Research Article
Research Article
Phylogeny and age of cockroaches: a reanalysis of mitogenomes with selective fossil calibrations
expand article infoXin-Ran Li§
‡ Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, Nanjing, China
§ University of Science and Technology of China, Hefei, China
Open Access


In spite of big data and new techniques, the phylogeny and timing of cockroaches remain in dispute. Apart from sequencing more species, an alternative way to improve the phylogenetic inference and time estimation is to improve the quality of data, calibrations and analytical procedure. This study emphasizes the completeness of data, the reliability of genes (judged via alignment ambiguity and substitution saturation), and the justification for fossil calibrations. Based on published mitochondrial genomes, the Bayesian phylogeny of cockroaches and termites is recovered as: Corydiinae + (((Cryptocercidae + Isoptera) + ((Anaplectidae + Lamproblattidae) + (Tryonicidae + Blattidae))) + (Pseudophyllodromiinae + (Ectobiinae + (Blattellinae + Blaberidae)))). With two fossil calibrations, namely, Valditermes brenanae and Piniblattella yixianensis, this study dates the crown Dictyoptera to early Jurassic, and crown Blattodea to middle Jurassic. Using the ambiguous ‘roachoid’ fossils to calibrate Dictyoptera+sister pushes these times back to Permian and Triassic. This study also shows that appropriate fossil calibrations are rarer than considered in previous studies.

Key Words

Blattaria, Blattodea, Dictyoptera, divergence time, mitochondrial DNA


The family-level relationships of cockroaches have been in dispute for decades (Fig. 1; see also McKittrick 1964, Klass 2001, Roth 2003). The debate recently intensified with many incongruent phylogenies emerging. Most recent studies are based on molecular data (e.g., Djernæs et al. 2015, Legendre et al. 2015, Wang et al. 2017, Bourguignon et al. 2018, Evangelista et al. 2019), or rarely on morphological and ethological data (Klass and Meier 2006, Djernæs et al. 2015 in part). Despite big data studies, the evolutionary pattern of cockroaches remains ambiguous: mitochondrial genomes (Bourguignon et al. 2018) suggest the basal splits as (Blaberoidea + Corydioidea) + the blattoid complex (i.e. Blattoidea nesting Isoptera), while the much bigger transcriptome data (Evangelista et al. 2019) suggest Blaberoidea + (Corydioidea + blattoid complex), not to mention more incongruent relationships of families and subfamilies.

Figure 1. 

Representative phylogenetic inferences of cockroaches based on various data and methods. McKittrick (1964) and McKittrick and Mackerras (1965): female and male genitalia, proventriculus and oviposition behaviour; discussion. Roth (1970): oothecal rotation; discussion. Klass and Meier (2006): male genitalia, accompanied by ethology etc.; parsimony. Wang et al. (2017): gene fragments (three mitochondrial and two nuclear), incorporating the data from Djernæs et al. (2015) and others; maximum likelihood. Bourguignon et al. (2018): mitogenome; maximum likelihood and Bayesian. Evangelista et al. (2019): transcriptome; maximum likelihood. Taxa are shown in currently recognized rank instead of original designation. Branches in orange, Blaberoidea; in green, Corydioidea; in purple, Blattoidea. Asterisk, paraphyly.

Calibration has a major impact on divergence time estimation (Inoue et al. 2010, Dos Reis and Yang 2012, Sauquet et al. 2012, Sauquet 2013, Magallón et al. 2013). Fossils are common calibrations for phylogenetic dating, while choosing a suitable fossil is difficult (Parham et al. 2011, Wolfe et al. 2016). The lack of justified fossil calibrations for dating cockroaches is particularly acute (Evangelista et al. 2017, 2019, Li and Huang 2019). Of the calibrations used, the ‘roachoid’ fossils are a particular point of contention (Tong et al. 2015 vs. Kjer et al. 2015, Bourguignon et al. 2018 per se).

Phylogenetic inference and time estimation can be improved by enlarging the dataset with new loci and new samples, but also by improving the quality of published data, calibrations and analytical procedure. The latter approach is emphasized and presented herein. In the present study, the mitochondrial genome is preferred as the only type of data for the following reasons. First, taxon coverage is comparatively high; second, missing data can be essentially avoided; third, the computation load (i.e. time investment) is acceptable, allowing multiple analyses for comparisons among datasets.

Material and methods


The present study focuses on true cockroaches (Blattaria), the major component of Dictyoptera. Taxa included in my analyses also cover other Dictyoptera, namely, termites (Isoptera) and mantises (Mantodea), and the living sister of Dictyoptera, namely Eukinolabia + Xenonomia, as suggested by transcriptome data (Misof et al. 2014, Evangelista et al. 2019, Wipfler et al. 2019). In order to reduce the difficulty and inaccuracy in alignment, more distant insect groups were not included in this study. All DNA sequences were collected from whole mitogenome sequencing data deposited in GenBank. Isolated fragments do not conform to the main idea of the present study because they leave missing cells in the alignments. A certain number of missing cells may be harmless to phylogenetic inferences (Wiens 2006, Wiens and Moen 2008), but there is no universal standard for different datasets and methods to avoid pitfalls. Using fully sequenced mitogenome (thus avoiding isolated fragments) can also prevent concatenation of sequences from different specimens, of which the genetic distance is unknown. This practice also automatically rules out specimens that are misidentified as conspecific. Fundamentally, concatenation of sequences from different specimens is an artefact and does not represent a natural organism. This artefact may cause unpredictable errors (pers. observ.); however, the influence of this issue seemingly has not yet been addressed in the literature.

The initial data pool comprises 169 mitogenomes, including all available cockroaches and selected other insects (Suppl. material 1). GenBank files were imported to PHYLOSUITE v1.2.1 (Zhang D et al. 2020), in which duplicates were removed. To save time and computation resources, only one species was kept in each genus, with the exception of genetically diverse and speciose genera (e.g., Cryptocercus, Allacta and Ischnoptera). The final taxon set comprises 95 species (Suppl. material 2: Table S1). The DNA data in the GenBank files were extracted using PHYLOSUITE. Most of the mitogenomes (68.4%) are from Bourguignon et al. (2018), and the remaining from Yamauchi et al. (2004), Cameron et al. (2005, 2012), Zhang YY et al. (2009), Kômoto et al. (2010, 2012), Chen (2012), Wang et al. (2014), Jeon and Park (2015), Tian et al. (2015), Cheng et al. (2016), Ye et al. (2016), Chen et al. (2017), Dumans et al. (2017), Ma et al. (2017), Gong et al. (2018), and Zhang LP et al. (2018).

The character set includes 13 protein coding genes while all RNA genes were excluded. Aligning RNA gene sequences is dependent on the prediction of secondary structure (Buckley et al. 2000, Stocsits et al. 2009), the accuracy of which clearly influences alignment and tree reconstruction (Letsch et al. 2010). This approach is unfeasible in the present study, because even predicting the structure of a small fragment of closely related species is hard and exhausting (e.g., Li et al. 2017). To avoid errors introduced by inaccurate alignment, I do not use them. Besides, RNA gene sequences only account for a minor proportion in the mitochondrial genome, therefore excluding them does not virtually reduce the size of dataset.

Alignment and quality check

Sequences of protein coding genes were aligned using MUSCLE in MEGA7 with default settings of codon mode (Edgar 2004, Kumar et al. 2016). Uneven ends were manually trimmed. 43 sequences were spotted containing missing or poorly-sequenced portions, which were deleted or question-marked (Suppl. material 3: Table S2). Then, all sequences were aligned again. The aligned sequences were translated into amino acids to check accuracy. Alignments of 13 genes were concatenated using PHYLOSUITE. The final alignment is 10932 bases long.

ALIGROOVE 1.05 (Kück et al. 2014) was used to assess alignment ambiguity for each gene, and DAMBE 7.2.7 (Xia 2018) to calculate the substitution saturation per codon position per gene (i.e. 3 bases × 13 genes). Amino acid alignments (translated from the nucleotide alignments) instead of nucleotide alignments were imported to ALIGROOVE, because the alignments were based on codon model. Saturation was calculated under GTR model by default, or under F84 model when an overflow error occurs (the error itself is a sign of saturation).

ALIGROOVE suggests high ambiguity in ATP8 alignment, followed by ND6 (Suppl. material 4). Other genes exhibit low general ambiguity, but a few taxa with too many missing cells in alignments show high ambiguity. Even if several incomplete sequences can be excluded in the following analyses, ATP8, in general, is still too ambiguous. On the other hand, the dataset as a whole is not significantly affected by scattered ambiguous alignments (Suppl. material 4).

