Weizhao YANG ,Tao ZHANG ,Zhongyi YAO ,Xiaolong TANG and Yin QI＊

1Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu 610041,Sichuan,China

2Department of Biology,Lund University,Lund 22362,Sweden

3Institute of Biochemistry and Molecular Biology,School of Life Science,Lanzhou University,Lanzhou 730000,Gansu,China

Abstract Understanding how organisms adapt to the environment is a compelling question in modern evolutionary biology.Genetic assimilation provides an alternative hypothesis to explain adaptation,in which phenotypic plasticity is first triggered by environmental factors,followed by selection on genotypes that reduce the plastic expression of phenotypes.To investigate the evidence of genetic assimilation in a high-altitude dweller,the toad-headed agama Phrynocephalus vlangalii,we conducted a translocation experiment by moving individuals from high-to low-altitude environments.We then measured their gene expression profiles by transcriptome sequencing in heart,liver and muscle,and compared them to two low-altitude species P.axillaris and P.forsythii.The results showed that the general expression profile of P.vlangalii was similar to its viviparous relative P.forsythii,however,the differentially expressed genes in the liver of P.vlangalii showed a distinct pattern compared to both the lowaltitude species.In particular,several key genes (FASN,ACAA2 and ECI2) within fatty acid metabolic pathway were no longer differentially expressed in P.valgnalii,suggesting the loss of plasticity for this pathway after translocation.This study provides evidence of genetic assimilation in fatty acid metabolism that may have facilitated the adaptation to high-altitude for P.vlangalii.

Keywords genetic assimilation,gene expression,plasticity,Phrynocephalus vlangalii

1.Introduction

Elucidating the mechanisms of adaptation is a compelling question in modern evolutionary biology (Rose,2001;Smith and Eyre-Walker,2002).In the past decades,a “mutation-first evolution” model was widely accepted to explain the process of adaptation,in which mutations linked to fit phenotypes are under natural selection,resulting in changes in phenotype frequencies,and ultimately,adaptation (Carroll,2008).However,a “plasticity-first evolution” model has also been proposed in which phenotypic plasticity is first triggered by environmental factors,followed by selection on genotypes influencing the plastic expression of phenotypes through genetic accommodation (Moczek

et al

.,2011;Jones and Robinson,2018).Genetic assimilation is a special case of such process,where the initially plastic phenotypes are gradually fixed,leading to reduced phenotypic plasticity and only adaptive phenotypes,even without environmental stimuli (Waddington,1953;Pigliucci,2006).Although a number of studies have shown evidence of genetic assimilation,such as cases in

Drosophila melanogaster

(Dworkin,2005;Ghalambor

et al

.,2010) and

Spea bombifrons

(Levis and Pfennig,2019),the role of genetic assimilation in adaptive evolution remains unclear and controversial.Gene expression profiles provide an excellent tool to investigate genetic assimilation in evolution.In general,phenotypic plasticity springs from environmentally sensitive regulation of gene expression,which alters the profiles of gene expression (Colinet and Hoffmann,2012;Beaman

et al

.,2016).As plastic phenotypes may evolve in response to selection with increased or decreased levels of plasticity,the gene expression associated with the phenotypes may evolve as well (Renn and Schumer,2013).Thus,variable patterns of gene expression mirror the evolution of phenotypic plasticity.In the case of genetic assimilation,plastically expressed genes in response to the environment may finally become fixed (Scoville and Pfrender,2010;Renn and Schumer,2013).Previous studies that have used gene expression profiles as a tool to investigate the pattern of genetic assimilation have mainly focused on eurociality in the honeybee (Toth

et al

.,2007;Bloch and Grozinger,2011);host specialization in

Drosophila

(Matzkin

et al

.,2006;Matzkin,2012);and character displacement in spadefoot toads (Levis

et al

.,2017).Toad-headed agamas (genus

Phrynocephalus

) at the Qinghai-Tibetan Plateau (Huey,1982;Zhao

et al

.,1982) provide an excellent model system to study genetic assimilation.As true dwellers of high-altitude environments,as high as 5 300 m above sea level (a.s.l),this group has experienced a long-term adaptive evolution under several extreme stressors,including hypobaric hypoxia,low ambient temperatures,and strong UV radiation (Scheinfeldt and Tishkoff,2010;Cheviron and Brumfield,2011).Phylogenetic studies indicated that a total of six high-altitude species in this genus formed a monophyletic viviparous clade,including another low-altitude species,

P.forsythii

(Figure 1A;Guo

et al

.,2002;Guo and Wang,2007;Wang

et al

.,2014).

