The authors have declared that no competing interests exist.
Conceived and designed the experiments: NS. Performed the experiments: NS. Analyzed the data: NS. Contributed reagents/materials/analysis tools: APL. Wrote the paper: NS.
The planthopper superfamily Fulgoroidea (Insecta: Hemiptera) is one of the most dominant groups of phytophagous insects. It comprises about 20 families, containing a total of 9000 species worldwide. Despite several recent studies, the phylogeny of Fulgoroidea is not yet satisfactorily resolved and the phylogenetic positions of several key families, especially Cixiidae, Delphacidae, Tettigometridae, Nogodinidae, Acanaloniidae and Issidae, are contentious. Here, we expand upon recent phylogenetic work using additional nuclear (
The planthopper superfamily Fulgoroidea (Insecta: Hemiptera) is among the dominant groups of phytophagous insects, which includes ∼20 families and more than 9000 species worldwide
Planthoppers are traditionally distinguished from other auchenorrhynchan insects by their antennal sensillae and hind coxae, which are synapomorphies. Muir
According to the study by Metcalf
At present, the fossil record for fulgoroids is very incomplete. The earliest Fulgoromorpha fossil record is found in deposits dating to the mid-Late Permian (260 Mya)
Molecular phylogenetic analyses concerning the superfamily Fulgoroidea have been based solely on either nuclear
In this paper, we expanded upon a recent phylogenetic study by adding more sequences and new taxa. This is the first comprehensive analysis incorporating both nuclear (
To provide a robust phylogenetic estimate for Fulgoroidea, we analyzed an expanded dataset including a larger number of
All adult specimens were morphologically identified, and preserved in 100% ethanol and stored at −80°C in the Key Laboratory of Animal Evolution and Systematics, Institute of Zoology, Chinese Academy of Sciences.
Total genomic DNA extraction was performed according to Aljanabi and Martinez
Two mitochondrial and two nuclear DNA markers were targeted for each sample. Sections of the large subunit
Up primers | Sequence (5′→3′) | Down primers | Sequence (5′→3′) |
Ful_18S_F |
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Ful_18S_R |
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Ful_28S_F |
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Ful_28S_R |
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Ful_16S_F |
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Ful_16S_R |
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Ful_cytb_F |
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Ful_cytb_R |
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Raw sequence files were proofread and aligned into contigs in BioEdit version 7.0.5.3
Nucleotide statistics and sequence analysis were performed in MEGA 5
The separate and combined datasets were used for phylogenetic analysis by maximum likelihood (ML) and Bayesian inference (BI). In view of the nucleotide saturation in the mitochondrial genes, the
In the ML analyses, tree searches with each dataset were performed under the best models using software PhyML v 3.0
BI analyses were conducted in MrBayes 3.2
Hypothesis testing was done in a maximum likelihood framework using the concatenated dataset. Based on the new results obtained in the current study, we tested several alternative positions for some interesting groupings or uncertain branching relationships (
Hypothesis | Item | Rank | ||
SH | AU | |||
ML (No constraint enforced) | 1 | 1 | 0.978 | 0.732 |
monophyly of Fulgoroidea | 2 | 2 | 0.978 | 0.732 |
(Delphacidae and, Cixiidae) as sister group to the rest of Fulgoroidea | 3 | 3 | 0.978 | 0.732 |
Tettigometridae as the clade branching off first in Fulgoroidea | 4 | 10 | 0.677 | 0.035* |
monophyly of Lophopidae, Eurybrachidae | 5 | 6 | 0.938 | 0.566 |
Lophopidae sister to Eurybrachidae | 6 | 7 | 0.938 | 0.566 |
Flatidae sister to Ricaniidae | 7 | 5 | 0.978 | 0.732 |
Caliscelidae sister to Tettigometridae | 8 | 9 | 0.836 | 0.433 |
((Achilidae, Achilixiidae),Derbidae) | 9 | 12 | 0.504 | 0.046* |
Kinnaridae sister to Meenoplidae | 10 | 11 | 0.601 | 0.083 |
Acanaloniidae sister to Issidae | 11 | 8 | 0.429 | 0.954 |
Fulgoridae sister to Dictyopharidae | 12 | 4 | 0.978 | 0.732 |
Asche (1987) | 13 | 16 | 0.000 | 6e−054** |
Emeljanov (1990) | 14 | 15 | 0.000 | 3e−052** |
Bourgoin (1993) | 15 | 14 | 0.000 | 0.001** |
Urban and Cryan (2007) | 16 | 13 | 0.000 | 0.001** |
The “Hypothesis” column indicates the constraints introduced in the phylogenetic analysis. The “Rank” column shows the order of each constraint compared with the ML tree inferred from our combined dataset.
