Dating in the Dark: Elevated Substitution Rates in Cave Cockroaches (Blattodea: Nocticolidae) Have Negative Impacts on Molecular Date Estimates.

Rates of nucleotide substitution vary substantially across the Tree of Life, with potentially confounding effects on phylogenetic and evolutionary analyses. A large acceleration in mitochondrial substitution rate occurs in the cockroach family Nocticolidae, which predominantly inhabit subterranean environments. To evaluate the impacts of this among-lineage rate heterogeneity on estimates of phylogenetic relationships and evolutionary timescales, we analyzed nuclear ultraconserved elements (UCEs) and mitochondrial genomes from nocticolids and other cockroaches. Substitution rates were substantially elevated in nocticolid lineages compared with other cockroaches, especially in mitochondrial protein-coding genes. This disparity in evolutionary rates is likely to have led to different evolutionary relationships being supported by phylogenetic analyses of mitochondrial genomes and UCE loci. Furthermore, Bayesian dating analyses using relaxed-clock models inferred much deeper divergence times compared with a flexible local clock. Our phylogenetic analysis of UCEs, which is the first genome-scale study to include all 13 major cockroach families, unites Corydiidae and Nocticolidae and places Anaplectidae as the sister lineage to the rest of Blattoidea. We uncover an extraordinary level of genetic divergence in Nocticolidae, including two highly distinct clades that separated ~115 million years ago despite both containing representatives of the genus Nocticola. The results of our study highlight the potential impacts of high among-lineage rate variation on estimates of phylogenetic relationships and evolutionary timescales.

Phylogenomic analyses of Blattodea combining traditional methods, incremental tree-building, and quality-aware support.

Despite the many advances of the genomic era, there is a persistent problem in assessing the uncertainty of phylogenomic hypotheses. We see this in the recent history of phylogenetics for cockroaches and termites (Blattodea), where huge advances have been made, but there are still major inconsistencies between studies. To address this, we present a phylogenetic analysis of Blattodea that emphasizes identification and quantification of uncertainty. We analyze 1183 gene domains using three methods (multi-species coalescent inference, concatenation, and a supermatrix-supertree hybrid approach) and assess support for controversial relationships while considering data quality. The hybrid approach-here dubbed "tiered phylogenetic inference"-incorporates information about data quality into an incremental tree building framework. Leveraging this method, we are able to identify cases of low or misleading support that would not be possible otherwise, and explore them more thoroughly with follow-up tests. In particular, quality annotations pointed towards nodes with high bootstrap support that later turned out to have large ambiguities, sometimes resulting from low-quality data. We also clarify issues related to some recalcitrant nodes: Anaplectidae's placement lacks unbiased signal, Ectobiidae s.s. and Anaplectoideini need greater taxon sampling, the deepest relationships among most Blaberidae lack signal. As a result, several previous phylogenetic uncertainties are now closer to being resolved (e.g., African and Malagasy "Rhabdoblatta" spp. are the sister to all other Blaberidae, and Oxyhaloinae is sister to the remaining Blaberidae). Overall, we argue for more approaches to quantifying support that take data quality into account to uncover the nature of recalcitrant nodes.

Molecular analyses of the Kalotermes dispar-complex (Blattodea: Kalotermitidae) from the Canary Islands reveal cryptic intraspecific divergence and a connection to a lone Nearctic congener.

The Canary Islands is a Macaronesian volcanic archipelago with a depauperate community of three species of Kalotermitidae, including Kalotermes dispar. A total of 54 Kalotermes colonies were collected from Gran Canaria, Tenerife, La Gomera, La Palma, and El Hierro islands. Soldiers and imagos were morphologically examined and sequenced for four mitochondrial markers. Although morphological differences could not be detected, phylogenetic analysis of both cox1/tRNA/cox2 and rrnL markers revealed two distinct clades of K. dispar, suggesting cryptic diversity. The diversification within the Canary Kalotermes lineage most likely occurred around 7.5 Mya, while the divergence within the two clades was reconstructed at about 3.6 Mya and 1.9 Mya. Kalotermes approximatus from the southeastern Nearctic constitutes a sister to the Canary Kalotermes, while the Palearctic K. flavicollis, K. italicus, and K. phoenicae form a separate clade. It is hypothesized that a faunal exchange of Kalotermes from the Nearctic to the Canary Islands occurred via transoceanic rafting during the mid-Miocene.

