First Report of the Lace Bug Neoplerochila Paliatseasi (Rodrigues, 1981) (Hemiptera: Tingidae) Infesting Cultivated Olive Trees

Zootaxa 4722 (5): 443–462 ISSN 1175-5326 (print edition) https://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2020 Magnolia Press ISSN 1175-5334 (online edition) https://doi.org/10.11646/zootaxa.4722.5.3 http://zoobank.org/urn:lsid:zoobank.org:pub:0183A47A-AA1E-4AAF-8802-54CB9CCDE58C

First report of the lace bug Neoplerochila paliatseasi (Rodrigues, 1981) (Hemiptera: Tingidae) infesting cultivated olive trees in South Africa, and its complete mitochondrial sequence

JETHRO LANGLEY1, MORGAN CORNWALL1, CHANTÉ POWELL1, CARLO COSTA2, ELLEUNORAH ALLSOPP3, SIMON VAN NOORT4,5, ERIC GUILBERT6 & BARBARA VAN ASCH1 1Department of Genetics, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa. 2Crop Development Division, Infruitec Campus, Agricultural Research Council, Private Bag X5013, Stellenbosch 7600, South Africa. 3Agricultural Research Council, Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch 7599, South Africa. 4Research and Exhibitions Department, Iziko South African Museum, P.O. Box 61, Cape Town 8000, South Africa. 5Department of Biological Sciences, University of Cape Town, Private Bag, Rondebosch 7701, South Africa. 6Département Adaptation du Vivant, Muséum National d’Histoire Naturelle, UMR 7179, CP50, 45 Rue Buffon, 75005 Paris, France. Barbara van Asch - [email protected]

ABSTRACT

Olive lace bugs are small phytophagous Hemipteran insects known to cause agricultural losses in olive production in South Africa. Plerochila australis (Distant, 1904) has been reported as the species responsible for damage to olive trees; however, the diversity of olive lace bug species in the region has lacked attention. Adult olive lace bugs were collected incidentally from wild and cultivated olive trees in the Western Cape Province, and identified as P. australis and Neoplerochila paliatseasi (Rodrigues, 1981). The complete mitochondrial genome of a representative specimen of N. paliatseasi was sequenced, and used for comparative mitogenomics and phylogenetic reconstruction within the family. Furthermore, the value of DNA barcodes for species identification in Tingidae was assessed using genetic clustering and estimates of genetic divergence. The patterns of genetic clustering and genetic divergence of COI sequences supported the morphological identification of N. paliatseasi, and the utility of DNA barcoding methods in Tingidae. The complete mitogenome sequence had the typical Metazoan gene content and order, including 13 PCGs, 22 tRNAs, two rRNAs, and an AT-rich non-coding region. A+T content was high, as commonly found in Tingidae. The phylogenetic reconstruction recovered Agramma hupehanum (Drake & Maa 1954) as basal to Tingini, and as a sister species to N. paliatseasi. Stephanitis Stål 1873 and Corythucha Stål 1873 were monophyletic, but Metasalis populi (Takeya 1932) was not recovered as sister to Tingis cardui (Linnaeus 1746), as expected. The mitochondrial phylogeny of the family Tingidae has been recovered inconsistently across different studies, possibly due to sequence heterogeneity and high mutation rates. Species diversity of olive lace bugs in South Africa was previously underestimated. The presence of P. australis was confirmed in both wild and cultivated olives, and N. paliatseasi is reported in cultivated olives for the first time. These results warrant further investigation on the diversity and distribution of olive lace bugs in the Western Cape to inform pest control strategies.

KEYWORDS: DNA barcoding, Olea europaea subsp. europaea, insect pest, lace bug, mitogenome, phylogeny

INTRODUCTION

The entomological fauna associated with wild and cultivated olive trees in South Africa is rich and specialized, including two olive fruit fly species, a variety of parasitoid and seed wasps, olive beetles, and olive lace bugs (Mkize, Hoelmer and Villet, 2008; Powell et al., 2019; Teixeira da Costa et al., 2019). This particular assemblage has most probably evolved in sub-Saharan Africa over time, along with the native African wild olive tree [Olea europaea subsp. cuspidata (Wall. ex G. Don) Cif.], as cultivated olive trees of European origin (Olea europaea subsp. europaea L.) were absent from the region until the colonial period. Due to its Mediterranean-like climate, South African commercial olive production is mostly concentrated in the Western Cape province. Olive orchards occupy approximately 3,000 ha of agricultural land interspersed with other crops and residual native Afromontane forest where

Accepted by K. Menard: 5 Dec. 2019; published: 16 Jan. 2020 443 the wild olive tree is common. Due to the similarity between the African wild olive and the cultivated olive, some insects associated with olives have been found on both plant species (Powell et al., 2019). Lace bugs (Hemiptera: Tingidae) are generally phytophagous, sap-sucking insects of small size (2 to 10 mm) distributed worldwide. The family Tingidae is classified in the infraorder Cimicomorpha, and currently comprises approximately 2,500 species across 300 genera (ITIS, 2019). Lace bugs are usually host-specific insects that feed on the underside of leaves, which leads to the development of chlorotic pinprick spots. Progressive damage with detrimental consequences to plant vitality occurs as the spots necrotize. Heavy lace bug infestations cause early mortality of young shoots, which results in bushy growth (Addison, Addison and Barnes, 2015). As severe lace bug infestation may lead to total defoliation of the host, control measures are necessary when heavier infestations occur (Costa, 1998). Economically important Tingidae attacking agricultural and ornamental plants include the avocado lace bug (Pseudacysta perseae Haidemann 1908), the tea lace bug (Stephanitis chinensis Drake 1948), and the black lace bug (Amblystira machalana Drake 1948) which feeds on cassava, an important South American crop (Arias and Bellotti, 2003). Corythucha gossypii (Frabicius 1794) is a serious pest of beans and cotton (Miller and Nagamine, 2005), and Corythucha ciliata (Say 1832), commonly known as the sycamore bug, is a pest of ornamental trees of the genus Platanus spp. (Oszi, Ladányi and Hufnagel, 2006). Many other lace bug species feed on ornamental plants, such as Stephanitis pyrioides (Scott 1874) on Rhododendron spp. (Nair and Braman, 2012), Corythauma ayyari (Drake 1933) on Jasminium spp. (Haouas, Guilbert and Halima-Kamel, 2015), and Teleonemia scrupulosa Stål 1873 on Lantana camara (Guidoti, Montemayor and Guilbert, 2015). Lace bugs affecting cultivated olives in South Africa, more commonly referred to as “olive tingids” by local growers, have been reported to be Plerochila australis (Costa, 1998; Addison, Addison and Barnes, 2015). The species is endemic to sub-Saharan Africa, and its distribution most likely overlaps the natural distribution of wild olive trees, as it has been reported from South Africa to northern Ethiopia (Deckert and Gollner-Scheiding, 2006; Yirgu, Getachew and Belay, 2012). Presently, P. australis is the only species reported as using both wild and cultivated olive trees as hosts. However, olive lace bugs have been poorly studied in South Africa, and the presence of species other than P. australis was suspected, as morphologically distinct specimens were incidentally collected during a re- cent survey of Hymenoptera associated with wild and cultivated olives in the Western Cape (Powell et al., 2019). Species identification in insects has increasingly made use of integrated approaches that include morphological and molecular analyses (Foottit and Adler, 2009; Pires and Marinoni, 2010). Analyses of DNA sequences, particularly the standard COI barcoding region, using genetic clustering methods and measures of genetic distances have become popular in the assessment of the genetic homogeneity of insect groups suggestive of biological species (Kress et al., 2015). Mitochondrial gene regions have been employed for the reconstruction of phylogenetic relationships at all taxonomic scales, due to the maternal pattern of inheritance and effectively haploid nature of the organelle genome. Complete mitochondrial sequences represent rich sources of data that have increasingly been used for a wide range of population genetic analyses, comparative genomics, and phylogenetics across a wide range of insect groups (Cameron, 2013). In this regard, the family Tingidae is poorly represented, with only 14 mitogenomes available on Genbank, as of August 2019. The objectives of this study were 1) to identify an unusual olive bug species found feeding on cultivated olives in South Africa, using morphology and DNA-barcoding methods, 2) to generate the complete mitochondrial genome for that species, and 3) to investigate its phylogenetic position within the family Tingidae using the new and publicly available mitogenome sequences.

