Abstract
The planthopper superfamily Fulgoroidea (Hemiptera: Fulgoromorpha) currently includes more than 14 000 described species in about 21 extant families. Despite multiple studies attempted, based on morphological characters or DNA sequence data, the phylogeny of this superfamily remains unsatisfactorily resolved. Here we reconstruct the phylogenetic relationship among the families of this superfamily based on the whole mitogenome sequences from 113 species representing 17 planthopper families and three families as outgroups, in which 41 species of Fulgoroidea were sequenced for this study. The phylogenetic trees were reconstructed based on three different datasets, both by maximum likelihood (ML, IQtree) and Bayesian inference (BI, MrBayes, and PhyloBayes). The tree topologies of ML and BI analyses were quite similar with only a few differences in some clades. The phylogenetic results showed that Cixiidae and Delphacidae were placed as sister-taxa at the base of this superfamily; the clade Fulgoridae + Dictyopharidae appeared younger than the Meenoplidae + Kinnaridae one, Derbidae and Achilidae were more recently derived than Fulgoridae + Dictyopharidae without depicting a monophyletic unit, and Tropiduchidae and Acanaloniidae appeared as sister-taxa. The divergence-time estimation analysis shows that most planthoppers underwent relatively rapid radiation during the Jurassic and Cretaceous. Divergence time between Cixiidae and Delphacidae can be traced back to the Middle Jurassic; Meenoplidae, Fulgoridae, and Issidae originated in the Lower Cretaceous; Lophopidae and Eurybrachidae can be traced to the Upper Cretaceous. This paper reconstructs the cladogram of Fulgoroidea based on more comprehensive mitogenome sequences so far, which could provide new clues for a better understanding of the evolution of this superfamily. However, the taxa sampling appears insufficient to address controversial groups in Fulgoroidea.
INTRODUCTION
The planthopper superfamily Fulgoroidea (Hemiptera: Fulgoromorpha) contains a rather diverse group of phytophagous insects, including 21 extant families with more than 14 000 described species worldwide (Bourgoin 2023). Many species in this group have been recorded as significant pests of economic plants (Wilson and O’Brien 1987, Wilson et al. 1994). Plant damage is caused by the direct feeding and ovipositing of the planthopper, causing plants to stunt and wilt, and possibly induce hopperburn (Backus et al. 2005); several species are vectors of bacterial, phytoplasma, or viral pathogens, causing huge damage to plant yield and quality (O’Brien and Wilson 1985, Wilson and O’Brien 1987, Wilson and Claridge 1991, Weintraub and Beanland 2006). Additionally, most planthopper species also secrete waxes and release honeydew; both might pollute the foliage and affect plant photosynthesis. Honeydew facilitates the growth of bacteria, often coating the plant surface with a black mould that can also hinder plant growth (Chou et al. 1985, O’Brien and Wilson 1985, Urban 2019).
Previous studies have explored phylogenetic relationships of Fulgoroidea, based either on morphological characters (Muir 1930, Asche 1987, Emeljanov 1990, Bourgoin 1993a, Chen and Yang 1995, Bourgoin et al. 1997) or by DNA sequence data (Bourgoin et al. 1997, Yeh et al. 1998, 2005, Yeh and Yang 1999, Urban and Cryan 2007, Song and Liang 2013, Bucher and Bourgoin 2019), all addressing the current planthopper families. Taking into account the fossil registers, a new classification of planthoppers was recently suggested (Bourgoin and Szwedo 2022, 2023), separating these current families into two superfamilies: the Delphacoidea Leach, 1815 (including the Delphacidae Leach, 1815 and the Cixiidae Stål, 1839) and the Fulgoroidea Latreille, 1857 (grouping all other current families). Also, in these last years, complete mitogenomes have been tested as molecular markers to analyse phylogenetic relationships within Fulgoroidea, but only with partial samplings, including the families Delphacidae, Fulgoridae Latreille, 1807, Flatidae Spinola, 1839, Achilidae Stål, 1866, Caliscelidae Amyot and Audinet-Serville, 1843, and Ricaniidae Amyot and Audinet-Serville, 1843 (Song et al. 2018, Xu et al. 2019, Huang et al. 2020, Ai et al. 2021, Gong et al. 2021, Wang et al. 2021, Zhang et al. 2022).
However, planthopper phylogeny, even at the family level, remains unsatisfactorily resolved. For example, whether Delphacidae originated within Cixiidae or they both should be sister-taxa needs further investigation (Muir 1923, Asche 1987, Urban and Cryan 2007, Ceotto and Bourgoin 2008, Ceotto et al. 2008, Song and Liang 2013, Bucher and Bourgoin 2019). While Dictyopharidae Spinola, 1839 and Fulgoridae were consistently recovered as sister-taxa (Muir 1923, 1930, Asche 1987, Emeljanov 1990, Bourgoin 1993a, Urban and Cryan 2007, Song and Liang 2013, Bucher and Bourgoin 2019), separating these two families remains an open discussion—particularly, the assignment of Dichopterinae Melichar, 1912 and Zanninae Metcalf, 1938 to one of the two families, or treated as separate families, remains controversial (Kirkaldy 1904, Muir 1923, Metcalf 1938, Emeljanov 1979, 2011, 2013, Urban and Cryan 2009). If sister-family relationships between Meenoplidae Fieber, 1872 and Kinnaridae Muir, 1925 seem well established (Bourgoin 1993a, b), the ones between Derbidae Spinola, 1839 and Achilidae, and Acanaloniidae Amyot and Audinet-Serville, 1843 and Flatidae remain under debate, along with the placement of Flatidae, Lophopidae Stål, 1866, Eurybrachidae Stål, 1862, Ricaniidae, and Caliscelidae, which still remain controversial (Muir 1923, Asche 1987, Urban and Cryan 2007, Song and Liang 2013). Even the monophyly of some of families such as Nogodinidae Melichar, 1898 (Yeh and Yang 1999, Yeh et al. 2005, Urban and Cryan 2007) or Issidae Spinola, 1839 (Wang et al. 2016, Gnezdilov et al. 2020, 2022) still remains debated.