Two New Species of the Genus Eucorydia (Blattodea: Corydiidae) from the Nansei Islands in Southwest Japan.

Two new species of the cockroach genus Eucorydia Hebard, 1929 from the Nansei Islands in Southwest Japan were compared to two closely related congeners, Eucorydia yasumatsui Asahina, 1971 and Eucorydia dasytoides (Walker, 1868). Eucorydia donanensis Yanagisawa, Sakamaki, and Shimano sp. nov. from Yonaguni-jima Island was characterized by an overall length of 12.5-14.5 mm in males. The dorsal side of the male abdomen was entirely dark purple and there was an obscure orange band running down the middle of the tegmen. Eucorydia tokaraensis Yanagisawa, Sakamaki, and Shimano sp. nov. was characterized by an overall length of 12.0-13.0 mm in males and a distinct orange band running down the middle of the tegmen. Eucorydia yasumatsui, E. donanensis, E. tokaraensis and the zonata population of E. dasytoides were divided into four lineages in a maximum-likelihood tree generated from a dataset concatenated from five (two nuclear, 28S rRNA, histone H3, and three mitochondrial, COII, 12S rRNA, 16S rRNA) genes. We recognized the three Japanese lineages E. yasumatsui, E. donanensis, and E. tokaraensis as distinct species, which were also supported by the pairwise genetic distances (5.4-7.8%, K2P) of the COI sequences. Morphometric analysis was performed on the genitalia. A principal component analysis plot revealed that the sizes of the genitalia in the three Japanese species were similar to each other and smaller than that of the zonata population of E. dasytoides. The analysis also revealed that the three Japanese species were distinguished from each other by combinations of the sizes of L3 and L7 sclerites and the shape of R2 sclerite, with some overlapping exceptions.

The power of neuropeptide precursor sequences to reveal phylogenetic relationships in insects: A case study on Blattodea.

Recent state-of-the-art analyses in insect phylogeny have exclusively used very large datasets to elucidate higher-level phylogenies. We have tested an alternative and novel approach by evaluating the potential phylogenetic signals of identified and relatively short neuropeptide precursor sequences with highly conserved functional units. For that purpose, we examined available transcriptomes of 40 blattodean species for the translated amino acid sequences of 17 neuropeptide precursors. Recently proposed intra-ordinal relationships of Blattodea, based on the analysis of 2370 protein-coding nuclear single-copy genes (Evangelista et al., 2019), were corroborated with maximum support. The functionally different precursor units were analyzed separately for their phylogenetic information. Although the degree of information was different in the different sequence motifs, all precursor units contained phylogenetic informative data at the ordinal level, and their separate analysis did not reveal contradictory topologies. This study is the first comprehensive exploitation of complete neuropeptide precursor sequences of arthropods in such a context and demonstrates the applicability of these rather short but conserved sequences for an alternative, fast and simple analysis of phylogenetic relationships.

An integrative phylogenomic approach illuminates the evolutionary history of cockroaches and termites (Blattodea).