MATERIALS AND METHODS

Specimen collection, morphological identification, and DNA extraction Lace bugs feeding on wild and cultivated olive trees in the Western Cape province of South Africa were incidentally collected between November 2015 and March 2018 during field surveys that focused on recovering species of olive-associated wasps and flies (Powell et al., 2019) (Table 1). Adult specimens were killed in 100% ethanol, and stored at -20°C until morphological identification, imaging and/or DNA extraction were performed. Adult specimens preserved in ethanol were identified as Plerochila australis and Neoplerochila paliatseasi, according to the current taxonomic keys (Göllner-Scheiding, 2007). Representative adult specimens for N. paliatseasi were imaged

444 · Zootaxa 4722 (5) © 2020 Magnolia Press LANGLEY et al. and deposited in the entomology collection of the Iziko South African Museum in Cape Town, South Africa (SAMC; Curator: Simon van Noort) for future reference. Codens of institutional depositories of voucher specimens follow Evenhuis 2019 (Evenhuis 2019). Images were acquired at SAMC with a Leica LAS 4.9 imaging system, comprised of a Leica® Z16 microscope (using either a 2X or 5X objective) with a Leica DFC450 Camera and 0.63x video objective attached. The imaging process, using an automated Z-stepper, was managed using the Leica Application Suite V 4.9 software installed on a desktop computer. Diffused lighting was achieved using a Leica LED5000 HDI dome. Total DNA was extracted from representative adult specimens using a standard phenol-chloroform method (Sambrook and Russell, 2012).

Table 1. List of adult specimens representative of the olive lace bug Neoplerochila paliatseasi (Hemiptera: Tingi- dae) used for DNA analyses (barcoding and sequencing of complete mitochondrial genome), and photographic imaging and deposit in the entomology collection of the Iziko Museums of South Africa (Cape Town). Cultivated host - Olea europaea subsp. cuspidata. *Museum collection coden; **Specimen presented in this report as representative image.

Sample code Species Collection date Region GPS Host Use in this study

Neoplerochila -33.890831, NP6 paliatseasi 19-Mar-18 Brackenfell 18.700360 Cultivated HEM-A011647* Neoplerochila -33.890831, NP11 paliatseasi 19-Mar-18 Brackenfell 18.700360 Cultivated HEM-A011646* Neoplerochila -33.890831, NP29 paliatseasi 19-Mar-18 Brackenfell 18.700360 Cultivated HEM-A011645* Neoplerochila -33.890831, NP44 paliatseasi 19-Mar-18 Brackenfell 18.700360 Cultivated HEM-A011644** Neoplerochila -33.890831, Complete NP2 paliatseasi 19-Mar-18 Brackenfell 18.700360 Cultivated mitogenome Neoplerochila -33.890831, NP1 paliatseasi 19-Mar-18 Brackenfell 18.700360 Cultivated DNA barcode Neoplerochila -33.890831, NP21 paliatseasi 19-Mar-18 Brackenfell 18.700360 Cultivated DNA barcode Neoplerochila -33.936937, P02 paliatseasi 11-Nov-15 Stellenbosch 18.819821 Cultivated DNA barcode Neoplerochila -33.936937, P03 paliatseasi 11-Nov-15 Stellenbosch 18.819822 Cultivated DNA barcode Neoplerochila -33.936937, P06 paliatseasi 11-Nov-15 Stellenbosch 18.819823 Cultivated DNA barcode

DNA barcoding and intra- and interspecific genetic distances To assess the intraspecific genetic diversity of N. paliatseasi, six adult individuals representative of the species, based on morphological identification, were sequenced for the standard COI barcoding region (702 bp). Species- specific primers (NEO-BAR-F 5’-CGACTAATCACAAAGACATCGG-3’ and NEO-BAR-R 5’-CTTCGGGAT- GTCCAAAGAATC-3’) were designed based on the new complete mitochondrial sequence of N. paliatseasi to replace the universal arthropod primers HLO/LCO (Folmer et al., 1994), as these produced non-specific amplicons due to imperfect annealing. PCR amplifications were performed in a total volume of 5 µL containing 1x of QIAGEN Multiplex PCR Kit (QIAGEN), 0.2 µM of each primer, 0.5 µL of MilliQ H2O, and 1.0 µL of template DNA. The thermal cycling program was as follows: 15 min at 95°C; 35 cycles of 30 s at 94°C, 90 s at 54°C, 90 s at 72°C; and 10 min at 72°C. PCR products were sequenced in the forward direction using the BigDye Terminator v3.1 Cycle Se- quencing Kit (Applied Biosystems), at the Central Analytical Facilities of Stellenbosch University, South Africa. To assess intraspecific and interspecific genetic divergence among Tingidae species, including N. paliatseasi, all barcoding sequences taxonomically assigned to the family were downloaded from BOLD Systems v3 (http:// v3.boldsystems.org/, accessed in August 2019). The BOLD dataset initially comprised a total of 933 Tingidae sequences, and was subsequently filtered for a) sequences longer than 500 bp and overlapping the standard COI barcoding region, b) sequences identified to the species level, and c) species represented by a minimum of three

Mitogenome of an olive lace bug Zootaxa 4722 (5) © 2020 Magnolia Press · 445 sequences. The final COI dataset used for genetic clustering analyses and estimates of genetic distances among Tingidae included 218 sequences, representing 25 species distributed among 16 genera. Multiple sequence alignments were performed using the MAFFT algorithm (Katoh and Standley, 2013) imple- mented in Geneious Prime v2019.2 (https://www.geneious.com). Genetic clustering was assessed using a Neigh- bor-Joining (NJ) tree constructed in MEGA X (Kumar et al., 2018) under the Kimura 2-parameter (K2P) model (Kimura, 1980). Intra- and interspecific genetic distances were calculated as percentage of p-distances in MEGA X, under the K2P model. Statistical support for the NJ tree and standard errors in p-distances were calculated from 1,000 bootstrap replicates.

