ABSTRACT

The Cerambycidae Oncideres impluviata (Germar, 1823) is an important insect pest for Acacia mearnsii De Wild in Southern Brazil. The damage caused by their girdling behavior reduces tree productivity, specially in the early years of plant establishment, when girdling is performed on the main trunk of trees. Here, we used a fragment of the mtDNA COI gene to analyze the genetic diversity, population structure and demography of O. impluviata in Southern Brazil, as well as to present the first hypothesis of phylogenetic relationships among species of the genus Oncideres. Our results identified five distinct haplotypes among the populations of O. impluviata, with the most common haplotype identified as O.imp_COI_01. The phylogenetic inferences corroborated the monophyly of O. impluviata with maximum statistical support. In addition, the phylogeny recovered three main population strains that are largely congruent with the haplotype network, which includes two lineages that are found in different edaphic regions of Rio Grande do Sul (Serra do Sudeste and Encosta Inferior do Nordeste). This is the first molecular phylogenetic assessment of O. impluviata. Our findings provide insights into the evolution of a significant species for the Brazilian forestry sector, as well as new resources for planning of pest management strategies.

Keywords:
mtDNA; Genetic diversity; Insect pest; Black wattle; Twig-girdling beetles; Forest pest management

Introduction

The Brazilian forestry sector has significantly contributed to the country’s economy, with eucalyptus and pine plantations occupying an estimated area of 7.53 and 1.93 million hectares, respectively (IBÁ, 2022Indústria Brasileira de Árvores - IBÁ, 2022. Relatório anual 2022, ano base 2021. Available in: https://www.iba.org/datafiles/publicacoes/relatorios/relatorio-anual-iba2022-compactado.pdf (accessed 26 December 2022).
https://www.iba.org/datafiles/publicacoe... ). The forestry sector also includes species from genus Acacia Martius, 1829 (Fabaceae), whose contribution has been noted mainly in Southern Brazil, with 51.000 hectares of Acacia mearnsii De Wild (2021) in the State of Rio Grande do Sul (AGEFLOR, 2022Associação Gaúcha de Empresas Florestais - AGEFLOR, 2022. O setor de base florestal no Rio Grande do Sul 2022 - Ano base 2021. Available in: http://www.ageflor.com.br/noticias/wp-content/uploads/2022/12/ANUARIO-AGEFLOR-2022-WEB.pdf (accessed 26 December 2022).
http://www.ageflor.com.br/noticias/wp-co... ). The cultivation of A. mearnsii, also known as black wattle, plays an important social and economic role, as it involves more than 35.000 family forest owners who work in the generation of products. These include plant extracts rich in tannins agents and phenols that originate tannins, which are used for leather tanning and animal nutrition; flocculants for water treatment processes (vegetable origin); dispersants used as additives where spraying of liquids is necessary; adhesives, resins and phenolic-based for industries; natural flavoring soluble in water for stimulating food consumption at all stages in the diet of pigs, chickens, sheep, cattle, fish and shrimp (AGEFLOR, 2022Associação Gaúcha de Empresas Florestais - AGEFLOR, 2022. O setor de base florestal no Rio Grande do Sul 2022 - Ano base 2021. Available in: http://www.ageflor.com.br/noticias/wp-content/uploads/2022/12/ANUARIO-AGEFLOR-2022-WEB.pdf (accessed 26 December 2022).
http://www.ageflor.com.br/noticias/wp-co... ).

The increase in acreage and homogeneous plantations of black wattle in Rio Grande do Sul has also promoted an upsurge of insect pests damaging the trees, resulting in an environmental imbalance, caused by the insufficient management of forest plantations, and leading to production losses (Silva et al., 2020Silva, B. C., Costa, E. C., Saldanha, M. A., Procknow, D., Souza, P. D., Croda, J. P., Capitani, L. C., 2020. Métodos de controle e prevenção de insetos-praga em povoamentos florestais. Braz. J. Dev. 6 (7), 48477-48496. http://dx.doi.org/10.34117/bjdv6n7-480.
http://dx.doi.org/10.34117/bjdv6n7-480... ). The most common insects causing damage in A. mearnsii populations are the twig-girdler long horned beetle Oncideres impluviata (Germar, 1823) (Coleoptera: Cerambycidae: Lamiinae) and the moth Adeloneivaia subangulata (Herrich-Schaeffer, 1855) (Lepidoptera: Saturniidae) (Oliveira and Costa, 2011Oliveira, L. S., Costa, E. C., 2011. Predação de sementes de Acacia mearnsii De Wild (Fabaceae: mimosoideae). Biotemas 22 (2), 39-44. http://dx.doi.org/10.5007/2175-7925.2009v22n2p39.
http://dx.doi.org/10.5007/2175-7925.2009... ; Costa et al., 2014Costa, E. C., D’Ávila, M., Cantarelli, E. D., 2014. Entomologia florestal. Editora UFSM, Santa Maria, 256 pp.; Ono et al., 2014Ono, M. A., Ferreira, E. N. L., Godoy, W. A., 2014. Black wattle insect pests currently in Brazil. Glob. Adv. Res. J. Agric. Sci. 3 (12), 409-414.).

