Ryan C. Garrick, Department of Biology, University of Mississippi, Oxford, MS 38677, USA

Garrick RC (2016) True syntopy between chromosomal races of the Cryptocercus punctulatus Scudder (Blattodea: Cryptocercidae) wood-roach species complex. Insectes Sociaux.

Ryan C. Garrick, Department of Biology, University of Mississippi, Oxford, MS 38677, USA

E-mail: ; Tel.: +1-662-915-2341; Fax: +1-662-915-5144

Online Resource 1. Locations from which members of the Cryptocercus punctulatus species complex were sampled (asterisk indicates the focal rotting log from Polk County, Tennessee).

Online Resource 2. Procedures for DNA extraction, PCR amplification and mtDNA sequencing.

Genomic DNA was extracted from three adult Cryptocercus punctulatus individuals per rotting log using a DNeasy Blood and Tissue Kit (Qiagen). Portions of the mtDNA cytochrome oxidase subunit I (COI) and subunit II (COII) genes were amplified via polymerase chain reaction (PCR) in 10 µL volumes comprised of 2 μL 5× PCR buffer (Promega), 0.8 μL MgCl2 (25 mM, Promega), 1.6 μL dNTPs (1.25 mM, Promega), 0.5 μL Bovine Serum Albumin (10mg/mL, New England BioLabs), 3 μL dH2O, 0.5 μL of each primer (10 μM), 0.1 μL Go-Taq (5U/μL, Promega), and 1 μL of DNA. Primers for COI were LCO-1490 (Folmer et al. 1994) and C1-N-2329 (Simon et al. 1994), and forward and reverse primers from Park et al. (2004) were used for COII. PCRs were performed in a Bio-Rad T100 Thermal Cycler as follows: 95°C for 2 min (1 cycle), 95°C for 30 sec, 50°C for 30 sec, and 72°C for 1 min (35 cycles), and final extension of 72°C for 2 min (1 cycle). Products were purified using ExoSAP-IT® (Affymetrix), then sequenced on an Applied Biosystems 3730x Genetic Analyzer at Yale University. Chromatograms were edited and aligned in MEGA v6.06 (Tamura et al., 2013), then translated into amino acids and compared to accessions in NCBI’s nucleotide and protein databases via the BLAST search function (Altschul et al., 1990). Sequences were A+T rich and closely matched COI and COII from Cryptocercus (and numerous other arthropods). Furthermore, alignments contained open reading frames and had no insertion-deletion mutations. Taken together, this indicates that data from true mitochondrial DNA (cf. nuclear pseudogenes) had been obtained. Final alignments for COI and COII were 762-bp and 363-bp, respectively.

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Online Resource 3. Details of phylogenetic analyses.

Chromosomal race assignments for samples from 70 sites were achieved via phylogenetic comparison to a “reference dataset” comprised of COII sequences from Lo et al. (2006; GenBank accessions DQ007628–DQ007642) and Everaerts et al. (2008; accessions AB425863–AB425884). This was done in two ways. First, a maximum likelihood gene tree was estimated in MEGA v6.06 (Tamura et al., 2013) using a GTR+I+G nucleotide substitution model as determined by jModelTest v2 (Darriba et al. 2012), a Neighbor Joining starting tree, and nearest-neighbor-interchange branch swapping. Node support was assessed via 500 bootstrap replicates. Cryptocercus clevelandi (Park et al. 2004, accession AB078557; Lo et al. 2006, accessions DQ007626 and DQ007627) was used as on out-group, as it is the sister lineage of the C. punctulatus species complex (Kambhampati et al. 1996; Burnside et al. 1999). Second, race assignments were performed by estimating statistical parsimony haplotype network (Templeton et al. 1992), implemented in TCS v1.21 (Clement et al. 2000). In order to minimize the number of disconnected sub-networks comprised of just a single haplotype, the connection limit was set at 15 mutational steps (i.e., a confidence criterion of ~85%).

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Online Resource 4. Statistical parsimony haplotype networks.

Haplotype networks estimated from COII sequence data. Colored circles are distinct haplotypes in the reference dataset (from Lo et al. 2006 and Everaerts et al. 2008, labeled with their GenBank accession number). Solid black circles are haplotypes from three cockroaches collected in the present study from the same rotting log in Polk County, Tennessee [samples 1, 2 and 3; lodged in the University of Mississippi Insect Collection (ID# UMIC-CpA42-1through -3, respectively)]. Each diagonal hash mark represents an inferred haplotype that was not sampled or is extinct, and a line between them represents one mutational step. Panels A–E: sub-networks that differ by >15 mutations; Panel F: key to chromosomal races.

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Supplementary References

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Burnside CA, Smith PT, Kambhampati S (1999) Three new species of the wood roach, Cryptocercus (Blattodea: Cryptocercidae), from the Eastern United States. J Kans Entomol Soc 72:361–378.

Clement M, Posada D, Crandall KA (2000) TCS: A computer program to estimate gene genealogies. Mol Ecol 9:1657–1659.

Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: More models, new heuristics and parallel computing. Nat Methods 9:772.

Everaerts C, Maekawa K, Farine JP, Shimada K, Luykx P, Brossut R, Nalepa CA (2008) The Cryptocercus punctulatus species complex (Dictyoptera: Cryptocercidae) in the eastern United States: Comparison of cuticular hydrocarbons, chromosome number, and DNA sequences. Mol Phylogenet Evol 47:950–959.

Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Marine Biol Biotechnol 3:294–299.

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Lo N, Luykx P, Santoni R, Beninati T, Bandi C, Casiraghi M, Lu WH, Zakharov EV, Nalepa CA (2006) Molecular phylogeny of Cryptocercus wood-roaches based on mitochondrial COII and 16S sequences, and chromosome numbers in Palearctic representatives. Zool Sci 23:393–398.

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Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P (1994) Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am 87:651–701.

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Templeton AR, Crandall KA, Sing CF (1992) A cladistics analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA sequence data. III. Cladogram estimation. Genetics 132:619–633.

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