According to the 39 saturation plots (Suppl. material 5), ATP8, ND4L, ND6 and, as expected, the third codon positions are saturated. These three genes are among the less informative ones considered in Talavera and Vila (2011). Excluding ATP8, ND4L and ND6, data incompleteness was calculated by counting Ns and question-marks, both of which are regarded as missing cells. Eight taxa with missing cells greater than 1% were grouped into ‘BadSeq’: Anallacta methanoides (9.17% missing), Aposthonia borneensis (1.80%), Beybienkoa kurandanensis (1.18%), Eublaberus distanti (2.87%), Galiblatta cribrosa (3.64%), Megaloblatta sp. (2.92%), Metallyticus sp. (8.84%), and Platyzosteria sp. (2.19%).

Bayesian phylogenetic inference

Phylogenetic inferences were performed in MRBAYES 3.2.7 (Ronquist et al. 2012). Data were divided into two partitions: the first and the second bases of the codon. The third position of codon was excluded from all analyses. I did not use programs to select a ‘best-fit’ model, not only because this is unnecessary (Nascimento et al. 2017, Abadi et al. 2019), but also because the ‘best-fit’ is not necessarily the best or accurate (Gatesy 2007, Kelchner and Thomas 2007, Luo et al. 2010). Instead, I used the empirically universal model, GTR, with Gamma rates (+G). The manual and tutorials of MRBAYES (among others) recommend GTR+I+G as the universal model, but the invariable-sites model (+I) generates a strong correlation between the proportion of invariable sites and the gamma shape parameter, and becomes undesirable (Sullivan et al. 1999, Yang 2014). Using PHYLOBAYES with the CAT-GTR model implemented therein accounting for more exhaustive heterogeneities may improve the resolution and accuracy of phylogenetic inference (Lartillot and Philippe 2004, Lartillot et al. 2009, Moran et al. 2015), but this approach is currently impossible given the computation resources available to this study. Instead, the alignment ambiguity of the final dataset (see below) was reported, because the alignment ambiguity assessed by ALIGROOVE is also a measure of heterogeneous sequence divergence (Kück et al. 2014). I did not perform maximum likelihood analysis because the interpretation of bootstrapping (the assessment of the uncertainty of maximum likelihood estimates) is vague (Yang 2014), in contrast to posterior probabilities of Bayesian estimates (Huelsenbeck and Rannala 2004). Instead, the maximum-likelihood tree sampled from MCMC is reported. Each MRBAYES analysis involved two runs, each of which comprises four chains, running 1.5–2.5 million iterations depending on the difficulty of converging. Samples were taken once every 500 iterations and the initial 1%–5% (depending on the difficulty of converging) of samples were discarded (burn-in). I used TRACER 1.7.1 (Rambaut et al. 2018) to ensure sufficient effective samples (200 at least, 300–1000 in general).

The first analysis utilized all 13 genes. The results are only used for comparison with the second step analyses, to observe the influence of ATP8, ND4L and ND6.

The second step is to compare the trees inferred from three taxon sets. All analyses excluded ATP8, ND4L and ND6. (1) All-species analysis using complete taxon set. (2) Good-species analysis, excluding ‘BadSeq’. (3) Short-species analysis, excluding long-branched taxa detected from the all-species analysis. This step aims to detect the impact of incomplete data and long branch.

The third step analysis used only the ‘safe’ taxa. In this step, all taxa within ‘BadSeq’ were excluded even if they do not virtually affect the topology of other species. It is learnt from experience that more missing or poorly-sequenced bases imply more potential errors in the superficially intact data. Potential pitfalls of incompleteness (e.g. erroneous positions of these taxa per se) violate the main idea of this study. Long-branched taxa with low support are also to be excluded. In the present study, they are Aposthonia borneensis, Aposthonia japonica and Nocticola sp.. ‘Safe’ taxa comprise 85 species (Suppl. material 2: Table S1).

The fourth, also the final, step yields the phylogeny that is regarded as the formal result. Prior to MRBAYES, sequences of ‘safe’ taxa were re-aligned and concatenated. This new, 9912-base-long alignment, as final dataset, was also imported to ALIGROOVE to assess alignment ambiguity. This 85-species dataset is less ambiguous than the original one (Suppl. material 4). The resulted tree was used as the fixed topology in dating analyses.

Fossil calibration and dating

As calibrations, only two fossils fulfill the criteria of Parham et al. (2011) and are suitable for the present study. The earliest known termite Valditermes brenanae calibrates the split between Cryptocercidae and Isoptera (minimum age 130.3 Ma, see Wolfe et al. 2016), as in Misof et al. (2014) and Evangelista et al. (2019). The earliest known blattelline cockroach Piniblattella yixianensis Gao et al., 2018 (Gao et al. 2018) calibrates the split between Blattellinae and Blaberidae. Piniblattella yixianensis is used for calibration for the first time, setting a minimum age as 120.9 Ma (see Discussion). To observe the effect of this new calibration, I also ran an analysis without this fossil (i.e. only calibrated by V. brenanae) to compare with the two-fossil analysis. The minimum bounds of calibrated nodes were set to the minimum age of fossils. The minimum root age was set to 130.3 Ma, the age of the older calibration fossil. All maximum bounds were set to 412 Ma, the oldest age of Rhynie Chert, as justified in Evangelista et al. (2019).

Some studies used the so-called ‘roachoids’ (Eoblattodea, see Li 2019) to calibrate Dictyoptera+sister (which is the root herein), based on the hypothesis that those ambiguous fossils are stem members of Dictyoptera (Legendre et al. 2015, Tong et al. 2015, Bourguignon et al. 2018, Evangelista et al. 2019). To detect the impact of such fossils, I performed another dating analysis with the earliest ‘roachoid’, namely Qilianiblatta namurensis Zhang et al., 2012 (Zhang ZJ et al. 2012, Guo et al. 2013), thus three fossil calibrations were used in this analysis. The radioisotopic age of the Q. namurensis-bearing stratum is unavailable, instead, a preliminary stratigraphic correlation gives latest Bashkirian to middle Moscovian (Trümper et al. 2020). Therefore, I used the top age of Moscovian (306.9 Ma).

I used the MCMCTREE program in PAML 4.9j (Yang 2007) to estimate divergence times. Dating analyses used autocorrelated relaxed clock model and GTR+G model. Rate prior was set to 1 substitution per site per 100 Ma by reference to the empirical estimations (Papadopoulou et al. 2010, Andújar et al. 2012). Estimation of divergence times used the approximate method implemented in MCMCTREE. The first 20000 iterations were discarded as burn-in. 5000 samples were gathered, once every 200 iterations. A replicating MCMC was performed to check for convergence in TRACER.

Figure preparation

Trees are visualized using FigTree 1.4.3 (Andrew Rambaut, and modified using Adobe Illustrator CC 2017.


For the reader’s convenience and to enable a comparison of studies, familial taxonomy of cockroaches in this paper follows recent studies that are compared (e.g., Djernæs et al. 2015, Legendre et al. 2015, Wang et al. 2017, Bourguignon et al. 2018, Evangelista et al. 2019). It is noteworthy that recent molecular studies focusing on Blaberoidea raised subfamilies of Ectobiidae to families (Djernaes et al. 2020, Evangelista et al. 2020). Consequently, their Ectobiidae is identical to the Ectobiinae herein.


The 13-gene tree recovers a sistergroup relationship between Aposthonia (Embioptera) and Nocticola (Blattaria), which is obviously erroneous regardless of posterior probability (Suppl. material 6). The 10-gene analyses removed this error (Suppl. material 79), and demonstrate that ATP8, ND4L and ND6 are detrimental to the analyses. The results from the all-species analysis (Suppl. material 7) and the good-species analysis (Suppl. material 8) are very close, with some divergences in small clades, and the posterior probabilities are similar in general. In the all-species analysis, three species have extremely long branches (Aposthonia borneensis, Aposthonia japonica and Nocticola sp.), and the posterior probabilities of corresponding nodes are low. Excluding these species increases the general supports (Suppl. material 9).

The ‘safe’-taxa analysis yields higher posterior probabilities (Suppl. material 10) than all analyses above. The final dataset, which is from the realignment of the ten genes of ‘safe’ taxa, yields the formal result (Fig. 2). The maximum-likelihood tree (log-likelihood = –155715.20) sampled from MCMC has only one topological difference with Fig. 2: Neostylopyga rhombifolia and Periplaneta brunnea are exchanged (not shown). The phylogeny recovers major splits in Dictyoptera as Mantodea + (Corydiinae + (Blaberoidea + Blattoidea-Isoptera complex)). These major splits have at least 95% posterior probability. Owing to the absence of Nocticolidae and Latindiinae, the relationships in Corydioidea are unknown.

