Introduction

Chrysomelidae is among the most diverse beetle families, totaling almost 40,000 described extant species in the world [1]. Chrysomelids are known as leaf beetles because most species in this group feed on the green part of the living plant. Some other leaf beetles feed on pollen, flowers, seeds and ant nests debris [2]. The great species diversity of leaf beetles has been ascribed to their co-radiation with the angiosperms [3–5]. Currently, most beetle systematists have reached a general consensus that the family Chrysomelidae includes 12 subfamilies, namely Bruchinae, Cassidinae, Chrysomelinae, Criocerinae, Cryptocephalinae, Donaciinae, Eumolpinae, Galerucinae, Lamprosomatinae, Sagrinae, Spilopyrinae and Synetinae [6,7].

Within the superfamily Chrysomeloidea, the Cerambycidae are often recovered as the sister group of Chrysomelidae [8–11]. The monophyly of Chrysomelidae was well supported by morphological [9] and molecular [10–14] studies. Although a number of previous phylogenetic studies have contributed significantly to our understanding of the evolution of leaf beetles [2,3,5,6,8–10,13–27], the interrelationships of subfamilies in Chrysomelidae remain incompletely resolved.

Gómez-Zurita et al. [12,13] conducted the comprehensive phylogenetic studies of the basal relationships in the Chrysomelidae, based on two nuclear (18S and 28S rDNA) and mitochondrial (rrnL) gene fragments for 167 taxa covering most major lineages and relevant outgroups. The Chrysomelidae was subdivided into three major subfamily groups: “sagrine” (Criocerinae, Donaciinae, Sagrinae and Bruchinae), “eumolpine” (Spilopyrinae, Eumolpinae, Cryptocephalinae and Cassidinae) and “chrysomeline” (Chrysomelinae and Galerucinae), with the basal “sagrine” as sister to the “eumolpine” plus “chrysomeline” clades [13] (Fig 1A). Within the “eumolpine” clade, Cassidinae was sister to Cryptocephalinae [13]. Although the three main chrysomelid lineages were distinguished based on the findings of the study by Gómez-Zurita et al. [13], the key nodes received no significantly statistical support across inference methods.

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Fig 1. Representations of previous hypotheses for the subfamily-level relationships within Chrysomelidae inferred from morphologically-based or molecular studies by authors cited.

(A) Gómez-Zurita at al. (2008) [13] based on molecular data; (B) Nie at al. (2020) [22] based on molecular data; (C) Reid (2000) [26] based on morphological data; (D) Bocak at al. (2014) [11] based on molecular data; (E) Jolivet at al. (2008) [22] based on morphological data; (F) Song at al. (2018) [27] based on molecular data.

https://doi.org/10.1371/journal.pone.0258587.g001

In the most recent molecular study of Nie et al. [14] (Fig 1B), most of the basal relationships are established, but some remain unclear. The relationships among the three chrysomelid main clades (i.e., sagrines, eumolpines, chyrsomelines) differed across the tree inference methods [14]. the reconstructions of basal relationships were complicated by the non-monophyly of sagrines and eumolpines (e.g., the RAxML tree in Fig 1 of Nie et al. [14]). In addition, the placements of Synetinae and Sagrinae were unresolved (PP = 0.51 and 0.84 in Fig 2 of Nie et al. [14], respectively). Many fundamental questions about Chrysomelidae systematics need to be addressed by additional sampling of taxa and characters.