Sequencing, assembly, and annotation of the complete mitogenome of Neoplerochila paliatseasi One adult specimen representative of N. paliatseasi, based on morphological identification, was used for recovering the complete mitochondrial sequence. Total DNA was extracted from the whole specimen, and then sequenced using the Ion Torrent Proton™ sequencing platform (ThermoFisher Scientific, Waltham, MA, USA), at the Central Analytical Facilities of Stellenbosch University, South Africa. Sequence libraries were prepared using the NEXTflex™ DNA Sequencing Kit for Ion Platforms (PerkinElmer, Waltham, MA, USA), according to the BI00 Scientific v15.12 protocol. Libraries were pooled and sequenced using the Ion PI HiQ™ Sequencing Solutions Kit (Life Technologies, CA, USA). The NGS reads were mapped to Agramma hupehanum, as this species was found to be the most similar to N. paliatseasi among the Tingidae for which complete mitogenomes were available, based on COI sequences (data not shown). The mapped reads were assembled into a consensus sequence using Geneious Prime. The open reading frames for the 13 typical Metazoan mitochondrial protein-coding genes (PCGs) were identified with Geneious Prime, using the invertebrate mitochondrial genetic code. Putative tRNAs were identified using ARWEN software (Laslett and Canbäck, 2008), and rRNAs were annotated manually by comparison with other Tingidae. The non-coding region between the 12s rRNA gene and the I-Q-M tRNA cluster was annotated as the AT-rich region containing the control region.

Mitogenome analyses Nucleotide composition and compositional biases [AT-skew = (A-T)/(A+T) and CG-skew = (G-C)/(G+C)] were calculated using Geneious Prime. Relative synonymous codon usage was calculated using DnaSP6 (Rozas et al., 2017). Start and stop codons, and overlapping and intergenic spaces were counted manually. Repeated regions were identified using Tandem Repeats Finder v4.09 (https://tandem.bu.edu/trf/trf.html) (Benson, 1999).

Phylogenetic analyses within Tingidae The phylogenetic relationships within Tingidae were reconstructed including the new N. paliatseasi mitogenome reported here and all of the complete mitogenomes available for the family in Genbank (August 2019) (Table S1). Apolygus lucorum (NC_023083.1) and Adelphocoris fasciaticollis (NC_023796.1) (Hemiptera: Miridae) were used as outgroups for rooting the trees. PCGs were extracted and aligned separately using the MAFFT algorithm in Geneious Prime. Stop codons, gaps, and poorly aligned regions were removed manually, and well-aligned blocks of each gene were selected using GBlocks v0.91b (Castresana, 2000). Genes were concatenated to produce a single alignment, from which three sub-datasets were generated: 1) PCG123 - all codons positions; 2) PCG12 – excluding the third codon position; and 3) AA - amino acid sequence. PCG12 and AA were used for Bayesian inference (BI) analysis under the GTR + GAMMA + I substitution model selected using jModelTest2 (Darriba et al., 2012). The PCG123 alignment was partitioned with PartitionFinder2 v2.1.1 (Lanfear et al., 2016), using the greedy algorithm for scheme search and the Bayesian Information Criterion for scheme selection. BI was performed on MrBayes v3.2.6 under the GTR + GAMMA + I substitution model (with four GAMMA categories), using two simultaneous runs, with four heated chains of 10,000,000 generations, with a subsampling frequency of 1,000 generations, and a burn-in length of 2,500,000 (25%). The confidence values for tree topology were estimated as Bayesian posterior probabilities (BPP). Model selection, partitioning schemes, and BI were run on the CIPRES Portal v.3.3 (https:// www.phylo.org/) (Miller, Pfeiffer and Schwartz, 2010). The new COI sequences and the complete mitogenome generated in this work were deposited in Genbank under the accession numbers MN794060to MN794065.

Species identification

The Western Cape province, the most important olive producing region in sub-Saharan Africa, is located within the Cape Floristic Region of South Africa, a remarkable global biodiversity hotspot for endemic plant species (Born, Linder and Desmet, 2007). Despite the climatic similarity with the Mediterranean Basin and California, South Af- rican olive groves have a distinct assemblage of insect pests and their natural enemies, and are relatively unaffected by the olive fruit fly. However, native olive seed wasps, olive flea beetles and olive lace bugs are known to have injurious activity. Olive lace bugs in South Africa are usually presumed to be Plerochila australis, a species native to sub-Saharan Africa (Addison, Addison and Barnes, 2015). In the course of field surveys of wild and cultivated olives aimed at recovering olive-associated wasps, we incidentally collected lace bugs feeding on the leaves of the trees. Preliminary morphological inspection of adult specimens suggested the presence of two distinct groups, subsequently identified as P. australis and Neoplerochila paliatseasi, based on classic taxonomy. No further analyses were performed on P. australis, and N. paliatseasi is reported here for the first time feeding on cultivated olive trees, where it was exclusively found during these field surveys (Figure 1).

Figure 1. Representative adult specimen of the olive lace bug Neoplerochila paliatseasi (Hemiptera: Tingidae), reported here for the first time as a pest of cultivated olives in the Western Cape Province of South Africa. South Africa Iziko Museum coden - SAM-HEM-A011644. A. Dorsal; B. Lateral; C. Ventral. Scale bars: 1 mm.

Mitogenome of an olive lace bug Zootaxa 4722 (5) © 2020 Magnolia Press · 447 No sequence data was available for N. paliatseasi or the genus Neoplerochila in BOLD Systems v3 and Gen- bank. Therefore, the standard COI barcoding region was sequenced for six representative individuals, and patterns of genetic clustering and genetic diversity within the family were assessed, using other Tingidae sequences available on BOLD Systems v3. Genetic clustering and genetic distances were compatible with N. paliatseasi representing a homogeneous genetic group, in agreement with the morphological identification of the species. The NJ tree showed that N. paliatseasi, and all other 24 Tingidae species formed monophyletic clusters with high statistical support (99%) (Figure 2). The intraspecific genetic divergence of N. paliatseasi was low (p-distance = 0.20%), similar to the other Tingidae (average p-distance = 0.55%) (Table S2; Figure 3). The highest intraspecific p-distances were found in Tingis crispata (Herrich-Schaeffer 1838) (2.60%), Tingis cardui (1.79%), and Acalypta elegans Horváth 1906 (1.78%). The interspecific p-distance between all species pairs was lowest for the congeneric pair Corythucha pallipes Parshley 1918/Corythucha ciliata (8.04%), in contrast with the average for the other species in the genus Corythucha (14.41%) (Table S3). Average p-distances among congeneric species were similar to the average over all pairs (20.01%) in the case of Tingis (19.25%) and Acalypta (19.15%). The highest p-distance was between the pair N. paliatseasi/A. elegans (26.52%). Overall, the patterns of genetic clustering and genetic divergence within and among Tingidae species supported the utility of barcoding methods for DNA-based species identification in the family, as each species formed a monophyletic cluster, and intraspecific distances were lower than interspecific distances, in all cases. However, the correctness of the inferences drawn from this type of approach greatly depends on the taxonomic accuracy of the available data. An assessment of the different types of data available on BOLD Sys- tems (v3 and v4) for burrower bugs (Hemiptera: Heteroptera: Cydnidae) identified errors in taxonomy, relevance of the names of the taxa, and species misidentifications affecting 7.55% of the specimens with barcodes (Lis, Lis and Ziaja, 2016). Although this error rate is concerning and potentially present in other taxonomic groups, the sequence dataset available in BOLD Systems v3 at the time of these analyses (August 2019) did not suggest misidentifications or taxonomic ambiguities in Tingidae. Although COI sequences are known to perform poorly for the recovery of the order of deep nodes (e. g. above the generic level), the marker has shown value in genetic clustering analyses for the assessment of the hypothetical conspecificity of individuals (Collins and Cruickshank, 2013). Intraspecific divergences among Tingidae were lower than 5%, a value above which specimen misidentification or cryptic speciation in Hemiptera may have to be considered (Park et al., 2011). However, the 5% threshold should not be interpreted as an absolute and universal value, as substitution rates may vary significantly between different evolutionary lineages. Therefore, genetic distances should be examined in the context of empirical data generated for the taxonomic group under study (Collins and Cruickshank, 2013). Average interspecific p-distances among congeneric species of Tingidae varied between 8.04% (C. ciliata/C. pallipes) and 22.50% (Acalypta musci (Schrank 1781)/Acalypta nigrina (Fallen 1807)), and were comparable with the range of values found in other families of Hemiptera. In Heteroptera, a previous study showed that the maximum interspecific genetic distance among pairs of congeneric species was similar (24.80%). The same study also reported the sharing of haplotypes between some individuals belonging to two different genera (Rhinocapsus vanduzeei Uhler 1890 and Plagiognathus fuscipes Fieber 1858), thus evidencing potential limita- tions of COI barcoding for species discrimination in this group (Park et al., 2011). This example highlights the importance of integrative approaches for species identification that combine molecular and expert morphological analyses, particularly in taxa with poor sequence coverage.