Figure 2. 

Bayesian phylogeny of Dictyoptera inferred from ten protein-coding genes of 85 mitogenomes, excluding the third base of codon. Posterior probabilities are shown in percentage otherwise are 100%. Clades of superfamilies or higher rank are numbered, as indicated by black background in the key. Species of major taxonomic identities (all are clades) are coloured, as indicated in the key. Subfamilies of Blattidae and Blaberidae are labeled; asterisked ones are not monophyletic. For comparison with trial analyses, see Suppl. material 610.

The dating result of two-fossil-calibration analysis (without Q. namurensis) is regarded as the formal result of this paper (Fig. 3: middle), suggesting that the age of crown Dictyoptera is 191.08 Ma (95% credibility interval 168.96–218.82 Ma), of crown Blattodea 171.2 Ma (95% CI 153.26–194.23 Ma). Qilianiblatta namurensis considerably pushes the ages back (Fig. 3: top): the age of crown Dictyoptera is 270.01 Ma (95% CI 236.69–309.31 Ma), of crown Blattodea 237.82 Ma (95% CI 204.46–276.04 Ma). In comparison, there is little difference in the estimated ages between the two-calibration analysis and the one-calibration one (Fig. 3: middle vs. bottom). Even though it is insufficient to conclude that P. yixianensis is a calibration as competent as V. brenanae, P. yixianensis is at least harmless to dating analyses. The time trees showing species are given in Suppl. material 1113.

Figure 3. 

Time trees of Dictyoptera estimated by MCMCTREE. Two-calibration result (middle) is regarded as the formal result of this study. Calibrated nodes are coloured, with vertical bars denoting bounds. Calibrations: Qilianiblatta namurensis (green), Valditermes brenanae (red), Piniblattella yixianensis (blue). Abbreviations: A[naplectidae], Dictyop[tera], L[amproblattidae], T[ryonicidae], Xyloph[agodea]. For detailed phylogenies showing species, see Suppl. material 1113.


Phylogeny of cockroaches

The relationship of major clades (suborder, superfamily, family, and subfamily) recovered herein is not identical to any previous studies. At the superfamily level, the sistergroup relationship of Corydioidea (only represented by Corydiinae) to the rest of Blattodea is consistent with that in Wang et al. (2017) and Djernæs et al. (2015, in part), both of which used three mitochondrial and at least two nuclear gene fragments, whereas conflicting with other recent phylogenies (Djernæs et al. 2015 in part, Legendre et al. 2015, Bourguignon et al. 2018, Evangelista et al. 2019). The superfamilial relationship of cockroaches is in dispute. On the other hand, the monophyletic Blaberoidea and the monophyletic blattoid complex (Blattoidea and Isoptera) are always supported.

Corydioidea are always undersampled. Species of Nocticolidae, Latindiinae, and Corydiidae incertae sedis (e.g. Ctenoneura) are lacking. Although one mitogenome of Nocticola is available, it is hardly serviceable unless the long branch is broken up by increased sampling (Poe 2003). Nonetheless, the transcriptome data support a monophyletic Corydioidea that include Corydiidae and Nocticolidae (Evangelista et al. 2019).

In the blattoid complex, only the sistergroup relationship between Cryptocercidae and Isoptera is universally recognized. These taxa constitute Xylophagodea (Engel 2011). The new phylogeny recovers Xylophagodea as sister to the remaining blattoid complex, of which the internal relationship is (Blattidae + Tryonicidae) + (Anaplectidae + Lamproblattidae). This is significantly different from other studies. Three major groups of Blattoidea are still undersampled, namely, Anaplectidae, Lamproblattidae and Tryonicidae. In addition, some mysterious taxa of Blattoidea incertae sedis (e.g. Oulopteryx) have not yet been sampled.

The paraphyly of Ectobiidae with respect to Blaberidae is a consensus among studies; the present study is not an exception. However, the relationships among Blaberidae and ectobiid subfamilies are conflicting among studies, especially in the positions of Ectobiinae and Pseudophyllodromiinae. The Ectobiinae contributes a weak point in the new phylogeny (pp = 79%), i.e. the node of Ectobiinae + (Blattellinae + Blaberidae). Regardless of the Nyctiborinae, which is not included in the final phylogeny herein, the sistergroup relationship between Blattellinae and Blaberidae is also supported in Bourguignon et al. (2018) and Evangelista et al. (2019, 2020). Although the considerably diversified Blaberidae are typically densely sampled, the phylogeny of them recovered by various studies is inconsistent.

A challenge to all molecular phylogenies is the reconciliation with morphological, behavioral, and other evidence. For example, oothecal property and rotation behavior are various and the taxonomic distribution of them in Blaberoidea is comparatively well known (McKittrick 1964, Roth 1967, 1968a, Bell et al. 2007). Note that the rotation behavior assigned to Ectobiinae in Evangelista et al. (2019) is contrary to the literature. The parsimonious scenario is that Pseudophyllodromiinae are sister to, or paraphyletic with respect to, the remaining Blaberoidea, which constitute a clade (e.g. McKittrick 1964, Klass and Meier 2006). The present study supports this scenario but the posterior probabilities of the “remaining Blaberoidea” node is relatively low (pp = 79%). Other studies, in which the phylogenies do not recover Pseudophyllodromiinae as sister to the remaining Blaberoidea, imply either parallel evolution of oothecal rotation in Ectobiinae and in Blattellinae + Nyctoborinae + Blaberidae, or an ancestral state of oothecal rotation in Blaberoidea and a loss in Pseudophyllodromiinae. At least, the relationship of Ectobiinae and Pseudophyllodromiinae to other Blaberoidea is debatable.

Fossil calibrations and divergence times

The only appropriate fossil calibration in Blattaria in the present study is Piniblattella yixianensis Gao et al., 2018 (Gao et al. 2018), which is used as a calibration for the first time. In the following, this fossil calibration is justified according to the five criteria in Parham et al. (2011).

Criteria 1 and 4. Information about the fossil-bearing stratum and museum collection is provided in Gao et al. (2018).

Criterion 2. Regardless of the determination of genus (which is in dispute, see Hinkelman 2019), P. yixianensis belongs in Blattellinae as evidenced by the oothecal rotation, reproduction type oviparity B, and the wing venation pattern, as explained below. Rotation feature and physical property of ootheca are crucial to the family-level phylogeny of cockroaches: the “advanced rotation” (i.e. rotating the ootheca and containing the anterior eggs inside vestibulum) is considered as a significant apomorphy in cockroaches, and distributed in Blaberoidea other than Pseudophyllodromiinae (McKittrick 1964, Roth 1967, 1968a, Bell et al. 2007). The rotation of the oothecae of P. yixianensis is unlikely due to taphonomic process: all preserved oothecae are horizontally positioned, none is perpendicularly or randomly positioned (Gao et al. 2018, Hinkelman 2019). The “primitive rotation” of Corydiidae is also ruled out: in the primitive rotation, the anterior eggs are outside the abdomen and the ootheca is obliquely positioned (Roth 1967) – this is not the case with P. yixianensis. Another difference between primitive and advanced rotation is the presence and absence of the flange, but which cannot be clearly observed in those fossils. In the phylogenies of the present study and some previous studies (e.g., McKittrick 1964, Klass and Meier 2006), Pseudophyllodromiinae are sister to or paraphyletic with the remaining Blaberoidea, supporting that the advanced rotation is autapomorphic for Blaberoidea excluding Pseudophyllodromiinae, and therefore P. yixianensis can at least calibrate the node of crown Blaberoidea (regardless of other evidence discussed below). However, other studies support several origins of the advanced rotation in Blaberoidea or loss of the advanced rotation in Pseudophyllodromiinae (e.g., Wang et al. 2017, Bourguignon et al. 2018, Evangelista et al. 2019). Under this hypothesis and regardless of other evidence (discussed below), P. yixianensis may only calibrate the split between Blaberoidea and the sister group with caution.

The reproduction type of P. yixianensis is oviparity B: (1) during reproduction, female cockroaches have a period of carrying the ootheca (if present) outside, but only the oviparity B carries the ootheca externally until hatching; other types only carry shortly before oviposition (oviparity A) or before retraction (ovoviviparity and viviparity) (Roth 1967, 2003, Bell et al. 2007), and have much less chance to leave fossils. Oviparity B likely contributes a lot to the preservation of ootheca-bearing fossils like P. yixianensis. (2) Based on the author’s observation during collecting, the oviparity A ootheca is easily dropped when the cockroach is caught, and almost certainly detached in the end. In comparison, some of the oviparity B oothecae remain attached in the abdomen even when the cockroach is preserved. This implies that it is unlikely that oviparity A cockroaches preserve fossils carrying oothecae, but oviparity B cockroaches may. (3) The oothecal keel of P. yixianensis is relatively underdeveloped and unornamented (Gao et al. 2018, Hinkelman 2019), and so accords with the characteristics of oviparity type B (Roth 1968a, 1971, Bell et al. 2007). Among living cockroaches, only some species of Blattella, Chorisia and Onycholobus are known of both the oviparity type B and the advanced rotation (McKittrick 1964, Roth 1967, 1968a, 1971, 1983, 2003, Bell et al. 2007). Chorisia and Onycholobus are not included in analyses here, but the former is considered as closest to Blattella (Roth 1983), while the latter has the ootheca resembling that of Blattella (Roth 1971).