The Mesozoic Fulgoromorpha fossils are quite numerous to date (Szwedo et al. 2004, Bucher and Bourgoin 2019, Bourgoin and Szwedo 2022, 2023). Several papers have already been provided but the divergence-time estimations of these taxa among Fulgoroidea is still inadequate. For instance, Song and Liang (2013) dated Delphacidae from the Early Cretaceous, around 129 Mya, while Huang et al. (2017) estimated Delphacidae originating in the Cretaceous (nearly 100 Mya). Bourgoin et al. (2018) suggested an Early Cretaceous origin of the Issidae with the basal split of the family occurring between Neotropical taxa (Thioniini) and the remaining issids, implying a possible Gondwanan vicariance scenario. However, Gnezdilov et al. (2022) still consider the Issidae as a more recent group of Fulgoroidea, most probably of Oriental origin with a subsequent dispersal into the Palaearctic, tropical Africa, New World, and Australia.
In this study, different mitogenomic-based matrices and evolutionary models were selected to reconstruct the phylogenetic relationships and to estimate the divergence time within Fulgoroidea; the characteristics of nucleic acid diversity and genetic distance were also explored. This study aims to provide clues for a better understanding of the evolution of this superfamily based on additional mitogenomic data.
MATERIAL AND METHODS
Sample preparation and DNA extraction
Four species collected outside of China between 2018 and 2021 were used in this study: Balduza sp. (Issidae) from Arizona (USA), Aplos simplex (Germar, 1830) (Issidae) from Moline (USA, Illinois), Paravarcia sp. (Nogodinidae) from Vietnam, and Hemisphaerius typicus Walker, 1857 (Issidae) from Malaysia Sabah. The remaining samples were collected from China. Collection information is summarized in Supporting Information, Table S1. All specimens were immediately preserved in 100% ethanol and thereafter stored at −20°C in the Entomological Museum of the Northwest A&F University. After morphological identification, the total genomic DNA was extracted from muscle tissues of the thorax using the DNeasy DNA Extraction kit (Qiagen).
Sequence assembly, annotation, and analysis
Whole mitogenomes were generated by next-generation sequencing (NGS) (Illumina Miseq platform with paired ends of 2 × 150 bp). The raw paired reads were quality-trimmed, assembled, and annotated using GENEIOUS 11.0.2 (Biomatters, Auckland, New Zealand) with default parameters (Kearse et al. 2012). All 13 protein-coding genes (PCGs) were identified as open reading frames and translated into amino acids based on the invertebrate mitochondrial genetic code. The 22 tRNAs were identified and secondary structures of tRNAs were predicted using the MITOS WebServer (Bernt et al. 2013). The rRNA genes and control regions were identified by the boundary of the tRNA genes and by alignment with other mitogenomes in Fulgoroidea. All 13 PCGs genes, 22 tRNA and two rRNA genes were determined by comparison with those of published insect mitochondrial sequences. A sliding window analysis based on 13 PCGs and two rRNA genes with 200 bp in a step size of 20 bp was performed by DnaSP v.5.0 to estimate nucleotide diversity (Pi) from 109 Fulgoroidea mitogenomes and to calculate the nucleotide diversity (Pi) of each PCG and rRNA (Librado and Rozas 2009). Genetic distances among these Fulgoroidea mitogenomes were calculated based on Kimura 2-parameter under MEGA–X (Kumar et al. 2018). Non-synonymous (Ka)/synonymous (Ks) rate ratios among the 13 PCGs were estimated with DnaSP v.5 (Librado and Rozas 2009).
Taxon sampling
We have selected 109 species in Fulgoroidea, including 28 species in Delphacidae, 20 in Issidae, 15 in Fulgoridae, seven in Ricaniidae, five in Cixiidae, Achilidae, Flatidae, and Caliscelidae, four in Meenoplidae, three in Dictyopharidae, two in Derbidae, Tropiduchidae Stål, 1866, Eurybrachidae, Lophopidae, and Nogodinidae, and one in Kinnaridae and Acanaloniidae (one Acanalonia species of Acanaloniidae was identified and sequenced based on one female from Guizhou, China, the genus Acanalonia, and the family Acanaloniidae are all new records in Chinese fauna). Two species from the family Membracidae (in the suborder Cicadomorpha) and each species of the two families Aphididae and Aphalaridae (in the suborder Sternorrhyncha) were selected as outgroups (Supporting Information, Table S1).