There are more than 131 species in the genus Oncideres (Monné, 2022Monné, M. A., 2022. Catalogue of the Cerambycidae (Coleoptera) of the Neotropical Region. Part II. Subfamily Laminae. Available in: https://cerambycids.com/catalog/Monne_Jun2022_NeotropicalCat_part_II.pdf (i).
https://cerambycids.com/catalog/Monne_Ju... ). Oncideres species are endemic to the Americas and many are classified as pests (Wang, 2017Wang, Q. 2017. Cerambycid pests in agricultural and horticultural crop. In: Wang, Q. (Ed.), Cerambycidae of the World: biology and pest management. CRC Press, Boca Raton, pp. 409-462.). Oncideres impluviata triggers significant damage in A. mearnsii plantations in Southern Brazil (Ono, 2015Ono, M. A., 2015. Dinâmica de infestação em Acacia mearnsii e ecologia de Oncideres impluviata (Coleoptera: Cerambycidae). Dissertação de Mestrado, Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo, Piracicaba, 68 pp.) and has been reported using more than 20 host plants, including native and exotic economically species, such as: Ilex paraguariensis Saint-Hilaire (Aquifoliaceae), Bauhinia forficata Link, Mimosa scabrella Bentham, Parapiptadenia rigida (Bentham) Brenan, Acacia spp., (Fabaceae), Croton floribundus Sprengel (Euphorbiaceae), Myrsine coriacea (Sq.) Roem. & Schult. (Myrsinaceae) (Ricketson and Pipoly, 1997Ricketson, J. M., Pipoly, J. J., 1997. Nomenclatural notes and a synopsis of the genus Myrsine (Myrsinaceae) in mesoamerica. SIDA Contrib. Bot. 17 (3), 579-589.) (Monné, 2022Monné, M. A., 2022. Catalogue of the Cerambycidae (Coleoptera) of the Neotropical Region. Part II. Subfamily Laminae. Available in: https://cerambycids.com/catalog/Monne_Jun2022_NeotropicalCat_part_II.pdf (i).
https://cerambycids.com/catalog/Monne_Ju... ).

Currently, the distribution of O. impluviata is restricted to southern South America, being reported in Argentina (Misiones), Brazil (South and Southeast regions), Paraguay, and Uruguay (Monné, 2022Monné, M. A., 2022. Catalogue of the Cerambycidae (Coleoptera) of the Neotropical Region. Part II. Subfamily Laminae. Available in: https://cerambycids.com/catalog/Monne_Jun2022_NeotropicalCat_part_II.pdf (i).
https://cerambycids.com/catalog/Monne_Ju... ). This insect has specific morphological characters for identification (Fig. 1), measuring 13 mm - 20 mm in length and 4 mm - 6 mm in width. Dorsal region has a yellowish-brown color with grayish hairs and yellowish spots on the entire surface of the elytra and glossy black dots near the pronotum (Bondar, 1953Bondar, G., 1953. Bondar, G. 1953. A biologia do gênero Oncideres (Coleoptera: Cerambycidae) e descrição de nova espécie. Agronomia 12 (2), 29-31.; Amante et al., 1976Amante, E., Berlato, M. A., Gessinger, G. I., 1976. Biologia do “Serrador” da acácia-negra, Oncideres impluviata (Germar, 1823) (Coleoptera: Cerambycidae) no Rio Grande do Sul: I Etologia. Agron Sulriograndense 12, 3-56.; Pedrozo, 1980Pedrozo, D. J., 1980. Contribuição ao estudo de Oncideres impluviata (Germar, 1823) e seus danos em bracatinga (Mimosa scabrella Benth). Tese de Doutorado, Universidade Federal do Paraná, Curitiba, 95 pp.). Adults of O. impluviata girdle the branches that are going to be used by females to lay eggs (Baucke, 1962Baucke, O., 1962. A inseto-fauna da acácia-negra no Rio Grande do Sul, biologia e controle às pragas mais importantes. Secretaria da Agricultura, Secção de Informações e Publicidade Agrícola, Porto Alegre, 32 pp.). This injury knocks down the branches, which become dry and suitable for feeding and development of larvae (Amante et al., 1976Amante, E., Berlato, M. A., Gessinger, G. I., 1976. Biologia do “Serrador” da acácia-negra, Oncideres impluviata (Germar, 1823) (Coleoptera: Cerambycidae) no Rio Grande do Sul: I Etologia. Agron Sulriograndense 12, 3-56.; Pedrozo, 1980Pedrozo, D. J., 1980. Contribuição ao estudo de Oncideres impluviata (Germar, 1823) e seus danos em bracatinga (Mimosa scabrella Benth). Tese de Doutorado, Universidade Federal do Paraná, Curitiba, 95 pp.). Girdled trees tend to bifurcate their trunk, which drastically reduces wood production (Costa et al., 2014Costa, E. C., D’Ávila, M., Cantarelli, E. D., 2014. Entomologia florestal. Editora UFSM, Santa Maria, 256 pp.). A high infestation of O. impluviata in the early years of forest establishment can result in the death of injured trees (Baucke, 1962Baucke, O., 1962. A inseto-fauna da acácia-negra no Rio Grande do Sul, biologia e controle às pragas mais importantes. Secretaria da Agricultura, Secção de Informações e Publicidade Agrícola, Porto Alegre, 32 pp.). Despite its economic importance, no study has been carried out with the aim of characterizing the population genetic structure of O. impluviata in Southern Brazil. Genetic structure studies of this economically important insect pest can contribute to understand biological and evolutionary aspects that can be applied to pest management.