However, oviparity B is homoplastic among Blaberoidea. Roth (1968b) found Lophoblatta, a pseudophyllodromiine genus, carrying the ootheca until the eggs hatch, but not rotating the ootheca. This discovery demonstrated that the oviparity B originated independently more than once within Blaberoidea. According to the reasonable hypothesis of Roth (1968a), oviparity B is the intermediate form between the ovoviviparity and the plesiomorphic oviparity A, i.e., ovoviviparity derived from oviparity B, which derived from oviparity A. Ovoviviparity occurs in most Blaberidae (with advanced rotation) but also, homoplastically, in two genera of Blattellinae, which rotate the ootheca (Roth 1982, 1984: Stayella, Roth 1995: Pseudoanaplectinia), and two genera of Pseudophyllodromiinae, which do not rotate the ootheca (Roth 1989: Sliferia, Roth 1997: Pseudobalta) (see also a review by Djernæs et al. 2020). Provided that Roth’s hypothesis is true, the homoplasy of ovoviviparity further demonstrates that the oviparity B is highly homoplastic.

Accordingly, it appears that Blaberoidea are preadapted to the advanced rotation and oviparity B (consequently ovoviviparity), but as far as known, these two features only co-occur in Blaberidae and Blattellinae. Blaberidae and Blattellinae were recovered as sister groups (Bourguignon et al. 2018 and the present study), or form a clade together with Nyctiborinae (Klass and Meier 2006, Evangelista et al. 2019), which is not included in analyses herein. This implies that species of the clade Blaberidae + Blattellinae (or Blaberidae + Blattellinae + Nyctiborinae) are more preadapted to allow (if not achieved) the co-occurrence of advanced rotation and oviparity B (consequently ovoviviparity). Therefore, the combination of advanced rotation and oviparity B may tentatively place P. yixianensis into that clade but outside of Blaberidae.

Other characters preserved in the fossils of P. yixianensis are barely discernible except the wing venation. The forewing of P. yixianensis conforms to the general form of Blattellinae (see Rehn 1951, Li et al. 2018): ScP with few branches, R pectinate proximally and dichotomous or irregular distally, M and CuA both developed and not essentially pectinate, claval furrow with sharp apical turn, and claval veins diagonal. These traits are distinct from other subfamilies of Ectobiidae. The hindwing has a simple ScP, a pectinate RA with four branches or so, a non-pectinate RP, a simple and feeble M, and a nearly pectinate CuA (Gao et al. 2018). This combination of hindwing traits is not characteristic of any taxon, although these traits are more common in Blaberoidea, particularly Blaberidae (see Rehn 1951, Li et al. 2018). Unfortunately, the polarity of wing venation characters above is barely clear, so that it is premature to conclude a phylogenetic position through these similarities in venation. It is noteworthy that both the tegmen and hindwing of P. yixianensis exhibit a developing characteristic posterior branch of R, i.e., the apicoposterior part of R is a branch with terminal branching only. Most cockroaches do not have a characteristic posterior branch (cpb), and this specialization is homoplastically derived among cockroaches, principally Ectobiidae (see Rehn 1951, Li et al. 2018). Nonetheless, the cpb and developing cpb vary in morphology, whereas the branching pattern of P. yixianensis is found in Blattella and related genera such as Episymploce, but not seen in others (Li et al. 2018, and unpublished observation). This evidence reinforces the hypothesis that P. yixianensis belongs in Blattellinae, although it is premature to conclude that P. yixianensis is a close relative of Blattella or even sister to Blattella.

So far, the evolution of reproduction type, ootheca handling behaviour and wing venation of cockroaches is not well understood, and might be more complicated than currently known. In view of this, the phylogenetic position of P. yixianensis is not securely settled. Nevertheless, P. yixianensis can be tentatively considered as a member of Blattellinae, and thus calibrates the node of Blattellinae + sister (Blaberidae herein). In summary, P. yixianensis as a calibration should be used with caution, and comparative analyses with/without this fossil should be performed to accommodate its uncertainty.

Criterion 3. Reconciliation between molecular and morphological phylogenies is partially achieved. As mentioned above, regardless of the Nyctiborinae that is not included in the final data, the sistergroup relationship between Blattellinae and Blaberidae is supported herein and in recent big data analyses (Bourguignon et al. 2018, Evangelista et al. 2019, 2020), and Ectobiinae and Pseudophyllodromiinae are not nested in the clade of Blattellinae + Blaberidae + Nyctiborinae. In the most comprehensive ever morphological (and ethological) phylogeny of Dictyoptera (Klass and Meier 2006), the above relationships within Blaberoidea are also recovered, except the absence of Ectobiinae. According to the phylogenetic discussion in McKittrick (1964), the Ectobiinae is nested in the clade of Blattellinae + Blaberidae + Nyctiborinae (Fig. 1), but that study is somewhat outdated and not strictly phylogenetic. There is a lack of recent morphological phylogeny covering all major groups; therefore, it is impossible to thoroughly compare the morphological phylogeny with the molecular phylogeny.

Criterion 5. Piniblattella yixianensis is from Huangbanjigou, Beipiao, Liaoning, northeastern China (Gao et al. 2018). Isotopic age of the fossiliferous layers in Huangbanjigou ranges from 121.2 Ma to 129.8 Ma (Swisher et al. 1999, Yang et al. 2007). However, the horizontal correlation between this fossil and the radiometric samples is unknown, and the radiometric sampling is insufficient, therefore the age range above does not necessarily represent the age of fossils. I conservatively use the age of the overlying stratum (of top Yixian Formation), 120.9 Ma (Smith et al. 1995), as the minimum age of P. yixianensis.

The other fossil for calibration, V. brenanae, has been frequently used for Xylophagodea (e.g., Misof et al. 2014, Bourguignon et al. 2018, Evangelista et al. 2019). Its identity as a termite is secured by the presence of basal suture (Jarzembowski 1981), one of the defining characters (autapomorphies) of Isoptera (Ax 1999, Krishna et al. 2013). Its validity as a calibration was justified by Wolfe et al. (2016), and I have no comments on this fossil.

Fossil calibrations contribute considerably to the discrepancy in the age estimation among studies. The ‘roachoid’ fossils, remarkably, were frequently assigned as “stem Dictyoptera” (e.g., Legendre et al. 2015, Tong et al. 2015, Bourguignon et al. 2018, Evangelista et al. 2019). Although the ambiguity of them and alternative interpretations were considered (e.g., Kjer et al. 2015, Bourguignon et al. 2018, Li and Huang 2019), a formal report on the impact of them is lacking. Because of the same assignment of ‘roachoid’ fossils, the age estimates with three calibrations herein (which is only for comparison) are close to that in Bourguignon et al. (2018) and Evangelista et al. (2019) (Permian origin of crown Dictyoptera and Triassic origin of crown Blattodea), and only somewhat younger than that in Legendre et al. (2015). ‘Roachoid’ fossils often play a decisive role in the dating of cockroaches, pushing the age estimates older. In comparison, the formal age estimates herein (two fossil calibrations) are close to that in Misof et al. (2014), both studies do not use ‘roachoid’ fossils as calibrations, suggesting Jurassic origins of crown Dictyoptera and crown Blattodea. Without the ‘roachoids’, other fossils will take over them as decisive calibrations and result in various, and usually younger, estimations (e.g., Wang et al. 2017, Bourguignon et al. 2018: fig. S12). A comparison of age estimates among studies is shown in Fig. 4.

Figure 4. 

Comparison among the ages estimated in various studies. The fossils are: Valditermes brenanae Jarzembowski, 1981; Piniblattella yixianensis Gao et al., 2018; Nodosigalea burmanica Li & Huang, 2018; Cretaperiplaneta kaonashi Qiu et al., 2020; Stegoblatta irmgardgroehni Anisyutkin & Gröhn, 2012. Abbreviation: ALTB, Anaplectidae + Lamproblattidae + Tryonicidae + Blattidae.