Figure 1 Translocation experiment and transcriptome sequencing.A:The phylogenetic relationship among Phrynocephalus axillaris,P.forsythii,and P.vlangalii.The red branch indicates the viviparous high-altitude clade leading to P.vlangalii.B:Principal component analysis (PCA) plot for gene expression profiles.C:Heatmap for gene expression profiles among P.vlangalii samples.D,E,and F:Heatmap for gene expression profiles among the three species for heart,liver,and muscle,respectively.In all the comparisons,P.vlangalii was clustered with P.forsythii,consistent with their phylogenetic relationship.

High-altitude toad-headed agamas have evolved a series of characteristics that underline their adaptations to extremely high-altitude environments.At DNA sequence level,a couple of genes have been identified with signature of positive selection associated with energy metabolism and DNA repair(Yang

et al

.,2014;Yang

et al

.,2015;Sun

et al

.,2018).Accordingly,physiological studies revealed that,compared to low-altitude species,high-altitude toad-headed agamas may have decreased basal metabolic rate and increased the utilization of nutrients(e.g.fatty acid) to balance the energy budget (Tang

et al

.,2013;Li

et al

.,2016;Zhang

et al

.,2018),which is a common strategy for high-altitude adaptation for ectothermic vertebrates (Cooper

et al

.,2002;Li

et al

.,2016).On the other hand,low-altitude toadheaded agamas exhibited the same direction of plastic response to highland environments (e.g.Tang

et al

.,2013).Qi

et al.

(unpublished) have conducted an experiment by translocating two low-altitude species

P.axillaris

(oviparous) and

P.forsythii

(viviparous) to highland environments and measured their transcriptomic,metabolomic,and behavioral responses.The two species showed a similar pattern,in which significantly plastic change occurred for core genes and metabolites within fatty acid metabolic pathway.However,whether the same plasticity still exists in high-altitude agamas remains unknown.For other taxa,several species have shown loss of plasticity while adapting to high-altitudes due to genetic assimilation,such as

Alnum glutinosa

(Kort

et al

.,2016),and montane butterfly

Colias eriphyle

(Kingsolver and Buckley,2017).Therefore,to investigate if toadheaded lizards have experienced the loss of plasticity at highaltitudes could provide new evidence of genetic assimilation in high-altitude adaptation for ectotherms.Here,we present a study to investigate the gene expression profiles and plasticity of a high-altitude species Qinghai toadheaded agama (

P.vlangalii

).This species inhabits in Qinghai-Tibetan Plateau at altitudes ranging from 2000 to 4600 a.s.l.(Zhao

et al

.,1999).We implemented a translocation experiment by moving

P.vlangalii

individuals from high-to low-altitude environment and measured the gene expression profiles for three organ tissues -heart,liver,and muscle,to investigate the plastic changes in gene expression.In addition,we compared

P.vlangalii

to two low-altitude species,

P.axillaris

(oviparous) and

P.forsythii

(viviparous),and revealed the pattern of expression plasticity for

P.vlangalii

that may indicate whether genetic assimilation has contributed to theadaptation to high-altitude environmentfor this species.

2.Materials and Methods

2.1.Ethical approval

All applicable international,national,and/or institutional guidelines for the care and use of animals were strictly followed.All animal sample collection protocols complied with the current laws of China.The sampling and experiment in this study were carried out with permission(Number 2017005) from the Ethical Committee for Animal Experiments in Chengdu Institute of Biology,Chinese Academy of Sciences.

