Positive Selection and Biological Traits Related

7/26/2019 Positive Selection and Biological Traits Related

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Positive selection and biological traits related to substitution rates in mitochondrial

genes in cyprinids (Teleostei: Cyprinidae)

Abstract

Mitochondriaplayscritical rolein energy metabolism and thermoregulation for organisms.

Increasing findings suggest that natural selection acts on the mitochondrial genes and

biological traits influence rate of mitochondrial molecular evolution. Cyprinid fishes, forming

the largest family of freshwater fish in the world, exhibit great diversity in living condition

and biological traits. Thus, to analyse the evolution of mitochondrial genes and correlations of

life history traits with substitution rates in mitochondrial genes of Cyprinidae species could

provide a useful model and a reference for molecular evolution of mitochondrial genomes

(mitogenomes) of vertebrates. In the present study, the substitution rates in a total of

!mitogenomes,including threemitogenomes that we se"uenced, of species or subspecies in

family Cyprinidae were analy#ed and the correlations of substitution rates with some

biological traits of cyprinids were investigated. The results showed that (i) based on the

concatenated nucleic acid se"uences of all $! mitochondrial protein%coding genes (&C's), the

principal phylogenetic branches coincide with the taxonomy of Cyprinidae, (ii)nucleotide

substitution rates exhibited clade%specific feature, and nonsynonymous substitution rate

(dN)showed to be gene%specific, but synonymous substitution rate (dS) were fairly uniform

among $!&C's in family Cyprinidae,(iii) five main biological traits, particularly maximum

absolute fecundity, are negatively significantly correlated with the substitution rates of

mitochondrial genes, which were "uite different from other vertebrates,( )signs of positive

selection were identified along four internal branches leading to the clade of Cyprininae,

anionini, 'obionini and euciscini, and the estimated divergence time indicated these

positive selections occurred *!+ Myr % $ Myr ago.-evertheless, the results provided more

molecular information for the evolution of mitochondrial - in family Cyprinidae.


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Key words/ mitochondrial protein%coding genes0 positive selection0 biological traits0

fecundity0 substitution rate0 evolution

Introduction

Cyprinidae, the largest family of fish, comprises about ++1 genera and about +,2+1 species

with widespread distributions across 3urasia, frica and -orth merica (-elson, +114).

Many of these species are the critical sources for food (e.g., the common carp, Cyprinus

carpio), or serve as model organisms for research such as the #ebrafish, (Danio rerio)

(Mayden et al., +115). Meanwhile, all of these species are fundamental elements in the

various biotic communities in fresh waters. 6ecause of a great variety of species, morphology,

niches and food habits, the family Cyprinidae is thought to be an ideal group for studying the

molecular evolution of mitochondrial genomes (mitogenomes) of vertebrates.

Many efforts have been made to understand the evolution of cyprinid fishes based on

morphological or anatomical characters and molecular data (e.g.7owes $55$0 Chen et al.

$5820 Chen et al. $5580'illes et al. $5580 iu and Chen +11!0 9ang et al. +11).

Mitochondrial - (mt-),as a type of nearly neutral molecular mar:er, played an

important role in reconstructing the phylogenetic relationship within the family Cyprinidae

(iu and Chen +11!0 9ang et al. +110Mayden et al. +1150 Tang et al +1$1), on account of

ease in - manipulation, rapid mutation rate, lac: of significant recombination, and

availability of universal primers for - amplification (vise $5520 Morit# et al. $580

;imon et al. $552). 7owever, with more and more information available, the

interrelationships inferred from mitochondrial mar:ers and nuclear loci exhibit some

incongruence in some cyprinid groups(e.g. &erea et al. +1$10 Mayden et al. +1150 Tao et al.

+1$1). Meanwhile, increasing findings suggest that mitochondrial genes commonly show

departures from neutral evolution (6allard and 9hitloc: +1120 6lier et al. +11$0 al#iel et al.