Mitochondrial DNA (mtDNA) has been used as molecular markers to elucidate genetic diversity in several species (Hebert et al., 2003Hebert, P. D. N., Cywinska, A., Ball, S. L., deWaard, J. R., 2003. Biological identifications through DNA barcodes. Proc. Biol. Sci. 270 (1512), 313-321. http://dx.doi.org/10.1098/rspb.2002.2218.
http://dx.doi.org/10.1098/rspb.2002.2218... ; Castalanelli et al., 2011Castalanelli, M. A., Mikac, K. M., Baker, A. M., Munyard, K., Grimm, M., Groth, D. M., 2011. Multiple incursions and putative species revealed using a mitochondrial and nuclear phylogenetic approach to the Trogoderma variabile (Coleoptera: Dermestidae) trapping program in Australia. Bull. Entomol. Res. 101 (3), 333-343. http://dx.doi.org/10.1017/S0007485310000544.
http://dx.doi.org/10.1017/S0007485310000... ; Goldstien et al., 2011Goldstien, S. J., Dupont, L., Viard, F., Hallas, P. J., Nishikawa, T., Schiel, D. R., Gemmell, N. J., Bishop, J. D. D., 2011. Global Phylogeography of the Widely Introduced North West Pacific Ascidian Styela clava. PLoS One 6 (2), e16755. http://dx.doi.org/10.1371/journal.pone.0016755.
http://dx.doi.org/10.1371/journal.pone.0... ; Valmorbida et al., 2019Valmorbida, I., Arnemann, J. A., Cherman, M. A., Bevilacqua, C., Perini, C. R., Ugalde, G. A., Guedes, J. C., 2019. Phylogeography Approach of Diloboderus abderus (Coleoptera: Melolonthidae) in the Southern Cone of America. Neotrop. Entomol. 48 (2), 332-339. http://dx.doi.org/10.1007/s13744-018-0637-0.
http://dx.doi.org/10.1007/s13744-018-063... ; Machado et al., 2020aMachado, D. M., Costa, E. C., Guedes, J. C., Barbosa, L. R., Martínez, G., Mayorga, S. I., Ramos, S. O., Branco, M., Garcia, A., Vanegas-Rico, J. M., Jiménez-Quiroz, E., Laudonia, S., Novoselsky, T., Hodel, D. R., Arakelian, G., Silva, H., Perini, C. R., Valmorbida, I., Ugalde, G. A., Arnemann, J. A., 2020a. One maternal lineage leads the expansion of Thaumastocoris peregrinus (Hemiptera: Thaumastocoridae) in the New and Old Worlds. Sci. Rep. 10 (1), 3487. http://dx.doi.org/10.1038/s41598-020-60236-7.
http://dx.doi.org/10.1038/s41598-020-602... ; Perini et al., 2020Perini, C. R., Sosa, V. I., Koda, V. E., Silva, H., Risso, A. A., Vasconcelos, W. N. F., Gonçalves, C. F., Ugalde, G. A., Machado, D. N., Bevilacqua, C. B., Ardisson-Araújo, D. M. P., Maebe, K., Smagghe, G., Valmorbid, I., Guedes, J. C., 2020. Genetic structure of two Plusiinae species suggests recent expansion of Chrysodeixis includes in the American continent. Agric. For. Entomol. 23 (3), 250-260. http://dx.doi.org/10.1111/afe.12427.
http://dx.doi.org/10.1111/afe.12427... ). The cytochrome c oxidase subunit 1 (COI) gene allows species identification due to its unique genetic signature and can be used in phylogenetic analysis (Wu et al., 2017Wu, Y., Trepanowski, N. F., Molongoski, J. J., Reagel, P. F., Lingafelter, S. W., Nadel, H., Myers, S. W., Ray, A. M., 2017. Identification of wood-boring beetles (Cerambycidae and Buprestidae) intercepted in trade-associated solid wood packaging material using DNA barcoding and morphology. Sci. Rep. 7 (1), 40316. http://dx.doi.org/10.1038/srep40316.
http://dx.doi.org/10.1038/srep40316... ; Torres-Vila and Bonal, 2018Torres-Vila, L. M., Bonal, R., 2018. DNA barcoding of large oak-living cerambycids: diagnostic tool, phylogenetic insights and natural hybridization between Cerambyx cerdo and Cerambyx welensii (Coleoptera: Cerambycidae). Bull. Entomol. Res. 109 (5), 583-594. http://dx.doi.org/10.1017/S0007485318000925.
http://dx.doi.org/10.1017/S0007485318000... ; Wang et al., 2019Wang, Z. L., Wang, T. Z., Zhu, H. F., Wang, Z. Y., Yu, X. P., 2019. DNA barcoding evaluation and implications for phylogenetic relationships in ladybird beetles (Coleoptera: coccinellidae). Mitochondrial DNA A. DNA Mapp. Seq. Anal. 30 (1), 1-8. http://dx.doi.org/10.1080/24701394.2018.1446950.
http://dx.doi.org/10.1080/24701394.2018.... ). The use of COI (DNA barcoding) as a tool for species identification and phylogenetic analysis has demonstrated to be able to discern close relative species (Wilson et al., 2011Wilson, J. J., Rougerie, R., Schonfeld, J., Janzen, D. H., Hallwachs, W., Hajibabaei, M., Kitching, I. J., Haxaire, J., Hebert, P. D. N., 2011. When species matches are unavailable are DNA barcodes correctly assigned to higher taxa? An assessment using sphingid moths. BMC Ecol. 11 (1), 18. http://dx.doi.org/10.1186/1472-6785-11-18.
http://dx.doi.org/10.1186/1472-6785-11-1... ; Grebennikov and Heiss, 2014Grebennikov, V. V., Heiss, E., 2014. DNA barcoding of flat bugs (Hemipte ra: Aradidae) with phylogenetic implications. Arthropod Syst. Phylogeny 72, 213-219.; Grebennikov et al., 2017Grebennikov, V. V., Jendek, E., Smirnov, M. E. D., 2017. Diagnostic and phylogenetic utility of the first DNA barcode library for longhorn beetles (Coleoptera: Cerambycidae) from the Russian Far East. Zootaxa 4276 (3), 441-445. http://dx.doi.org/10.11646/zootaxa.4276.3.9.
http://dx.doi.org/10.11646/zootaxa.4276.... ).