The subfamily Bruchinae, with about 1700 known species [28,29], are specialized internal feeders of bean seeds. This group of beetles has traditionally been treated as a separate family Bruchidae [29–34]. Numerous phylogenetic studies converged on supporting the group as a chrysomelid subfamily [8–11,14,35]. Within Chrysomelidae, the phylogenetic placement of Bruchinae varied between analyses. Reid [25,26] recovered Sagrinae as the sister group of Bruchinae based on morphological characters (Fig 1C). Farrell and Sequira [15], based on a combined analysis of molecular (18S rDNA) and morphological data, also retrieved a sister group relationship between Bruchinae and Sagrinae. In a multi-gene analysis of Hunt et al. [8], Bruchinae was sister to the clade Donaciinae + Criocerinae. In the ML tree of Gómez-Zurita et al. [12], Synetinae was placed as the sister group of Bruchinae. In the further analyses based on multi-locus ribosomal RNA data [13] (Fig 1A), the relative position of Bruchinae remained unclear, though it was placed in a basal “sagrine” clade also containing Donaciinae, Criocerinae and Sagrinae. The morphological study of Lawrence et al. [9] grouped Bruchinae and Cryptocephalinae in a clade. Bocak et al. [11] also recovered a sister-group relationship of Bruchinae to Sagrinae (Fig 1D). In the molecular study of McKenna et al. [10], Bruchinae and Criocerinae clustered together. Some recent analyses, based on the mitogenome sequence data [14,27], recovered Bruchinae as sister to all remaining Chrysomelidae. As reviewed above, most of previous studies have tended to recover a close relationship between Bruchinae and Sagrinae [11,13,15,25,26].

An earlier study suggested a close relationship of the Cassidinae with the “sagrine” clade [3]. In the study of Duckett et al. [36], Cassidinae was recovered as the sister group of the clade Chrysomelinae + Galerucinae. In the study of Hunt et al. [8], the sister group Cassidinae + Hispinae was placed in an intermediate position between the clade Bruchinae + (Criocerinae + Donaciinae) and the clade Sagrinae + ((Galerucinae + Chrysomelinae) + ((Lamprosomatinae + (Cryptocephalinae + Eumolpinae))). Marvaldi et al. [37], based on the secondary structural information of 18S and 28S rDNA, also recovered Cassidinae as the sister group of Cryptocephalinae. In the morphological analysis of Lawrence et al. [9], Cassidinae was placed as sister to all other Chrysomelidae. Bocak et al. [11] recovered Cassidinae in a more derived position and as the sister group of a clade including Spilopyrinae, Lamprosomatinae, Eumolpinae, Cryptocephalinae and Clytrinae.

Besides the phylogenetic placements of Bruchinae and Cassidinae, the affinity of Alticinae relative to Galerucinae is another focus of debate on the higher-level phylogeny of Chrysomelidae. Traditionally, based on the presence of the jumping apparatus, flea beetles were distinguished from the closely related Galerucinae [38–40] and considered as an independent subfamily (Alticinae). Several recent studies suggested Alticini as a tribe within Galerucinae s.l. [36,41]. The studies of Ge et al. [16,17] based on the combined analyses of multi-locus sequence data (18S, 28S rDNA, rrnL and cox1) and morphological characters showed that the traditionally defined Alticinae or Alticini was non-monophyletic. The metafemoral spring is prone to convergence and not sufficient for classification at the subfamily level [17]. In more recent studies by Nie et al. [14,24], the placements of some genera have been transferred between Alticinae and Galerucinae. As a result, two reciprocally monophyletic lineages corresponding to the subfamily level were recognized. Thus, in the new classification of the chrysomelid subfamilies, flea beetles can be classified as a separate subfamily. In this study, we follow the new definition of Alticinae as Nie et al. [14,24].

Mitogenome as a class of molecular marker has been demonstrated to be informative in resolving higher-level phylogeny of Chrysomelidae [14,24,42]. Recently, next-generation sequencing has been effectively used for phylogenetic studies in Coleoptera [14,27,43,44]. This has resulted in rapid increase in the number of mitogenomes of leaf beetles. As of June 2020, there are more than 300 complete or partial mitogenome sequences of Chrysomelidae published in GenBank. In this study, we obtained 16 new mitogenomes of Chrysomelidae by using the next-generation sequencing method.

Given the above-outlined uncertainties in the phylogenetic relationships of the subfamilies of Chrysomelidae, we attempt to resolve the major lineages of the group using the expanded mitogenome sequence data. Specifically, we aim to (1) assess the validity of the subfamily groups of [13], and (2) investigate the placements of Bruchinae, Spilopyrinae, Cassidinae, and Timarcha.