The mitogenome of Neoplerochila paliatseasi

The Ion Torrent run generated 12,196,060 reads with an average size of 170 bp, and 76,051 reads were assembled to the reference sequence (A. hupehanum) into a final circular sequence with high coverage (838X). The complete mitochondrial sequence of N. paliatseasi was 15,348 bp long, similar to the average found in Tingidae (15,724 bp). The mitogenome had the typical Metazoan complement of 37 genes (13 PCGs, 22 tRNAs and two rRNAs), and a putative control region (AT-rich region) (Table 2; Figure 4). Twenty-three genes were located in the majority (J) strand, and 14 genes in the minority (N) strand. Mitochondrial gene architecture seems to be conserved in Tingidae, as it was identical among all species analyzed, and identical to the hypothetical ancestral organization for Arthropoda (Boore, 1999).

448 · Zootaxa 4722 (5) © 2020 Magnolia Press LANGLEY et al. Figure 2. Neighbor-Joining (K2P) tree of lace bugs (Hemiptera: Tingidae) based on an alignment of COI sequences (512 bp). The analysis included 218 sequences belonging to 25 species in 16 genera retrieved from BOLD, except for the newly reported Neoplerochila paliatseasi (in bold). Triangles represent collapsed groups of sequences belonging to the same species. Nodal statistical support was based on 1,000 replicates (only values > 85% are shown).

Mitogenome of an olive lace bug Zootaxa 4722 (5) © 2020 Magnolia Press · 449 Figure 3. Percentage of maximum intragroup p-distances (K2P) in 25 species of lace bugs (Hemiptera: Tingidae). The analysis was based on a 512 bp alignment of COI barcoding sequences (n = 218).

Figure 4. Linear map of the complete mitochondrial genome of the olive lace bug Neoplerochila paliatseasi (Hemiptera: Tingidae). The arrows represent the direction of the genes (right – majority strand; left – minority strand).

Intergenic spaces, overlaps and AT-rich region

The mitogenome of N. paliatseasi was highly compact, with 20 short gene overlaps mostly involving tRNAs, and a longer overlap (14 bp) between ATP6 and COIII. Intergenic regions were found at nine locations representing 39 nucleotides, the longest of which between tRNATyr and COI (12 bp). The largest non-coding region (854 bp) between the 12s rRNA and the I-Q-M tRNA cluster, where the control of replication and transcription is putatively located, was annotated as the AT-rich region. The length of the AT-rich region in most Tingidae was in the order of 1,000 bp but it was longer in S. chinensis (2,215 bp) and shorter in T. cardui (287 bp). Repeated elements in the control region of Tingidae seem to be common, but variable in size and structure. For example, Phatnoma laciniatum Fieber 1844 had two perfect repeats of a 280-bp (40.1% of the AT-rich region) in the 3’-end, whereas A. hupehanum had 6.5 perfect repeats of a 68-bp motif (30.0% of the AT-rich region) in the middle of the region. Neoplerochila paliatseasi had two repeats of a 156 bp long motif in the 3’-end of the AT-rich region, separated by 18 bp, and representing 36.5% of the region. Tandem repeats in the 3’-end of the control region of P. persae and C. ciliata have also been identified (Kocher et al., 2015). Conserved structures thought to be involved in the initiation of replication, e. g. the homopolymeric A and T stretches observed in some Diptera species (Teixeira da Costa et al., 2019), were not found in Tingidae.

Nucleotide composition and codon usage

The complete sequence had the high A+T content typical of insect mitogenomes, with values higher than 75.5% (complete sequence) in all genes except COI, COII, COIII, CYTB and ND1 (average = 70.8%) (Table 3). The A+T content of the AT-rich region was 76.6%, higher than the average for the complete sequence but lower than the combined tRNAs (77.4%), the two rRNAs (79.3%), and six of the PCGS (ATP8, ND2, ND3, ND4L, ND5, ND6; average 79.5%). A lower A+T-content in the AT-rich region than in other mitogenomic regions was also found in C. ciliata (Yang, Yu and Du, 2013). The complete sequence had a positive AT-skew and a negative GC-skew, a trend that was apparent in most genes, except for a negative AT-skew in COI, ND6 and CYTB. The nucleotide bias towards A and T was reflected in the codon usage, with the AT-rich codons (UUU, UUA, AUU, AUA, UAU, AAU and AAA) representing 42.8% of all codons, and relative synonymous codon usage (RSCU) higher than 1.0 among all synonymous

450 · Zootaxa 4722 (5) © 2020 Magnolia Press LANGLEY et al. Table 2. Gene composition and order of the complete mitochondrial genome of the olive lace bug Neoplerochila paliatseasi (Hemiptera: Tingidae). N – majority strand; J – minority strand; IGN – number of intergenic nucleotides (negative values indicate overlapping between genes). Gene/region Code Coordinates Strand Size (bp) Anticodon Start Stop IGN COI - 1-1536 J 1536 - ATG TAA - tRNALeu2 L2 1538-1605 J 68 TAA - - 1 COII - 1605-2283 J 679 - ATT T-- -1 tRNALys K 2285-2358 J 74 CTT - - 1 tRNAAsp D 2361-2430 J 70 GTC - - 2 ATP8 - 2430-2585 J 156 - ATC TAA -1 ATP6 - 2579-3250 J 672 - ATG TAA -7 COIII - 3237-4025 J 789 - ATG TAA -14 tRNAGly G 4028-4094 J 67 TCC - - 2 ND3 - 4094-4445 J 352 - ATA T-- -1 tRNAAla A 4448-4508 J 61 TGC - - 2 tRNAArg R 4517-4582 J 66 TCG - - 8 tRNAAsn N 4580-4647 J 68 GTT - - -3 tRNASer1 S1 4643-4715 J 73 GCT - - -5 tRNAGlu E 4719-4784 J 66 TTC - - 3 tRNAPhe F 4782-4849 N 68 GAA - - -3 ND5 - 4849-6523 N 1675 - ATG T-- -1 tRNAHis H 6524-6587 N 64 GTG - - 0 ND4 - 6594-7919 N 1326 - ATG TAA 6 ND4L - 7913-8206 N 294 - ATA TAA -7 tRNAThr T 8199-8261 J 63 TGT - - -8 tRNAPro P 8258-8325 N 68 TGG - - -4 ND6 - 8326-8826 J 501 - ATT TAA 0 CYTB - 8826-9968 J 1143 - ATG TAG -1 tRNASer2 S2 9967-10035 J 69 TGA - - -2 ND1 - 10033-10980 N 948 - ATG TAA -3 tRNALeu1 L1 10981-11047 N 67 TAG - - 0 16s rRNA - 11048-12274 N 1227 - - - 0 tRNAVal - 12276-12349 N 74 TAC - - 1 12s rRNA - 12350-13125 N 776 - - - 0 AT-rich region - 13126-13979 - 854 - - - 0 tRNAIle I 13980-14042 J 63 GAT - - 0 tRNAGln Q 14040-14107 N 68 TTG - - -3 tRNAMet M 14106-14172 J 67 CAT - - -2 ND2 - 14172-15143 J 972 - ATT TAA -1 tRNATrp W 15145-15215 J 71 TCA - - 1 tRNACys C 15205-15267 N 63 GCA - - -11 tRNATyr Y 15280-15343 N 64 GTA - - 12

