Limnogeton scutellatum Mayr MNHN

Limnogeton scutellatum Mayr MNHN View in CoL /MRAC

Weberiella rhomboides (Menke) * INPA/DZRJ/LEBIP /MNHN

NEPIDAE

Curicta borellii Montandon LEBIP

Curicta cf. pelleranoi LEBIP

Curicta volxemi (Montandon) * LEBIP

Laccotrephes japonensis (Scott) LEBIP

Laccotrephes pfeiferiae (Ferrari) LEBIP

Laccotrephes sp. * LEBIP

Nepa cinerea Linnaeus LEBIP

Nepa hoffmanni Esaki * LEBIP

Ranatra brevicauda Montandon LEBIP

Ranatra chinensis Mayr * LEBIP

Ranatra heydeni Montandon * LEBIP

Ranatra robusta Montandon LEBIP

Ranatra sattleri De Carlo LEBIP

CHINA: Chekiang Province; JAPAN: Kumamoto, Tokyo, Alpes de Nikko; LAOS: Baudan

BRAZIL: Rio de Janeiro; Rio Grande do Sul

SOMALIA: Glohar

BRAZIL: Rio Grande do Sul

FRENCH GUYANA: Route de Pointe Combi REPUBLIC OF THE CONGO: Brazzaville; CAMEROON:

Batouri, Nyaounderé; TANGANYIKA: Mlingano EGYPT; SUDAN; CAMEROON: Garoua

SENEGAL: Kolda; EGYPT; REPUBLIC OF THE CONGO;

CAMEROON: Yaoundé; UGANDA: Victoria Nyanza EGYPT; ETHIOPIA; CENTRAL AFRICAN REPUBLIC:

Lamaboké; DEMOCRATIC REPUBLIC OF THE CONGO:

Molindi River; REPUBLIC OF THE CONGO: Brazzaville;

ZAMBIA: Muliba

BRAZIL: Amazonas, Mato Grosso; FRENCH GUIANA: Piste

Coralie

BRAZIL: Rio Grande do Sul

BRAZIL: Mato Grosso do Sul

BRAZIL: Mato Grosso do Sul

JAPAN: Kumamoto

CHINA: Tianjin (Wuqing Country)

CHINA: Tianjin (Wuqing Country)

FRANCE: Vayral

JAPAN: Hyogo

BRAZIL: Mato Grosso do Sul

JAPAN: Kumamoto

BRAZIL: Mato Grosso do Sul

BRAZIL: Mato Grosso do Sul; Rio Grande do Sul

BRAZIL: Mato Grosso do Sul

†List of specimen depositories is based on Arnett, Samuelson & Nishida (1993), except for DZRJ, LEBIP and MNRJ. DZRJ, Departamento de Zoologia , Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil ; LEBIP, Laboratório de Estudos da Biodiversidade do Pampa , São Gabriel, Universidade Federal do Pampa, Rio Grande do Sul, Brazil ; MNRJ, Museu Nacional , Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil .

such material was examined at 5 kV, without a coat of gold–palladium.

DNA PREPARATION

Insect specimens collected in the field were placed directly in 95–100% ethanol and stored at −20 °C until processing. To amplify genes, genomic DNA was extracted from a single foreleg and associated muscles using a modified ethanol precipitation/resuspension protocol ( Bender, Spierer & Hogness 1983) or the DNEasy tissue kit (Qiagen Inc.). In some cases, multiple individuals from the same species were extracted. All belostomatid vouchers were stored in 99.5% ethanol pro-analysis (PA) at −20 °C deposited at the LEBIP (Laboratório de Estudos da Biodiversidade do Pampa, Universidade Federal do Pampa, Rio Grande do Sul, Brazil), except for W. rhomboides , which was dried and is deposited at the MNHN (Museum National d’Histoire Naturelle, Paris, France). For amplification of genes from W. rhomboides , genomic DNA was extracted following protocols adapted from those of Gilbert et al. (2007), Thomsen et al. (2009) and Lis, Ziaja & Lis (2011), which allowed us to recover amplifiable DNA from dried museum specimens that were up to 10 years old. The specimens were pinned again and redeposited in the collection as vouchers.

PCR, SEQUENCING AND ALIGNMENT

Modified primers based on Simon et al. (1994), von Dohlen & Moran (1995) and Simon et al. (2006) were used to amplify parts of the mitochondrial gene COI (~730 bp) and 16S rDNA (~480 bp). Nuclear 18S sequences were amplified using the primers Ns1 and Ns2a (Barker et al., 2002) (~600 bp) (see Table 2). Amplification was carried out in a 25-µL volume reaction, with 5 µL of Taq and Load Mastermix, 0.5 µL of each primer at 25 µM and 2 µL of extraction product. For 16S loci, all reactions were performed using HifiTaq DNA Polymerase – an enzyme mixture that greatly increases fidelity and amplification of genomic targets. For COI reactions, a ‘step-up procedure’ was used: 2 min at 95 °C, five amplification cycles to improve DNA stock of 1 min at 95 °C, 15 min at 45 °C and 1 min at 72 °C; followed by 35 cycles of 40 s at 95 °C, 1 min at 51 °C and 1 min at 72 °C, with a final extension at 72 °C for 5 min. The protocol for the 16S rDNA region was: 94 °C for 3 min and 80 °C for 20 min (hot start), followed by 35 cycles of 94 °C for 1 min, 50 °C for 1 min and 72 °C for 2 min, with a final extension at 72 °C for 7 min. The protocol for 18S rDNA was 94 °C for 2 min, 35 cycles of 94 °C for 30 s, 49 °C for 30 s and 72 °C for 30 s, with a final extension at 72 °C for 7 min. Negative controls (no template) were always run simultaneously with our PCR experiments. All reaction mixtures were discarded when any DNA appeared in the negative control.

Sequences were sent to the Genoscope (Centre National de Séquençage, Evry). The sequences obtained were read and cleaned with CodonCode (CodonCode Corporation, 2002–2015). All sequences were aligned using MUSCLE (Edgard, 2004), and MAFFT v.7 ( Katoh & Standley, 2013) for non-coding sequences. In all regions, gaps were checked manually and treated as missing data. This procedure yielded approximately 1830 bp per taxon, although sequences for some taxa were not complete ( Table 1). All Belostomatidae and Nepidae sequences are original accessions, except Abedus breviceps Stål, 1862 , Curicta scorpio Stål, 1862 and Nepa apiculata Uhler, 1862 , which were taken from GenBank. NCBI accession numbers and references are summarized in Table 3.

PHYLOGENETIC ANALYSES