+1140 6rown +1180 ;hen et al.+1150 ;un et al. +1$$). nd substitution rates of mitochondrial

genes can be affected by certain species characteristics including biological traits such as

body si#e and longevity (9elch et al. +1180 6romham +11+0 -unn and ;tanley $558), or


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ecological factors such as geographic niches (;un et al. +1$$0iu et al. +1$+). espite the

predominance of negative selection against deleterious mutations acting on protein%coding

mitochondrialgenes (&C's), a number of evidences suggested that there exists positive

selection as the mitochondrial evolution (e.g.p to now,

whether the correlation between biological traits and mitochondrial evolution exists and how

the positive selection of mitochondrial evolution is, to our :nowledge, are still not

investigated elaborately in cyprinids.

In order to clarify the above issues and provide a relatively panoramic view of the

evolution of &C's in cyprinids, we compared then ucleotide substitution rates of $! &C'son

mitochondrial genomes (mitogenomes) of ! cyprinid fishes, including threemitogenomes

that we se"uenced, and studied the correlations of substitution rates with five biological traits,

as well as the possibility of positive selection acting on mitochondrial genes of these clades.

Material and Methods

Three mitogenomes sequencing

To enrich the information of mitogenomes of genus Megalobrama of Cyprinidae,

mitogenomes of three breams, Megalobrama skolkovii,Megalobrama terminalis,

andMegalobrama pellegrini, were se"uenced. M.skolkovii and M. terminalis were sampled

from the &earl =iver drainage, and M. pellegriniwas collected from ongxi =iver on the

upper reaches of the


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had been were deposited in the 'en6an: with accession numbers from of @A+2++8 to

@A+2+!1.

Source of data and primary treatment

3xcept the ! mitogenomes we se"uenced, the mitogenomes for other 1 fish species or

subspecies in family Cyprinidae were downloaded from 'en6an: and 36I (available in @uly+1$+). Information on life history traits for cyprinid species was collected from Bish base

(Broese and &auly +115) and literature sources. The accession numbers of mitogenome

se"uences and the available life history traits were listed in Table ;+.

Phylogenetic analysis

Concatenated nucleic acid se"uences of all $! &C's were used to reconstruct a maximum

li:elihood (M) phylogenetic tree for cyprinids by using =xM 9eb%servers (;tamata:is et

al. +118). Myxocyprinus asiaticus (Cypriniformes/ Catostomidae) and Misgurnus

anguillicaudatus (Cypriniformes/ Cobitidae) were used as outgroups. 7owever, before

constructing M tree, Modeltest !. (&osada and Crandall $558) was used to select the best

model of evolution for subse"uent M analysis.

6ayesian analyses (M=.6


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phylogenetic tree using the codon%based Model $ (freeDratios for branches) implemented in

&M (


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lineages, and a li:elihood ratio test (=T) was constructed by comparing a model that allows

positive selection on the foreground lineages with a model that did not allow such positive

selection (Ehang et al. +11). 9hen positively selected sites were detected, &M will

identify these sites using 6ayes empirical 6ayes (636) approach to calculate posterior

probabilities (


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was used as the wor:ing topology in the subse"uent analyses as Tincayielded the low degree

of support in the M tree and the ambiguous taxonomicstatusin previous studies(9ang et al.

+110 Tang et al. +1$1).

Substitution rate variation among protein-coding mitochondrial genes in Cyprinidae

ThedS and dNvalues of all $! &'Cs along the terminal branches in cyprinids ranged from

1.11$$to $.28, and from 1to 1.1288, respectively. The average values of dSwerefrom

(AT!) to $!2 times (C"#$) of dN among different genes. The dNdS values of all $! &'Cs

along the terminal branches in cyprinids are lower than $, which indicates the genes were

under purifying selection or negative selection. Three genes of the cytochrome oxidase (CFA

or complex IG),C%T& of ubi"uinoneHcytochrome c oxidoreductase(complex III), andND'(

of -7 dehydrogenase (- or complex I) were turned out to be slow%evolving with lower

dN and dNdS values, whereas most of - genes and two T& synthase (B1B$%T&ase or

complex G)genes were classified as fast%evolving genes with higher dNand dNdS(Big.+).>%test

results further confirmed that significant difference exists in dN and dNdSbetween most of

mitochondrial &'Cs and the combined se"uences of mitochondrial genes for three complexes

(complex I, IG and G) (Table ;!).-o significant difference in dS was found among $!

individual &C's or among the ! complexes between clades although the mean values of dS

varied. The absence of divergence indicates thatthe synonymous rate is fairly uniform over

the &C's in cyprinids.