2.2.Translocation experiment

A total of 12 male adults of

P.vlangalii

were sampled from Zoige,Sichuan Province of China,at an altitude of 3 400 meters a.s.l.All samples were randomly and evenly assigned into an ‘origin group’ and ‘translocation group’.For the origin group,we measured their morphological traits and then performed euthanasia using decapitation and collected tissues of heart,liver,and muscle,by preserved in RNA later (Invitrogen,USA) in the field.For the translocation group,we moved the individuals to a workstation in Chengdu City,Sichuan Province of China,with an altitude of 650 meters a.s.l.All individuals had been kept in an outdoor enclosure similar to their native environment for six weeks to make the individuals acclimate to translocation environment.Then,the same procedure was conducted for the translocation group to collect tissue samples.To alleviate the potential bias stimulated by the environment,the field sites were standardized in three aspects:1) sand obtained from the origin sites of

P.vlangalii

to the translocation site as substrate;2) mealworms were provided as food every three days for both sites;and 3) fishing net above enclosures to reduce the risks of bird predation.

2.3.Transcriptome sequencing

Total RNA was extracted from each tissue sample according to TRIzol protocols(Invitrogen,USA).Transcriptome sequencing was implemented on Illumina HiSeq 2500 platform with paired-end 150 base pair (bp) by Novogene (Beijing,China).Sequence data were deposited in NCBI Short Reads Archive (SRA) with BioProject accession number PRJNA718616.Raw sequence reads were first cleaned by removing the adapter sequences and low-quality base calls using a Novogene pipeline.Trimmomatic v0.35(Bolger

et al

.,2014) was used to trim the reads with LEADING:3,TRAILING:3,SLIDINGWINDOW:4:5,MINLEN:70,and default parameters.We checked for reads quality before and after filtering with FASTQC version 0.11.8 (Andrews,2010).One individual with all three types of tissues (E1,K1,and Q1;details in Table S1) was used for

de novo

assembly of transcriptome via Trinity v2.8.4 after

in silico

read normalization (Grabherr

et al

.,2011;Haas

et al

.,2013).We then used kallisto version 0.44.0 (Bray

et al

.,2016) to quantify the abundance of the assembly and build the transcripts expression matrices.Assembled transcripts with ‘transcripts per million transcripts’ (TPM) less than three were filtered to generate the final assembly for each species.To compare to other two low-altitude species

P.axillaris

and

P.forsythii

(Qi

et al

.,unpublished),a best reciprocal hit (BRH)method was applied to identify 1:1:1 orthologous sequences among the three species (Camacho

et al

.,2009).

2.4.Differential expression analysis

The clean reads for each sample were mapped against the

P.vlangalii

transcriptome assembly by using STAR version 2.6 (Dobin

et al

.,2013).The number of reads matched to the same transcripts was counted by HTSeq-count tool with the ‘union’ resolution mode (Anders

et al

.,2015).The overall similarity among tissue samples was measured by Euclidean distance and visualized by clustering heatmap through ‘pheatmap’ package after regularized log transformation (rlog) of normalized counts via DESeq2 version 1.20 (Love

et al

.,2014).Principal component analysis (PCA) was also used to assess the relationship among different samples.Differentially expressed genes (DEGs) were estimated through generalized linear models in edgeR package version 3.22.5(Robinson

et al

.,2010;McCarthy

et al

.,2012).We adopted a strict criterion to identify DEGs,with fold-value ≥ 2 and adjusted

P

-value (FDR) ＜ 0.05.Functional annotation was performed by mapping the transcripts against the UniProtKB/Swiss-Prot database (release“2018_08”) with blast hits E-value cut-off greater than 10.For those transcripts with annotation information,functional over-representations of DEGs were performed using the clusterProfiler package (Yu

et al

.,2012) in R with annotation to GO category and KEGG pathway database.The minimum number of genes required for each test of a given category was 5.All tests were corrected by false discovery rate (FDR).To compare the plastic pattern with

P.axillaris

and

P.forsythii

for genes associated with fatty acid metabolism,we also checked the expression profiles of 12 diagnostic genes (Table S2),which showed differential expression in liver for the two low-altitude species,including key genes regulating the synthesis (Fatty Acid Synthase,FASN) and catabolic process (Acetyl-Coenzyme A Acyltransferase 2,ACAA2,and Enoyl-CoA Delta Isomerase 2,ECI2) of fatty acid.We calculated the absolute fold change of expression from low-to high-altitude for those genes among the three species,and tested the significance of differences by two-tailed Student’s

t

test.