In general, mitochondrial genes have an advantage of high rates of evolution, compared to the nuclear DNA, and have relatively conserved regions (Meyer, 1994Meyer, A., 1994. Shortcomings of the cytochrome b gene as a molecular marker. Trends Ecol. Evol. 9 (8), 278-280. http://dx.doi.org/10.1016/0169-5347(94)90028-0.
http://dx.doi.org/10.1016/0169-5347(94)9... ; Sosa-Gómez et al., 2012Sosa-Gómez, D. R., Carvalho, M. C. C. G., Guimarães, F. C. M., Hoffmann-Campo, C. B., 2012. A biotecnologia, o melhoramento e o manejo de pragas da soja. In: Hoffmann-Campo, C.B., Corrêa Ferreira, B.S., Moscardi, F. (Eds.), Soja: manejo integrado de insetos e outros artrópodes-praga. Cap. 11. EMBRAPA, Brasília, pp. 725-788.); therefore, are remarkably useful for organisms succinctly investigated (Guo et al., 2020Guo, L., Gao, F., Cheng, Y., Gao, C., Chen, J., Li, Z., Wang, T., Xu, J., 2020. Mitochondrial COI Sequence Variations within and among Geographic Samples of the Hemp Pest Psylliodes attenuata from China. Insects 11 (6), 370. http://dx.doi.org/10.3390/insects11060370.
http://dx.doi.org/10.3390/insects1106037... ). In the present study, we used a fragment of the mtDNA COI gene to analyze the genetic diversity, population structure, and demography of O. impluviata in the State of Rio Grande do Sul, Brazil. In addition, the phylogenetic relationships among the populations of O. impluviata, as well as among the species of the genus Oncideres Lacordaire, 1830 were explored.

Materials and methods

Sampling of adult specimens of O. impluviata

The sampling procedure was performed in five sites of A. mearnsii plantations in Rio Grande do Sul, Brazil. Adults of O. impluviata were randomly collected from recently girdled branches which were lying on the ground (Fig. 2a, 2b; Table 1). Five to 15 adults were collected per site. Adult specimens were chosen to be captured instead of larvae because the branches girdled by O. impluviata often host other cerambycid opportunistic species, which could lead to inaccurate species identification (Paulino- Neto, 2004).

The samples were placed into 50 mL Falcon type plastic tubes containing 96% ethanol. The species was identified by comparison with the photo of the lectotype (Nearns and Tavakilian, 2015Nearns, E. H., Tavakilian, G. L., 2015. Seven new species of Oncideres Lacordaire, 1830 (Coleoptera, Cerambycidae, Lamiinae, Onciderini) from South America, with notes on additional taxa. Arq. Zool. 46 (2-11), 87-113. http://dx.doi.org/10.11606/issn.2176-7793.v46i2-11p87-113.
http://dx.doi.org/10.11606/issn.2176-779... ; Nearns and Nascimento, 2019Nearns, E. H., Nascimento, F. E. L., 2019. Onciderini Thomson, 1860 (Coleoptera: Cerambycidae: Lamiinae) types of the Museum für Naturkunde - Leibniz Institute for Evolution and Biodiversity Science, Berlin (ZMHB). Insecta Mundi 0725, 1-7.), as well morphological characteristics (Bondar, 1953Bondar, G., 1953. Bondar, G. 1953. A biologia do gênero Oncideres (Coleoptera: Cerambycidae) e descrição de nova espécie. Agronomia 12 (2), 29-31.; Amante et al., 1976Amante, E., Berlato, M. A., Gessinger, G. I., 1976. Biologia do “Serrador” da acácia-negra, Oncideres impluviata (Germar, 1823) (Coleoptera: Cerambycidae) no Rio Grande do Sul: I Etologia. Agron Sulriograndense 12, 3-56.; Pedrozo, 1980Pedrozo, D. J., 1980. Contribuição ao estudo de Oncideres impluviata (Germar, 1823) e seus danos em bracatinga (Mimosa scabrella Benth). Tese de Doutorado, Universidade Federal do Paraná, Curitiba, 95 pp.). After identification, O. impluviata adults, both males and females, were stored at -20 °C until extraction of genomic DNA.

DNA extraction, amplification and sequencing

Genomic DNA extraction was performed using parts of adult specimens (head, legs or thoracic muscles). We used the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), following manufacturer's instructions. Specimens were carefully removed from the Falcon tubes, placed on paper towel and air dried at room temperature. Specimens used for DNA extraction were vouchered and deposited in the Entomological Collection 'Padre Jesus Santiago Moure' (Universidade Federal do Parana, Curitiba, Brazil; DZUP), the Entomological Collection of the National Museum (Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; MNRJ) and Integrated Pest Management Laboratory (Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil).

Amplification of the partial mtDNA COI gene was performed using primers LCO1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') and HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') (Folmer et al., 1994Folmer, 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. Mar. Biol. Biotechnol. 3 (5), 294-299.). PCRs were processed with a final volume of 24μL containing 0.25 μL of Taq DNA Polymerase JumpStart ™ (5 U / μL); 2.5 μL of JumpStart ™ 10x Buffer; 1.25μL of the dNTP mixture (10 mM each); 2μL of each primer (10µM); 1μL of template DNA (70-120 ng / µL); and 15μL of ultra-pure water. PCR amplification comprised the following steps: initial denaturation at 95 °C (5 min), followed by 34 cycles at 94 °C (30 s), 48 °C (30 s) and 72 °C (1, 5 min) and a final extension at 72 °C (5 min). The quality of the PCR products was verified on 1.0% agarose electrophoresis gel stained with Nancy-520 DNA Gel Stain fluorescent (Sigma-Aldrich, St. Louis, MO, USA) using 3 µL of PCR product, and visualized on a gel documentation system. The amplified samples were sequenced by ABI 3500 Genetic Analyzer. All sequences obtained in this study were deposited in GenBank (Accession numbers: from MZ408185 to MZ408227; from MZ427518 to MZ4275528).