Phylogenetic relationships among subgroups of cockroaches and termites are still matters of debate. Their divergence times and major phenotypic transitions during evolution are also not yet settled. We addressed these points by combining the first nuclear phylogenomic study of termites and cockroaches with a thorough approach to divergence time analysis, identification of endosymbionts, and reconstruction of ancestral morphological traits and behaviour. Analyses of the phylogenetic relationships within Blattodea robustly confirm previously uncertain hypotheses such as the sister-group relationship between Blaberoidea and remaining Blattodea, and Lamproblatta being the closest relative to the social and wood-feeding Cryptocercus and termites. Consequently, we propose new names for various clades in Blattodea: Cryptocercus + termites = Tutricablattae; Lamproblattidae + Tutricablattae = Kittrickea; and Blattoidea + Corydioidea = Solumblattodea. Our inferred divergence times contradict previous studies by showing that most subgroups of Blattodea evolved in the Cretaceous, reducing the gap between molecular estimates of divergence times and the fossil record. On a phenotypic level, the blattodean ground-plan is for egg packages to be laid directly in a hole while other forms of oviposition, including ovovivipary and vivipary, arose later. Finally, other changes in egg care strategy may have allowed for the adaptation of nest building and other novelties.

Historical biogeography of the termite clade Rhinotermitinae (Blattodea: Isoptera).

Termites are the principal decomposers in tropical and subtropical ecosystems around the world. Time-calibrated molecular phylogenies show that some lineages of Neoisoptera diversified during the Oligocene and Miocene, and acquired their pantropical distribution through transoceanic dispersal events, probably by rafting in wood. In this paper, we intend to resolve the historical biogeography of one of the earliest branching lineages of Neoisoptera, the Rhinotermitinae. We used the mitochondrial genomes of 27 species of Rhinotermitinae to build two robust time-calibrated phylogenetic trees that we used to reconstruct the ancestral distribution of the group. Our analyses support the monophyly of Rhinotermitinae and all genera of Rhinotermitinae. Our molecular clock trees provided time estimations that diverged by up to 15.6 million years depending on whether or not 3rd codon positions were included. Rhinotermitinae arose 50.4-64.6 Ma (41.7-74.5 Ma 95% HPD). We detected four disjunctions among biogeographic realms, the earliest of which occurred 41.0-56.6 Ma (33.0-65.8 Ma 95% HPD), and the latest of which occurred 20.3-34.2 Ma (15.9-40.4 Ma 95% HPD). These results show that the Rhinotermitinae acquired their distribution through a combination of transoceanic dispersals and dispersals across land bridges.

The evolution of sociality in termites from cockroaches: A taxonomic and phylogenetic perspective.

Despite multiple studies and advances, sociality still puzzles evolutionary biologists in numerous ways, which might be partly addressed with the advent of sociogenomics. In insects, the majority of sociogenomic studies deal with Hymenoptera, one of the two groups that evolved eusociality with termites. But, to fully grasp the evolution of sociality, studies must obviously not restrict to eusocial lineages. Multiple kinds of social system transitions have been recorded and they all bring complementary insights. For instance, cockroaches, the closest relatives to termites, display a wide range of social interactions and evolved convergently subsocial behaviors (i.e., brood care). In this context, we emphasize the need for natural history, taxonomic, and phylogenetic studies. Natural history studies provide the foundations on which building hypotheses, whereas taxonomy provides the taxa to sample to test these hypotheses, and phylogenetics brings the historical framework necessary to test evolutionary scenarios of sociality evolution.

Topological support and data quality can only be assessed through multiple tests in reviewing Blattodea phylogeny.

Assessing support for molecular phylogenies is difficult because the data is heterogeneous in quality and overwhelming in quantity. Traditionally, node support values (bootstrap frequency, Bayesian posterior probability) are used to assess confidence in tree topologies. Other analyses to assess the quality of phylogenetic data (e.g. Lento plots, saturation plots, trait consistency) and the resulting phylogenetic trees (e.g. internode certainty, parameter permutation tests, topological tests) exist but are rarely applied. Here we argue that a single qualitative analysis is insufficient to assess support of a phylogenetic hypothesis and relate data quality to tree quality. We use six molecular markers to infer the phylogeny of Blattodea and apply various tests to assess relationship support, locus quality, and the relationship between the two. We use internode-certainty calculations in conjunction with bootstrap scores, alignment permutations, and an approximately unbiased (AU) test to assess if the molecular data unambiguously support the phylogenetic relationships found. Our results show higher support for the position of Lamproblattidae, high support for the termite phylogeny, and low support for the position of Anaplectidae, Corydioidea and phylogeny of Blaberoidea. We use Lento plots in conjunction with mutation-saturation plots, calculations of locus homoplasy to assess locus quality, identify long branch attraction, and decide if the tree's relationships are the result of data biases. We conclude that multiple tests and metrics need to be taken into account to assess tree support and data robustness.