Unfortunately, many of the fossil calibrations other than ‘roachoids’ are also unjustified. Subsequently, comparisons among the age estimates from various studies could be pointless. For example, the “stem MantodeaHomocladus grandis (Djernæs et al. 2015, Bourguignon et al. 2018) is highly questionable (Evangelista et al. 2019 and references therein); the “oldest Mantoidea fossil” Prochaeradodis enigmaticus (Djernæs et al. 2015, Wang et al. 2017) may be a cockroach (Cui et al. 2018); the “blattid” Balatronis libanensis is unlikely a true cockroach (Blattaria), not to mention Blattidae (Evangelista et al. 2017, Qiu L et al. 2020a); the “Diploptera fossils” (Bourguignon et al. 2018) cannot be identified to Diploptera and the higher-rank placement of those fossils also remains undetermined (Evangelista et al. 2017, Li et al. 2017); the “first modern cockroach” Zhujiblatta anofissilis (Bourguignon et al. 2018) is phylogenetically unsettled and has to be redescribed, which I am preparing elsewhere. Even more surprisingly, an unnamed “Epilampra fossil” found in an extant cockroach database was used (Bourguignon et al. 2018).

A critical review of cockroach fossil calibrations was not achieved until Evangelista et al. (2017), who recommended four cockroach fossils for node calibration. Evangelista et al. (2019) discarded one of them and retained corydiid Cretaholocompsa montsecana Martinez-Delclos, 1993, blaberid “Gynaobesa (Piton, 1940) and ectobiid Ectobius kohlsi Vršanský et al., 2014. However, these fossils are still debatable.

Cretaholocompsa montsecana was determined as a close relative of extant Holocompsa (Martinez-Delclos 1993), and used as a calibration for corydiid nodes (Legendre et al. 2015, Wang et al. 2017, Evangelista et al. 2019). However, Cretaholocompsa significantly differs from Holocompsa in the presence of large spines along the ventral margin of midfemora (other legs unknown) (Qiu L et al. 2020b). Besides, according to recent accounts of Corydiidae, such spines are absent from all femora in this family (e.g., Estrada-Alvarez and Guadarrama 2012, Hopkins 2014, Crespo et al. 2015, Qiu L 2017, Qiu L et al. 2017, 2019a, 2019b, 2020b).

Gynaobesa was used as a calibration for blaberid nodes (Bourguignon et al. 2017, Evangelista et al. 2019). Evangelista et al. (2017) identified this fossil to Blaberidae based on (1) the stout cerci, (2) approximately parallel edges of tegmina, (3) elongated CuP, (4) shape of the pronotum, (5) asymmetrical male subgenital plate and (6) large body size. Although this fossil appears to be blaberid in overall appearance, the evidence above is weak or invalid to place “Gynaobesa in Blaberidae. First, as acknowledged by Evangelista et al. (2017), traits 1, 2 and 3 are homoplastic with Blattidae. Second, the shape of the pronotum is not entirely clear (Evangelista et al. 2017). Third, the subgenital plate is not clearly discernible: according to the figures in Evangelista et al. (2017), the “concave margin” is implausible in favour of poor preservation. Besides, the subgenital plate appears to be large and cover three segments as in extant female cockroaches, in comparison to the male subgenital plate that is as small as one normal segment; therefore, the specimen may be a female. Fourth, large-sized species are common in Blattidae and Corydiidae in addition to Blaberidae.

Ectobius kohlsi was identified based on a comparison with extant species (Vršanský et al. 2014). However, the preserved characters are not unique enough to indicate the genus; i.e., diagnostic characters of Ectobius or of Ectobiinae are not clearly observed, e.g., elongate male genital elements (Roth 2003) and a pinnate R+M+CuA system of tegmina (unpublished observation). Instead, spot-and-line macula patterns on the pronotum and forewings are common in Ectobiinae and Pseudophyllodromiinae. Vršanský et al. (2014) reported a female with valvate subgenital plate. If this is true, then this species likely belongs to Pseudophyllodromiinae because suchlike females do exist in Pseudophyllodromiinae (e.g. Euphyllodromia, Anisyutkin 2011). If this fossil is a male, then it is more likely to be a member of Pseudophyllodromiinae, some genera of which have a valvate subgenital plate in males (e.g. Balta, Qiu ZW et al. 2017).

Interestingly, the fossil discarded by Evangelista et al. (2019), Cariblattoides labandeirai Vršanský et al., 2011, is likely to be a genuine pseudophyllodromiine species, even though the genus is uncertain. According to Vršanský et al. (2011b), the forewing of C. labandeirai bears venational characters in common with most Pseudophyllodromiinae: ScP simple and short, R essentially pectinate, M pectinate and more developed than CuA, claval veins oblique or diagonal (see Rehn 1951, Li et al. 2018). The venation alone is a weak indicator of the taxonomic identity, whereas the combination of venation and macula pattern is stronger reasoning. Unfortunately, C. labandeirai is not suitable for calibrating Pseudophyllodromiinae + sister (= Blaberoidea herein) even if its inclusion in Pseudophyllodromiinae is proven, because it would be suppressed by the older fossil, P. yixianensis, which already calibrates an internal node.

Only one true-cockroach fossil is used as a calibration in the present study, but this does not imply that other fossils are substandard. Every informative fossil (with high phylogenetic resolution and ascertained geological context) has the potential to be a competent calibration, but the incorporation of them is hampered by the fact that relevant living species are under-sampled or have not yet been sequenced. Noteworthy examples of fossils include those of extant genus, e.g. Supella (Nemosupella) miocenica Vršanský et al., 2011 (see Vršanský et al. 2011a), and those of Corydioidea, e.g., Proholocompsa fossilis (Shelford, 1910) (see Gorokhov 2007), Paraeuthyrrhapha groehni Anisyutkin, 2008 (see Anisyutkin 2008), Crenocticola Li & Huang, 2019 (see Li and Huang 2019). These fossils could become powerful calibrations for smaller clades (younger nodes) if the data of related extant species are available, otherwise they can only calibrate larger clades (older nodes), and become ineffective when older fossils (e.g. those used herein) calibrate the same or internal nodes.


Based on published mitochondrial genomes, the present study infers a phylogeny of cockroaches and termites as Corydiinae + (((Cryptocercidae + Isoptera) + ((Anaplectidae + Lamproblattidea) + (Tryonicidae + Blattidae))) + (Pseudophyllodromiinae + (Ectobiinae + (Blattellinae + Blaberidae)))). The sistergroup relationship between (Cryptocercidae + Isoptera) and (Anaplectidae + Lamproblattidae + Tryonicidae + Blattidae) is recovered for the first time. This study suggests that the phylogenetic reconstruction of cockroaches is in urgent need of the data of Corydioidea (particularly the Nocticolidae), of which the phylogenetic relationships are poorly known. This study dates the crown Dictyoptera to early Jurassic, and crown Blattodea to middle Jurassic. Using the ambiguous ‘roachoid’ fossils to calibrate Dictyoptera+sister pushes these times back to Permian and Triassic. Given currently available data and fossils, few nodes within true cockroaches can be calibrated. This can be overcome by discovering more fossils, or by sampling fossil-related species to allow the incorporation of well-justified fossils. In view of the scarcity of suitable fossils for calibration, the latter approach may be more promising.


I deeply thank Dr Klaus Klass and Dr Dominic Evangelista for constructive comments and critiques, and thank the authors of mitogenome data which are fundamental to the present study. This study was motivated by my interest and not funded.