Pattern of substitution rate variation among the six clades of Cyprinidae

mong the six clades, Aenocypridini was involved in the maority of those pairwise

comparisons with significant difference, and exhibited considerably lower substitution rates in

most of the analy#ed genes and concatenated se"uences than other clades, particularly the

clades of anionini and 'obionini, (Big.+0 Table;!). Meanwhile, significant divergences in


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some &C's or combined se"uences were also detected between anionini and the clade of

Cyprininae, 'obionini and anionini, and euciscini and anionini, as well as 'obionini and

cheilognathini. 7owever, no significant divergence was found between euciscini and other

three clades, i.e. cheilognathini, 'obionini and the clade of Cyprininae. cheilognathini

showed no significant differences in dS between other five clades(Table ;!).Burthermore,

significant divergences in dNdSbetween clades of cyprinids were only found in 2 genes

(ND'(,C"# $$, C"# $$$ andAT!), two complexes (complex IG and complex G) and the

concatenated se"uence of all $! &C's (Table ;!). The clade of Aenocypridini still exhibited

pronounced difference from other clades except the clade of Cyprininae, and its mean dNdS

values were obviously lower than other clades (Big.+). dditionally, 'obionini also showed

much more significant differences from some other clades. 6ut no sign of significant

divergence in dNdSwas found in the pairwise comparisons among euciscini, cheilognathini

and the clade of Cyprininae.

dditionally, considering the difference in divergence time of clades in family Cyprinidae,

the absolute substitution rates (substitution rate per codon per million years) of the six clades

in Cyprinidae were also compared. The results were "uite similar to those from the relative

substitution rates described above (shown in Table ;!).

Positive selection analysis

The four clades of Cyprininae, anionini, 'obionini, and euciscini, respectively, hadhave

experienced significant positive selection, while various amounts of putative positively

selective sites were identified along these four internal branches (Table $).long the four

internal branches in the phylogenetic tree, positive selection has been detected in C"#$$,

C%T& andND). The divergent time estimates suggest the positive selections have occurred

during the period from the Fligocene to the middle of Miocene(from *!+ Mry ago to $ Myr

ago) (Big.$).

;trong positive selection were also identified at sites in C"# $$(++7, 2


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(branch 6 in Big.$0 Table $). Meanwhile, significant adaptive selection was also found in

branch C leading to 'obionini in ND) and in branch (euciscini). Fne site under

significant positive selection was tested inND)(+8) along branch C and in C%T&($58;)

along branch , respectively(Table $0 Big.$). -o positive selection and site under positive

selection were identified along the branches leading to cheilognathini (branch ' in Big.$)

and Aenocypridini (branch 7 in Big.$).

mong the seven sites under strong adaptive selection in CFAII of the branch 6, three sites

(7is++, Tyr2, and Gal$) were located at CFAII transmembrane domain, and other four sites

were on the periplasmic domain according to the information from &fam. These sites in the

extant species of anionini are "uite different from those in their ancestor lineage.

The uni"ue site under positive selection (;+58) in C


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Correlations of substitution rates with life history traits

The single variable regressions showed that all five biological traits, especially maximum

absolute fecundity exhibited significantly negative correlation with both d-and d; (Table +0

Big.;$). 7owever, only maximum absolute fecundity showed higher d-d; (p 1.1) in

Cyprinids whereas no significant correlations with d-d;were identified for other four

predictor traits(p 1.1). Burthermore, a multiple regression analysis including all five

biological traits also showed that only maximum absolute fecundity remained significant (dN/

n L $+, slope L %1.11+!2!, =+ L 1.282!, p L 1.1$$50 dS/ n L $+, slope L %1.121, =+L

1.!54,p L 1.1!5+5).

!iscussion

Mitochondria are the power plant of cells, which generate adenosine triphosphate (T&) for

energy%consuming processes and produce more heat for organisms in cold climates by

oxidative phosphorylation (FA&7F;) (=ui#%&esini et al. +1120 ;un et al. +1$1). The $!

peptides encoded by vertebrate mitochondrial genome are essential subunits of en#ymes in

FA&7F;. ;everal reports showed evidence of purifying selection and the physiological or

functional constraints on mt- evolution (6allard and 9hitloc: +1120 Mei:leohn et al.