Mitogenome of an olive lace bug Zootaxa 4722 (5) © 2020 Magnolia Press · 451 Table 3. Nucleotide composition of the complete mitochondrial sequence of the olive lace bug Neoplerochila paliatseasi. AT-skew = (A - T)/(A + T); CG-skew = (G - C)/(G + C). Gene/region A% C% G% T% A+T% G+C% AT-skew GC-skew Size (bp) % (size) COI 32.7 17.8 15.1 34.2 66.9 32.9 -0.02 -0.08 1536 10.01 COII 36.5 17.5 11.2 34.8 71.3 28.7 0.02 -0.22 679 4.42 COIII 37.1 15.6 13.4 33.9 71.0 29.0 0.05 -0.08 789 5.14 CYTB 34.6 16.5 12.9 35.9 70.5 29.4 -0.02 -0.12 1143 7.45 ATP6 40.2 14.1 9.2 36.3 76.5 23.3 0.05 -0.21 672 4.38 ATP8 49.4 15.4 7.1 28.2 77.6 22.5 0.27 -0.37 156 1.02 ND1 50.1 15.3 10.3 24.4 74.5 25.6 0.34 -0.20 948 6.18 ND2 44.2 11.8 7.6 36.2 80.4 19.4 0.10 -0.22 972 6.33 ND3 42.6 12.8 8.8 35.8 78.4 21.6 0.09 -0.19 352 2.29 ND4 52.0 14.0 10.0 24.0 76.0 24.0 0.37 -0.17 1326 8.64 ND4L 52.0 12.6 6.1 29.3 81.3 18.7 0.28 -0.35 294 1.92 ND5 52.5 13.6 8.7 25.3 77.8 22.3 0.35 -0.22 1675 10.91 ND6 38.7 11.2 8.2 41.9 80.6 19.4 -0.04 -0.15 501 3.26 16s rRNA 43.6 13.1 7.7 35.5 79.1 20.8 0.10 -0.26 1227 7.99 12s rRNA 44.1 12.4 8.0 35.6 79.7 20.4 0.11 -0.22 776 5.06 PCGs 43.1 14.8 10.6 31.5 74.6 25.4 0.16 -0.17 11043 71.95 tRNAs 39.9 12.2 10.4 37.5 77.4 22.6 0.03 -0.08 1482 9.66 rRNAs 43.8 12.8 7.8 35.5 79.3 20.6 0.10 -0.24 2003 13.05 AT-rich region 38.2 12.1 10.4 38.4 76.6 22.5 0.00 -0.08 854 5.56 Complete mtDNA 42.6 14.2 10.2 32.9 75.5 24.4 0.13 -0.16 15348 100.00 codons (Table 4). A+T content of PCGs was particularly high at the third codon position (83.5% of all codons). The PCGs of Tingidae have been found to be more AT-rich than other families of Cimicomorpha (Anthocoridae, Miri- dae, Nabidae and Reduviidae) (Liu et al., 2018), and one of the highest in true bugs (Yang et al., 2018).

Protein-coding genes

The combined length of the 13 PCGs was 11,043 bp, with an A+T content of 74.6%, ranging from 66.9% in COI to 81.3% in ND4L. All start codons in N. paliatseasi were ATN: seven genes initiated with ATG (COI, COIII, CYTB, ATP6, ND1, ND4, and ND5), three genes with ATT (COII, ND2, and ND6), two genes with ATA (ND3 and ND4L), and one gene with ATC (ATP8) (Table S4). Most PCGs of N. paliatseasi terminated with the typical complete TAA stop codon (COI, COIII, ATP6, ATP8, ND1, ND2, ND4, ND4L, and ND6), but TAG (in CYTB) and T (in COII, ND3 and ND5) were also identified. All PCGs in the other mitogenomes of Tingidae here analysed started with ATN codons except for ND5 in Corythucha marmorata, which was initiated with GTG. The most frequent start codon across all species was ATG, which was fully conserved in COI and CYTB (Figure 5). The relative frequencies of stop codons across Tingidae were not calculated because the annotation of the terminator codon may vary according to the criteria used by the authors, who may or may not choose to minimize gene overlapping, as incomplete stop codons are thought to be completed by posttranscriptional adenylation (Salinas-Giegé, Giegé and Giegé, 2015).

Ribosomal RNAs and transfer RNAs

The large ribosomal RNA gene (16s rRNA; 1,227 bp) was located between tRNALeu1 and tRNAVal, and the small ribosomal RNA gene (12s rRNA; 776 bp) was located between tRNAVal and the AT-rich region. The typical complement of 22 tRNAs was identified with ARWEN software. tRNA sizes varied between 61 bp (tRNAAla) and 74 bp (tRNALys and tRNAVal). The typical clover-leaf structure was predicted for all tRNAs including tRNASer1, in which the DHU arm is usually reduced to a simple loop in many Metazoans (Bernt et al., 2013) (Figure 6). A complete DHU arm in tRNASer1 was also reported in Pseudacysta perseae (Kocher et al., 2015), and in C. ciliata, which additionally had a different anticodon (TTC) from other insects.

452 · Zootaxa 4722 (5) © 2020 Magnolia Press LANGLEY et al. Table 4. Codon usage in the mitochondrial genome of Neoplerochila paliatseasi. Amino acids are labelled according to the IUPAC-IUB single letter codes. N - the total number of occurrences in all protein coding genes, RSCU - relative synonymous codon usage. Scaled Chi-squared = 0.649.

Amino acid Codon N RSCU Amino acid Codon N RSCU F UUU 300 1.68 Y UAU 123 1.59 UUC 57 0.32 UAC 32 0.41 L UUA 325 3.74 H CAU 45 1.36 UUG 55 0.63 CAC 21 0.64 CUU 66 0.76 Q CAA 38 1.49 CUC 5 0.06 CAG 13 0.51 CUA 62 0.71 N AAU 155 1.48 CUG 9 0.1 AAC 55 0.52 I AUU 295 1.62 K AAA 95 1.58 AUC 70 0.38 AAG 25 0.42 M AUA 315 1.83 D GAU 44 1.57 AUG 30 0.17 GAC 12 0.43 V GUU 103 2.16 E GAA 83 1.8 GUC 4 0.08 GAG 9 0.2 GUA 73 1.53 C UGU 31 1.55 GUG 11 0.23 UGC 9 0.45 S UCU 112 2.57 W UGA 70 1.54 UCC 21 0.48 UGG 21 0.46 UCA 93 2.13 R CGU 19 1.52 UCG 8 0.18 CGC 1 0.08 P CCU 55 1.82 CGA 26 2.08 CCC 19 0.63 CGG 4 0.32 CCA 41 1.36 S AGU 28 0.64 CCG 6 0.2 AGC 2 0.05 T ACU 65 1.34 AGA 83 1.9 ACC 25 0.52 AGG 2 0.05 ACA 93 1.92 G GGU 80 1.64 ACG 11 0.23 GGC 4 0.08 A GCU 40 1.62 GGA 73 1.5 GCC 19 0.77 GGG 38 0.78 GCA 33 1.33 GCG 7 0.28

Phylogeny of Tingidae

Phylogenetic relationships among Tingidae were inferred including all the mitogenomes available, except Eteoneus sigillatus Drake & Poor 1936 (KU896784, unverified sequence). This dataset represents the highest mitogenomic coverage of Tingidae so far, although it represents a mere 0.6% of the family (14 species in 12 genera), all belonging to the subfamily Tinginae. We tested three datasets: PCG123 with each codon position partitioned, PCG12 (only the 1st and the 2nd codon positions, and AA (amino acid sequence. PCG12 and AA were tested as this approach was shown to decrease the impact of the high compositional heterogeneity typical of Heteroptera mitogenomes (Liu et al., 2018; Yang et al., 2018).