3.Results

3.1.Transcriptome sequencing

A total of 44 793 768-70 718 484 raw reads were generated for

P.vlangalii

by Illumina sequencing.After filtering,42 722 956-67 578 714 reads were retained.One sample (R6) was excluded from the following analyses due to low quality of sequencing.Overall,36 336 transcripts were obtained with N50 size of 2 232 bp and mean length of 1 093 bp.By BRH method,8 892 orthologous transcripts were identified among

P.axillaris

,

P.forsythii

,and

P.vlangalii

.

3.2.Gene expression profile

Both principal component analysis (PCA) and clustering analysis demonstrated that each tissue type presented a distinct expression signature and all samples were unambiguously grouped by tissue origin (Figure 1B).Within each tissue,samples were also separated by origin and translocation groups.In addition,samples from heart and muscle expressed closer than from liver,which may reflect that a large part of the heart is composed of cardiac muscle tissue(Figure 1C).By comparing the expression profiles of

P.vlangalii

to the other two low-altitude species

P.axillaris

and

P.forsythii

,we found that the

P.vlangalii

was closer to

P.forsythii

in all the three tissues,consistent with their phylogenetic relationship(Figure 1D,E,F).

3.3.Differential expression analysis

Given low-altitude samples as reference,a total of 875 differentially expressed genes(DEGs) were identified in the heart for

P.vlangalii

,with 400 down-regulated and 475 up-regulated.Similarly,688 DEGs were identified in muscle,with 345 down-regulated and 343 up-regulated.We identified 1 220 DEGs in liver,the greatest number of DEGs among all three tissues,with 612 downregulated and 608 up-regulated.

Through functional annotation,we found that no GO category and KEGG pathway was over-represented by DEGs in heart.A total of 41 GO categories and 13 KEGG pathways were identified over-represented by DEGs in liver (Figure S1).Network clustering of the GO categories indicated that most of the categories were associated with monocarboxylic acid metabolic process,which could be linked to antibiotic catabolic process and detoxification (Figure 2A).In muscle,7 GO categories and 1 KEGG were over-represented by DEGs(Figure S2),which were associated with extracellular matrix organization and carbohydrate derivative catabolic process.

For the 12 diagnostic genes associated with fatty acid metabolism,the absolute fold change of expression in liver of

P.vlangalii

was significantly lower (ΔlogFC=0.8300) than that of

P.axillaris

(ΔlogFC=2.1298;

P-value

=2.84×10) and

P.forsythii

(ΔlogFC=1.7397;

P-value

=1.31×10),along with no significant difference between the latter two species (

P-value

=0.44) (Figure 2B).In addition,none of the key genes FASN,ACAA2 and ECI2 was differentially expressed between low-and high-altitude(Figure 2C).

4.Discussion

In this study,we conducted a translocation experiment by moving

P.vlangalii

from a high-to low-altitude environment and measured their expression profiles and plasticity via transcriptome sequencing.Our results clearly illustrated that each tissue type of

P.vlangalii

presented an unambiguous expression signature,with hundreds of DEGs up-regulated or down-regulated in each tissue,respectively.In comparison to the other two low-altitude species

P.axillaris

(oviparous) and

P.forsythii

(viviparous),although the expression profile of

P.vlangalii

was still closer to

P.forsythii

,DEGs in liver exhibited a distinct pattern from the other two species,with reduced plasticity in expression of genes associated with fatty acid metabolism.A common strategy for ectothermic vertebrates in response to extreme environments at high-altitude is to suppress basal metabolism and increase utilization of nutrients to balance the energy budget (Cooper

et al

.,2002;Li

et al

.,2016;Zhang

et al

.,2018).Tang

et al.