Materials and methods

Results

Genome sequencing and characteristics of mitogenomes

Eight new mitogenomes with a genome size of more than 15 kb included the full set of 37 mitochondrial genes and the complete control region (e.g., the Callosobruchus maculatus in Fig 2). The remaining eight leaf beetles newly sequenced had the partial mitogeomes (often < 10 kb), in which some gene regions and/or the control region were missing due to failure of genome sequencing and assembling. With regard to the completeness of mitogenomes, the individual species library construction presented the better results than the multiple species library construction. In addition, sequencing depth was correlated with the full assembly of mitogenomes. The detailed statistics of the sequencing of mitogenomes using next-generation sequencing technology was shown in Table 1.

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Fig 2. Organization of the mitogenome of Callosobruchus maculatus.

https://doi.org/10.1371/journal.pone.0258587.g002

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Table 1. Statistics associated to the sequencing of mitogenomes.

https://doi.org/10.1371/journal.pone.0258587.t001

The majority of the new mitogenomes showed the similar genome organization and gene content with the putatively ancestral insect mitogenome [68,69], with the exception of C. maculatus and Plagiodera versicolora. In C. maculatus, a large intergenic spacer region (2,070 bp) was identified between nad2 and trnW (Fig 2). This also resulted in a lager genome size of C. maculatus (18,380 bp). In addition, the trnQ gene (typically between trnI and trnM) was translocated to the downstream of trnS2 on the heavy strand. For the partial mitogenome of P. versicolora, we detected a tRNA translocation for the trnL1 (typically between nad1 and rrnL), which was translocated to the position between trnY and cox1.

Phylogenetic inference

To compare with the prior studies, we used the delimitation of three major sublineages (i.e., the “chrysomelines”, “eumolpines” and “sagrines”) of Gómez-Zurita et al. [13] (Fig 1A) to discuss the subfamily relationships within Chrysomelidae. Our analyses constantly recovered a monophyletic Chrysomelidae, with strong nodal support (BP ≥ 99, PP ≥ 0.96) (Figs 3–5 and S1–S8 Figs). Chrysomelidae was subdivided into two main clades: (clade 1) Bruchinae, Sagrinae, Donaciinae, Synetinae, Criocerinae, Spilopyrinae, Cassidinae, Eumolpinae, Lamprosomatinae and Cryptocephalinae; and (clade 2) Chrysomelinae, Alticinae and Galerucinae. In some analyses, Synetinae was placed in the clade 1. Other discrepancies among analyses were restricted to the relationships of subfamilies in the “sagrine” and “eumolpine” clades, in which the branching order varied depending on the datasets and methods.

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Fig 3. ML tree inferred from the dataset PCGaa_Dayhoff using IQ-TREE under the MK+FQ+I+G4 model.

Lineages have been collapsed for clarity. The lengths of the triangles correspond to the longest terminal branches in the collapsed lineages. Node numbers show the bootstrap support values. The full tree with all branches is available in S1 Fig.

https://doi.org/10.1371/journal.pone.0258587.g003

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Fig 4. Bayesian tree inferred from the dataset PCG_aa using PhyloBayes under the site-heterogeneous CAT model.

Node numbers show the posterior probability values.

https://doi.org/10.1371/journal.pone.0258587.g004

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Fig 5. Bayesian tree inferred from the dataset PCGnt+RNA using PhyloBayes under the site-heterogeneous CAT-GTR model.

