IsAedes nr. albopictussynonymous withAedes pseudalbopictus?

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View ORCID Profile Om P. Singh , Radhika Mittal , Rajnikant Dixit doi: https://doi.org/10.1101/2025.06.28.661682 Om P. Singh 1 ICMR-National Institute of Malaria Research , Dwarka, New Delhi-110075 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Om P. Singh For correspondence: dr.opsingh{at}gmail.com Radhika Mittal 1 ICMR-National Institute of Malaria Research , Dwarka, New Delhi-110075 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rajnikant Dixit 1 ICMR-National Institute of Malaria Research , Dwarka, New Delhi-110075 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Preview PDF Abstract A recent study reported a putative cryptic species of Aedes albopictus , provisionally named Aedes nr. albopictus , from Tripura, India, based on molecular data from mitochondrial cytochrome c oxidase subunit I (COI) and nuclear internal transcribed spacer 2 (ITS2) regions. However, our reanalysis, incorporating COI and ITS2 sequences of the co-occurring species Aedes pseudalbopictus and employing maximum likelihood phylogenetic analysis, albeit with a limited number of available sequences, suggests that this lineage is conspecific with Ae. pseudalbopictus . Given the morphological similarities and overlapping habitat use of Ae. albopictus and Ae. pseudalbopictus in India and Southeast Asia, we hypothesize that the reported cryptic species could potentially be attributed to Ae. pseudalbopictus . Our findings underscore the need for comprehensive taxonomic assessments, integrating morphological and molecular analyses of all sympatric Stegomyia species, to ensure accurate species delimitation and support effective vector surveillance and control. Background Biswas et al . (2025) [ 1 ] recently identified Aedes nr. albopictus , a putative cryptic species sympatric with Aedes albopictus in Tripura, India, based on molecular analysis of mitochondrial cytochrome c oxidase subunit I (COI) and nuclear internal transcribed spacer 2 (ITS2) regions. Their findings underscore its possible role in arbovirus transmission, with significant implications for vector control strategies. Biswas et al . noted that Ae. nr. albopictus shares genetic similarities with cryptic Ae. albopictus lineages previously reported by Minard et al . (2017) in Vietnam [ 2 ] and Guo et al . (2018) in China [ 3 ], suggesting a broader regional presence of this lineage. Biswas et al . asserted that Ae. nr. albopictus is “genetically distinct from Ae. albopictus, Ae. subalbopictus, Ae. pseudalbopictus , Ae. flavopictus, Ae. aegypti , and Ae. vittatus ” [ 1 ] but their claim of genetic distinction from Ae. pseudalbopictus is unsubstantiated, as their phylogenetic analyses did not include genetic data Ae. pseudalbopictus . Biswas et al .’s assertion appears to be an assumption, as their study lacks both molecular sequences and detailed morphological comparisons with this species. This critical omission undermines the validity of their conclusion regarding Ae. pseudalbopictus , leaving open the possibility that Ae. nr. albopictus may be conspecific with or closely related to Ae. pseudalbopictus , particularly given their morphological and ecological similarities in shared habitats across India and Southeast Asia [ 4 - 6 ]. Recently, Hide et al . (2024) [ 7 ] also described Aedes unalom sp. nov. as a new Stegomyia species in Cambodia, distinguishing it from Ae. albopictus and other species including Ae. pseudalbopictus using COI, 5.8S-ITS2, and morphological traits, such as an interrupted white stripe on the hind femur [ 7 ]. While Hide et al . included Ae. pseudalbopictus sequences from their Cambodian collections, they did not include sequences from the cryptic lineages reported by Minard et al . and Guo et al . in their phylogenetic analyses of Ae. unalom , leaving the phylogenetic relationship between new Ae. albopictus lineage (designated as Ae. nr. albopictus by Biswas et al .) and Ae. pseudalbopictus unresolved. Given the morphological and ecological overlap between Ae. albopictus and Ae. pseudalbopictus , we