3.5 Molecular evolution of rps3
The rps3 gene was identified in the rnl intron of Hypocreales fungi，except Acremonium fuci.The rps3 in A.fuci is freestanding.The length of rps3 ranged from 1131bp in A.implicatum to 1584bp in O.sinensis.In O.sinensis，rps3 was located in the IA intron of rnl(rnl_I8).A BLASTx search against the NCBI database showed that the rps3 was fused to a novel LAGLIDADG HE gene.Half of rnl-18 was homologous to rps3 in H.rhossiliensis and half was similar to the HE gene from Ophiostoma novo-uimi subsp.americana(Gibb and Hausner，2005).
The phylogenetic relationships among the Hypocreales inferred from rps3 genes were different from that based on 14 PCGs.The sub-clusters(Hypocreaceae，Cordycipitaceae，Clavicipitaceae，and Ophiocordycipitaceae)in Hypocreales were similar between these two phylogenetic trees，except Nectriaceae.In the phylogenetic tree based on rps3，Fusarium oxysporum，belonging to Nectriaceae，was clustered with M.anisopliae rather than Fusarium spp.Moreover，the Cordycipitaceae cluster was separated from the Ophiocordycipitaceae cluster in the phylogenetic tree inferred from rps3.To measure the selective pressure of the rps3 genes in Hypocreales，d N/dS values were calculated.The values were 0.2400，0.1075，0.1075，0.1023，and 0.0848，when the adopted models were M0(one-ratio)，M1a(neutral)，M2a(selection)，M7(beta)，and M8(beta&ω)，respectively.However，the LRT statistic for comparing M7(ln L(log likelihood value)=—10，788.91)and M8(ln L=—10，702.06)is 173.70(2Δ=2*(10，788.91 10，702.06)=173.70)，with a P-value＜0.01 using the Chisquare test(with d.f.=2).To further confirm whether rps3 genes in Hypocreales are evolving under positive selection or not，the values of d N/dS were calculated in relation to the outgroup Penicillium nordicum.The values of dN/dS were above 1.0 in Hypocreales.Moreover，we explored a total of 36 sites in rps3 with the values of dN/dS＞1(Table 4；Fig.3)，and 16 sites were statistically significant(P≥0.95)(Table 4；Fig.3).
Fig.3 Positive selection sites across rps3 in Hypocreales(https://www.daowen.com)
Positive selection sites were identified by Bayes Empirical Bayes dN/dS values and labeled with symbols of“.”，“*”(P≥0.95)and“#”(P≥0.99).
Table 4 Log-likelihood values and parameter estimates for rps3 genes in Hypocreales
p is the number of parameters in theωdistribution.Positive selection sites with P≥95% were shown in bold.