  • Andújar C, Serrano J, Gómez-Zurita J (2012) Winding up the molecular clock in the genus Carabus (Coleoptera: Carabidae): assessment of methodological decisions on rate and node age estimation. BMC Evolutionary Biology 12: e40.
  • Anisyutkin LN (2008) Paraeuthyrrhapha groehni gen. et sp. nov., a new genus of the family Polyphagidae (Dictyoptera) from Baltic amber and its phylogenetical position. Alavesia 2: 77–85.
  • Ax P (1999) Das System der Metazoa II. Ein Lehrbuch der phylogenetischen Systematik. Gustav Fischer, Stuttgart/Jena/Lübeck-Ulm, 384 pp.
  • Bell WJ, Nalepa CA, Roth LM (2007) Cockroaches: Ecology, Behavior, and Natural History. Johns Hopkins University Press, Baltimore, 230 pp.
  • Bourguignon T, Lo N, Cameron SL, Šobotník J, Hayashi Y, Shigenobu S, Watanabe D, Roisin Y, Miura T, Evans TA (2014) The evolutionary history of termites as inferred from 66 mitochondrial genomes. Molecular Biology and Evolution 32(2): 406–421.
  • Bourguignon T, Tang Q, Ho SYW, Juna F, Wang ZQ, Arab DA, Cameron SL, Walker J, Rentz D, Evans TA, Lo N (2018) Transoceanic dispersal and plate tectonics shaped global cockroach distributions: evidence from mitochondrial phylogenomics. Molecular Biology and Evolution 35: 970–983.
  • Buckley TR, Simon C, Flook PK, Misof B (2000) Secondary structure and conserved motifs of the frequently sequenced domains IV and V of the insect mitochondrial large subunit rRNA gene. Insect Molecular Biology 9(6): 565–580.
  • Cameron SL, Barker SC, Whiting MF (2005) Mitochondrial genomics and the new insect order Mantophasmatodea. Molecular Phylogenetics and Evolution 38(1): 274–279. [print version 2006]
  • Cameron SL, Lo N, Bourguignon T, Svenson GJ, Evans TA (2012) A mitochondrial genome phylogeny of termites (Blattodea: Termitoidae): Robust support for interfamilial relationships and molecular synapomorphies define major clades. Molecular Phylogenetics and Evolution 65(1): 163–173.
  • Chen AH (2012) Complete mitochondrial genome of the double-striped cockroach Blattella bisignata (Insecta: Blattaria: Blaberoidea). Mitochondrial DNA 24(1): 14–16. [print version 2013]
  • Chen Z-T, Lü L, Lu M-X, Du Y-Z (2017) Comparative mitogenomic analysis of Aposthonia borneensis and Aposthonia japonica (Embioptera: Oligotomidae) reveals divergent evolution of webspinners. Scientific Reports 7: 8279.
  • Cheng XF, Zhang LP, Yu DN, Storey KB, Zhang JY (2016) The complete mitochondrial genomes of four cockroaches (Insecta: Blattodea) and phylogenetic analyses within cockroaches. Gene 586(1): 115–122.
  • Crespo FA, Di Iorio O, Valverde ADC (2015) Contributions to the knowledge of Hypercompsa, new register from Argentina (Blattaria: Corydiidae). Revista de la Sociedad Entomológica Argentina 74(3–4): 203–207.
  • Djernæs M, Klass KD, Eggleton P (2015) Identifying possible sister groups of Cryptocercidae + Isoptera: A combined molecular and morphological phylogeny of Dictyoptera. Molecular Phylogenetics and Evolution 84: 284–303.
  • Djernæs M, Varadínová ZK, Kotyk M, Eulitz U, Klass K-D (2020) Phylogeny and life history evolution of Blaberoidea (Blattodea). Arthropod Systematics & Phylogeny 78(1): 29–67.
  • Dumans ATN, Grimaldi DB, Furtado C, Machado EA, Prosdocimi F (2017) The complete mitochondrial genome of the subsocial cockroach Nauphoeta cinerea and phylogenomic analyses of Blattodea mitogenomes suggest reclassification of superfamilies. Mitochondrial DNA Part B 2(1): 76–78.
  • Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32(5): 1792–1797.
  • Estrada-Alvarez JC, Guadarrama CA (2012) Primeros registros de Homoegamia mexicana Burmeister, 1838 (Blattaria: Polyphagidae) para el Estado de México. Dugesiana 19(1): 11–12.
  • Evangelista DA, Djernæs M, Kohli MK (2017) Fossil calibrations for the cockroach phylogeny (Insecta, Dictyoptera, Blattodea), comments on the use of wings for their identification, and a redescription of the oldest Blaberidae. Palaeontologia Electronica 20: 1FC. [23 pp.]
  • Evangelista DA, Wipfler B, Béthoux O, Donath A, Fujita M, Kohli MK, Legendre F, Liu S, Machida R, Misof B, Peters RS, Podsiadlowski L, Rust J, Schuette K, Tollenaar W, Ware JL, Wappler T, Zhou X, Meusemann K, Simon S (2019) An integrative phylogenomic approach illuminates the evolutionary history of cockroaches and termites (Blattodea). Proceedings of the Royal Society B: Biological Sciences 286: 20182076.
  • Evangelista D, Simon S, Wilson MM, Kawahara AY, Kohli MK, Ware JL, Wipfler B, Béthoux O, Grandcolas P, Legendre F (2020) Assessing support for Blaberoidea phylogeny suggests optimal locus quality. Systematic Entomology 46(1)[bound in 2021]: 157–171.
  • Gao T, Shih C, Labandeira CC, Liu X, Wang Z, Che Y, Yin X, Ren D (2018) Maternal care by Early Cretaceous cockroaches. Journal of Systematic Palaeontology 17(5): 379–391. [bound in 2019]
  • Gong R, Guo X, Ma J, Song X, Shen Y, Geng F, Price M, Zhang X, Yue B (2018) Complete mitochondrial genome of Periplaneta brunnea (Blattodea: Blattidae) and phylogenetic analyses within Blattodea. Journal of Asia-Pacific Entomology 21(3): 885–895.
  • Guo Y, Béthoux O, Gu JJ, Ren D (2013) Wing venation homologies in Pennsylvanian ‘cockroachoids’ (Insecta) clarified thanks to a remarkable specimen from the Pennsylvanian of Ningxia (China). Journal of Systematic Palaeontology 11: 41–46.
  • Hennig W (1969) Die Stammesgeschichte der Insekten. Waldemar Kramer, Frankfurt am Main, 436 pp.
  • Hinkelman J (2019) Spinaeblattina myanmarensis gen. et sp. nov. and Blattoothecichnus argenteus ichnogen. et ichnosp. nov. (both Mesoblattinidae) from mid-Cretaceous Myanmar amber. Cretaceous Research 99: 229–239.
  • Hopkins H (2014) A revision of the genus Arenivaga (Rehn) (Blattodea, Corydiidae), with descriptions of new species and key to the males of the genus. ZooKeys 384: 1–256.
  • Huelsenbeck JP, Rannala B (2004) Frequentist properties of Bayesian posterior probabilities of phylogenetic trees under simple and complex substitution models. Systematic Biology 53(6): 904–913.
  • Inoue J, Donoghue PCJ, Yang Z (2010) The impact of the representation of fossil calibrations on Bayesian estimation of species divergence times. Systematic Biology 59(1): 74–89.
  • Jeon MG, Park YC (2015) The complete mitogenome of the wood-feeding cockroach Cryptocercus kyebangensis (Blattodea: Cryptocercidae) and phylogenetic relations among cockroach families. Animal Cells and Systems 19(6): 432–438.
  • Kjer KM, Ware JL, Rust J, Wappler T, Lanfear R, Jermiin LS, Zhou X, Aspöck H, Aspöck U, Beutel RG, Blanke A, Donath A, Flouri T, Frandsen PB, Kapli P, Kawahara AY, Letsch H, Mayer C, McKenna DD, Meusemann K, Niehuis O, Peters RS, Wiegmann BM, Yeates DK, von Reumont BM, Stamatakis A, Misof B (2015) Response to Comment on “Phylogenomics resolves the timing and pattern of insect evolution”. Science 349: 487.
  • Klass KD (2001) Morphological evidence on blattarian phylogeny: “phylogenetic histories and stories” (Insecta, Dictyoptera). Deutsche Entomologische Zeitschrift 48: 223–265.
  • Klass KD, Meier R (2006) A phylogenetic analysis of Dictyoptera (Insecta) based on morphological characters. Entomologische Abhandlungen 63: 3–50.
  • Kômoto N, Yukuhiro K, Ueda K, Tomita S (2010) Exploring the molecular phylogeny of phasmids with whole mitochondrial genome sequences. Molecular Phylogenetics and Evolution 58(1): 43–52. [bound in 2011]
  • Kômoto N, Yukuhiro K, Tomita S (2012) Novel gene rearrangements in the mitochondrial genome of a webspinner, Aposthonia japonica (Insecta: Embioptera). Genome 55(3): 222–233.
  • Krishna K, Grimaldi DA, Krishna V, Engel MS (2013) Treatise on the Isoptera of the World. Bulletin of the American Museum of Natural History 377, 2704 pp.
  • Kück P, Meid SA, Groß C, Wägele JW, Misof B (2014) AliGROOVE – visualization of heterogeneous sequence divergence within multiple sequence alignments and detection of inflated branch support. BMC Bioinformatics 15: 294.
  • Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33(7): 1870–1874.
  • Lartillot N, Philippe H (2004) A Bayesian mixture model for across-site heterogeneities in the amino-acid replacement process. Molecular Biology and Evolution 21(6): 1095–1109.
  • Legendre F, Nel A, Svenson GJ, Robillard T, Pellens R, Grandcolas P (2015) Phylogeny of Dictyoptera: Dating the origin of cockroaches, praying mantises and termites with molecular data and controlled fossil evidence. PLoS ONE 10: e0130127.
  • Letsch HO, Kück P, Stocsits RR, Misof B (2010) The Impact of rRNA Secondary Structure Consideration in Alignment and Tree Reconstruction: Simulated Data and a Case Study on the Phylogeny of Hexapods. Molecular Biology and Evolution 27(11): 2507–2521.
  • Li XR, Huang D (2019) A new mid-Cretaceous cockroach of stem Nocticolidae and reestimating the age of Corydioidea (Dictyoptera: Blattodea). Cretaceous Research 106: 104202. [bound in 2020]
  • Li XR, Li M, Wang ZQ (2017) Preliminary molecular phylogeny of beetle cockroaches (Diploptera) and notes on male and female genitalia (Blattodea: Blaberidae: Diplopterinae). Zootaxa 4320(3): 523–534.
  • Li XR, Zheng YH, Wang CC, Wang ZQ (2018) Old method not old-fashioned: parallelism between wing venation and wing-pad tracheation of cockroaches and a revision of terminology. Zoomorphology 137: 519–533.
  • Luo A, Qiao H, Zhang Y, Shi W, Ho SYW, Xu W, Zhang A, Zhu C (2010) Performance of criteria for selecting evolutionary models in phylogenetics: a comprehensive study based on simulated datasets. BMC Evolutionary Biology 10: 242.
  • Ma J, Du C, Zhou C, Sheng Y, Fan Z, Yue B, Zhang X (2017) Complete mitochondrial genomes of two blattid cockroaches, Periplaneta australasiae and Neostylopyga rhombifolia, and phylogenetic relationships within the Blattaria. PLoS ONE 12(5): e0177162.
  • Magallón S, Hilu KW, Quandt D (2013) Land plant evolutionary timeline: Gene effects are secondary to fossil constraints in relaxed clock estimation of age and substitution rates. American Journal of Botany 100(3): 556–573.