Distribution of Karyotypes of the Cryptocercus punctulatus Species Complex (Blattodea: Cryptocercidae) in Great Smoky Mountains National Park.

During the period between 1999 and 2006, wood-feeding cockroaches in the Cryptocercus punctulatus Scudder species complex were collected throughout Great Smoky Mountains National Park, USA. The chromosome numbers of insects from 59 sites were determined, and phylogenetic analyses were performed based on mitochondrial COII and nuclear ITS2 DNA. The distribution of the three male karyotypes found in the park (2n = 37, 39, and 45) is mapped and discussed in relation to recent disturbances and glacial history. Clades of the three karyotype groups meet near the ridgeline separating North Carolina from Tennessee in the center of the park, suggesting that these may have originated from separate lower elevation refugia after the last glacial maximum. The timing of divergence and a significant correlation between elevation difference and genetic distance in two of the clades supports this hypothesis. The ecological role of the cockroaches in the park is discussed.

The Complete Mitogenome of the Wood-Feeding Cockroach Cryptocercus meridianus (Blattodea: Cryptocercidae) and Its Phylogenetic Relationship among Cockroach Families.

In this study, the complete mitochondrial genome of Cryptocercus meridianus was sequenced. The circular mitochondrial genome is 15,322 bp in size and contains 13 protein-coding genes, two ribosomal RNA genes (12S rRNA and 16S rRNA), 22 transfer RNA genes, and one D-loop region. We compare the mitogenome of C. meridianus with that of C. relictus and C. kyebangensis. The base composition of the whole genome was 45.20%, 9.74%, 16.06%, and 29.00% for A, G, C, and T, respectively; it shows a high AT content (74.2%), similar to the mitogenomes of C. relictus and C. kyebangensis. The protein-coding genes are initiated with typical mitochondrial start codons except for cox1 with TTG. The gene order of the C. meridianus mitogenome differs from the typical insect pattern for the translocation of tRNA-SerAGN, while the mitogenomes of the other two Cryptocercus species, C. relictus and C. kyebangensis, are consistent with the typical insect pattern. There are two very long non-coding intergenic regions lying on both sides of the rearranged gene tRNA-SerAGN. The phylogenetic relationships were constructed based on the nucleotide sequence of 13 protein-coding genes and two ribosomal RNA genes. The mitogenome of C. meridianus is the first representative of the order Blattodea that demonstrates rearrangement, and it will contribute to the further study of the phylogeny and evolution of the genus Cryptocercus and related taxa.

A global molecular phylogeny and timescale of evolution for Cryptocercus woodroaches.

Cryptocercus is a genus of sub-social wood-feeding cockroaches that represents the sister group to the eusocial termites. We generated mitochondrial (12S+16S rRNA, COII), nuclear (28S rRNA) and Blattbacterium endosymbiont (16S+23S rRNA) sequence data for 8 new Chinese species, and combined these with previously available data to undertake the most extensive analysis of phylogenetic relationships within the genus to date. As expected, phylogenetic relationships among Blattabacterium strains were found to be congruent with those of their hosts. Three major clades were found to exist in Asian populations, one representing taxa from the Hengduan mountains in Southwestern China, a second including taxa from Russia, Korea, Northeastern China, and Yunnan in the Hengduan Mountains, and a third including taxa from the Qinling Mountains and Daba Mountains in Central China. A molecular dating analysis using 7 termite fossils to calibrate the molecular clock indicated that the divergence of American and Asian Cryptocercus occurred 55.09Ma (41.55-72.28Ma 95% CI), and that the radiations of American and Asian taxa occurred 28.48Ma (20.83-37.95Ma 95% CI) and 20.97Ma (15.78-27.21Ma 95% CI) respectively. Reconstruction of ancestral geographic distributions using S-DIVA suggested Cryptocercus was originally distributed across both continents, as opposed to ancestral migration of Cryptocercus from one continent to the other. The last common ancestor of Asian Cryptocercus was inferred to have existed in Central China. An examination of male chromosome numbers in Asian Cryptocercus showed that diploid numbers vary from 2n=15 to 2n=41, and indicates the presence of eight new species. Our study represents the most comprehensive phylogenetic and biogeographic study yet performed for this important group of cockroaches.