Phylogenetic analysis
Phylogenetic analyses were performed based on 13 protein-coding genes and two rRNA genes among 109 species of ingroups and two species of outgroups. Forty-one mitochondrial sequences were sequenced for this study; the remaining mitogenomes were acquired from GenBank. MAFFT was used to align each PCG gene individually in codon-alignment mode and G–INS–i (accurate) strategy, and RNA sequences were aligned using the Q–INS–i strategy normal alignment mode. Results were concatenated into PhyloSuite (Zhang et al. 2020). Poorly aligned regions in the alignments were removed using GBlocks v.0.91b (Castresana 2000, Zhang et al. 2020), the codon data-type was selected for the PCG gene, the nucleotide type was selected for RNA sequences, and default values were used for other parameters. The optimal nucleotide substitution models and partition strategies were recommended by ModelFinder (Zhang et al. 2020). Substitution saturation of each sequence segment was tested using DAMBE 5 (Xia 2013) by comparing the index of substitution saturation (Iss) with critical values (Iss.c). Alignments of individual genes were concatenated to generate three different datasets: (i) the PCG123R matrix with all three codon positions of PCGs and the two rRNA genes; (ii) the PCG123R–ATP8 matrix with all three codon positions of PCGs but without the ATP8 genes; and (iii) the PCG12R–ATP8 matrix with only the first and second codon positions of PCGs and the two rRNA genes but without the ATP8 genes. Both ML and BI analyses were used to infer the phylogenies. The ML analyses were conducted using IQ–TREE (Nguyen et al. 2015), under an ML + rapid bootstrap (BS) algorithm with 1000 replicates. The Bayesian analyses were performed using MrBayes 3.2.6 (Ronquist et al. 2012). Two simultaneous runs were performed, each with one cold chain and three hot chains. Markov Chain Monte Carlo (MCMC) sampling estimated the posterior distributions using the settings for 5 × 106 MCMC generations, with a relative burn-in of 25%, and MCMC termination when the average standard deviation of split frequencies fell below 0.01. Phylogenetic reconstruction of concatenation data based on the site‐heterogeneous model CAT+GTR was performed using PhyloBayes MPI v.1.5a on CIPRES (Miller et al. 2010). The two independent trees were searched and the analysis was terminated when the likelihood of the sampled trees had stabilized and the two runs reached convergence (maxdiff < 0.3 and minimum effective size > 50). The initial 25% of each run was discarded as burn‐in, and a consensus tree was then generated from the remaining trees combined from the two runs.
Divergence-time estimation
MCMCTREE in paml v.4.9h was used to estimate the divergence time (Brown and Yang 2010). Two fossil records were selected as the reference for time calibration: Karebodopoides aptianus (Fennah, 1987) (129.4–125.0 Mya) (Luo et al. 2021), the most recent common ancestor of Cixiidae, and Fulgoridiella raeticaBecker-Migdisova, 1962 (Fulgoridiidae, 199.3–190.8 Mya), the most recent common ancestor of the Delphacoidea + Fulgoroidea (Bourgoin and Szwedo 2023). Karebodopoides (125.0 Mya) was used as a mimimum time constraint for Cixiidae and Fulgoridiella (190.8 Mya) as a mimimum time constraint for the Delphacoidea + Fulgoroidea.
The Supporting Information, Table S2 shows the configuration files mcmctree.ctl (Brown and Yang 2010). TRACER v.1.7.1 (Rambaut et al. 2018) was used to test the convergence of the chains and effective sample size (ESS) for each parameter, which were all >200 (Brown and Yang 2010).
RESULTS
Nucleotide diversity
Nucleotide diversity is based on 13 PCGs and two rRNA genes from 109 sequenced Fulgoroidea species (Fig. 1). The nucleotide diversity values range from 0.139 (rrnL) to 0.379 (ATP8). ND2 (Pi = 0.364), ND6 (Pi = 0.351), and ND3 (Pi = 0.340) appear to show the highest variability after ATP8, while cytochrome c oxidase subunit I (COI) with a Pi value of 0.215 appear as the most conserved gene within the 13 PCGs. The two rRNA genes are also highly conserved, showing lower Pi values (rrnL = 0.139 and rrnS = 0.261).
Sliding window analysis. The red curve shows the value of nucleotide diversity (Pi). Pi value of each PCG is shown below the arrows.
The evolutionary rate analysis is estimated by non-synonymous (Ka)/synonymous (Ks), and based on 13 PCGs among the 109 mitogenomes from Fulgoroidea species (Fig. 2). We find that the genetic distance values range from 0.255 (COI, with the shorter genetic distance) to 0.548 (ATP8, with the largest genetic distance). Cytochrome c oxidase subunit I (COI) with the lowest Ka/Ks value present the strongest purifying selection, while ND4L exhibited the higher Ka/Ks value.
Genetic distance (on average) and ratio of non-synonymous (Ka) to synonymous (Ks) substitution rates.