+110 ;hen et al. +1150 ;un et al. +1$1). Thus, in the present study, we analy#ed the evolution

of &'Cs based on the genome information from ! species or subspecies in family

Cyprinidae.

&arameter dNdScan provide a measure of selective pressure at protein level. The dNdSvalue

less than $ significantly indicates that nonsynonymous mutations are deleterious and fixed in

the population at a lower rate than synonymous mutations, called purifying selection or

negative selection (i et al. $580


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phylogenetic tree, some genes (ND), C"#$$ and C%T&) have undergone positive selection.

;ome advantageous nonsynonymous mutations might retain by adaptive selection would be

enriched in internal branches relative to terminal branches (=ui#%&esini et al. +112). The

universally detected significant divergences in dNdS of terminal branches among these

mitochondrial genes indicate diverse purifying selection on mitochondrial genes. 'enes under

stronger purifying selection usually encode the subunits forming the catalytic core (e.g.C"#$,

C"#$$and C%T&) (6runori et al. $58, 7altia et al. $55$, T#u:ihara $554) or playing critical

roles in structural stability of complexes (e.g.C"#$$$andND'() (T#u:ihara $5540 ervinen

+112) in FA&7F;, whereas, genes encoding for - and T& synthase are generally under

wea:er purifying selection in cyprinids. similar trend also has been observed in avian and

other fish taxa (;hen +1150 Marsall +1150 ;un +1$1). The uniform distribution of dSamong

genes highlights that significantly different dNbetween the two groups of genes is attributed to

the pronounced divergence in dNdSin cyprinids. 6ecause of the important roles of some genes

playing in functions of FA&7F;, nonsynonymous mutations are usually deleterious, even

lethalto organisms, which were then swept by purifying selection.

ivergent time estimates suggest the positive selections, which have been detected in

C"#$$, C%T& andND),have occurred from the Fligocene to the middle of Miocene. uring

this period of time, the maor geographical events are the uplift of Tibetan plateau and the

land%sea redistributions associated with the continental collision of India and sia, which are

estimated to start from the early Ceno#oic at least ! Myr ago (n et al. +11$0 =owley and

Currie +1140 7arris +114). These geographical events are mainly attributed to the significant

continental aridification and cooling in sia (n et al. +11$0 'uo et al. +11+0'uillaume et al.

+110 =amstein et al. $55). =ecent findings suggest that C"#$$may have more important

roles for heat generation than for energy production in teleost (;un et al. +1$$).There were

studies showing that the adaptive mutations in C%T& genes are associated with the

adaptability to colder climates in humans (=ui#%&esini et al. +112). - encodes -7

dehydrogenase in mitochondria, which is shown to play an important role in proton or


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sodium ion translocation in overall respiration regulation in mammals (-a:amaru%Fgiso et al.

+11!) (6ai et al. +112).

Moreover, our results indicate the ancestral lineages of six cyprinid clades distributing in

different regions have experienced to various degrees selection pressure originating from the

abrupt changes in palaeo environmental conditions from the Fligocene to the middle of

Miocene. -o positive selection site has been detected in the branches leading to

Aenocypridini and cheilognathini and a few numbers of positively selective sites occurred in

the branches leading to euciscini and 'obionini living in high latitude while much more

selection sites involved in more genes has been found in the clades of anionini and

Cyprininae. The maority of the extant species of anionini are small%si#ed fishes inhabiting

in hilly streams and rivers, or shallow, slow%moving and standing waters such as rice paddies

and other temporally%inundated environments in southwest of China and southern sia. The

various habitats of anionini might suggest their ancestral lineages adapt to the temporary

waters caused by aridification orand swift streams or rivers attribute to the tectonic processes.

Combined the fossil records and the distributions of the extant species in the clade of

Cyprininae (6anarescu and Coad $55$0 -elson +1140 ;chul#%Mirbach and =eichenbacher,

+1140 Chen et al. $558), the main origin of the Cyprininae sensu latomight be related to the

uplift of Tibetan plateau and the continental collision of India and sia from the early

Fligocene to the early Miocene and one of the lineages of the primitive barbins could

accumulate more adaptive mutations in genes and differentiate into the extant species in latter

Cyprininae.