Sequence editing and alignments

Sequence analysis and editing were performed using Pregap and Gap4 programs within the Staden package (Staden et al., 2000Staden, R., Beal, K. F., Bonfield, J. K., 2000. The Staden package, 1998. Methods Mol. Biol. 132, 115-130. http://dx.doi.org/10.1385/1-59259-192-2:115.
http://dx.doi.org/10.1385/1-59259-192-2:... ). Partial mtDNA COI sequences were aligned using the software BioEdit version 7.2.6 (Hall, 1999Hall, T. A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41, 95-98.). These sequences were then translated into protein sequences to check for any translation problems and confirm the absence of premature stop codons.

Statistical analyses and genetic differentiation

The number of polymorphic sites (S), number of haplotypes (h), haplotype diversity (Hd ± SE), nucleotide diversity (π ± SE) and neutrality tests (Tajima's D and Fu FS) were calculated individually for each population and for the total populations using the DnaSP v. 5.10.01 (Librado and Rozas, 2009Librado, P., Rozas, J., 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25 (11), 1451-1452. http://dx.doi.org/10.1093/bioinformatics/btp187.
http://dx.doi.org/10.1093/bioinformatics... ). The nucleotide composition was calculated using MEGA version 7.0.26 (Kumar et al. 2016Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33 (7), 1870-1874. http://dx.doi.org/10.1093/molbev/msw054.
http://dx.doi.org/10.1093/molbev/msw054... ) and statistical analysis of parsimony was performed using TCS 1.21 (Clement et al., 2000Clement, M., Posada, D. C., Crandall, K. A., 2000. TCS: a computer program to estimate gene genealogies. Mol. Ecol. 9 (10), 1657-1659. https://doi.org/10.1046/j.1365-294x.2000.01020.
https://doi.org/10.1046/j.1365-294x.2000... ), within the PopART program to generate a haplotype network (Leigh and Bryant, 2015Leigh, J. W., Bryant, D., 2015. Popart: full-feature software for haplotype network construction. Methods Ecol. Evol. 6 (9), 1110-1116. http://dx.doi.org/10.1111/2041-210X.12410.
http://dx.doi.org/10.1111/2041-210X.1241... ).

Paired population differentiation (F_ST) and analysis of molecular variance (AMOVA) were performed on Arlequin 3.5.2.2 (Excoffier and Lischer, 2010Excoffier, L., Lischer, H. E., 2010. Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and windows. Mol. Ecol. Resour. 10 (3), 564-567. http://dx.doi.org/10.1111/j.1755-0998.2010.02847.x. [x]
http://dx.doi.org/10.1111/j.1755-0998.20... ), and the significant values were estimated with 10,000 permutations. F_ST values were used to estimate the number of female migrants per generation (1-F_ST / 2F_ST) (Wright, 1943Wright, S., 1943. Isolation by distance. Genetics 28 (2), 114-138. http://dx.doi.org/10.1093/genetics/28.2.114.
http://dx.doi.org/10.1093/genetics/28.2.... ). For the AMOVA, the data were grouped according to the physiographic regions of the State of Rio Grande do Sul (http://coralx.ufsm.br/ifcrs/fisiografia.htm). The populations of Capela de Santana and Montenegro are in the physiographic region Encosta Inferior do Nordeste; General Câmara in the region known as Depressão Central; Canguçu and Encruzilhada do Sul in the Serra do Sudeste (Fig. 3).

The relationship between genetic distance (F_ST) and geographic distance (km) was used to test isolation by distance (IBD). The geographical distance between each population pair was estimated using the Google Maps Distance Calculator (https://www.daftlogic.com/projects-advanced-google-maps-distance-calculator.htm#), and analysis was performed using IBD version 1.52 with 10.000 randomizations (Bohonak, 2002Bohonak, A. J., 2002. IBD (Isolation By Distance): a program for analyses of isolation by distance. J. Hered. 93 (2), 153-154. http://dx.doi.org/10.1093/jhered/93.2.153.
http://dx.doi.org/10.1093/jhered/93.2.15... ).

Phylogenetic analyses

Phylogenetic analyses were performed to infer the relationships among the populations of O. impluviata and included 17 species of Oncideres and representatives of five genera of Onciderini (Supplementary material, Table S1), which were used as outgroups. The trees were rooted in Hypsioma hezia Dillon & Dillon, 1945 in all analyses. The data matrix used for the phylogenetic analyses comprised a total of 95 terminals and 612 aligned positions of COI. The alignment included both sequences newly generated for this study and sequences obtained from Barcode of Life Data System (BOLD) (http://www.boldsystems.org/). Sequences obtained from BOLD were only reduced to suit them to the length of our alignments. Phylogenetic analyses were performed under Bayesian and Maximum Likelihood criteria.