The full tree with all branches is available in S8 Fig.

https://doi.org/10.1371/journal.pone.0258587.g005

For the ML analyses, we recovered a tree topology comparable with the hypothesis proposed by Gómez-Zurita et al. [13], when recording the amino acid matrix with Dayhoff categories (Figs 3 and S1). The ML tree with Dayhoff recoding had the log-likelihood score of -218718.087, which was higher than other ML trees. In clade 1, we recovered a deep subdivision of analysed taxa into two groups corresponding to the “sagrine” and “eumolpine” clades. Differences between the Dayhoff ML tree and the hypothesis of Gómez-Zurita et al. [13] lied mainly in the placements of Sagrinae and Spilopyrinae. In the Dayhoff ML tree, Spilopyrinae was nested within the “sagrine” clade and as sister to Donaciinae (BP = 72). Sagrinae represented by a single species of Sagra femorata appeared as sister to Cassidinae, both of which were sister to the major “eumolpine” clade. Last but not least, the “chrysomeline” clade was sister to the “sagrine” plus “eumolpine” clade. In the remaining ML analyses, Bruchinae was sister to all other taxa of the clade 1. The “eumolpine” clade including Spilopyrinae, Cassidinae, Eumolpinae, Lamprosomatinae and Cryptocephalinae was retrieved as monophyletic. However, the “sagrine” clade was not supported with respect to Bruchinae (e.g., S2 Fig).

The BI analyses under the empirical site-heterogeneous CAT mixture model provided more clearly resolved relationships in Chrysomelidae. In three out of four BI analyses, each of the three subfamily groups proposed by Gómez-Zurita et al. [13] were strongly supported (e.g., PCG_aa BI tree in Fig 4). The phylogenetic placement of Synetinae remained unresolved across the BI analyses. The BI tree from the dataset PCG_aa recovered Synetinae as sister to the “chrysomeline” clade, but without the significantly statistical support (PP = 0.8).

The similar situation occurred in the BI tree from the dataset PCGnt+RNA (Fig 5). For the inter-subfamily relationships in the “eumolpine” clade, the BI trees were concordant with the majority of ML trees. The following subfamily relationships were consistently found: (Spilopyrinae + (Cassidinae + (Eumolpinae + (Lamprosomatinae + Lamprosomatinae)))). Within the “sagrine” clade, the BI tree from the dataset PCG_aa placed Bruchinae as the most basal lineage, followed by Sagrinae, and Donaciinae + Criocerinae. In contrast, the alternative branching pattern was retrieved in the BI tree from the nucleotide datasets PCG_nt and PCGnt+RNA. In both analyses, Sagrinae formed a sister group of Bruchinae, which in turn was sister to the clade Donaciinae + Criocerinae. The “sagrine” clade was recovered as the sister group of the “eumolpine” clade in all BI analyses except for that with Dayhoff recoding.

The FcLM analysis testing the relationships among the three major chrysomelid clades showed conflicting phylogenetic signal present in the dataset PCG_aa. Support for the relationship between the “sagrine”, “eumolpine” and “chrysomeline” clades was divided, though the highest percentage of data points fell in favor of a branching pattern of ((“sagrine” + “eumolpine”) + “chrysomeline”) (38.2%, in Fig 6).

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Fig 6. Results obtained from the four-cluster likelihood-mapping analysis based on the dataset PCG_aa showing conflicting signal for the alternative hypotheses.

The above triangle picture shows the possible relationships of four clusters. The below triangle picture on the left is the three posterior probabilities for the three possible unrooted trees of four clusters. The below triangle pictures on the right shows the seven areas supporting different evolutionary information from the dataset PCG_aa.

https://doi.org/10.1371/journal.pone.0258587.g006

Discussion

Mitochondrial DNA as a phylogenetic marker has its potential shortcomings, for example, the substitutional saturation (at a single site or some gene regions) [70] and lineage-specific compositional heterogeneity [71]. Saturation tests showed that the third codon positions of PCGs, as well as the rrnL and rrnS gene regions were saturated in our data sets (S3 Table).

The sequence heterogeneity analyses indicated that the greatest degree of heterogeneity occurred at third codon positions of PCGs, and that some heterogeneity also occurred in the other data partitions (Figs 7 and S9).

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Fig 7. AliGROOVE heat maps of pairwise sequence comparisons for the data partitions PCG_1st-codon, PCG_2nd-codon, PCG_3rd-codon and the dataset PCG_aa.