Selective sweeps in Cryptocercus woodroach antifungal proteins.

We identified the antifungal gene termicin in three species of Cryptocercus woodroaches. Cryptocercus represents the closest living cockroach lineage of termites, which suggests that the antifungal role of termicin evolved prior to the divergence of termites from other cockroaches. An analysis of Cryptocercus termicin and two ß-1,3-glucanase genes (GNBP1 and GNBP2), which appear to work synergistically with termicin in termites, revealed evidence of selection in these proteins. We identified the signature of past selective sweeps within GNBP2 from Cryptocercus punctulatus and Cryptocercus wrighti. The signature of past selective sweeps was also found within termicin from Cryptocercus punctulatus and Cryptocercus darwini. Our analysis further suggests a phenotypically identical variant of GNBP2 was maintained within Cryptocercus punctulatus, Cryptocercus wrighti, and Cryptocercus darwini while synonymous sites diverged. Cryptocercus termicin and GNBP2 appear to have experienced similar selective pressure to that of their termite orthologues in Reticulitermes. This selective pressure may be a result of ubiquitous entomopathogenic fungal pathogens such as Metarhizium. This study further reveals the similarities between Cryptocercus woodroaches and termites.

Origin of origami cockroach reveals long-lasting (11 Ma) phenotype instability following viviparity.

Viviparity evolved in bacteria, plants, ˃141 vertebrate lineages (ichthyosaurs, lizards, fishes, mammals, and others), and in 11 of 44 insect orders. Live-birth cockroaches preserved with brood sac (3D recovered two times optically) included Diploptera vladimir, Diploptera savba, Diploptera gemini spp.n., D. sp.1-2, and Stegoblatta irmgardgroehni from Green River, Colorado; Quilchena, Republic; McAbee, Canada; and Baltic amber, Russia (49, 54, and 45 Ma). They evolved from rare and newly evolved Blaberidae; they radiated circumtropically, later expanded into SE Asia, and have now spread to Hawaii and the SE USA. Association of autapomorphic characters that allow for passive and active protections from parasitic insects (unique wing origami pleating identical with its egg case-attacking wasp) suggest a response to high parasitic loads. Synchronized with global reorganization of the biota, morphotype destabilization in roaches lasted approximately 11-22 Ma, including both the adaptation of novel characters and the reduction of others. Thus, while viviparity can be disadvantageous, in association with new Bauplans and/or behaviors, it can contribute to the evolution of taxa with viviparous representatives that are slightly selectively preferred.

Identifying possible sister groups of Cryptocercidae+Isoptera: a combined molecular and morphological phylogeny of Dictyoptera.