Phylogeny
To test whether 13PCG and 2rRNA genes, especially ATP8, ND2, ND6, and ND3 with high variability, can be used to construct phylogenetic trees, Xia’s (2013) Iss index of substitution saturation (with Iss < Iss.c, critical values) was used to detect substitution saturation of the 13 PCG genes and two rRNA genes. The results detected little saturation in all genes except for ATP8. To detect the bias of the third codon, the third codon of all PCG genes was deleted and the substitution saturation of the genes was re-tested. The same result was observed. Hence, ATP8 saturated gene and third codon position were removed to reduce the impact of sequence heterogeneity, and three different matrices, PCG123R, PCG123R–ATP8, and PCG12R–ATP8 matrices, were analysed. The phylogenetic trees were constructed based on homogenous and heterogenous models. Meanwhile, the differences between different matrices and models are compared.
The Supporting Information, Table S3 shows the best site-homogenous partitioning scheme and models. The tree topologies are quite similar (Figs 3–5; Supporting Information, Figs S1–S6) with only a few differences in some clades. After gene screening, we used the results based on datasets PCG12R–ATP8 (Figs 3–5). ML and BI analyses based on data from the three different datasets yielded a well-resolved phylogeny with most branches receiving moderate to strong bootstrap support.
ML analysis based on PCG12R–ATP8. Numerals at nodes are bootstrap values (BS). ‘–’ indicates different clades.
BI analysis (MrBayes) based on PCG12R–ATP8. Numerals at nodes are Bayesian posterior probabilities (PP). ‘–’ indicates different clades.
BI analysis (PhyloBayes) based on PCG12R–ATP8. Numerals at nodes are Bayesian posterior probabilities (PP). ‘–’ indicates different clades.
All tree topologies show that the monophyly of the group of the current planthoppers is strongly supported. In Fulgoroidea sec. Bourgoin and Szwedo 2023, sister-clade to Delphacoidea (=Cixiidae–Delphacidae), Meenoplidae appears as a sister to Kinnaridae. Under the homogeneity model, all topologies show Meenoplidae + Kinnaridae forming the first 148.56 Mya clade (Figs 3, 4, 6; Supporting Information, Figs S1, S2, S4, S5). However, under the heterogeneity model, all the trees (PCG123R, PCG123R–ATP8, and PCG12R–ATP8) depict the Meenoplidae + Kinnaridae clade as the most basal in the Fulgoroidea, although its support value remains weak (Fig. 5; Supporting Information, Figs S3, S6). In all trees, Meenoplidae is monophyletic except for Suva longipennaYang and Hu (1985) that moved into the Derbidae clade (Figs 3–5; Supporting Information, Figs S1–S6).
Chronogram of Fulgoroidea estimated using the Bayesian phylogenetic by MCMCTREE in paml. Time units are in millions of years. Estimated divergence times are shown near nodes.
Derbidae sister-relationship with Achilidae was rejected (Fig. 3) or confirmed (Figs 4, 5). However, this position is challenged by the Dictyopharidae–Fulgoridae clade originating first (Fig. 5) and the fact that the monophyly is always strongly supported (Figs 3–5; Supporting Information, Figs S1–S6).
The monophyly and sister-relationship of Dictyopharidae and Fulgoridae are strongly supported in all trees (Figs 3–5; Supporting Information, Figs S1–S6) with Zanna robusticephalica Liang, 2017 and Dichoptera sp. being the more basal genera in Fulgoridae.
Acanaloniidae and Tropiduchidae have always been found as sister-taxa, basal to all remaining families: Caliscelidae, Lophopidae, Eurybrachidae, Nogodinidae, Ricaniidae, Flatidae, and Issidae (Figs 3–5; Supporting Information, Figs S1–S6).
All results support the sister-relationship of (Caliscelidae + (Lophopidae + Eurybrachidae)) forming a younger 121.12 Mya clade, sister to the 122.88 Mya clade of the Nogodinidae, Ricaniidae, Flatidae, and Issidae families (Figs 3–6; Supporting Information, Figs S1–S6). The latter form the last major branch with the couple Ricaniidae–Flatidae positioned as sister-groups when the ATP8 gene is removed, while the topology (Nogodinidae + Ricaniidae) + (Flatidae + Issidae) is observed without removing ATP8 (Figs 3–5; Supporting Information, Figs S1–S3).
The monophyly of the family Issidae is strongly supported, but the monophyly of the subfamilies Issinae and Hysteropterinae (sec. Gnezdilov, 2022) is not supported in all tree topologies (Figs 3–5; Supporting Information, Figs S1–S6).
Divergence dates
Divergence and diversification time estimates are illustrated in Figure 6 and Table 1. The divergence time of Cixiidae + Delphacidae was traced back into Middle Jurassic, around 173 Mya; Meenoplidae + Kinnaridae into the Upper Jurassic (148.56 Mya); Dictyopharidae + Fulgoridae separated at approximately the Lower Cretaceous (130.16 Mya); Lophopidae + Eurybrachidae into the Lower Cretaceous (101.3 Mya).