  • Martínez-Delclòs X (1993) Blátidos (Insecta, Blattodea) del Cretácico Inferior de España. Familias Mesoblattinidae, Blattulidae y Poliphagidae. Boletín Geológico y Minero 104(5): 516–538.
  • Misof B, Liu SL, Meusemann K, Peters RS, Donath A, Mayer C, Frandsen PB, Ware J, Flouri T, Beutel RG, Niehuis O, Petersen M, Izquierdo-Carrasco F, Wappler T, Rust J, Aberer AJ, Aspöck U, Aspöck H, Bartel D, Blanke A, Berger S, Böhm A, Buckley TR, Calcott B, Chen JQ, Friedrich F, Fukui M, Fujita M, Greve C, Grobe P, Gu SC, Huang Y, Jermiin LS, Kawahara AY, Krogmann L, Kubiak M, Lanfear R, Letsch H, Li YY, Li ZY, Li JG, Lu HR, Machida R, Mashimo Y, Kapli P, McKenna DD, Meng GL, Nakagaki Y, Navarrete-Heredia JL, Ott M, Ou YX, Pass G, Podsiadlowski L, Pohl H, von Reumont BM, Schütte K, Sekiya K, Shimizu S, Slipinski A, Stamatakis A, Song WH, Su X, Szucsich NU, Tan MH, Tan XM, Tang M, Tang JB, Timelthaler G, Tomizuka S, Trautwein M, Tong XL, Uchifune T, Walzl MG, Wiegmann BM, Wilbrandt J, Wipfler B, Wong TKF, Wu Q, Wu GX, Xie YL, Yang SZ, Yang Q, Yeates DK, Yoshizawa K, Zhang Q, Zhang R, Zhang WW, Zhang YH, Zhao J, Zhou CR, Zhou LL, Ziesmann T, Zou SJ, Li YR, Xu X, Zhang Y, Yang HM, Wang J, Wang J, Kjer KM, Zhou X (2014) Phylogenomics resolves the timing and pattern of insect evolution. Science 346: 763–767.
  • Moran RJ, Morgan CC, O’Connell MJ (2015) A guide to phylogenetic reconstruction using heterogeneous models—a case study from the root of the placental mammal tree. Computation 3: 177–196.
  • Papadopoulou A, Anastasiou I, Vogler AP (2010) Revisiting the insect mitochondrial molecular clock: the mid-Aegean trench calibration. Molecular Biology and Evolution 27(7): 1659–1672.
  • Parham JF, Donoghue PCJ, Bell CJ, Calway TJ, Head JJ, Holroyd PA, Inoue JG, Irmis RB, Joyce WG, Ksepka DT, Patané JSL, Smith ND, Tarver JE, van Tuinen M, Yang Z, Angielczyk KD, Greenwood JM, Hipsley CA, Jacobs L, Makovicky PJ, Müller J, Smith KT, Theodor JM, Warnock RCM, Benton MJ (2011) Best practices for justifying fossil calibrations. Systematic Biology 61: 346–359.
  • Qiu L (2017) Taxonomic revision of the family Corydiidae from China (Blattodea: Corydioidea). M.S. thesis, Southwest University, Chongqing, China.
  • Qiu L, Che YL, Wang ZQ (2017) Contribution to the cockroach genus Ctenoneura Hanitsch, 1925 (Blattodea: Corydioidea: Corydiidae) with descriptions of seven new species from China. Zootaxa 4237: 265–299.
  • Qiu L, Wang ZQ, Che YL (2019a) New and little known Latindiinae (Blattodea, Corydiidae) from China, with discussion of the Asian genera and species. ZooKeys 867: 23–44.
  • Qiu L, Yang ZB, Wang ZQ, Che YL (2019b) Notes on some corydiid species from China, with the description of a new genus (Blattodea: Corydioidea: Corydiidae). Annales de la Société entomologique de France (N.S. ) 55(3): 261–273.
  • Qiu L, Liu YC, Wang ZQ, Che YL (2020a) The first blattid cockroach (Dictyoptera: Blattodea) in Cretaceous amber and the reconsideration of purported Blattidae. Cretaceous Research 109: 104359.
  • Qiu L, Wang ZQ, Che YL (2020b) Discovery of the second Asian Holocompsa species from China, and supplemental description of the male of H. debilis (Blattodea: Corydiidae: Euthyrrhaphinae). Annales de la Société entomologique de France (N.S. ) 56(6): 481–487.
  • Qiu ZW, Che YL, Zheng YH, Wang ZQ (2017) The cockroaches of Balta Tepper from China, with the description of four new species (Blattodea, Ectobiidae, Pseudophyllodromiinae). ZooKeys 714: 13–32.
  • Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA (2018) Posterior summarisation in Bayesian phylogenetics using Tracer 1.7. Systematic Biology 67(5): 901–904.
  • Rehn JWH (1951) Classification of the Blattaria as indicated by their wings (Orthoptera). Memoirs of the American Entomological Society 14: 1–134.
  • Ronquist F, Teslenko M, Der Mark PV, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539–542.
  • Roth LM (1968b) Oviposition behavior and water changes in the oöthecae of Lophoblatta brevis (Blattaria: Blattellidae: Plectopterinae). Psyche 75: 99–106.
  • Roth LM (1971) Additions to the oöthecae, uricose glands, ovarioles, and tergal glands of Blattaria. Annals of the Entomological Society of America 64(1): 127–141.
  • Roth LM (1982) Ovoviviparity in the blattellid cockroach, Symploce bimaculata (Gerstaecker) (Dictyoptera: Blattaria: Blattellidae). Proceedings of the Entomological Society of Washington 84(2): 277–280.
  • Roth LM (1984) Stayella, a new genus of ovoviviparous cockroaches from Africa (Dictyoptera: Blattariae, Blattellidae). Entomologica Scandinavica 15: 113–139.
  • Roth LM (1989) Sliferia, a new ovoviviparous cockroach genus (Blattellidae) and the evolution of ovoviviparity in Blattaria (Dictyoptera). Proceedings of the Entomological Society of Washington 91(3): 441–451.
  • Roth LM (1995) Pseudoanaplectinia yumotoi, a new ovoviviparous myrmecophilous cockroach genus and species from Sarawak (Blattaria: Blattellidae; Blattellinae). Psyche 102: 79–87.
  • Sauquet H, Ho SYW, Gandolfo MA, Jordan GJ, Wilf P, Cantrill DJ, Bayly MJ, Bromham L, Brown GK, Carpenter RJ, Lee DM, Murphy DJ, Kale Sniderman JM, Udovicic F (2012) Testing the impact of calibration on molecular divergence times using a fossil-rich group: the case of Nothofagus (Fagales). Systematic Biology 61(2): 289–313.
  • Smith PE, Evensen NM, York D, Chang M, Jin F, Li J, Cumbaa S, Russell D (1995) Dates and rates in ancient lakes: 40Ar–39Ar evidence for an Early Cretaceous age for the Jehol Group, northeast China. Canadian Journal of Earth Sciences 32: 1426–1431.
  • Stocsits RR, Letsch H, Hertel J, Misof B, Stadler PF (2009) Accurate and efficient reconstruction of deep phylogenies from structured RNAs. Nucleic Acids Research 37(18): 6184–6193.
  • Swisher CC III, Wang Y, Wang X, Xu X, Wang Y (1999) Cretaceous age for the feathered dinosaurs of Liaoning, China. Nature 400(6739): 58–61.
  • Talavera G, Vila R (2011) What is the phylogenetic signal limit from mitogenomes? The reconciliation between mitochondrial and nuclear data in the Insecta class phylogeny. BMC Evolutionary Biology 11: 315.
  • Tian X, Ma G, Cui Y, Dong P, Zhu Y, Gao X (2015) The complete mitochondrial genomes of Opisthoplatia orientalis and Blaptica dubia (Blattodea: Blaberidae). Mitochondrial DNA Part A 28(1): 139–140. [bound in 2017]
  • Trümper S, Schneider JW, Nemyrovska T, Korn D, Linnemann U, Ren D, Béthoux O (2020) Age and depositional environment of the Xiaheyan insect fauna, embedded in marine black shales (Early Pennsylvanian, China). Palaeogeography, Palaeoclimatology, Palaeoecology 538: 109444.
  • Vršanský P, Cifuentes-Ruiz P, Vidlička Ľ, Čiampor F, Vega F (2011a) Afro-Asian cockroach from Chiapas amber and the lost Tertiary American entomofauna. Geologica Carpathica 62(5): 463–475.
  • Vršanský P, Vidlička Ľ, Čiampor Jr F, Marsh F (2011b) Derived, still living cockroach genus Cariblattoides (Blattida: Blattellidae) from the Eocene sediments of Green River in Colorado, USA. Insect Science 19(2): 143–152. [bound in 2012]
  • Vršanský P, Oružinský R, Barna P, Vidlička Ľ, Labandeira CC (2014) Native Ectobius (Blattaria: Ectobiidae) from the Early Eocene Green River Formation of Colorado and its reintroduction to North America 49 million years later. Annals of the Entomological Society of America 107: 28–36.
  • Wang T, Yu P, Ma Y, Cheng H, Zhang J (2014) The complete mitochondrial genome of L. albella (Mantodea: Iridopterygidae). Mitochondrial DNA Part A 27: 465–466. [bound in 2016]
  • Wang ZQ, Shi Y, Qiu ZW, Che YL, Lo N (2017) Reconstructing the phylogeny of Blattodea: robust support for interfamilial relationships and major clades. Scientific Reports 7: 3903.
  • Wiens JJ, Moen DS (2008) Missing data and the accuracy of Bayesian phylogenetics. Journal of Systematics and Evolution 46(3): 307–314.
  • Wipfler B, Letsch H, Frandsen PB, Kapli P, Mayer C, Bartel D, Buckley TR, Donath A, Edgerly-Rooks JS, Fujita M, Liu SL, Machida R, Mashimo Y, Misof B, Niehuis O, Peters RS, Petersen M, Podsiadlowski L, Schütte K, Shimizu S, Uchifune T, Wilbrandt J, Yan E, Zhou X, Simon S (2019) Evolutionary history of Polyneoptera and its implications for our understanding of early winged insects. Proceedings of the National Academy of Sciences 116: 3024–3029.
  • Xia X (2018) DAMBE7: New and improved tools for data analysis in molecular biology and evolution. Molecular Biology and Evolution 35: 1550–1552.
  • Yamauchi MM, Miya MU, Nishida M (2004) Use of a PCR-based approach for sequencing whole mitochondrial genomes of insects: two examples (cockroach and dragonfly) based on the method developed for decapod crustaceans. Insect Molecular Biology 13(4): 435–442.
  • Yang W, Li S, Jiang B (2007) New evidence for Cretaceous age of the feathered dinosaurs of Liaoning: zircon U-Pb SHRIMP dating of the Yixian Formation in Sihetun, northeast China. Cretaceous Research 28(2): 177–182.
  • Yang Z (2014) Molecular Evolution: A Statistical Approach. Oxford University Press, Oxford, 492 pp.
  • Ye F, Lan XE, Zhu WB, You P (2016) Mitochondrial genomes of praying mantises (Dictyoptera, Mantodea): rearrangement, duplication, and reassignment of tRNA genes. Scientific Reports 6: 25634.
  • Zhang YY, Xuan WJ, Zhao JL, Zhu CD, Jiang GF (2009) The complete mitochondrial genome of the cockroach Eupolyphaga sinensis (Blattaria: Polyphagidae) and the phylogenetic relationships within the Dictyoptera. Molecular Biology Reports 37(7): 3509–3516. [bound in 2010]
  • Zhang ZJ, Schneider JW, Hong YC (2012) The most ancient roach (Blattodea): a new genus and species from the earliest Late Carboniferous (Namurian) of China, with a discussion of the phylomorphogeny of early blattids. Journal of Systematic Palaeontology 11: 27–40. [bound in 2013]
  • Zhang L-P, Yu D-N, Storey KB, Cheng H-Y, Zhang J-Y (2018) Higher tRNA gene duplication in mitogenomes of praying mantises (Dictyoptera, Mantodea) and the phylogeny within Mantodea. International Journal of Biological Macromolecules 111: 787–795.
  • Zhang D, Gao F, Jakovlić I, Zou H, Zhang J, Li WX, Wang GT (2020) PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Molecular Ecology Resources 20(1): 348–355.