The AliGROOVE graph shows the mean similarity scores between sequences. AliGROOVE scores range from −1 (indicating great difference in rates from the remainder of the data set, i.e. red coloring implies the significant heterogeneity) to +1 (indicating rates match all other comparisons).

https://doi.org/10.1371/journal.pone.0258587.g007

In this study, most phylogenetic analyses conducted on the concatenated matrices revealed a well-supported topology for deep nodes in the chrysomelid tree of life. The monophyly of Chrysomelidae was strongly supported in all analyses. This was concordant with previous studies [9–14]. Nevertheless, the relationships among subfamilies in Chrysomelidae varied across analyses. The major concerns in phylogenetic reconstruction based on the insect mitogenomes have been the substitutional saturation and compositional heterogeneity as mentioned above. Because both factors may have negative effects on the accuracy of the reconstructed phylogeny. To reduce the effect of substitution saturation, we translated nucleotide sequences into amino acid sequences. Furthermore, we recoded the matrix of amino acids into Dayhoff categories, by which the 20 character states of amino acids were reduced down to six states [53]. As a result, the sequence heterogeneity was reduced greatly by Dayhoff recoding. In the ML analysis with Dayhoff recoding under the homogeneous model, three main clades recovered largely matched the three major subfamily groups of Chrysomelidae proposed by Gómez-Zurita et al. [13]. In contrast, the ML analysis of PCG_aa without Dayhoff recoding and the ML analyses with nucleotide datasets did not produce a clear relationship of the subfamilies corresponding to the “sagrine” and “eumolpine” clades. The discrepancies across datasets in the ML analyses demonstrated that reducing the heterogeneity of mitogenome data can improve the recovery of a reasonable relationship in Chrysomelidae, even under the homogeneous model of evolution.

Under the site-heterogeneous model, nucleotide and amino acid data were basically congruent, with nucleotide datasets also strongly supporting the three major subfamily groups of Chrysomelidae [13]. These results showed that applying the site-heterogeneous model in the BI analyses lessened the effect of compositional heterogeneity. The BI tree based on the amino acid dataset was preferred (Fig 4), due to the strong support for deep nodes. Although each of the “sagrine”, “eumolpine” and “chrysomeline” clades was supported in the majority of BI analyses, their interrelationships conflicted with the study of Gómez-Zurita at al. [13]. In the present study, the “sagrine” clade was always recovered as the sister group of the “eumolpine” clade, together forming the sister group of the “chrysomeline” clade. In contrast, the branching pattern of “sagrine” + (“eumolpine” + “chrysomeline”) was supported in the study of Gómez-Zurita at al. [13]. Result of the FcLM analysis testing the major nodes connecting the three subfamily groups revealed conflicting signal for the inferred relationship on the dataset PCG_aa (Fig 7). Further study is required to provide resolution in nodes of the major subfamily groups, emphasizing denser taxon sampling in the “sagrine” and “eumolpine” clades.

In this study, bruchid seed beetles were consistently recovered as members of Chrysomelidae, consistent with recent phylogenetic analyses focusing on this clade [8–11,14,35]. Thus, the current mitogenome data supported the subfamily rank of Bruchinae within Chrysomelidae. In the “sagrine” clade, Bruchinae was sister to Sagrinae in the BI trees from the datasets PCG_nt (PP = 0.91) and PCGnt+RNA (PP = 0.97). This result was congruent with previous studies [11,15,25,26]. In addition, the sister-group relationship between Donaciinae and Criocerinae [8,11,36] received strongly support in most analyses under both homogeneous and heterogeneous models.

The status of Spilopyrinae was controversial in prior studies [26]. Reid [26] and Marvaldi et al. [37] proposed to elevate Spilopyrini to subfamily rank (as Spilopyrinae). However, Jolivet & Verma [22] placed Spilopyrinae within Eumolpinae (Fig 1E), based on morphological data. Gómez-Zurita et al. [12,13] recovered Spilopyrinae as the most-basal lineage in the “eumolpine” clade. In the present study, seven out of eight phylogenetic analyses retrieved Spilopyrinae as sister to all other “eumolpine” clade. This supported the subfamily status of Spilopyrinae.