Estimated divergence and diversification times and 95% credibility intervals. NA: not applicable
| Family | Estimated divergence–diversification times | 95% credibility intervals |
|---|---|---|
| Delphacidae | 173.00 Mya–165.15 Mya (Middle Jurassic) | [163.28, 181.60]–[155.24, 174.24] |
| Cixiidae | 173.00 Mya–164.63 Mya (Middle Jurassic) | [163.28, 181.60]–[153.73, 174.54] |
| Kinnaridae | 148.56 Mya–NA (Upper Jurassic) | [131.03, 161.85]–NA |
| Meenoplidae | 148.56 Mya–117.65 Mya (Lower Cretaceous) | [131.03, 161.85]–[99.76, 132.74] |
| Achilidae | 138.58 Mya–120.75 Mya (Lower Cretaceous) | [125.82, 148.98]–[107.00, 132.22] |
| Derbidae | 138.58 Mya–112.71 Mya (Lower Cretaceous) | [125.82, 148.98]–[96.34, 126.12] |
| Fulgoridae | 130.16 Mya–113.32 Mya (Lower Cretaceous) | [118.13, 143.10]–[101.14, 123.13] |
| Dictyopharidae | 130.16 Mya–97.39 Mya (Upper Cretaceous) | [118.13, 143.10]–[79.71, 114.27] |
| Caliscelidae | 121.12 Mya–98.56 Mya (Upper Cretaceous) | [111.00, 130.33]–[84.68, 111.13] |
| Issidae | 118.72 Mya–110.79 Mya (Lower Cretaceous) | [109.52, 126.91]–[101.75, 118.97] |
| Flatidae | 118.72 Mya–98.84 Mya (Upper Cretaceous) | [109.52, 126.91]–[87.21, 109.62] |
| Nogodinidae | 115.91 Mya–105.34 Mya (Lower Cretaceous) | [106.10, 124.55]–[93.86, 115.17] |
| Ricaniidae | 115.91 Mya–83.54 Mya (Upper Cretaceous) | [106.10, 124.55]–[73.00, 94.33] |
| Acanaloniidae | 115.77 Mya–NA (Lower Cretaceous) | [100.65, 127.73]–NA |
| Tropiduchidae | 115.77 Mya–105.33 Mya (Lower Cretaceous) | [100.65, 127.73]–[89.67, 118.55] |
| Lophopidae | 101.30 Mya–73.30 Mya (Upper Cretaceous) | [88.80, 112.73]–[58.72, 87.41] |
| Eurybrachidae | 101.30 Mya–69.03 Mya (Upper Cretaceous) | [88.80, 112.73]–[52.43, 84.77] |
| Family | Estimated divergence–diversification times | 95% credibility intervals |
|---|---|---|
| Delphacidae | 173.00 Mya–165.15 Mya (Middle Jurassic) | [163.28, 181.60]–[155.24, 174.24] |
| Cixiidae | 173.00 Mya–164.63 Mya (Middle Jurassic) | [163.28, 181.60]–[153.73, 174.54] |
| Kinnaridae | 148.56 Mya–NA (Upper Jurassic) | [131.03, 161.85]–NA |
| Meenoplidae | 148.56 Mya–117.65 Mya (Lower Cretaceous) | [131.03, 161.85]–[99.76, 132.74] |
| Achilidae | 138.58 Mya–120.75 Mya (Lower Cretaceous) | [125.82, 148.98]–[107.00, 132.22] |
| Derbidae | 138.58 Mya–112.71 Mya (Lower Cretaceous) | [125.82, 148.98]–[96.34, 126.12] |
| Fulgoridae | 130.16 Mya–113.32 Mya (Lower Cretaceous) | [118.13, 143.10]–[101.14, 123.13] |
| Dictyopharidae | 130.16 Mya–97.39 Mya (Upper Cretaceous) | [118.13, 143.10]–[79.71, 114.27] |
| Caliscelidae | 121.12 Mya–98.56 Mya (Upper Cretaceous) | [111.00, 130.33]–[84.68, 111.13] |
| Issidae | 118.72 Mya–110.79 Mya (Lower Cretaceous) | [109.52, 126.91]–[101.75, 118.97] |
| Flatidae | 118.72 Mya–98.84 Mya (Upper Cretaceous) | [109.52, 126.91]–[87.21, 109.62] |
| Nogodinidae | 115.91 Mya–105.34 Mya (Lower Cretaceous) | [106.10, 124.55]–[93.86, 115.17] |
| Ricaniidae | 115.91 Mya–83.54 Mya (Upper Cretaceous) | [106.10, 124.55]–[73.00, 94.33] |
| Acanaloniidae | 115.77 Mya–NA (Lower Cretaceous) | [100.65, 127.73]–NA |
| Tropiduchidae | 115.77 Mya–105.33 Mya (Lower Cretaceous) | [100.65, 127.73]–[89.67, 118.55] |
| Lophopidae | 101.30 Mya–73.30 Mya (Upper Cretaceous) | [88.80, 112.73]–[58.72, 87.41] |
| Eurybrachidae | 101.30 Mya–69.03 Mya (Upper Cretaceous) | [88.80, 112.73]–[52.43, 84.77] |
Estimated divergence and diversification times and 95% credibility intervals. NA: not applicable
| Family | Estimated divergence–diversification times | 95% credibility intervals |
|---|---|---|
| Delphacidae | 173.00 Mya–165.15 Mya (Middle Jurassic) | [163.28, 181.60]–[155.24, 174.24] |
| Cixiidae | 173.00 Mya–164.63 Mya (Middle Jurassic) | [163.28, 181.60]–[153.73, 174.54] |
| Kinnaridae | 148.56 Mya–NA (Upper Jurassic) | [131.03, 161.85]–NA |
| Meenoplidae | 148.56 Mya–117.65 Mya (Lower Cretaceous) | [131.03, 161.85]–[99.76, 132.74] |
| Achilidae | 138.58 Mya–120.75 Mya (Lower Cretaceous) | [125.82, 148.98]–[107.00, 132.22] |
| Derbidae | 138.58 Mya–112.71 Mya (Lower Cretaceous) | [125.82, 148.98]–[96.34, 126.12] |
| Fulgoridae | 130.16 Mya–113.32 Mya (Lower Cretaceous) | [118.13, 143.10]–[101.14, 123.13] |
