Biodiversity Data Journal :
Research Article
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Corresponding author: Steffan Hansen (shansen.rsa@gmail.com)
Academic editor: Jennifer C. Girón Duque
Received: 24 Mar 2021 | Accepted: 09 Jun 2021 | Published: 30 Jun 2021
© 2021 Steffan Hansen, Pia Addison, Laure Benoit, Julien Haran
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Hansen S, Addison P, Benoit L, Haran JM (2021) Barcoding pest species in a biodiversity hot-spot: the South African polyphagous broad-nosed weevils (Coleoptera, Curculionidae, Entiminae). Biodiversity Data Journal 9: e66452. https://doi.org/10.3897/BDJ.9.e66452
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Polyphagous broad nosed weevils (Curculionidae: Entiminae) constitute a large and taxonomically challenging subfamily that contains economically significant agricultural pests worldwide. South Africa is a hot-spot for biodiversity and several species of indigenous and endemic genera of Entiminae have shifted on to cultivated plants, with some being phytosanitary pests. The sporadic pest status of many species (where the species has an occasional economic impact on the agricultural industry, but is not encountered often enough that is is readily recognisable by researchers and agricultural extension workers) and the presence of pest complexes and cryptic species represent an identification challenge to non-specialists. Furthermore, no comprehensive identification tools exist to identify immature stages that may be found in crops/soil. In this paper, a curated barcoding database with 70 COI sequences from 41 species (39 Entiminae, 2 Cyclominae) is initiated, to assist with the complexity of identification of species in this group.
Entiminae, PBNW, agricultural pests, identification, COI barcode
Curculionidae Latreille, 1802 is a hyperdiverse family of beetles (Coleoptera), containing approximately 17 subfamilies and more than 51000 described species (
Most entimine weevil species have loose host plant associations, being oligo- or polyphagous in the larval and adult stages; several species constitute some of the most economically important agricultural pests worldwide (
South Africa has a rich native fauna of Entiminae, notably with considerable species diversity across a number of tribes, including those such as Oosomini Lacordaire, 1863, Embrithini Marshall, 1942, Tanyrhynchini Schönherr, 1826 and Otiorhynchini Schönherr, 1826 that contain agricultural pest species (
Several PBNW species have successfully spread and established outside their native range, where they may become important pests. To current knowledge, three South African native species have become pests overseas. Phlyctinus callosus is a major introduced pest on vegetable and orchard/vineyard crops in New Zealand, Tasmania and Western Australia (
Barcode sequences of the mitochondrial gene cytochrome oxidase I (COI) have been shown to be an accurate and powerful tool in species identification of most animals, including insects (
This study initiates a curated barcode database of PBNW found in crops and disturbed habitats in South Africa to assist in rapid and robust identification of species, irrespective of sex or life stage of specimens.
Specimens were collected alive from agro-ecosystems with recorded damage of weevils, disturbed roadside habitat and natural environments in South Africa (with focus on the Western Cape Province), between 2017 and 2020 (Suppl. material
Species were identified, based on external morphology using the keys and descriptions of
The right hind leg of each prepared specimen was used for DNA extraction. The DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). PCR amplification was done for the COI standard barcoding region (
PCR primers and conditions. M13 tails from Ivanova et al. (2007) are in bold.
Gene |
Primer |
Primer Sequence |
Annealing temperature |
Reference |
COI |
HCO2198 |
CAGGAAACAGCTATGACTAAACYTCDGGATGBCCAAARAATCA |
52°C |
modified in |
CAGGAAACAGCTATGACTAAACYTCAGGATGACCAAAAAAYCA |
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CAGGAAACAGCTATGACTAAACTTCWGGRTGWCCAAARAATCA |
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LCO1490 |
TGTAAAACGACGGCCAGTTTTCAACTAAYCATAARGATATYGG |
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TGTAAAACGACGGCCAGTTTTCAACWAATCATAAAGATATTGG |
The barcode sequences were aligned and manually checked using CodonCode Aligner ver. 3.7.1 (CodonCode Corporation, Centerville, MA, USA), verifying the absence of pseudogenes using standard detection methods (
The collection and voucher data of all specimens used in this study, including identification of specimens, images, primer cocktails used in amplification, sequences and trace files are deposited at BOLD (
http://dx.doi.org/10.5883/DS-CURSA1 (
The COI sequence data of the Phlyctinus spp. (JHAR0941-0101, JHAR0755-0101, JHAR2288-0101, JHAR2290-0101, JHAR2086-0101, JHAR2101-0101, JHAR0822-0101, JHAR1353-0101, JHAR2252-0101, JHAR2252-0101, JHAR0819-0101, JHAR1016-0101, JHAR2173-0101, JHAR1304-0101, JHAR1195-0101, JHAR0973-0101, JHAR1300-0101, JHAR1082-0101, JHAR2264-0101) and Oosomus sp. (JHAR01073-0101) obtained in
The COI sequence data obtained by
A total of 70 COI barcode sequences > 500 bp, from 41 morphospecies (39 Entiminae, 2 Cyclominae) are presented in this study. Of these, all have been identified to genus level, 29 to species level and 12 of the morphospecies were either not possible to identify or can not be identified with certainty (Suppl. material
Of the 14 species for which more than one sequence was obtained, eight had K2P intraspecific variation ≥ 1%. Of these, five had intraspecific K2P variation of 2% or higher and were divided into haplotypes (Suppl. materials
Amongst the present dataset of barcode sequences, the mean intraspecific distance is 2.1% (max 9.2%, min 0.0%), the mean of the maximum intraspecific distances is 2.2% and the mean distance to the nearest heterospecific is 13.9%. The smallest interspecific distance is 4.3%. There is some overlap between intra- and interspecific distances and a small barcoding gap (mean distance to nearest heterospecific being 6.3-fold higher than the mean of maximum intraspecific distances) with this conservative method of calculation. Out of the 14 species for which two or more sequences were obtained, six had maximum intraspecific distances of ≤ 10% that of the distance to the closest heterospecific and 10 had intraspecific distances ≤ 12% that of the distance to the closest heterospecific. Except for Phlyctinus xerophilus, the distance to the nearest heterospecific was always larger than the maximum intraspecific value.