Termites (Isoptera) offer an alternative model for the development of eusociality which is not dependent on a high degree of relatedness as found between sisters in hymenopterans (bees, wasps, ants). Recent phylogenetic studies have established that termites belong within the cockroaches as sister to the subsocial Cryptocercidae. Cryptocercidae shares several important traits with termites, thus we need to understand the phylogenetic position of Cryptocercidae+Isoptera to determine how these traits evolved. However, placement of Cryptocercidae+Isoptera is still uncertain. We used both molecular (12S, 16S, COII, 18S, 28S, H3) and morphological characters to reconstruct the phylogeny of Dictyoptera. We included all previously suggested sister groups of Cryptocercidae+Isoptera as well as taxa which might represent additional major cockroach lineages. We used Bayes factors to test different sister groups for Cryptocercidae+Isoptera and assessed character support for the consensus tree based on morphological characters and COII amino acid data. We used the molecular data and fossil calibration to estimate divergence times. We found the most likely sister groups of Cryptocercidae+Isoptera to be Tryonicidae, Anaplecta or Tryonicidae+Anaplecta. Anaplecta has never previously been suggested as sister group or even close to Cryptocercidae+Isoptera, but was formerly placed in Blaberoidea as sister to the remaining taxa. Topological tests firmly supported our new placement of Anaplecta. We discuss the morphological characters (e.g. retractable genitalic hook) that have contributed to the previous placement of Anaplecta in Blaberoidea as well as the factors that might have contributed to a parallel development of genitalic features in Anaplecta and Blaberoidea. Cryptocercidae+Isoptera is placed in a clade with Tryonicidae, Anaplecta and possibly Lamproblattidae. Based on this, we suggest that wood-feeding, and the resultant need to conserve nitrogen, may have been an important factor in the development of termite eusociality. Nocticolidae was placed as sister group to Latindia+Paralatindia (both Corydiidae), this clade was in turn placed as sister group to the remaining Corydiidae. The Nocticolidae+Corydiidae clade is supported by both morphological and COII amino acid changes. Our divergence time estimates placed the split between Mantodea and Blattodea at 273mya (middle Permian) and the splits between the major blattodean lineages no later than 200mya (end of Triassic).

Two new species of Blaberidae (Blattaria) collected in the Santa Lúcia Station, Espírito Santo State, Brazil.

This contribution presents two new species belonging to the genera Audreia (Shelford, 1910) and Epilampra Burmeister, 1838, subfamily Epilamprinae (Blattaria, Blaberidae). The Blaberidae include large and medium-bodied species with a high adaptive capacity. The new taxa were collected in Espírito Santo State and evaluated based on the analysis of subgenital and supranal plates and genital parts, by means of standard dissection techniques, by comparison with specimens of the corresponding genera deposited in the collection of the Museu Nacional and with published descriptions.

Cockroaches (Blattaria) of Ecuador-checklist and history of research.

Cockroaches are an understudied group and the total number of described taxa increases every year. The last checklist of Ecuador species was published in 1926. The main aim of this study was to complete a new checklist of cockroach species recorded in Ecuador supplemented with a research history of cockroaches (Blattaria) on the territory of continental Ecuador. In addition, the checklist contains comments on Ecuadorian faunistic records, including the Galápagos Islands. A total of 114 species (105 in continental Ecuador and 18 in Galápagos Islands) belonging to 6 families and 44 genera are listed. Forty species (38.1 %) occur solely in continental Ecuador and five (27.8 %) are endemic on Galápagos Islands. The results indicate that further research on the cockroach fauna of Ecuador as well as determination of museum collections from this territory is needed.

A new species of Dendroblatta Rehn, 1916 from northern Brazil (Blattaria: Ectobiidae) collected in wasp nests.

Currently sixteen species of Dendroblatta Rehn, 1916 are known, twelve of them occurring in Brazil. In this paper, one new species, D. spiculata sp. nov., is described from material collected from the Acre and Amazonas states in northern Brazil, in wasp nests. The new species differs from all other members of the genus by the male genitalia pieces. Illustrations of the genital pieces are presented. In Brazil, the genus is now known from the states of Acre, Amazonas, Bahia, Mato Grosso, Minas Gerais, Pará, Rio de Janeiro, and Rondônia.

One new genus of cockroach in the Neotropical subfamily Nyctiborinae (Dictyoptera: Blattodea: Ectobiidae).

The genus Nyctantonina gen. nov. and its two species Nyctantonina breviclasma sp. nov. and Nyctantonina pteromacrotata sp. nov. are described and illustrated. Dichotomous key to identify the species of genus is given.