Mitogenome of an olive lace bug Zootaxa 4722 (5) © 2020 Magnolia Press · 453 Figure 5. Usage of start codons (ATG, ATA, ATT, ATC and GTG) found in the complete complement of 13 mitochondrial protein-coding genes from 15 lace bug species belonging to the family Tingidae.

Statistical support was higher in the PCG12 tree (BPP = 1, all nodes) than in the AA or the PCG123 trees, where several nodes had a BPP lower than 1 (Figure 7). The PCG12 and AA trees showed a very similar topology, differ- ing only in the positions of Tingis cardui and Dictyla platyoma (Fieber 1861), which also had the lowest statistical support of all nodes in the AA tree. Although the order of some deeper nodes differed between the PCG123, PCG12 and AA trees, all topologies showed Phatnoma laciniatum (Phatnomini) in the basal position to all other species (Tingini), as expected, and Stephanitis and Corythucha, the only genera represented by more than one species, as monophyletic. These two features were also recovered in previous phylogenies (Guilbert, Damgaard and D’Haese, 2014; Lin et al., 2017). Metasalis populi was not recovered as sister to Tingis cardui, in disagreement with their putative close relationship, as M. populi was previously described as a Tingis species. Instead, M. populi was sister to the Stephanitis clade, and Cysteochila chiniana Drake 1954 was sister to Trachypeplus jacobsoni Horváth 1926, as previously found (Yang et al., 2018). Ammianus toi (Drake 1938) and Perissonemia borneenis (Distant 1909) were also sister taxa in the all trees. Neoplerochila paliatseasi was most closely related to Agramma hupehanum (BPP = 1, in all trees), in accordance to the high similarity between the COI sequences of the two species, which was the criteria we used for selecting A. hupehanum as the reference mitogenome for the mapping and assembly of N. paliatseasi. Agramma Stephens 1829 is considered to be basal to Tingini in traditional systematics works (see Peìricart, 1983), and A. hupehanum was also recovered as basal in phylogenies based on shorter gene regions and morphological characters (Guilbert, Damgaard and D’Haese, 2014). However, this was not the case in our study, similarly to Liu et al. 2018, and the basal position in in the PCG12 and AA trees was occupied by Pseudacysta perseae (as in Ko- cher et al. 2015). Several studies have presented reconstructions of the phylogeny of true bugs, including Tingidae, based on mitochondrial genomes (Yang, Yu and Du, 2013; Kocher et al., 2015; H. Li et al., 2017; Lin et al., 2017; P. W. Li et al., 2017; Liu et al., 2018; Yang et al., 2018). However, the relationships within Tingidae could not be discussed as too few terminals for the family were included. Mitogenomic phylogenies have recovered different topologies compared to phylogenies based on shorter mitochondrial regions combined with morphological characters (Guilbert, Damgaard and D’Haese, 2014), and the results have been contradictory. This could be explained by the large number of characters (up to 15,000 bp) generally analyzed in mitogenomic phylogenies on a few terminal taxa, in contrast with the analysis of a much higher number of taxa using shorter sequences (up to 5,000 bp) (Guilbert, Damgaard and D’Haese, 2014). Additionally, the inconsistent recovery of the phylogeny of Tingidae could also be explained by the high sequence heterogeneity and the highest evolutionary rate characteristic of this family among Cimicomorpha, and the highest family-level nonsynonymous substitutions per non-synonymous site (Ka) among

454 · Zootaxa 4722 (5) © 2020 Magnolia Press LANGLEY et al. true bugs (Liu et al., 2018). In line with previous studies, our phylogenetic reconstruction confirmed that the order of the deeper nodes varies depending on the type of analyses performed. In the future, it may be necessary to include nuclear DNA, as mitochondrial data does not produce consistent results.

Figure 6. Predicted structure of the 22 tRNAs in the complete mitochondrial genome of Neoplerochila paliatseasi (Hemip- tera: Tingidae). Inferred canonical Watson-Crick bonds are represented by lines, and non-canonical bonds (U-U, A-A, G-U, G-A, G-G, C-C, A-C, and C-U) are represented by dots.

Wild and cultivated olive trees and olive fruit in the Western Cape host a diverse and specialized assemblage of insects, including two olive fruit flies (B. oleae and B. biguttula), parasitoid and seed wasps, flea beetles, and lace bugs (Powell et al., 2019). Despite the economic significance of this particular entomofauna, most species have been poorly characterized with regards to their biology, ecology, and impact on commercial olive production. Olive lace bug infestations are known to affect olive tree health and productivity, but no systematic assessment of the species involved has been performed in the region, where they are generally referred to as “olive tingids” and presumed to be P. australis. This species can disseminate rapidly and become devastating, as reported in a study performed in natural stands of wild olive trees in Ethiopia (Yirgu, Getachew and Belay, 2012). The authors suggested that dissemination of P. australis was mainly due to accidental transport of specimens stuck on clothes and domestic animals, as the insect was not observed to fly distances of more than three meters. Interestingly, the report stated that recently introduced cultivated olives were relatively free from the pest, which is not the case in South Africa. In addition to the previously known presence of P. australis in wild and cultivated olives in the Western Cape, we report here N. paliatseasi as a pest of cultivated olives for the first time. The biology and ecology of the species is poorly understood, as well as the extent to which it affects the olive industry in South Africa. It is possible that N. paliatseasi has co-infested cultivated olives along with P. australis since the introduction of cultivated olives to the Western Cape, and that it remained unidentified until now due to the local habit of designating olive lace bugs generically as “olive tingids”. However, it is also possible that N. paliatseasi has recently moved from wild olive trees, or other unknown wild hosts, to cultivated olives. Surveys of wild and cultivated olives in the region will be necessary to gain further biological and ecological insights into this potential threat and its natural enemies. Inter- estingly, the Mediterranean Basin and California, two of the most important olive-producing world regions are free from host-specific olive lace bugs. On the contrary, olive growers in Western Australia regard Froggattia olivina Froggatt 1901, a native species that has moved from its native host Notelaea longifolia to the cultivated olive, as one of the most serious threats to the local industry (Spooner-Hart, 2005). The species is currently restricted to Australia, and has not been reported anywhere else in the world. It would be interesting to assess the phylogenetic relationships among P. australis, N. paliatseasi and F. olivina, as these three species are presently the only known lace bugs well- adapted to feeding on Olea europaea spp.

