Lateral and dorsal views of female (a) and male (b). c-f. Feeding damage to leaves and quiotes of Agave inaequidens by adults. d. Branches of the quiote with damaged floral pods. g-h. Larvae feeding inside Agave inaequidens quiotes.

To strengthen this study, we also used molecular data to confirm the identity of P. polymitus and provide an additional tool for its identification. For this, 10 adults from the same samples were preserved in 96 % alcohol for molecular analysis. The molecular characterization of the spotted agave weevil involved sequencing the mitochondrial gene Cytochrome Oxidase subunit I (COI) from two adults of approximately 6.5 to 8.5 mm in length. We amplified the COI gene using primers designed by Folmer et al. (1994) (LCO-1490f and HCO-2198r). DNA extraction was conducted with a 2 % CTAB solution (Doyle, 1991) and sodium acetate buffer, while purification was performed using the Wizard® Genomic DNA Purification Kit. For DNA quantification, we used spectrophotometry with a NanoDrop 2000 (Thermo Scientific RMA, Wilmington, Delaware, USA). PCR amplification of the product was carried out under specific conditions. The master mix included 7.86 μL of ultra-pure water, 3 μL of 10X buffer, 0.6 μL of dNTPs, 0.18 μL of LCO-HCO primers, 0.18 μL of Taq DNA polymerase, and 3 μL of DNA (20 ng). The cycling program featured an initial denaturation at 94 °C for 3 minutes, followed by 35 cycles of 94 °C for 30 seconds, annealing at 45 °C for 1.5 minutes, and 72 °C for 1 minute for the final extension, utilizing a C1000 Touch thermal cycler (Bio-Rad, Foster City, California, USA). Amplified products were visualized by electrophoresis on a 1.5 % agarose gel (SeaKem, Lonza, Greenwood, South Carolina, USA), and the results were documented using an Infinity system 3026/WL/LC/26 MX X-Press (Vilber Lourmat, Deutschland GmbH, Eberhardzell, Germany). For DNA sequencing, the amplified products were cleaned with ExoSAP (Affymetrix, Santa Clara, California, USA) and sequenced using a 3130 DNA 4-capillary Genetic Analyzer (Applied Biosystems, Foster, California, USA). When the sequences were obtained, they were assembled using BioEdit v.7.0.5 software (Hall, 1999) and compared with multiple zygopines sequences through the Basic Local Alignment Search Tool (BLASTN, developed by Altschul et al. [1997] on the National Center for Biotechnology Information [NCBI] platform). This alignment method incorporated gaps (Higgins et al., 2005). The generated sequence was compared with those of American Zygopini species available at NCBI, using the Copturus aguacatae Kissinger (Lechriopini) sequence (from Mexico) to test the monophyly of the group and as an external conoderine (Table I). A phylogenetic analysis was performed with MEGA v. 11 software, using the neighbor-joining tree method (with 1000 bootstrap replicates). Phylogenetic distances were calculated using the Jukes-Cantor method (1969) and were measured in the number of base substitutions per site. All ambiguous positions were removed from each sequence pair (pairwise deletion option).

Table I

Note: Length sequences in all species were between 600 and 660 base pairs (bp).

This finding constitutes the first record of adults and larvae of P. polymitus feeding on leaves and floral scapes of maguey alto, in the state of Michoacán. Notches or punctures in the leaves and floral scapes created by adults, as well as galleries in the floral scapes created by larvae (Fig. 1c-h), are consistent with the damage recorded on A. angustifolia, A. cupreata, A. duranguensis, A. kerchovei, and A. tequilana plants (Brena-Bustamante, 2012; González-Hernández et al., 2015; Reyes-Muñoz et al., 2020). Current evidence suggests that while P. polymitus does not represent a serious threat to cultivated agave species in Michoacán; its feeding behavior on the quiotes of maguey alto may potentially impact seed production and sexual reproduction in wild populations.

In this study, we produced the first sequence of P. polymitus with COI gene, which consists of approximately 656 bp. This sequence was submitted to the GenBank database under accession number PV790451. Molecular phylogenetic analysis revealed a close evolutionary relationship between P. polymitus from Mexico and Peltophorus sp. from the United States, supported by a high bootstrap value (99/100). This analysis also showed that the zygopine species Hemicolpus abdominalis Hustache, Cylindrocopturus sp., and Zygops wiedii (Germar) are lineages related to Peltophorus , which evolved early from a common ancestor (Fig. 2). The presence in Mexico of species belonging to the genera Hemicolpus, Cylindrocopturus , and Zygops (Maes & O´Brien, 1990; Hespenheide, 2018; Palemón-Alberto et al., 2022) suggests that P. polymitus and those species probably share the same biogeographic origin.

Figure 2.

Analysis showing the evolutionary relationships between Peltophorus polymitus and other zygopine species. The numbers on the nodes indicate bootstrap support values (based on 1000 replicates). The scale represents genetic distance.

Within the Zygopini tribe there are at least 83 species known from the New World (Anzaldo, 2017), relatively few of them have been sequenced. This is probably the reason why zygopine species have been poorly represented in molecular phylogenies (Anzaldo, 2019). Although it is too early to draw conclusions about Zygopini phylogeny, we conclude that further studies involving sequence data from zygopine species are required to clarify the tribe´s taxonomic status and evolutionary relationships.