| Dictyopharidae | 130.16 Mya–97.39 Mya (Upper Cretaceous) | [118.13, 143.10]–[79.71, 114.27] |
| Caliscelidae | 121.12 Mya–98.56 Mya (Upper Cretaceous) | [111.00, 130.33]–[84.68, 111.13] |
| Issidae | 118.72 Mya–110.79 Mya (Lower Cretaceous) | [109.52, 126.91]–[101.75, 118.97] |
| Flatidae | 118.72 Mya–98.84 Mya (Upper Cretaceous) | [109.52, 126.91]–[87.21, 109.62] |
| Nogodinidae | 115.91 Mya–105.34 Mya (Lower Cretaceous) | [106.10, 124.55]–[93.86, 115.17] |
| Ricaniidae | 115.91 Mya–83.54 Mya (Upper Cretaceous) | [106.10, 124.55]–[73.00, 94.33] |
| Acanaloniidae | 115.77 Mya–NA (Lower Cretaceous) | [100.65, 127.73]–NA |
| Tropiduchidae | 115.77 Mya–105.33 Mya (Lower Cretaceous) | [100.65, 127.73]–[89.67, 118.55] |
| Lophopidae | 101.30 Mya–73.30 Mya (Upper Cretaceous) | [88.80, 112.73]–[58.72, 87.41] |
| Eurybrachidae | 101.30 Mya–69.03 Mya (Upper Cretaceous) | [88.80, 112.73]–[52.43, 84.77] |
| Family | Estimated divergence–diversification times | 95% credibility intervals |
|---|---|---|
| Delphacidae | 173.00 Mya–165.15 Mya (Middle Jurassic) | [163.28, 181.60]–[155.24, 174.24] |
| Cixiidae | 173.00 Mya–164.63 Mya (Middle Jurassic) | [163.28, 181.60]–[153.73, 174.54] |
| Kinnaridae | 148.56 Mya–NA (Upper Jurassic) | [131.03, 161.85]–NA |
| Meenoplidae | 148.56 Mya–117.65 Mya (Lower Cretaceous) | [131.03, 161.85]–[99.76, 132.74] |
| Achilidae | 138.58 Mya–120.75 Mya (Lower Cretaceous) | [125.82, 148.98]–[107.00, 132.22] |
| Derbidae | 138.58 Mya–112.71 Mya (Lower Cretaceous) | [125.82, 148.98]–[96.34, 126.12] |
| Fulgoridae | 130.16 Mya–113.32 Mya (Lower Cretaceous) | [118.13, 143.10]–[101.14, 123.13] |
| Dictyopharidae | 130.16 Mya–97.39 Mya (Upper Cretaceous) | [118.13, 143.10]–[79.71, 114.27] |
| Caliscelidae | 121.12 Mya–98.56 Mya (Upper Cretaceous) | [111.00, 130.33]–[84.68, 111.13] |
| Issidae | 118.72 Mya–110.79 Mya (Lower Cretaceous) | [109.52, 126.91]–[101.75, 118.97] |
| Flatidae | 118.72 Mya–98.84 Mya (Upper Cretaceous) | [109.52, 126.91]–[87.21, 109.62] |
| Nogodinidae | 115.91 Mya–105.34 Mya (Lower Cretaceous) | [106.10, 124.55]–[93.86, 115.17] |
| Ricaniidae | 115.91 Mya–83.54 Mya (Upper Cretaceous) | [106.10, 124.55]–[73.00, 94.33] |
| Acanaloniidae | 115.77 Mya–NA (Lower Cretaceous) | [100.65, 127.73]–NA |
| Tropiduchidae | 115.77 Mya–105.33 Mya (Lower Cretaceous) | [100.65, 127.73]–[89.67, 118.55] |
| Lophopidae | 101.30 Mya–73.30 Mya (Upper Cretaceous) | [88.80, 112.73]–[58.72, 87.41] |
| Eurybrachidae | 101.30 Mya–69.03 Mya (Upper Cretaceous) | [88.80, 112.73]–[52.43, 84.77] |
DISCUSSION
Phylogeny
Although the nucleotide diversity results show that ATP8, ND2, ND6, and ND3have the highest variability, the gene substitution saturation test shows that ND2, ND6, and ND3 are not saturated contrarily to ATP8 genes, which ATP8 genes was removed from our analyses. The phylogenetic relationships in the superfamily Fulgoroidea attempted in this study were based on three different datasets (PCG123R, PCG123R–ATP8, and PCG12R–ATP8) with the homogeneity and heterogeneity model CAT+GTR combined. Once the saturation gene ATP8 was removed, the topology of some branches in the trees changed, mainly in the younger branches of Nogodinidae, Ricaniidae, Flatidae, and Issidae. Topologies of the younger branches ((Nogodinidae + Ricaniidae) + (Flatidae + Issidae)), generated from the PCG123R dataset under both homogeneity and heterogeneity analysis, are more stable. After the removal of the saturated ATP8 gene, the topologies of these younger branches changed significantly for Nogodinidae (Figs 3–5; Supporting Information, Figs S1–S3), and the sister-relationship of Ricanidae and Flatidae was supported (Figs 3–5; Supporting Information, Figs S1–S3), a view consistent with Ai et al. (2021) and Gong et al. (2021). Therefore, it appears that including ATP8 sequences in a molecular dataset may affect the topological structure of the young branches and might not be suitable for addressing Fulgoroidea phylogeny at the young family level. However, the deletion of the ATP8 gene appeared to have little impact on the topology of the base branches, showing the importance of screening genes to construct the phylogenetic tree.