Figure 7. Phylogenetic relationships among 15 lace bug species (Hemiptera: Tingidae) based on 13 mitochondrial protein- coding genes, using Bayesian inference. PCG123 was constructed using DNA sequences with partitioned codon positions. PCG12 was constructed using DNA sequences, excluding the 3rd codon position. AA was constructed using amino acid sequences. Trees were rooted by the outgroups Adelphocoris fasciaticollis and Apolygus lucorum (Miridae). Nodal statistical support is given as Bayesian posterior probability.

CONCLUSION

The diversity of lace bug species associated with wild and cultivated olive trees in South Africa has been poorly characterized, and the species deemed responsible for olive production losses in the Western Cape has been reported

456 · Zootaxa 4722 (5) © 2020 Magnolia Press LANGLEY et al. as P. australis. We showed that N. paliatseasi is also present in cultivated olives, thus contributing to the general knowledge on olive pests in the region. This work adds the complete mitochondrial of N. paliatseasi to the family Tingidae, a group with low representation in Hemiptera mitogenomics. Furthermore, the dataset of COI barcoding sequences for Tingidae currently available on BOLD Systems v3 supports the utility of this genetic marker for species identification in the family, and no taxonomic errors were detected in the database. The phylogenetic reconstruction within the family recovered N. paliatseasi as closely related to A. hupehanum, the species generally regarded as basal to Tingini. This study was based on specimens collected incidentally during haphazard sampling of olives aimed at recovering olive-associated Diptera and Hymenoptera. Therefore, it does not represent a comprehensive assessment of olive lace bugs in South Africa, but rather warrants deeper investigation into the diversity and distribution of these species across their range of wild and cultivated olive hosts.

ACKOWLEDGEMENTS

This work was supported by the Foundational Biodiversity Information Programme (FBIP) – Small Grants and Biodiversity Surveys, and by the University of Stellenbosch. Jethro Langley was funded by the NRF Honours and Final Year B-Tech Bursary (Reference: HBG181023379536).

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First report of the lace bug Neoplerochila paliatseasi (Rodrigues, 1981) (Hemiptera: Tingidae) infesting cultivated olive trees in South Africa, and its complete mitochondrial sequence. LANGLEY J, CORNWALL M, POWELL C, COSTA C, ALLSOPP E, VAN NOORT S, GUILBERT E, VAN ASCH B.

Table S1. List of the 15 complete and near-complete mitochondrial sequences used in the phylogenetic reconstruction within the family Tingidae, with common name, broad geographic distribution, Genbank accession numbers and references. Complete – sequence includes putative control region; Partial – sequence does not include putative control region. Species Distribution Common name GenBank Reference Size (bp) Status Agramma India na NC_037146.1 Liu et al., 15,992 Complete huperhanum 2018 Ammianus toi China, Java, Vietnam na JQ739178.1 Li, H et al., 14,230 Partial 2017 Corythucha Canada, USA Sycamore lace NC_022922.1 Yang et al., 15,257 Complete ciliata bug 2013 Corythucha Canada, USA, Mexico, Chrysanthemum MG479390.1 Lin et al., 15,635 Complete marmorata Jamaica lace bug 2017 Cysteochila China, Taiwan, Japan na NC_037833.1 Yang et al., 15,475 Complete chiniana 2018 Dictyla Belarus, Caucasus, China, na NC_037834.1 Yang et al., 15,649 Complete platyoma Eastern Europe, Greece, 2018 Middle Asia, Moldova, Russia, Russia (Siberia), Russian Far East, Southeastern Europe, Turkey, Ukraine Metasalis Europe & Northern Asia Poplar lace bug NC_037835.1 Yang et al., 15,257 Complete populi (excluding China), Southern 2018 Asia Neoplerochila South Africa, Namibia Olive lace bug MN794065 This study 15,348 Complete paliatseasi Perissonemia China, Indonesia (Borneo), na KU896785.1 Liu et al., 15,479 Complete borneensis Malaysia (Borneo), Papua New 2018 Guinea, Philippines, Singapore Phatnoma India, Sri Lanka na NC_037148.1 Liu et al., 15,821 Complete laciniatum 2018 Pseudacysta USA, Mexico, Caribbean, Avocado lace bug NC_025299.1 Direct 15,850 Complete perseae Guatemala, Venezuela, French submission Guiana Stephanitis China Tea lace bug MF498769.1 Li, P et al., 16,667 Complete chinensis 2017 Stephanitis Japan na JQ739184.1 Li, H et al., 14,045 Partial mendica 2017 Tingis cardui Europe, North Africa Spear thistle lace NC_037836 Yang et al., 14,711 Complete bug 2018 Trachypeplus India, Sumatra na NC_037837.1 Yang et al., 15,712 Complete jacobsoni 2018

460 · Zootaxa 4722 (5) © 2020 Magnolia Press LANGLEY et al. Table S2. Minimum, maximum and mean intraspecific p-distances (K2P) in 25 species in the family Tingidae, based on a 512 bp alignment of COI barcoding sequences (n = 218). Standard errors were calculated using 1,000 bootstrap replicates. Number of Haplotype Nucleotide Species n haplotypes diversity diversity Min Max Mean±SE Acalypta elegans 32 9 0.804 0.0051 0.00 1.78 0.51±0.192 Acalypta musci 4 2 0.667 0.0026 0.00 0.39 0.26±0.171 Acalypta nigrina 3 1 0.000 0.0000 0.00 0.00 0.00±0.000 Acalypta parvula 10 3 0.511 0.0011 0.00 0.20 0.11±0.071 Catoplatus fabricii 4 1 0.000 0.0000 0.00 0.00 0.00±0.000 Copium clavicorne 4 1 0.000 0.0000 0.00 0.00 0.00±0.000 Corythucha ciliata 7 3 0.762 0.0039 0.00 0.79 0.39±0.220 Corythucha immaculata 3 2 0.667 0.0039 0.00 0.59 0.39±0.204 Corythucha marmorata 38 16 0.881 0.0080 0.00 0.59 0.81±0.196 Corythucha pallipes 3 2 0.667 0.0013 0.00 0.20 0.13±0.131 Derephysia foliacea 3 1 0.000 0.0000 0.00 0.00 0.00±0.000 Dictyla humuli 5 3 0.700 0.0031 0.00 0.59 0.31±0.170 Dictyonota strichnocera 4 2 0.500 0.0020 0.00 0.39 0.19±0.125 Gargaphia opacula 5 4 0.900 0.0023 0.00 0.39 0.23±0.123 Gargaphia tiliae 8 4 0.821 0.0024 0.00 0.39 0.24±0.146 Hesperotingis fuscata 4 3 0.833 0.0046 0.00 0.59 0.46±0.248 Kalama tricornis 10 3 0.378 0.0008 0.00 0.20 0.08±0.055 Lasiacantha capucina 5 3 0.800 0.0027 0.00 0.59 0.27±0.156 Neoplerochila paliatseasi 6 2 0.600 0.0012 0.00 0.20 0.12±0.114 Oncochila simplex 7 3 0.667 0.0015 0.00 0.39 0.15±0.099 Physatocheila variegata 31 9 0.725 0.0026 0.00 0.99 0.26±0.094 Stephanitis takeyai 3 1 0.000 0.0000 0.00 0.00 0.00±0.000 Tingis cardui 10 5 0.800 0.0098 0.00 1.79 0.99±0.259 Tingis crispata 6 5 0.933 0.0129 0.00 2.60 1.31±0.300 Tingis reticulata 3 1 0.000 0.0000 0.00 0.00 0.00±0.000 Average 9 3.56 0.545 0.0029 0.00 0.55 0.29±0.123