Prior the phylogenetic analyses, the dataset was partitioned considering codon positions separately and the best-fitting model of nucleotide substitution for each partition was selected under Akaike Information Criterion (AIC) in PartitionFinder 2.1.1 (Lanfear et al., 2017Lanfear, R., Frandsen, P. B., Wright, A. M., Senfeld, T., Calcott, B., 2017. PartitionFinder 2: new methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Mol. Biol. Evol. 34 (3), 772-773. http://dx.doi.org/10.1093/molbev/msw260.
http://dx.doi.org/10.1093/molbev/msw260... ). Bayesian inference (BI) was carried out using MrBayes 3.2.6 (Ronquist et al., 2012Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M. A., Huelsenbeck, J. P., 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61 (3), 539-542. http://dx.doi.org/10.1093/sysbio/sys029.
http://dx.doi.org/10.1093/sysbio/sys029... ), using partitioned data with their corresponding best-fitting evolutionary model (SYM+G, HKY+I, TRN+G for 1st, 2nd and 3rd codon position, respectively) with eight chains of 50 million generations each and sampling trees every 1000 generations. The analysis convergence was assessed in Tracer 1.7.1 (Drummond and Rambaut, 2007Drummond, A. J., Rambaut, A., 2007. BEAST: bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7 (1), 214. http://dx.doi.org/10.1186/1471-2148-7-214.
http://dx.doi.org/10.1186/1471-2148-7-21... ) and the maximum clade credibility tree was generated discarding the output of the 10% initial generations in TreeAnnotator 1.8.4 (Heled and Bouckaert, 2013Heled, J., Bouckaert, R. R., 2013. Looking for trees in the forest: summary tree from posterior samples. BMC Evol. Biol. 13 (1), 221. http://dx.doi.org/10.1186/1471-2148-13-221.
http://dx.doi.org/10.1186/1471-2148-13-2... ). Posterior probability values (PP) were used to infer branch support in the BI tree. Maximum likelihood (ML) analysis was performed in RAxML 7.2.6 (Stamatakis, 2006Stamatakis, A., 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22 (21), 2688-2690. http://dx.doi.org/10.1093/bioinformatics/btl446.
http://dx.doi.org/10.1093/bioinformatics... ) using unlinked GTR + I + G nucleotide substitution models for each partition with 1.000 bootstrap (BS) pseudo-replicates. Both BI and ML analyses were implemented in CIPRES (Miller et al., 2010Miller, M. A., Pfeiffer, W., Schwartz, T., 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: 2010 Gateway Computing Environments Workshop (GCE), 2010, New Orleans, LA, USA. Proceedings. New York: IEEE, pp. 1-8.).

Results

Genetic diversity and phylogenetic analyses

A total of 48 sequences of COI (with 612 bp) from five populations of O. impluviata from Rio Grande do Sul, Brazil, were analyzed in the genetic diversity study. Twenty-six polymorphic sites (S), haplotype diversity (Hd) of 0.520 ± 0.073 and nucleotide diversity (π) of 0.01479 ± 0.00206 were observed when combining all sequences (Table 2). Hd in the municipalities of CA and MN varied from 0.564 to 0.800, and π ranged from 0.01014 to 0.01827. In addition, the neutrality tests (Tajima´s D: -0.34857, P = 0.70; and Fu´s Fs: 2.06, P = NA) were not significant, suggesting a pattern of constant size in populations (Table 2). The average nucleotide composition among the 48 sequences of O. impluviata was 38.3% thymine (T), 17.2% cytosine (C), 28.5% adenine (A) and 15.9% guanine (G).

Five haplotypes were identified and designated as: O.imp_COI_01, O.imp_COI_02, O.imp_COI_03, O.imp_COI_04 and O.imp_COI_05). The O.imp_COI_01 haplotype was the most common, being present in 32 specimens, and exclusively found in three populations: Capela de Santana (CS = 10), Encruzilhada do Sul (ES = 12) and General Câmara (GC = 10)). The populations of Canguçu (CA) and Montenegro (MN) shared two haplotypes, O.imp_COI_02 (CA = 02 and MN = 03) and O.imp_COI_03 (CA = 07 and MN = 02). In addition, the following haplotypes were represented by only one specimen: O.imp_COI_04 (CA = 01) and O.imp_COI_05 (MN = 01) (Fig. 4).

Both BI and ML phylogenetic inferences generated congruent results, supporting the monophyly of O. impluviata with strong statistical support and recovering three well-supported clades within the species: two closely related including the O.imp_COI_02 and O.imp_COI_03, both comprising samples from CA and MN; and one containing O.imp_COI_01, O.imp_COI_04 and O.imp_COI_05, comprising all samples from CS, ES, GC and one sample from each CA and MN (Fig. 4). In addition, our analyses recovered a clade containing all samples of a close related species, Oncideres saga (Dalman, 1823) (PP = 0.83; BO = 71). Oncideres was recovered as paraphyletic in relation to Psyllotoxus griseocinctus Thomson, 1868 (PP = 1; BO = 95), which was recovered as sister group of Oncideres cervina Thomson, 1868. Except for Oncideres digna Bates, 1865 and Oncideres putator brevifasciata Dillon & Dillon, 1946 all other Oncideres species sampled by more than one specimen in the analyses were strongly supported as monophyletic (Fig. 4).

Genetic distance between populations

Pairwise genetic distances among the populations of O. impluviata evidenced significant distances (Table 3). However, the analysis of female migrants per generation showed a weak and low evidence for this hypothesis. Furthermore, O. impluviata populations showed no statistically significant correlation of genetic distance (F_ST) vs geographical distance (P = 0.572).

Molecular analysis of variance (AMOVA)

The analysis of molecular variance (AMOVA) was conducted grouping populations according to the physiographic regions of Rio Grande do Sul: Serra do Sudeste, Depressão Central and Encosta Inferior do Nordeste. The results showed significant genetic differentiation among populations within groups (Φ_SC = 0.85304; P < 0.0001) and within populations (Φ_ST = 0.72641; P < 0.0001). No statistical significance differences were observed by grouping within physiographic regions (Φ_CT = -0.8617; P = 1.0000) (Table 4).

Discussion

Genetic diversity, genetic distance, and molecular analysis of variance

Specimens of O. impluviata, an important insect pest of A. mearnsii in Southern Brazil, were collected in five Acacia plantation sites in Rio Grande do Sul. Analyses of COI resulted in five haplotypes, representing the first molecular characterization and genetic diversity study of O. impluviata. In addition, our data are limited to an analysis of a partial mtDNA cytochrome Oxidase I fragment.