Under the homogeneity model, the phylogenetic trees generated by ML and BI analyses based on the three datasets show that Cixiidae and Delphacidae are placed at the base of the superfamily, consistent with the previous studies of Bourgoin (1993a), Yeh et al. (2005), Urban and Cryan (2007), and Song and Liang (2013). However, the tree generated under the heterogeneity model based on different datasets showed Meenoplidae + Kinnaridae located as most basal of Fulgoroidea, the nodes had lower support values. Results under the heterogeneity model depict a (Meenoplidae + Kinnaridae) clade basal to Delphacoidea and other fulgoroidea families. This unusual position might be due to the poor sampling of Meenoplidae and Kinnaridae in this study. In agreement with the previous studies on the phylogeny of Fulgoroidea based on morphology and molecular fragments (Bourgoin 1993a, Yeh et al. 2005, Urban and Cryan 2007, Bourgoin and Szwedo 2022), and the topology generated based on mitogenomes and the results of divergence-time estimation in this study, we still favour an earlier branch for Cixiidae + Delphacidae (173 Mya) than for Meenoplidae + Kinnaridae (148.56 Mya). The phylogenetic analysis here is consistent with previous investigations (Yeh et al. 2005, Urban and Cryan 2007, Ceotto et al. 2008), which fully support the monophyly of Delphacidae, sister to Cixiidae. The restricted sampling of the cixiid taxa in this study does not allow, however, further discussion of the respective position of these two families.
Yang and Hu (1985) described two species of the genus Suva Kirkaldy in Meenoplidae in China, i.e. S. flavimaculata Yang and Hu, 1985 and S. longipenna Yang and Hu, 1985. These two species were later placed in Derbidae, and the genus Suva was considered problematic and even polyphyletic (Bourgoin 1997). The phylogenetic analysis based on mitogenome evidence here shows Suva longipenna clustered with two other species in Derbidae, confirming the placement of this species in Derbidae by Bourgoin (1997). The generic placements of S. flavimaculata and S. longipenna need to be re-evaluated. In this study the (Meenoplidae + Kinnaridae) clade is placed in a relatively basal position in Fulgoroidea sec. Bourgoin and Szwedo 2023, confirming previous results of Asche (1987), Emeljanov (1990) and Urban and Cryan (2007).
Bourgoin (1993a) proposed a sister-relationship between Achilidae and Derbidae based on female genitalia, which was challenged by Urban and Cryan (2007), with paraphyletic Achilidae, in which Achilixiidae nested. Derbids are morphologically diverse and tribes included in this family are easily recognizable by their peculiar appearance, while the most basal taxa of the family often look like Achilidae (Emeljanov and Shcherbakov 2020). A sister-relationship between Achilidae and Derbidae is supported by the trees under MrBayes (all three matrices) and PhyloBayes (PCG12R–ATP8 matrix) analyses only.
These two families are younger than Dictyopharidae and Fulgoridae and their monophyly is uncertain or not supported in this study. The oldest fossils of Achilidae and Derbidae, known since the Cretaceous (Szwedo 2004, Emeljanov and Shcherbakov 2020), are consistent with the results of the divergence-time estimation in this study.
The monophyly of Fulgoridae and the sister-relationship between Fulgoridae and Dictyopharidae are strongly supported in this study, consistent with previous morphological and molecular phylogenies. All the results showed that Dichoptera sp. (in Dichopterinae) and Zanna robusticephalica (in Zanninae) branched off much earlier than other species in Fulgoridae (Figs 3–5). The phylogenetic position of these sister-families within Fulgoroidea is in line with the previous results of Urban and Cryan (2007).
Tropiduchidae and Acanaloniidae are closely related in this study, which is also consistent with Urban and Cryan’s (2007) analysis. Nogodinidae monophyly and positioning were not addressed in this study due to insufficient sampling.
