Codon usage of PCGs and gene selection pressure
Total PCGs nucleotide length ranges from 10 848 bp to 11 142 bp, and the
AT contents ranges from 60.7% to 81.0%. Most of the PCGs initiate with
the typical start codon ATN and TTG, whereas the special start codons
AAC, AAT, AAA and TCA are found for COX1 ; AAA for COX2 ;
GTG for ND1 and ND4L ; AGG and AAA for ND2 and GTG
for ND4 . The most frequently used stop codons are TAA and TAG,
followed by the incomplete stop codons T and TA. The most frequently
used codons are UUA (Leu2), UCU and UCA (Ser2), CGA (Arg), whereas AGC
(Ser1), ACG (Thr), GCG (Ala) and CUG (Leu1) are the least used (Fig. 4).
For each PCG, the Ka/Ks ratio is less than one, and the ATP8 has
the highest Ka/Ks ratio (0.33-0.67), followed by seven genes (ND6,
ND5, ND4, ND2, ND4L, ND1, ND3 ) with Ka/Ks ratios of 0.17-0.42. Complex
IV (COX1, COX2 and COX3 ), Complex III (CYTB ) andATP6 have low Ka/Ks ratios with range from 0.01 to 0.17 (Fig. 5).
These results imply all of these 13 PCGs experienced purifying
selection, especially Complex IV and Complex III.
Phylogenetic
relationships
Substitution saturation tests show no saturation for
three datasets AA, PCG12 and
PCG12 + rRNAs (Iss < Iss.cSym or Iss.cAsym, p< 0.05) (Table 2), which proposes that these three datasets be
appropriate for phylogenetic construction based on ML and BI. Six trees
generated using these three datasets and both ML and BI are slightly
different in topology (Figs. 6-7; Figs. S3-S6).
The Ciidae is located at base
of all phylogenetic trees, followed
by families
Mordellidae + Ripiphoridae. The
Mordellidae + Ripiphoridae, Mordellidae and Ripiphoridae all looks
monophyletic (PP = 1; BP = 100) and the later two appear sister groups
each other. All remaining families also appear monophyletic, and the
family Aderidae seems sister with
“Meloidae clade” +
“Tenebrionidae clade”. Both “Tenebrionidae clade” and
“Meloidae clade” looks
monophyletic with both of them being sister groups (Fig. 6). In the
“Meloidae clade”, the families Meloidae + Anthicidae look a
monophyletic (PP = 1; BP = 100), and it appears sister to the
“Oedemeridae clade”, and the Meloidae and Anthicidae look monophyletic
(PP = 1; BP = 100), and a sister each other. In the “Oedemeridae
clade”, the Oedemeridae, Pyrochroidae, Salpingidae and Scraptiidae seem
monophyletic (PP = 1; BP = 100), and Salpingidae looks a sister with
Scraptiidae. In the “Tenebrionidae clade”, the family Lagriidae and
Tenebrionidae are monophyletic (PP = 1; BP = 90-100), and both of them
are sister groups each other. In Lagriidae, the subfamily Adeliinae is
based at the subfamilies
Lagriinae and Statininae, both of which look monophyletic (PP = 1; BP =
100) and are sister groups each other. In Tenebrionidae, the subfamily
Pimeliinae appears monophyletic
group (PP = 1; BP = 100) and is located at the base of Tenebrionidae.
The Alleculinae and Stenochiinae look monophyletic (PP = 1; BP = 100),
and the subfamilies Tenebrioninae and Diaperinae appears polyphyletic
groups.
Six trees generated using AA, PCG12 and PCG12 + 2 rRNAs datasets and
both ML and BI are slightly different in topology. For AA dataset, the
topologies using ML and BI are different in the positions of
Prostomidae. Prostomidae and Tetratomidae are clustered as one clade in
ML tree, but not in BI tree. For PCG12 dataset,
two same topologies of trees from
BI and ML differ from two topologies of AA dataset in phylogenetic
relationship of the “Oedemeridae clade”. For PCG12 + 2 rRNAs dataset,
the positions of major families are the same as the four topologies of
AA and PCG12 datasets, with only a few differences in the “Oedemeridae
clade”.
Divergence time
The AA dataset was used to estimate divergence time because AA had
higher node support values than others in the initial phylogenetic
assessment using Bayesian appraoch. Based on three fossil calibrations
points of Protoripidius burmiticus (166.6 Mya),Camelomorpha longicervix (145 Mya) and Alphitopsis
initialis (143.6 Mya) (filled red cycles in Fig. 8), the superfamily
Tenebrionoidea was inferred to originate in the early Jurassic (192.6
Mya, 95% confidence interval (CI): 179.3-208.7 Mya), with most families
subsequently diverging in the Jurassic and early Cretaceous (Fig. 8).
The family Mordellidae and Ripiphoridae is among the earliest diverged
families in the superfamily, and is estimated to originate
at
115.7 and 126.6 Mya in the Cretaceous, respectively. In the “Meloidae
clade”, the family Meloidae is estimated to be derived at 105 Mya in
the Cretaceous, the Anthicidae at 123.8 Mya in the early Cretaceous, and
the Oedemeridae at 100.9 Mya in the middle Cretaceous. In the
“Tenebrionidae clade”, the Lagriidae is estimated to originate at
134.3 Mya in the early Cretaceous, and the Tenebrionidae at 128.9 Mya in
the early Cretaceous. In the family Lagriidae, the subfamily Statiriinae
diverged 97.6 Mya in the late Cretaceous, and the Lagriinae diverged
78.6 Mya in the late Cretaceous. In the family Tenebrionidae, the
subfamilies Pimeliinae, Alleculinae and Stenochiinae originated at 71.1,
53.6 and 53.3 Mya in the Paleogene, respectively. All of these
families/subfamilies are proposed to be monophyletic and confirmed in
relationships in the phylogenetic analyses using different data or
inferring methods, and others are not determined for their monophyly or
relationships or have a few species included in the phylogenetic
analyses, and therefore they are not given an inferring of divergence
time.