The dating analysis based on combined dataset was performed under the GTR+I+G substitution model using BEAST 1.7.1
The number of taxa included in the separate
Loci | Length (bp) | New sequences obtained | SequencesfromGenBank | Total number of sequences | Conserved sites | Variable sites | Parsim-Info sites | |||
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1321 | 45 | 26 | 71 | 781 | 540 | 332 | |||
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1306 | 39 | 21 | 60 | 587 | 719 | 469 | |||
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497 | 44 | 7 | 51 | 100 | 397 | 346 | |||
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1st | 196 | − | − | − | 63 | 133 | 119 | |||
2nd | 196 | − | − | − | 82 | 114 | 79 | |||
3rd | 196 | − | − | − | 0 | 196 | 190 | |||
1st+2nd | 392 | − | − | − | 145 | 247 | 198 | |||
All | 588 | 44 | 7 | 51 | 145 | 443 | 388 | |||
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24.23 | 24.75 | 21.95 | 29.07 | 46.18 | 55.01 | 1.000 | |||
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20.65 | 27.01 | 20.64 | 31.70 | 41.29 | 72.09 | 1.000 | |||
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32.05 | 16.43 | 43.00 | 8.52 | 75.05 | 376.80 | 0.000 | |||
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1st | 30.1 | 15.82 | 38.27 | 15.82 | 68.37 | 111.65 | 0.992 | ||
2nd | 45.92 | 23.47 | 20.41 | 10.20 | 66.33 | 31.85 | 1.000 | |||
3rd | 37.25 | 9.69 | 49.49 | 3.57 | 86.73 | 403.84 | 0.000 | |||
1st+2nd | 38.01 | 19.64 | 29.34 | 13.01 | 67.35 | 87.40 | 1.000 | |||
All | 37.76 | 16.33 | 36.05 | 9.86 | 73.81 | 240.94 | 0.000 | |||
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0.038 | 0.699 | 0.0000 | 0.373 | 0.0000 | |||||
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0.058 | 0.713 | 0.0000 | 0.385 | 0.0000 | |||||
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0.421 | 0.702 | 0.0000 | 0.377 | 0.1290 | |||||
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1st | 0.309 | 0.697 | 0.0000 | 0.390 | 0.0477 | ||||
2nd | 0.185 | 0.697 | 0.0000 | 0.390 | 0.0000 | |||||
3rd | 0.720 | 0.697 | 0.4335 | 0.389 | 0.0000 | |||||
1st+2nd | 0.246 | 0.687 | 0.0000 | 0.358 | 0.0000 | |||||
All | 0.372 | 0.707 | 0.0000 | 0.379 | 0.7658 |
Index of substitution saturation assuming a symmetrical true tree.
Probability of significant difference between Iss and Iss.cSym (two-tailed test).
Index of substitution saturation assuming an asymmetrical true tree.
Probability of significant difference between Iss and Iss.cAsym (two-tailed test).
A chi-square test for base compositional homogeneity revealed significant heterogeneity among taxa for the combined dataset (p<0.0001). To detect the source of this heterogeneity within the data, all loci and each codon position of
Because saturation in substitutions can lead to confusing effects on phylogenetic inferences
The results indicated that GTR+I+G is the best-fit nucleotide substitution model for
The separate analyses of
For the
The trees estimated from
By combining all nuclear and mitochondrial data, we obtained a well-supported phylogenetic hypothesis for Fulgoroidea. Bootstrap support and posterior probabilities are high for almost all nodes. Moreover, shallow nodes were better resolved by the combined dataset than individual
Values above branches denote ML bootstrap support (≥70). The red numbers in circles correspond to ten major clades discussed in the text.
Values above branches denote posterior probabilities (≥0.9).