The sister-relationship between Lophopidae and Eurybrachidae is strongly supported here and is consistent with the analyses of Emeljanov (1990) and Urban and Cryan (2007). But the clade Lophopidae + Eurybrachidae, sister of the Caliscelidae clade, is inconsistent with Urban and Cryan (2007) who depicted Caliscelidae as sister to Ricaniidae.
The monophyly of the family Issidae is strongly supported, but the monophyly of the subfamilies Issinae and Hysteropterinae following Gnezdilov et al. (2020, 2022) is not supported in all tree topologies in this paper. The phylogenetic trees within Issinae support the previous study (Wang et al. 2016, Gnezdilov et al. 2020, 2022, Yang et al. 2021), which includes the tribes Thioniini, Kodaianellini, Sarimini, Parahiraciini, and Hemisphaeriini (Wang et al. 2016, Gnezdilov et al. 2020, 2022, Yang et al. 2021). However, Celyphoma yangi Chen, Zhang and Chang, 2014 in Hysteropterinae is nested within Issinae; this is probably because of the limited samples in Hysteropterinae. The phylogenetic relationships in Issidae still need further validation.
Most of our results support a Ricaniidae–Flatidae sister-group relationship when the ATP8 gene was removed. In addition, the results support Flatidae, Issidae, Ricaniidae, and Nogodinidae belonging to the younger branch in the superfamily Fulgoroidea. This placement is similar to Emeljanov (1990), Bourgoin (1993a), Yeh et al. (2005), and Urban and Cryan (2007).
Divergence and diversification-time estimation
The divergence-time estimation in this analysis shows that most species of Fulgoroidea underwent relatively rapid radiation during the Jurassic and Cretaceous, which is consistent with the previous statements of Szwedo et al. (2004) and Johnson et al. (2018). The families Cixiidae and Delphacidae represent the older taxa in current Fulgoromorpha. The origin of Delphacidae is about 165.15 Mya, much earlier than that of the Cretaceous (100 Mya) as estimated by Huang et al. (2017).
The families Meenoplidae and Kinnaridae appear to have originated in the Lower Cretaceous and Upper Jurassic, which is inconsistent with the results of Song and Liang (2013) (Kinnaridae: 120 Mya and Meenoplidae: 60 Mya). Fulgoridae originated from Lower Cretaceous.
Ricaniidae and Dictyopharidae date from the Upper Cretaceous, consistent with the estimation of Song and Liang (2013). Lophopidae can be traced into the Upper Cretaceous (73.3 Mya), slightly earlier than that estimated by Soulier-Perkins (2000), who considered Lophopidae to have originated 65 Mya in South-East Asia.
The family Issidae originated from the Lower Cretaceous. This estimated diversification time is consistent with the result of Bourgoin et al. (2018), and slightly earlier than the oldest Issidae fossil (Upper Cretaceous) (Bourgoin et al. 2020). However, this result is different from that of Gnezdilov et al. (2020, 2022) who considered that Issidae belonged to a more recent Eocene group of Fulgoroidea and does not support the family’s origin in the Lower Cretaceous. However, based on the oldest known fossil data (Bourgoin et al. 2020) (Upper Cretaceous) of Issidae, the origin of this family should predate at least the Upper Cretaceous.
CONCLUSION
This study reconstructed the phylogenetic relationship of the superfamily Fulgoroidea based on the whole mitogenome sequences from 113 species representing 17 planthopper families and three families as outgroups. The phylogenetic analyses recovered that Delphacoidea (=Cixiidae + Delphacidae) was sister to all current planthopper families (i.e. Fulgoroidea sec. Bourgoin and Szwedo (2022, 2023)). At the base of this superfamily, the clade (Meenoplidae + Kinnaridae) first separated from the other current families. (Fulgoridae + Dictyopharidae) appears as a younger clade than the Derbidae, which grouping appears uncertain or not supported, Tropiduchidae and Acanaloniidae are depicted as sister-taxa.
In addition, the results of the nucleotide diversity and evolutionary rate analysis of 13 protein-coding genes of the mitogenomes sequence of Fulgoroidea show that ND2, ND3, and ND6 have a faster evolutionary rate, and may be selected as potential DNA markers to study closely related taxa within Fulgoroidea families. Divergence-time estimation shows that most fulgoroids underwent relatively rapid radiation during the Jurassic and Cretaceous, and the divergence time of Delphacoidea can be traced back to the Middle Jurassic. The three families Meenoplidae, Fulgoridae, and Issidae originated in the Lower Cretaceous, and the four families Ricaniidae, Dictyopharidae, Lophopidae, and Eurybrachidae occurred at about the Upper Cretaceous.
The phylogenetic analyses presented here provide some new perspectives in addressing planthopper phylogeny by using complete mitogenome sequences and, particularly, the nucleotide diversity analysis and saturation test provide bases for molecular marker selection in phylogenetic investigations at the lower level of the planthopper classification. However, the problem of the respective position of Delphacidae to Cixiidae remains unresolved, the assignments of some families such as Derbidae or (Meenoplidae + Kinnaridae) remain unstable, and the monophyly of some families is still in doubt. This uncertainty could be addressed by improved mitogenomic sampling of these fast-evolving genes.
FUNDING
This study was supported by the National Natural Science Foundation of China (32170475, 31750002).
CONFLICT OF INTEREST
All authors declare no conflicting interests.