&resently, it has become increasingly clear that numerous aspects of its biology in

mammals, birds and reptiles affected their genome evolution (6romham +11+0 -unn and

;tanley $5580 9elch et al. +118). ;imilar to other vertebrates, life history traits, such as

lifespan and body si#e, in cyprinids have been also shown significantly negative relationships

with the rate of their mitochondrial molecular evolution in our study. 7owever, the

relationship between fecundity of cyprinid fishes and the evolutionary rates of mitochondrial

&C's are "uite different from other vertebrates. In mammals, the fecundity has strongly


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positive association with both synonymous and non%synonymous rates for six nuclear genes,

(9elch et al. +118). In reptiles, a positive correlation between clutch si#e and total genetic

distance for cytochrome bhas been found (6romham et al. +11+). In ourstudy, the fecundity

of cyprinid fishes arenot only isthe strongest predictor of mitochondrial - substitution

rate variations, but also showed significant negative relationships with both synonymous and

non%synonymous rates for the concatenated se"uence of $! mitochondrial protein%coding

genes.6romham(+1$$) mentioned that the strongly positive relationship between fecundity

and genetic variation in mammals waswereresulted from the number of genome copies per

generation scales. s we :now, hundreds of thousands of eggs of fishes are derived from a

finite amount of oogonial stem cells that can generate a new cohort of oocytes each year

('ilbert +114), yet the higher fecundity seems toplay no more roles in increasing copy error

mutations in cyprinids. Cyprinid fishes are a group of primitive teleostean that are strictly

distributed in freshwater and their living conditions including hydrological regimes, spawning

grounds,and feeding conditions, etc, fluctuate fre"uently. Moreover, the vast maority of

cyprinids practice external fertili#ation and external development of #ygotes, and the #ygotes

and the young offspring are lac: of parental protection (9eyl and 6ooth $5550 Ehang et al.

+1$$). 7ence, N%ecological or nearly N%ecological strategy with high fecundities, high

mortalities of early life stages, and young age at maturation, which adopted by the vast

maority of cyprinids (9eyl and 6ooth $555), would be another cause for the significant

negative relationships between the fecundity of cyprinid fishes with both synonymous and

non%synonymous rates.

In conclusion, the evolution of mitochondrial genes and correlations of life history traits

with substitution rates in mitochondrial genes in Cyprinidae enriched and provided a

reference for molecular evolution of mitogenomes of vertebrates. The cyprinids had have

experienced significant positive selection, which has beenweredetected in C"#$$, C%T& and

ND), while various amounts of putative positively selective sites were identified. The

relationship between fecundity of cyprinid fishes and the evolving rates of mitochondrial

&C's were significantly negative, which are"uite different from other vertebrates.


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"upporting in#ormation

Table "$mplification primers for mitogenomes of ! breams Megalobrama s:ol:ovii, M.

terminalis, and M. pellegrini.

Table "%The accession numbers of mitogenome se"uences from 'en6an: and the available

life history traitsfrom Bishbase.

Table "&The divergence in substitution rates in individual mitochondrial gene, three

complexes and the combined se"uence of the $! genes.

'ig"$Correlations of substitution rates with biological traits. , correlations of substitution

rates with Max absolute fecundity0 6,correlations of substitution rates with Max body mass0C,correlations of substitution rates with Max body length0 , correlations of substitution rates

with length at first maturity0 3, Max lifespan.

Acnowledgements

This study was supported by grants from the Modern groindustry Technology=esearch

;ystem, OO;taple Breshwater Bishery Industry Technology ;ystemPP (-o. C=;%24%

1),Bundamental =esearch Bunds for the Central >niversities (-o.

+1$$&


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6allard @9F, 9hitloc: MC. +112. The incomplete natural history of mitochondria.Mol *co.

$!/ +5H22.

6anarescu &, Coad 69. $55$. Cyprinidae of 3urasiaIn Cyprinid fishes, ;ystematics, 6iology

and 3xplotation (9infield I@ and -elson @3, eds), $st edition. ondon, Chapman and 7all/

$+H$.

6lier &>, ufresne B, 6urton =;. +11$. -atural selection and the evolution of mt-%

encoded peptides/ evidence for intergenomic co%adaptation. Trends +enet. $/ 211H214.