The members of Syneta were traditionally classified within Eumolpinae as a tribe (Synetini) [15,25]. Syneta was placed as an early separated lineage in Eumolpinae [19,26,70]. Some authors have proposed the subfamily rank (Synetinae) for this group and supported the exclusion of Syneta from Eumolpinae [22,70]. The close relationship of Synetinae to Eumolpinae was not retrieved in the present study. In the preferred tree (PCG_aa BI tree in Fig 4), Synetinae represented by a single species of Syneta adamsi was sister to the “chrysomeline” clade. Most of other analyses retrieved a close affinity of Synetinae to Timarcha. Together, these taxa were sister to either the “eumolpine” clade or the “chrysomeline” clade. In fact, the phylogenetic placement of Synetinae was unstable in the previous study [71], because different inference methods produced conflicting results on Syneta. Morphological analyses could not address this problem [19,70], either. Based on the current mitogenome data, the phylogenetic position of Synetinae is still ambiguous.

The phylogenetic placement of Cassidinae in Chrysomelidae has been the subject of debate. Wilf et al. [72] hypothesized a single origin of monocot feeding in Chrysomelidae. The monocot feeding groups included the Donaciinae, Cassidinae and Cryptocephalinae. However, the morphological characters uniting the monocot feeding groups were considered to be convergent [13]. In the study of Gómez-Zurita et al. [13], the Cassidinae was kept separate from the other major monocot feeding groups in the “sagrine” clade and as sister to Cryptocephalinae s.l. in the “eumolpine” clade. Our results consistently recovered Cassidinae nested within the “eumolpine” clade and as sister to the clade comprising Eumolpinae, Lamprosomatinae and Cryptocephalinae.

Crownson [1] established the subfamily Clytrinae, which was composed by the tribes Lamprosomatini, Cryptocephalini, Clytrini and Chlamisini. The subsequent studies elevated the former Lamprosomatini to subfamily status (as Lamprosomatinae) [25,73]. The remaining tribes Cryptocephalini, Clytrini and Chlamisini constituted the subfamily Cryptocephalinae [25,73]. The monophyletic Cryptocephalinae was further supported by Gómez-Zurita et al. [13]. In the present study, the sister-group relationship between Cryptocephalinae and Lamprosomatinae was consistently recovered by the mitogenome data. This arrangement supported the hypothesis of Gómez-Zurita et al. [13].

The Chrysomelinae was non-monophyletic with respect to Timarcha. This result was consistent with the previous studies [2,13,14,18]. Thus, our analyses reinforced the point that Timarcha should be regarded as a separate clade in Chrysomelidae [2,18]. As for Galerucinae and Alticinae, the relationships recovered by the current mitogenome data were consistent with the prior mitogenomic study [14].

Conclusions

Expanded mitogenome data resulted in the improved resolution of the higher-level phylogeny of Chrysomelidae, as the deep notes having the generally high nodal support values. When we used the mixture heterogeneous CAT model in the BI analyses, the three major subfamily groups recognized by Gómez-Zurita et al. [13] were strongly supported (Fig 3, sagrine clade: PP = 0.96, eumolpine clade: PP = 1, chrysomeline clade: PP = 1). In addition, the phylogenetic placements of Bruchinae, Spilopyrinae and Cassidinae were resolved with confidence. Despite this, we acknowledged the potential pitfalls of mitochondrial DNA sequences in reconstructing the phylogenetic relationships in Chrysomelidae. The heterogeneous sequence divergence may lead to conflicting signals for the alternative hypothesis of the interrelationships among three major subfamily clades of Chrysomelidae. The placement of Syneta remained questionable. Further mitogenome studies should sequence more species from Syneta and other taxa from the “sagrine” and “eumolpine” clades.

Supporting information