As with
Clade 5 was composed of Flatidae and Ricaniidae. These formed a well-supported sister group (BS = 95). Clade 6 included the families Achilidae, Tropiduchidae and part of Nogodinidae. Among these, Tropiduchidae and part of Nogodinidae were recovered as sister groups. Part of Nogodinidae, part of Lophopidae, the family Caliscelidae and the Tettigometridae composed Clade 7. Of these, Caliscelidae and Tettigometridae were retrieved as sister groups.
The representative of Achilixiidae, part of Nogodinidae, and the family Derbidae constituted the sister clade to all others in the second major group. Clade 9 was a monophyletic and strongly supported lineage that comprised Dictyopharidae and Fulgoridae. The sister grouping of Fulgoridae and Dictyopharidae was consistently recovered in both inference methods. The remaining species made up Clade 10, which included Acanaloniidae and Issidae. Acanaloniidae was the sister family to Issidae.
Alternative hypotheses were compared based on previous studies or unstable branching in the present analyses. Results of comparisons are summarized in
The alternative hypotheses from previous studies were all rejected by SH and AU tests. Nevertheless, the results of the “Rank” generated by CONSEL clearly showed that our preferred topology was more similar to previous molecular studies than to those based on morphology (
Divergence time estimates are illustrated in
Time units are in millions of years before present. Estimated divergence times are shown near nodes, while blue bars represent 95% credibility intervals. The values on the branches denote the posterior probabilities (≥0.9).
Fulgoroidea is of economic importance, because many species in this group are pests on agricultural crops. However, the relationships among the main lineages of the Fulgoroidea are poorly understood, and previous analyses yielded conflicting hypotheses. Even the family-level classification of Fulgoroidea has remained controversial. As mentioned by Bourgoin et al.
Our molecular study has produced a set of well-supported phylogenetic relationships based on an expanded dataset, which includes both nuclear and mitochondrial sequences for the main clades. Phylogenetic analysis of
Several previous morphology-based studies using a largely unstable character set (e.g., wing morphs, and the position of the median ocellus) have resulted in different topologies for planthoppers. The influential classification by Muir
Basal branching patterns have been the focus of past studies of the phylogeny of Fulgoroidea. Due to morphological characters shared with Cicadoidea, Tettigometridae had been treated as the sister lineage to all other families within Fulgoroidea by Muir
The ML analysis of the combined dataset recovered the grouping of Kinnaridae and Meenoplidae as the sister group to all fulgoroids excluding Cixiidae and Delphacidae. In previous studies, Kinnaridae was assigned as a sister group to Meenoplidae
The sister relationship between Ricaniidae and Flatidae was strongly supported by the concatenated dataset with different analytical methods. Although this sister relationship is a novel placement inferred from molecular data, morphological evidence supporting this arrangement has been noted by previous authors
Most of the previous morphological studies have proposed a close relationship between Achilixiidae and Achilidae. Though Urban and Cryan
Monophyly of Tropiduchidae was supported by different methods (BS = 100 and PP = 1.0). Among the trees generated in this study, Tropiduchidae and partial Nogodinidae always clustered together. In previous studies, the exact relative phylogenetic position of Tropiduchidae remained uncertain
The placement of Tettigometridae among higher Fulgoroidea was decisively demonstrated by our data and has been corroborated by other genetic studies
Fossil evidence shows that Derbidae may be a group branching off early in Fulgoroidea
Morphologists agree that Fulgoridae is a sister group to Dictyopharidae, because both lineages share distinctive male genitalia structure
The monophyly of Issidae has been questioned by previous studies
The superfamily Fulgoroidea started to diversify in the mid-Late Permian (260 Mya), and their presumed ancestors, Coleoscytoidea, extended back into the mid-Permian (270 Mya)
Our ML analysis of combined nuclear and mitochondrial markers recovered some relationships with strong support among major lineages of Fulgoroidea, but our Bayesian analysis only resolved the basal relationships reliably. Individual analyses of nuclear
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We thank Dr Jie Liu and Dr Xing Zhao (Institute of Zoology, Chinese Academy of Sciences, Beijing, China) for technical assistance and advice on the experiments. Thanks Dr Zhi-shun Song, Dr Rong-rong Wang and Dr Ming-xia Sun (Institute of Zoology, Chinese Academy of Sciences, Beijing, China) for help collecting insect specimens.