6romham . +11+. Molecular Cloc:s in =eptiles/ ife 7istory Influences =ate of Molecular

3volution.Mol &iol *vol. $5(!)/ !1+%!15.

6rown . +118. Bish mitochondrial genomics, se"uence, inheritance and functional

variation., -ish &iol. +/ !H!2.

6runori M, ntonini ', Malatesta B, ;arti &, 9ilson MT. $58. Cytochrome%c

oxidase.;ubunit structure and proton pumping.*ur , &iochem. $45/$H8.

Chang M, Chen &. +111. &hanero#oic succession of fish faunas in mainland China. In/ Chow


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rummond , =ambaut . +11. 63;T/ 6ayesian evolutionary analysis by sampling trees.

&MC *vol &iol. / +$2.

Broese =, &auly . +115. Bish6ase.9orld 9ide 9eb electronic publication.vailable from

www.fishbase.org, version ($$+115).

'ilbert ;B. +114. evelopmental 6iology.;underland/ ;inauer ssociates.

'illes , ecointre ', Baure 3, Chappa# =, 6run '. $558. Mitochondrial phylogeny of

3uropean cyprinids/ implications for their systematics, reticulate evolution, and

coloni#ation time.Mol hylogenet *vol. $1/ $!+H$2!.

'uillaume , rigsman 9, angereis C', bels 7, ai ;, Ban A. +11. Tibetan plateau

aridification lin:ed to global cooling at the 3oceneHFligocene transition. Nature. 22/

4!H4!8.

'uo ET, 9illiam B=, 7ao JE, 9u 76, Jiao


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i 97, 9u CI, uo CC. $58. new method for estimating synonymous and

nonsynonymous rates of nucleotide substitutions considering the relative li:elihood of

nucleotide and codon changes.Mol &iol *vol. +/$1H$2.

ittle ', ougheed ;C, Moyes C. +1$+. 3volution of mitochondrial%encoded cytochrome

oxidase subunits in endothermic fish/ the importance of taxon%sampling in codon%based

models.Mol hylogenet *vol. 4!/ 45H482

iu 7, Chen


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i


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;chul#%Mirbach T, =eichenbacher 6. +114. =econstruction of Fligocene and -eogene

freshwater fish faunas% an actualistic study on cypriniforms otoliths.Acta aleontol ol.

$(+)/ +8!H!12.

;hen


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'igure legends

'ig $ The phylogenetic tree o# cyprinids based on $& protein*coding genes #rom

mitogenomes and molecular dating o# the +% cyprinid species or subspecies 6ootstrap

support values and 6ayesian posterior probabilities are shown at nodes and separated by

slashes for maximum li:elihood and Mrbayes, respectively.6ranch , 6, C, , ', 7 represent

the branches leading to the clades of Cyprininae, anionini, 'obionini, euciscini,

cheilognathini and Aenocypridini, respectively. 6ranch 3 and B indicate the ancestral

lineages of Cyprinini and extant genus Carassius, respectively. The fossil%based constraints

are indicated with diamonds. 6ranch lengths are proportional to divergence times (in million

of years). The names of species shadowed by gray belong to the clade of Cyprininae. The

names of species in box belong to Carassias.

'ig % Comparisons o# median and the averagevalues o# dN,dS- dNand dS#or $& individual

genes- genes involved in comple.es I- I/- and /- and $& protein*coding genes set in +%

cyprinid species among si. clades o# cyprinids6oxes include 1K of the distributions. (a)

Comparison of median and the average values ofdNdS. (b) Comparison of median and theaverage values ofdN. (c) Comparisons of median and the average values ofdS.


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Table $ Parameter estimates- lielihood ratio testsand positively selected sites under 0ranch*site ModelA test %

0ranch or

lineage

1enes Model A test % 2stimates o# parameters 3d# %3ln4 P value Positively selected sites

branch A ND3 alternative model p1 L 1.8+,p$ L 1.1!, D1L 1.122, D+L $.!$4 $ 1.1+5 p X 1.1 !!+I

null model p1 L 1.81,p$ L 1.1$, D1L 1.122,D+L $ -one

ND' alternative model p1 L 1.54, p$ L 1.1+2, D1L 1.1+!, D+L !.884 $ $.1+ pX 1.1 !5

null model p1 L 1.5,p$ L 1.1+2, D1L 1.1+!,D+L $ -one

ND) alternative model p1 L 1.84,p$ L 1.14, D1L 1.124, D+L .!1 $ +2.$8 pY 1.11$ 56, &7!, &89, !M, 2$T,