(2013) revealed that a high-altitude toadheaded agama,

P.erythrurus

,behaved similarly,with lower mitochondrial respiratory rate but higher fat utilization in liver compared to a low-altitude species

P.przewalskii

.Qi

et al.

(unpublished) further found that both oviparous (

P.axillaris

)and viviparous (

P.forsythii

) species in this genus showed a very similar plastic pattern of gene expression and metabolites associated with fatty acid metabolism in liver by translocating from low-to high-altitude.However,our study suggests that the true high-altitude species

P.vlangalii

has a distinct pattern.Although the general expression profile of

P.vlangalii

was still closer to

P.forsythii

,consistent with their phylogenetic relationship,DEGs in liver of

P.vlangalii

were mostly concentrated in functional categories like monocarboxylic acid metabolic process,antibiotic catabolic process and detoxification.Furthermore,the expression patterns of 12 diagnostic genes associated with fatty acid metabolism,which showed significantly differentially expression in

P.axillaris

and

P.forsythii

,illustrated reduced plastic expression in

P.vlangalii

,including key genes FASN,ACAA2,and ECI2.FASN gene encodes an enzyme fatty acid synthase that plays a core role in fatty acid synthesis (Jayakumar

et al

.,1995);ACAA2 and ECI2 encode proteins which are key mitochondrial enzymes involved in beta-oxidation,a step in the catabolic process of fatty acid(Abe

et al

.,1993;Geisbrecht

et al

.,1999).None of these genes were differentially expressed between low-and high-altitude groups for

P.vlangalii

,suggesting no significant difference in fatty acid metabolism after translocation.The result indicated that

P.vlangalii

may have lost the capacity of plasticity in fatty acid metabolism due to genetic assimilation.Plasticity in fatty acid metabolism is thought to contribute to maintaining life activities for organisms moving to high altitude (Hammond

et al

.,2001;Storz

et al

.,2010;Refsnider

et al

.,2018).As a true dweller adapted to high altitude,

P.vlangalii

may have evolved other traits to trade-off the reduction of capacity for plasticity in fatty acid metabolism,similar to the case of

P.erythrurus

(Tang

et al

.,2013).However,the detailed mechanism for

P.vlangalii

still needs further research.Our study provided special evidence of genetic assimilation that may have facilitated the high-altitude adaptation for

P.vlangalii

.Genetic assimilation refers to the process in which initially plastic phenotypes are gradually fixed,leading to reduced phenotypic plasticity and only adaptive phenotypes,even without environmental stimuli (Waddington,1953;Pigliucci,2006).At gene expression level,plastically expressed genes may finally become fixed,given that gene expression mirrors phenotypic plasticity (Scoville and Pfrender,2010;Renn and Schumer,2013).Genetic assimilation provides an alternative hypothesis to explain the mechanism of adaptive evolution,where phenotypic plasticity is first triggered by environmental factors,followed by selection on genotypes influencing the plastic expression of phenotypes (Moczek

et al

.,2011;Jones and Robinson,2018).In fact,species such as

Alnum glutinosa

(Kort

et al

.,2016) and

Colias eriphyle

(Kingsolver and Buckley,2017) also showed similar loss of plasticity in adaptation to high-altitude environments,which indicated that the genetic assimilation might be an effective way for organisms to adapt to extreme environments.However,more studies especially on modeling of phenotypic plasticity are still required to elucidate the role of genetic assimilation in adaptation.

Acknowledgements

We are grateful to Cuoke,Erga,X.Qiu,Y.Wu,and X.Zhu for logistic assistance in field station,and J.Ramos for English language editing.We have obtained the permits from Zoige National Wetland Nature Reserve,where we have a long-term field research station on ecology and evolution of lizards.This work was supported by National Natural Science Foundation of China (No.31501855) and the Second Tibetan Plateau Scientific Expedition and Research Program (STEP) with Grant No.2019QZKK0402.

Appendix

Figure S1 Top 20 GO categories over-represented by DEGs in liver.

Figure S2 GO categories over-represented by DEGs in muscle.

Table S1 Sample information.

Table S2 Diagnostic genes associated with fatty acid metabolism.