First report of non-native bamboo longhorn beetle, Chlorophorus annularis (Fabricius, 1787) (Coleoptera: Cerambycidae) from Madagascar

The bamboo longhorn beetle Chlorophorus annularis has been recently intercepted in several countries and becoming an emerging invasive species of concern. Here, we report the presence of C. annularis in Madagascar based on a verified specimen and citizen science data. The establishment of C. annularis in Madagascar is highly likely, given the multiple independent observations of the species at different locations within close proximity. Molecular evidence supports the morphological identification of the species, with the specimen collected in Madagascar showing closest affinity to a sample from Japan. However, due to limited samples and sequence length, further research is needed to determine the introduction pathway. We also briefly discuss the potential risks associated with the establishment of C. annularis in Madagascar and the need for coordinated actions to prevent its establishment


Introduction
Chlorophorus annularis (Fabricius, 1787), commonly referred to as the bamboo borer or bamboo longhorn beetle, is a widespread cerambycid native to subtropical and temperate Southeast and East Asia (Kariyanna et al. 2017).This species primarily infests various bamboo species, such as Bambusa, Dendrocalamus, and Phyllostachys.Chlorophorus annularis has also been reported to infest other plant families, including Aceraceae, Anacardiaceae, Betulaceae, Dipterocarpaceae, Hamamelidaceae, Juglandaceae, Lamiaceae, Malvaceae, Mimosaceae, Paeoniaceae, Rosaceae, Rutaceae, Ulmaceae, and Vitaceae, although some of these records have yet to be validated (Friedman et al. 2008;Monné 2005, Yu et al. 2010).To date, C. annularis has been intercepted in 12 European countries, Israel, Australasia (Australia, New Zealand, New Caledonia), Oceania (Guam, Hawaii), North America (USA, Canada), South America (Brazil, Uruguay), and South Africa (Monné 2005;Friedman et al. 2008;Suma and Bella 2018;Seidel et al. 2021).Despite being initially considered a minor economic pest in previous studies (Freidman et al. 2008;Yu et al. 2010), the repeated introduction of C. annularis to various regions and its ability to affect a wide range of host plants has elevated it to the status of an emerging invasive species of concern (Suma and Bella 2018;Seidel et al. 2021).This species is particularly concerning in areas where the bamboo industry is well-developed or bamboo is heavily utilized, as it has the potential to pose a serious threat as a major pest.In Madagascar, bamboo holds significant importance for many households in both rural and urban areas (Bystriakova et al. 2004).Furthermore, natural bamboo forests play a crucial role in Malagasy ecosystem, serving as the habitat for many endangered endemic species such as the Bamboo lemur (Hapalemur aureus Meier et al., 1987) and the Angonoka tortoise (Astrochelys yniphora Vaillant, 1885).
This report provides evidence of the presence of C. annularis in Madagascar, based on a specimen collected by the first author and verified through citizen science data.The specimen was identified using both morphological and molecular evidence.Our evidence suggests that the establishment of C. annularis in Madagascar is likely as multiple independent observations of the species were recorded at different locations within close proximity.The potential risks associated with the establishment of C. annularis in Madagascar and the likely introduction pathway are discussed.

Materials and methods
Andasibe-Mantadia National Park, a protected primary growth forest region in the eastern part of Madagascar, is situated in the middle of the largest national highway that connects Toamasina, the largest port in the country, and the capital city Antannarivo.This park boasts a diverse bamboo species composition, with 6-7 recorded species, similar to other Eastern mountain regions (Bystriakova et al. 2004).In December 2022, two specimens of C. annularis were encountered by chance at the Mantadia Lodge in Andasibe (18.918691°S: 48.415555°E).One specimen was collected, while the other managed to escape.
The collected specimen was easily identified based on their external morphologies, such as the yellowish pubescence on the dorsum, the glabrous regions forming oval markings at the anterolateral margins and reverse Y-shaped marking on the pronotum, and the characteristic patterns on elytra.Photographs of the specimen were taken using a Canon digital camera (EOS 80d) and macro lens (Canon MP-E 65 mm f/2.8 1-5x), with the use of Cognisys Stackshot for control.The specimen was deposited at the University of Antananarivo insect collection.
Additionally, we have searched the citizen science platform iNaturalist.orgusing the keywords "Chlorophorus annularis" and "Cerambycidae" and sorted relevant occurrence data of this species in Madagascar and neighboring countries.
To provide molecular evidence, we adopted both tree-based molecular identification (e.g., Wu et al. 2017) and BLAST search that has been widely used in the previous researches (Stover and Cavalcanti 2017).Genomic DNA was extracted from the thoracic muscles of the beetle using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany), and the mitochondrial cytochrome oxidase subunit I (COI) was targeted using universal primer sets.Both strands were assembled and examined using Seqman Pro v.7.1.0(DNASTAR, Inc., Madison, WI, USA) and manually adjusted with MEGA X (Kumar et al. 2016) using the amino acid translation option.The COI sequences generated in this study is deposited in Genbank (Accession No. OQ592181).Additionally, 34 public COI sequences of various Chlorophorus species, including four sequences of C. annularis sourced from China, India, and Japan, were obtained from GenBank (Supplementary material Table S1).The phylogenetic analysis was conducted using the maximum likelihood method and W-IQ-TREE software (Trifinopoulos et al. 2016).The optimal substitution model (TPM2 + F + I + G4) was determined using Modelfinder (Kalyaanamoorthy et al. 2017) based on the Bayesian information criterion.The nodal support value was determined through ultrafast bootstrap with 1,000 replicates.

