

94 100 PL-FR-ANT[II] PP-FR-MAR 100 93 PL-FR-YON[III] PP-JP-ROK[OM] 100 PP-JP-BIW[BW] 46 PP-JP-BIW[BW]

100 PP-FI-HEL 88 PP-JP-NAK[FW]

85 PP-FR-MAR PS-CN-LIU

0.02 0.02

Pungitius

pyrad | P. pungitius and P. sinensisP. pungitius and P. kaibarae- - P. pungitius and P. laevisP. sin- - ensisP. pungitius P. hellenicusP. laevis or P. pungitius D- P. sinensisP. pungi- sinensis- tius P. sinensis P. sinensis and P. pun- P. pungitiusP. kaibarae gitius P. pungitiuspungi- DP. kaibarae tiusinensis and P. pungitius P. laevis P. laevis P. pungitius P. pungitius- P. pungitius Pungitius. P. pungitius-

D D - Pungitius tymensis- Pungitius P. sinensis | GUO et al.

Pungitius

Two taxa under test P1 P2 P3 O Z (min.–max.) nSig/n F (%)

P. pungitius–P. sin- 1.6–31.38 30/39 0.75–5.73 ensis 0.32–2.90 0/18 P. pungitius–P. kai- 0.61–15.52 17/26 0.43–4.26 barae 0.23–2.97 0/12 P. pungitius–P. lae- 21.57–43.98 5/5 2.11–7.61 vis 1.63 0/1 P. tymensis–P. sin- 3.65–13.14 3/3 3.00–4.00 ensis 0.08–11.59 2/4 2.00–3.00 P. tymensis–P. kai- 20.8–29.74 2/2 8.00–12.00 barae

Note: D- DZZ Dn FCulaea inconstansP. pungitius P. pungitiusP. pungitiusP. pungitiusP. pungitiusPungitius hellenicus; P. kaibaraeP. laevisP. pungitiusP. sinensisP. tymensisP. pungitius.

Culaea inconstans Pungitius

D and D D

Pungitius kaibarae D

D Pungitius tymensis

Pungitius sinensis

Pungitius platygaster

Pungitius hellenicus

Pungitius pungitius

Pungitius laevis

P. tymensisP. sinensis Pungitius ty- P. pungitius mensis P. kaibarae

P. sinensis The D P. pungitiusPungitius tymensis GUO et al. |

P. pungitius P. sinensisP. kaibarae and P. tymensis. P. pungitius P. laevisP. pungitius - and P. tymensis - | - Pungitius - - Pungitius - Pungitius - sinensis pungitius |Pungitius P. pungitiusP. laevis - - - - P. laevis P. pungitius - P. laevis- - P. pungitius- P. laevis - - Pungitius sinensis - - P. sinensis - P. pungitius - - P. pungitius - | GUO et al.

- - 1 × 10 Pungitius- inensis-clade. |Pungitius sticklebacks P. pungitiusP. sinensis- - Pungitius sinensis- and pungitius - PungitiusCulaea inconstans P. sinensis P. pungitius P. sinensis- sinensis pungitius P. sinensis Pungitius P. tymensis and P. kaibarae P. kaibarae - P. kaibarae sinensis-clade P. kaibarae- pungitius- - P. pungitiusP. sin- P. pungitiusP. occidentalis ensis- P. pungitius sinensis pungitius P. sinensis-clade. P. tymen- (P. platygaster and P. hellenicus) sis and P. sinensis P. sinensis P. pungitius P. ty- - mensis and P. sinensis P. laevis pungitius - - P. laevisP. pungitius P. laevis GUO et al. |

P. pungitius oc- P. pungitius pungitius sinensis pungitiu P. sinensis- pungitius P. tymensis and - P. kaibarae- - sinensis- P. pungitius sinensis- P. pungitius - - P. tymensis P. kaibarae - P. pungitius P. sinensis - - P. pungitius - |Pungitius stickleback - - - Pungitius. Pungitius | GUO et al.

80 1% 3% 12.5% 1% 3% 12.5% Within Within Populations Populations 60 40 20 (a) (e) 0

80 Among Among 60 Populations Populations 40 Pungitius pungitius 20 (b) (f) 0 P. pungitius 80 Frequency Among Among P. sinensis Species 60 Species 40 P. platygaster and P. hellenicus [Colour figure can be viewed at 20 (c) (g) wileyonlinelibrary.com] 0 80 Among Among 60 Genera Genera

40

20 (d) (h) 0 0510 15 20 0510 15 20 Sequence divergence (%)

Pungitius P. laevis lin- - P. laevis - - P. laevis P. hellenicus and P. platy- gaster Pungitius P. tymen- P. pungitius sisP. kaibaraeP. sinensisP. hellenicus P. platygaster P. laevis and P. pungitius- P. pungitius P. sinensis - - , P. occidentalis- P. kaibarae P. pungitius P. kaibarae GUO et al. |

- P. pungitius - P. pungitius P. pungitiusP. sinensis Pungitius - P. sinensis and P. pun- gitius P. kaibarae - Pungitius - Pungitius - Pungitius - - P. pungitiusP. sinensis- Pungitius P. bussei and P. stenurus P. polyakovi P. polyakovi were P. sinensis- P. polyakovi and P. sinensisP. polyakoviP. sinensis. P. bussei and P. stenurus | - - | GUO et al.

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