8&K, 5G, $%T, ++!, &85',7+8;, 7$M, ,


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lineage o# C"# $ alternative model p1 L 1.55$ ,p$ L1.11+ , D1L 1.11, D+L $.111 $ 1.!8+ & X 1.1 285M

Cyprinini null model p1 L 1.55$,p$ L 1.11+, D1L1.11 ,D+L $.111 -one

C"#

$$

alternative model p1 L 1.52!,p$ L 1.1$+, D1L 1.1$ , D+L$.111 $ 2.+8+ & Y 1.1 7&I

null model p1 L1.525,p$ L 1.1$!, D1L 1.1$,D+L $.111 -one

C"#

$$$

alternative model p1 L 1.51,p$L 1.1$2 ,D1L 1.1$+, D+L $.111 $ 1.!5 & X 1.1 $$

-one

null model p1 L 1.51,p$ L1.1$2, D1L 1.1$+,D+L $.111

ND5 alternative model p1 L 1.52,p$ L 1.1$8 , D1L 1.1+$,D+L $.111 $ 1.!1 & X 1.1 $=8Inull model p1 L 1.52,p$ L 1.1$8, D1L 1.1+$,D+L $.111 -one

ND3 alternative model p1 L 1.858,p$ L 1.14, D1L 1.12!,D+L $.111 $ 1.41 & X 1.1 T,%=&I,++8;,%$I,&&$/

null model p1 L 1.858,p$ L 1.14, D1L 1.12!,D+L $.111

ND' alternative model p1 L 1.541,p$ L 1.1+2, D1L 1.1++,D+L $.111 $ 1.52 & X 1.1 &84,&


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Table % "ingle variable regression analyses on substitution rates and biological traits

ates Trait n Coe##icients: slope % p*

value

dN6dS Max absolute

fecundity

+4 1.2$$ 1.$45 1.1![

Max body mass + 1.!2 1.$+ 1.1[

Max body length 41 1.$+ 1.1! 1.$8

length at first maturity +5 1.+4 1.14 1.$44

Max lifespan +$ 1.! 1.18+ 1.$$+

dN Max absolute

fecundity

+4 %1.45 1.28 1[[

Max body mass + %1.215 1.$4 1.1!2[Max body length 41 %1.2!$ 1.$84 1.1$[

length at first maturity +5 %1.248 1.+$5 1.115[

Max lifespan +$ %1.22 1.+54 1.1$$[

dS Max absolute

fecundity

+4 %1. 1.25 1[[

Max body mass + %1.2$$ 1.$45 1.1!![

Max body length 41 %1.22 1.+$+ 1[[

length at first maturity +5 %1.22$ 1.$52 1.1$[

Max lifespan +$ %1.!5 1.+5 1.1$+[ [p1.1, [[p1.11.


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'ig $ The phylogenetic tree o# cyprinids based on $& protein*coding genes #rom mitogenomes and molecular dating o# the +% cyprinid

species or subspecies 6ootstrap support values and 6ayesian posterior probabilities are shown at nodes and separated by slashes for maximum

li:elihood and Mrbayes, respectively.6ranch , 6, C, , ', 7 represent the branches leading to the clades of Cyprininae, anionini, 'obionini,euciscini, cheilognathini and Aenocypridini, respectively. 6ranch 3 and B indicate the ancestral lineages of Cyprinini and extant genusCarassius, respectively. The fossil%based constraints are indicated with diamonds. 6ranch lengths are proportional to divergence times (in millionof years). The names of species shadowed by gray belong to the clade of Cyprininae. The names of species in box belong to Carassias.


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(a)

(b)

(c)

'ig % Comparisons o# median and the averagevalues o# dN,dS- dNand dS#or $& individual

genes- genes involved in comple.es I- I/- and /- and $& protein*coding genes set in +%

cyprinid species among si. clades o# cyprinids6oxes include 1K of the distributions. (a)Comparison of median and the average values ofdNdS. (b) Comparison of median and the

average values ofdN. (c) Comparisons of median and the average values ofdS.

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