Although the specimens of O. impluviata were collected in its native range, the genetic diversity among the sampled populations was relatively low, contrary to our expectations of higher genetic diversity which is usually expected from native populations (Puillandre et al., 2008Puillandre, N., Dupas, S., Dangles, O., Zeddam, J. L., Capdevielle-Dulac, C., Barbin, K., Torres-Leguizamon, M., Silvain, J. F., 2008. Genetic bottleneck in invasive species: the potato tuber moth adds to the list. Biol. Invasions 10 (3), 319-333. http://dx.doi.org/10.1007/s10530-007-9132-y.
http://dx.doi.org/10.1007/s10530-007-913... ). We also found that O.imp_COI_01 was the most common haplotype in the localities with the larger areas of cultivation of A. mearnsii, such as General Câmara, Capela de Santana and Encruzilhada do Sul. The two lineages (O.imp_COI_04 and O.imp_COI_05) found in different edaphic regions, Serra do Sudeste and Encosta Inferior do Nordeste, should be monitored in further landscape adaptation studies. The force of gene flow in each landscape can balance the genetic diversity and form frequency of specific lineages (Dong et al., 2021Dong, Z., Wang, Y., Li, C., Li, L., Men, X., 2021. Mitochondrial DNA as a molecular marker in insect ecology: current status and future prospects. Ann. Entomol. Soc. Am. 114 (4), 470-476. http://dx.doi.org/10.1093/aesa/saab020.
http://dx.doi.org/10.1093/aesa/saab020... ).

The reduction of native areas, the expansion of acaciculture at Rio Grande do Sul, along with the proximity of plantations A. mearnsii associated with unappropriated management strategies, have contributed significantly to the increase in population density and dispersion of O. impluviata. Older plantations of black wattle can be important bridges of migration of O. impluviata to new plantations, especially in areas where legislative measures are not taken for this insect pest management (Costa et al., 2014Costa, E. C., D’Ávila, M., Cantarelli, E. D., 2014. Entomologia florestal. Editora UFSM, Santa Maria, 256 pp.). This same pattern was observed in old plantations of Mimosa scabrella Bentham (Baucke, 1958Baucke, O., 1958. Biologia e controle do serrador da Acácia-Negra. Tese de Doutorado, Escola de Agronomia “Eliseu Maciel”, Porto Alegre, 67 pp.) which is a tree species from the same botanical family as A. mearnsii.

The preference of O. impluviata for A. mearnsii may be associated to the volatile compounds present in this plant species (Berkov et al., 2000Berkov, A., Meurer-Grimes, B., Purzycki, K. L., 2000. Do Lecythidaceae specialists (Coleóptera, Cerambycidae) shun fetid tree species? Biotropica 32 (3), 440-451. http://dx.doi.org/10.1111/j.1744-7429.2000.tb00491.x.
http://dx.doi.org/10.1111/j.1744-7429.20... ). These are fundamental to stimulate feeding, copulation, and oviposition (Paulino-Neto, 2004Paulino-Neto, H. F., 2004. Lenhadores da natureza. Cienc. Hoje 35, 67-69.). Studies should be carried out to know which compounds emitted by A. mearnsii, particularly in the initial establishment in the field, are attractive to this insect.

Low diversity of O. impluviata can be associated with biotic and biotic factors, in addition to its limited samples. Oncideres impluviata is a univoltine insects (Costa et al., 2014Costa, E. C., D’Ávila, M., Cantarelli, E. D., 2014. Entomologia florestal. Editora UFSM, Santa Maria, 256 pp.; Ono, 2015Ono, M. A., 2015. Dinâmica de infestação em Acacia mearnsii e ecologia de Oncideres impluviata (Coleoptera: Cerambycidae). Dissertação de Mestrado, Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo, Piracicaba, 68 pp.), as well polyphagous, and reproduces through sexual reproduction, it is worth to note that females of this genus usually oviposit one egg per incision, but there are cases of multiple eggs per branch (Rogers, 1977Rogers, C. E., 1977. Bionomics of Oncideres cingulata (Coleoptera: Cerambycidae) on mesquite. J. Kans. Entomol. Soc. 50, 222-228.; Solomon, 1995Solomon, J. D., 1995. Guide to Insect Borers in North American Broadleaf Trees and Shrubs. U.S. Department of Agriculture, Forest Service, Washington, 747 pp.; Paulino-Neto, 2016Paulino-Neto, H. F., 2016. A review of the biology and ecological interactions of Oncideres (Cerambycidae): brazilian wood borers species. J. Ecosyst. Ecography 6 (4), 223. http://dx.doi.org/10.4172/2157-7625.1000223.
http://dx.doi.org/10.4172/2157-7625.1000... ). They seek to lay eggs on plants that, due to their characteristics (branch diameter, age, height, vigor, nutritional quality, and others), provides greater fitness to their offspring (Paulino-Neto, 2004Paulino-Neto, H. F., 2004. Lenhadores da natureza. Cienc. Hoje 35, 67-69.). Therefore, it is possible that more than one specimen emerged from the same branch or other branches of the same tree, all of which are maternally related (Machado et al., 2020bMachado, D. N., Costa, E. C., Carvalho, A. G., Puntel, L., Pozebon, H., Arnemann, J. A., 2020b. DNA barcoding de Oncideres saga (Coleoptera: Cerambycidae) do Brasil. Cienc. Florest. 30 (3), 937-943. http://dx.doi.org/10.5902/1980509841046.
http://dx.doi.org/10.5902/1980509841046... ), as possibly is the case of insects found in Capela de Santana, Encruzilhada do Sul and General Câmara.