Supplementary materials

Supplementary material 1 

Initial pool of 169 mitochondrial genomes

Xin-Ran Li

Data type: document/list (pdf. file)

Explanation note: Initial pool of 169 mitochondrial genomes found in GenBank.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (400.50 kb)
Supplementary material 2 

Table S1

Xin-Ran Li

Data type: Table (pdf. file)

Explanation note: Metadata of the 95 selected mitochondrial genomes.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (109.96 kb)
Supplementary material 3 

Table S2

Xin-Ran Li

Data type: Table (pdf. file)

Explanation note: Sequence correction record.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (102.79 kb)
Supplementary material 4 

Figure S1

Xin-Ran Li

Data type: statistic plot (jpeg. image)

Explanation note: Pairwise similarity scores calculated by ALIGROOVE.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (4.64 MB)
Supplementary material 5 

Figure S2

Xin-Ran Li

Data type: statistic plot (tiff. image)

Explanation note: Substitution saturation plots per codon position per gene.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (950.72 kb)
Supplementary material 6 

Figure S3

Xin-Ran Li

Data type: phylogram (tiff. image)

Explanation note: Bayesian phylogeny of 13 protein-coding genes of 95 species, excluding the third base of codon.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (585.89 kb)
Supplementary material 7 

Figue S4

Xin-Ran Li

Data type: phylogram (tiff. image)

Explanation note: Bayesian phylogeny of 10 protein-coding genes of 95 species, excluding the third base of codon.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (606.64 kb)
Supplementary material 8 

Figure S5

Xin-Ran Li

Data type: phylogram (tiff. image)

Explanation note: Bayesian phylogeny of 10 protein-coding genes of 87 species (Good-species analysis), excluding the third base of codon.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (566.29 kb)
Supplementary material 9 

Figure S6

Xin-Ran Li

Data type: phylogram (tiff. image)

Explanation note: Bayesian phylogeny of 10 protein-coding genes of 92 species (Short-species analysis), excluding the third base of codon.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (595.73 kb)
Supplementary material 10 

Figure S7

Xin-Ran Li

Data type: phylogram (tiff. image)

Explanation note: Bayesian phylogeny of 10 protein-coding genes of 85 species (Safe-species analysis), excluding the third base of codon.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (571.07 kb)
Supplementary material 11 

Figure S8

Xin-Ran Li

Data type: time tree (tiff. image)

Explanation note: Time tree estimated by MCMCTREE with three fossil calibrations (incl. Qilianiblatta namurensis).

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (716.34 kb)
Supplementary material 12 

Figure S9

Xin-Ran Li

Data type: time tree (tiff. image)

Explanation note: Time tree estimated by MCMCTREE with two fossil calibrations (excl. Qilianiblatta namurensis).

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (682.99 kb)
Supplementary material 13 

Figure S10

Xin-Ran Li

Data type: time tree (tiff. image)

Explanation note: Time tree estimated by MCMCTREE with only one fossil calibration (Valditermes brenanae).

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (2.98 MB)
login to comment