The NJ tree of the obtained sequences show distinct, non-monopheletic groupings amongst species in the genera Eremnus Schönherr, 1826, Afroleptops Oberprieler, 1988 and Sciobius and non-monopheletic groupings amongst genera and/or species in the tribes Tanyrhynchini (genera Eremnus, Tanyrhynchus Schönherr, 1826 and Afroleptops in this study), Embrithini (genera Afrophloeus Borovec and Oberprieler, 2013 and Ellimenistes Boheman, 1843, in this study) and Otiorhynchini (genus Sciobius in this study) (Fig.
Barcoding PBNW species from South Africa provides a valuable tool in rapidly and robustly identifying species of potential economic concern, including highly sporadic pest species that only rarely rise to population levels of economic concern. This study also reveals some challenges with the application of this approach to PBNW in South Africa. A small barcoding gap between the mean of maximum intraspecific genetic distance and the mean distance to closest heterospecific is observed, even without comprehensive sampling and multiple sequences per species for most of the PBNWs in this study. However, this result is not unexpected for Coleoptera (
The greatest challenge to successful barcoding of PBNW is undoubtedly inadequate taxonomic coverage of this group. The taxonomy of South African Entiminae is complex and many tribal/genus/species classification is still in the process of being resolved, with new species, genera and even tribes constantly being described (
The NJ tree, calculated using the COI sequences, itself provides some preliminary signs that some South African genera and tribes, previously described only on morphological characteristics, might not form naturally monophyletic groupings (Fig.
A further complication is produced by the potential presence of parthenogenic lineages in PBNWs in South Africa. Of the tribes treated here, Otiorhynchini and Naupactini are well known for containing species with parthenogenic lineages and the exotic species Naupactus leaucoloma, Pantomorus cervinus (Entiminae: Naupactini) and Listroderes costirostris (Cyclominae: Rhythirrinini) are confirmed parthenogenic species (
The fourth challenge is insufficient sampling to adequately cover genetic diversity across the target groups' distributional range (
A curated barcoding database (as on BOLD and GenBank) will enable the addition of sequences and species with every new study, allowing for taxonomic amendment. It can include location data, such that the robustness and accuracy in identifying specimens of the group of interest can continuously be improved (
The ability to accurately identify agricultural pest insects is key in their successful management. The PBNWs of South Africa are a diverse and taxonomically understudied group that contains a number of important pest species and strong potential for future pest emergence, due to their polyphagous nature. Their identification provides a challenge for non-specialists, an issue compounded by the presence of cryptic species in some taxa. The present curated barcode database provides a quick and simple identification tool that allows for a better understanding of their taxonomy, biology and distribution. This database aims at being continually expanded and improved as more species and specimens are sequenced and as taxonomic work progresses and improves current classifications, increasing the diagnostic power of barcode identifications in this challenging group.
This project was funded in partial fulfilment of a thesis by HortGro Pome and Hortgro Stone and the South African Table Grape Industry (SATI). We thank the Iziko and SANC Pretoria Museum’s for the loan of valuable reference specimens, from which many of the identifications were made. We thank Noémie Hévin (CIRAD) for creating the sampling point map. This project would not have been possible without the hospitality of growers and land-owners who graciously allowed us to collect on their properties. Specimens were collected under permits from Cape Nature, Ezemvelo KZN Wildlife and Cape Research Centre SAN Parks; permits numbers CN44 30 4229, OP 1382/2019 and CRC/2019-2020/012--2012/V1, respectively.
Voucher specimen BOLD codes, GenBank accession codes (where applicable) for COI sequences and collection data of the polyphagous broad-nosed weevils in the study
The Kimura-2-Parameter pairwise genetic distances between COI sequences for polyphagous broad-nosed weevils obtained/used in study