The correlation between genetic distance (FST) and geographical distance didn’t show any statistically significance, which suggests absence of distance isolation. However, pairwise genetic distance shows significant population structure when comparing the collection sites (GC x MN, GC x CA, MN x CS, MN x ES, CS x CA, and CA x ES), regardless of its geographic distance. This suggests that most populations are not interbreeding freely, excepting some low FST value (GC x CS, GC x ES, CS x ES, and MN x CA). These pairwise comparisons agree with the AMOVA results, which showed that the populations presented considerable degree of differentiation, within populations lacking haplotype sharing. The number of female migrants of O. impluviata between the collection sites was not relevant in this study.

Still, new specimen collections should be carried out in order to expand the possibilities of finding more haplotypes in another areas in Rio Grande do Sul state and Brazil.

Phylogenetic analyses

Our results support the monophyly of O. impluviata, recognizing three main population strains that are largely congruent with the haplotype network (clades A, B and C), but the statistical support among the clades was negligible to infer with confidence their relationships. Oncideres impluviata is morphologically close to Oncideres guttulata Thomson, 1868. Both species present rounded yellowish pubescent spots on the elytra and a white pubescent macula on the metaventrite (Dillon and Dillon, 1946Dillon, L. S., Dillon, E. S., 1946. The tribe Onciderini (Coleoptera: Cerambycidae). Part II. Sci. Public Read. Pub. Mus. 6, 189-413.). However, our analyses did not recover an immediate relationship between these species. Instead, O. saga was found to be the closest relative of O. impluviata, although with moderate statistical support.

This is the first molecular phylogenetic study in the genus Oncideres. Our results reveal two important questions about the evolution of Onciderini: (1) the placement of Psyllotoxus within Oncideres, corroborating the close relationship between these genera, as suggested by some authors in the past (Thomson, 1868Thomson, J., 1868. Révision du groupe des oncidérites (Lamites, Cérambycides, Coléoptères). Physis Recl. Hist. Nat. 2, 41-92.; Lacordaire, 1872Lacordaire, J. T., 1872. Histoire Naturelle des Insectes. Genera des Coléoptères, ou exposé méthodique et critique de tous les genres proposés jusqu’ici dans cet ordre d’insectes. Libr. Encyclopedique Roret 9, 411-930.), and (2) hypotheses about the relationship between the species of Oncideres. However, it is still premature to propose taxonomic changes based on our sampling. These findings suggest evidence to redefine the systematic limits of Oncideres and must be taken into consideration in future phylogenetic and systematic studies.

Implications and insights for pest management

In the agricultural area, insect pests as the Lepidoptera Chrysodeixis includens (Walker, 1858) (Lepidoptera: Noctuidae) can get lineages adaptation not only at the local landscapes (Silva et al., 2020bSilva, C. S., Cordeiro, E. M. G., Paiva, J. B., Dourado, P. M., Carvalho, R. A., Head, G., Martinelli, S., Correa, A. S., 2020b. Population expansion and genomic adaptation to agricultural environments of the soybean looper, Chrysodeixis includens. Evol. Appl. 13 (8), 2071-2085. http://dx.doi.org/10.1111/eva.12966.
http://dx.doi.org/10.1111/eva.12966... ) but also between countries (Perini et al., 2020Perini, C. R., Sosa, V. I., Koda, V. E., Silva, H., Risso, A. A., Vasconcelos, W. N. F., Gonçalves, C. F., Ugalde, G. A., Machado, D. N., Bevilacqua, C. B., Ardisson-Araújo, D. M. P., Maebe, K., Smagghe, G., Valmorbid, I., Guedes, J. C., 2020. Genetic structure of two Plusiinae species suggests recent expansion of Chrysodeixis includes in the American continent. Agric. For. Entomol. 23 (3), 250-260. http://dx.doi.org/10.1111/afe.12427.
http://dx.doi.org/10.1111/afe.12427... ). Thus, edaphic regions in Rio Grande do Sul can force and create lineages of O. impluviata adapted to specific conditions that should be kept under investigation in further years.

Understanding the occurrence and ecology of O. impluviata in areas where new A. mearnsii tree are being cultivated is essential for the management of this pest species. Main strategies to manage O. impluviata populations includes a rotation every 6-7 years, without thinning or pruning and population density of 2-2.5 thousand trees per hectare (AGEFLOR, 2022Associação Gaúcha de Empresas Florestais - AGEFLOR, 2022. O setor de base florestal no Rio Grande do Sul 2022 - Ano base 2021. Available in: http://www.ageflor.com.br/noticias/wp-content/uploads/2022/12/ANUARIO-AGEFLOR-2022-WEB.pdf (accessed 26 December 2022).
http://www.ageflor.com.br/noticias/wp-co... ). Besides our finding of five genetic lineages, pest management strategies at a local-scale management of O. impluviata might help to reduce local populations which will not be boosted by long-distance dispersal of adults to another regions.

Furthermore, the destruction of twigs girdled by this species seems not to be efficient, since that O. impluviata populations continues to increase in some regions of Rio Grande do Sul state (2006 to 2013), affecting the black wattle plantations (Ono, 2015Ono, M. A., 2015. Dinâmica de infestação em Acacia mearnsii e ecologia de Oncideres impluviata (Coleoptera: Cerambycidae). Dissertação de Mestrado, Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo, Piracicaba, 68 pp.). This requires, besides legislative tactics, other integrated pest management strategies, including chemical and biological control, plant resistance, behavior control and cultural activities should be performed to manage this pest.

Supplementary material

The following online material is available for this article:

Table S1 Taxon sampling Oncideres impluviata and other species of genus Oncideres.

Acknowledgments

Authorization for activities for the scientific purpose of collecting O. impluviata specimens was obtained from the Instituto Chico Mendes de Conservação da Biodiversidade - ICMBio, number: 71112-1 and the authorization to access genetic patrimony, in the National Management System of the Genetic Patrimony and Associated Traditional Knowledge (SisGen), number: A90AB15. The Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) [grant number E-26/201.917/2020].

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