Results
In total, one specimen was collected from Madagascar (Figure 1A-C), and one iNaturalist observation from Madagascar and four observations from South Africa were verified (Figure 1D, Table S2).Both records in Madagascar are from the Andasibe-Mantadia National Park region, which the records are about 11km apart from each other (Figure 1D).The collected specimen (Figure 1A) was found on the indoor side of the hotel window during the day.A bamboo forest is located right near the collection site (Figure 1B), and the hotel official confirmed that no imported bamboo products were present at the lodge.The single observation posted on iNaturalist.org,observed in January 02, 2014 (18.928932°S; 48.518827°E), was photographed while sitting on a natural substrate with no apparent association with bamboo.
The identification of the sample collected in Madagascar was supported through Blast search, with 99.09% identification and 100% query cover with the sample from Japan (LC492867.1).In the phylogenetic analysis, C. annularis formed a single clade with high supporting values (Ultrafast bootstrap values > 90) at all nodes (Figure 2).The molecular phylogenetic analysis revealed that the C. annularis specimen collected in Madagascar exhibited the closest affinity to the Japanese sample LC492867.1,which is consistent with the BLAST result.While the resulting phylogenetic tree lacks strong support for deeper nodes, it does show good supporting values for shallow nodes, which increases the credibility of species-level clustering.S2.RN2: National Road number 2, RN5: National Road number 5, Ship Icon: Toamasina, Castle Icon: Antananarivo.Photo by Seunghyun Lee.

Discussion
The discovery of C. annularis at two different location and date indicates high probability of the local establishment, since the likelihood of a random encounter of the initially introduced individuals is low given the limited survey capacity of non-resident observers.Most of the interception records of C. annularis are related to imported bamboo goods (Cocquempot 2007;Friedman et al. 2008;Suma and Bella 2018;Seidel et al. 2021), although there are some records outside its natural range that are not associated with imported bamboos (Vives 1995;Seidel et al. 2021).The absence of imported bamboo products, the presence of suitable host species nearby, and two independent observations suggest the likely establishment of C. annularis in Andasibe.Our analysis shows that the Malagasy sample is closest to the Japanese sample based on COI sequence (Figure 2), but due to limited samples and short sequences, it is difficult to conclude that it originated from Japan, especially given that this species is widely distributed in Southeast Asia (Kariyanna et al. 2017).Nonetheless, it is clear that C. annularis was introduced to Madagascar from somewhere outside the country.Assuming a human-mediated introduction via Toamasina, the province that has the largest port in Madagascar, Andasibe would be the most likely intermediate settlement, as it is only 130km away from Toamasina and located along the only national highway to the capital, Antananarivo (Figure 1D).Two possible means of species spread include slow natural dispersal through the eastern bamboo forest from Andasibe to the north and south, and human-mediated spread primarily along National Route 2 towards Antananarivo (Figure 1D).If this species is also established in Toamasina, there is also a possibility of spread along National Routes 5 to the northern coastal area (Figure 1D).
The usage of bamboo in Madagascar is diverse, covering construction, agriculture, industry, and transportation (Bystriakova et al. 2004;Ramananantoandro et al. 2013).The establishment of bamboo longhorn beetle can impede the use of this important resource and limit opportunities (see Ramananantoandro et al. 2013).Evaluation of potential negative effect that this species might cause to local bamboo species will be crucial, since Madagascar harbors more than 30 native bamboo species of conservational importance (Bystriakova et al. 2004).Our study indicates a high probability of establishment, but more direct evidence such as larvae and exit holes in nature should be identified.Investigating the current distribution of this species in Madagascar is also necessary to prevent further spread.A population genetic study with dense sampling, both in native and invaded areas, including Madagascar, will reveal the invasive history and origin of the Malagasy population(s).

Figure 1 .
Figure 1. A. Dorsal habitus of Chlorophorus annularis collected from Madagascar.B. Site of collection.Red marking below the red arrow: neighboring bamboo forest, yellow dots: exact point where two beetles were observed.C. Photograph of C. annularis upon collection.D. Distribution map of C. annularis across Africa.Orange circles: iNaturalist observations, Yellow circles: specimen collected, Numbers in the circles correspond to the observation records in TableS2.RN2: National Road number 2, RN5: National Road number 5, Ship Icon: Toamasina, Castle Icon: Antananarivo.Photo by Seunghyun Lee.

Figure 2 .
Figure 2. Phylogenetic relationship using IQ-tree.Nodes with high Ultrafast bootstrap support values (> 90) marked with black dots.