Mitogenome of an olive lace bug Zootaxa 4722 (5) © 2020 Magnolia Press · 461 25 ― 2.02 2.07 1.97 1.89 2.64 2.32 2.24 2.02 2.13 1.81 2.21 2.23 2.17 2.52 2.18 2.50 2.44 2.70 2.34 2.08 1.71 2.43 2.19 2.30 24 ― 2.01 1.86 2.25 2.38 2.33 2.06 2.07 2.21 2.25 2.05 2.29 2.21 2.38 2.26 2.45 2.24 1.93 2.47 2.39 2.44 2.60 2.04 2.30 22.14 23 ― 2.07 1.99 1.80 1.89 2.38 1.95 2.23 1.88 1.79 1.99 2.07 1.85 1.82 2.12 2.03 2.09 2.02 2.20 1.89 2.08 2.35 1.96 22.30 21.80 22 ― 2.11 2.20 1.99 2.17 2.10 2.07 1.70 1.97 2.19 2.19 1.88 1.82 1.94 1.80 2.01 2.15 1.96 2.40 2.01 2.44 2.27 19.80 19.88 24.62 21 ― 1.83 2.09 2.13 1.99 2.55 2.34 2.14 2.01 1.97 1.65 1.63 1.67 1.87 2.16 2.23 2.35 2.17 1.87 2.10 1.79 20.94 22.22 23.31 19.06 20 ― 1.77 1.62 1.81 1.80 2.16 2.17 1.87 2.15 1.92 2.06 1.89 1.80 1.94 2.13 1.95 2.37 2.05 1.90 1.65 19.54 22.26 18.68 26.52 19.10 19 ― 2.20 1.70 1.92 2.00 2.21 2.13 2.24 2.31 1.91 2.46 1.72 1.80 1.77 2.05 2.14 2.20 2.06 2.04 13.91 18.82 19.49 19.13 22.28 22.25 18 ― 2.11 1.93 2.00 2.02 2.04 2.18 2.12 1.99 1.89 1.93 2.08 1.83 1.84 2.09 2.05 2.03 1.90 19.51 17.70 16.79 21.49 21.44 23.79 23.37 barcoding sequences (n = 218). Standard errors Standard 218). = (n sequences barcoding 17 ― 2.00 1.76 2.30 1.95 2.08 1.90 1.98 2.06 1.95 2.37 2.20 1.83 1.92 2.18 2.23 2.13 19.35 20.70 24.09 20.51 19.19 23.19 18.99 23.61 COI 16 ― 2.11 1.93 2.03 1.90 2.23 2.09 1.99 1.84 2.08 2.04 2.31 1.82 2.10 1.84 1.93 19.56 22.22 22.86 24.10 21.14 23.22 23.69 22.13 24.66 15 ― 1.73 2.00 1.96 1.97 1.98 2.02 2.04 2.13 2.05 2.16 2.10 1.86 1.93 1.88 18.78 20.75 19.75 20.17 19.55 20.67 18.97 20.74 21.99 23.00 14 ― 2.09 1.94 1.76 1.84 2.07 1.52 1.86 2.38 2.00 2.20 1.88 1.94 2.07 18.11 19.05 18.35 18.73 18.87 19.61 20.14 17.45 21.34 20.56 24.16 13 ― 1.71 1.61 2.12 1.85 2.19 1.92 2.15 1.94 1.90 2.09 1.87 1.67 21.00 19.39 22.52 19.47 20.31 18.25 20.98 18.04 19.21 20.64 22.87 21.63 12 ― 1.67 1.89 1.89 1.72 1.99 1.98 2.17 1.79 1.76 1.62 1.45 15.13 18.49 17.88 21.07 18.45 18.27 17.38 19.39 14.62 18.00 18.46 20.80 21.24 11 ― 1.75 1.79 1.93 1.85 1.94 2.09 1.79 1.95 1.87 2.16 13.70 17.32 14.49 18.21 20.32 20.48 17.40 17.85 17.86 16.59 16.82 19.70 21.31 22.49 10 ― 1.97 2.17 1.92 1.92 2.22 2.18 2.09 1.95 2.00 19.41 14.20 18.81 20.15 18.45 20.14 21.19 18.26 21.47 19.60 15.29 18.85 20.87 19.35 22.12 9 ― 1.59 1.65 1.90 1.90 1.97 1.99 1.86 1.95 21.26 17.90 16.48 17.71 20.70 18.59 23.06 22.32 19.33 20.29 19.28 20.67 20.67 17.50 22.43 21.85 8 ― 1.63 1.78 2.05 1.73 1.95 2.03 2.24 26.11 20.08 18.41 18.37 17.33 20.70 18.99 19.93 19.27 19.22 19.35 21.01 20.19 18.80 19.23 18.70 21.08 7 ― 1.91 1.80 2.18 1.84 1.54 1.40 19.94 19.12 19.28 18.46 17.13 18.73 17.74 18.39 21.16 18.64 18.71 19.64 19.12 19.21 19.54 21.40 18.37 23.90 6 ― 1.93 1.69 1.77 1.71 1.27 11.18 20.16 18.96 20.54 19.01 18.82 19.51 17.03 18.54 22.86 16.81 20.70 19.24 21.23 19.93 19.12 21.29 17.16 23.35 5 ― 1.89 1.77 1.87 1.92 8.04 12.88 19.06 19.42 21.35 17.63 18.30 22.30 18.12 19.41 23.49 19.19 20.64 21.52 21.16 21.36 22.19 22.97 19.29 24.57 4 ― 1.73 1.67 1.72 16.23 16.80 14.97 17.93 19.95 17.72 18.47 16.37 18.99 17.38 21.45 21.23 19.17 19.00 19.73 19.45 19.13 19.56 21.15 21.47 21.88 3 ― 1.78 1.78 14.24 16.01 16.26 19.78 18.95 20.35 16.64 18.85 16.40 21.13 17.18 19.68 21.87 21.00 19.45 19.25 19.46 18.36 22.54 19.98 21.66 21.54 2 ― 1.44 17.53 14.17 15.61 15.96 16.12 17.15 15.26 18.31 17.03 15.86 17.07 17.44 19.92 21.44 17.05 18.43 16.57 16.93 18.76 20.93 20.70 17.70 20.84 1 ― 14.16 16.19 15.35 18.53 17.92 18.41 15.21 17.91 17.33 16.82 16.35 17.56 20.80 16.90 19.91 18.28 17.14 20.15 18.52 18.28 20.55 20.68 20.60 23.04 Interspecific p-distances (K2P) among 25 species of the family Tingidae based on a 512 bp alignment of alignment bp 512 a on based Tingidae family the of species 25 among (K2P) p-distances Interspecific asiacantha capucina L asiacantha Species Tingis reticulata Tingis Stephanitis takeyai Oncochila simplex Corythucha marmorata Corythucha pallipes Corythucha ciliata Corythucha immaculata Dictyla humuli Dictyonota strichnocera Physatocheila variegata opacula Gargaphia Copium clavicorne Kalama tricornis tiliae Gargaphia Catoplatus fabricii crispata Tingis cardui Tingis Acalypta nigrina Acalypta elegans foliacea Derephysia fuscata Hesperotingis Acalypta parvula Neoplerochila paliatseasi Acalypta musci S3. able 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 T were calculated using 1,000 bootstrap replicates, and are shown above the diagonal.