hypothesize that Ae. nr. albopictus and the cryptic lineages reported by Minard et al . and Guo et al . may be conspecific with Ae. pseudalbopictus . To test this, we conducted a maximum likelihood phylogenetic analysis, incorporating COI and ITS2 sequences of Ae. nr. albopictus, Ae. pseudalbopictus (five COI from BOLD Systems [ 8 ] originating from China [ 9 ] and Cambodia [ 7 ] and three sequences of ITS2 [ 7 ]), Ae. unalom, Ae. albopictus , and Ae. aegypti as an outgroup, to resolve their genetic relationships and clarify species boundaries. Methods Sequences of COI and ITS2 for Aedes nr. albopictus from Tripura [ 1 ], Ae. pseudalbopictus from Cambodia [ 7 ] and China [ 9 ], Ae. unalom [ 7 ] were retrieved from GenBank. COI sequences of Ae. albopictus , and Ae. aegypti were obtained from VectorBase and ITS2 sequences from GenBank. To ensure data quality, flanking sequences potentially containing primer-derived region or sequencing artifact were trimmed in case of COI sequence with accession number JQ728197 [ 9 ]. Sequences for each marker were aligned independently using ClustalW in MEGA X v10.2.2 [ 10 ] with default parameters (gap opening penalty: 15, gap extension penalty: 6.66) and manually adjusted to ensure accuracy. Alignments were trimmed to uniform lengths for downstream analysis. Phylogenetic analysis was conducted using IQ-TREE2 [ 11 ] under a maximum likelihood framework. Nucleotide substitution models for COI and ITS2 datasets were selected with ModelFinder Plus based on the Akaike Information Criterion (AIC), Corrected AIC (AICc), and Bayesian Information Criterion (BIC). For the COI dataset, the AIC and AICc favored TVM+F+G4, but the K3Pu+F+G4 model was selected based on BIC, incorporating unequal base frequencies (+F) and gamma-distributed rate variation with four categories (+G4). For the ITS2 dataset, the AIC favoured K3P+I, while AICc and BIC selected K2P+G4, which was chosen as the best-fit model per BIC, incorporating Kimura’s two-parameter model with gamma-distributed rate variation (+G4). Results and Discussion Our phylogenetic analysis revealed that Aedes nr. albopictus , along with cryptic lineages reported by Minard et al . and Guo et al . cluster with Aedes pseudalbopictus for both loci—COI and ITS2 loci ( Figure 1 , panel A and B, respectively). Furthermore, the ITS2 sequences of Ae. nr. albopictus , despite the known intragenomic variation in ITS2, were 100% identical to those of Ae. pseudalbopictus reported from Cambodia [ 7 ]. Notably, both studies [ 1 , 7 ] identified two identical haplotypes each differing only in a number of GC repeats (microsatellite), a pattern also observed in ITS2 of other mosquito species, Anopheles stephensi [ 12 ]. These findings strongly suggest that the proposed cryptic species may be Ae. pseudalbopictus . Further, our inclusion of Ae. unalom sequences in analysis confirms a distinct clade from Ae. albopictus and Ae. pseudalbopictus / Ae. nr. albopictus ( Figures 1 ), supporting its status as a novel species. Download figure Open in new tab Figure 1. Maximum likelihood phylogenetic trees based on COI (panel A) and ITS2 (panel B) sequences of Aedes species. Ae. nr. albopictus from Tripura clustered with Ae. pseudalbopictus from Cambodia and China in both trees. Only bootstrap values ≥70% (1,000 replicates) are shown for clarity; scale bars indicate substitutions per site. The misidentification of Ae. pseudalbopictus as Ae. albopictus likely stems from their morphological similarity. Huang (1968) [ 5 ] described subtle diagnostic characters, such as the presence of broad, flat white scales on the lateral margin of the scutum in Ae. albopictus , contrasted with narrow, curved white scales in Ae. pseudalbopictus . These differences are easily overlooked without meticulous examination. Additionally, Huang [ 5 ] noted that Ae. pseudalbopictus was frequently mistaken for Ae. albopictus , a confusion compounded by their sympatric distribution [ 5 - 6 ]. While Hide et al . explicitly identified Ae. pseudalbopictus in their collections using standard morphological taxonomic keys [ 14 - 15 ], the studies reporting new species [ 1 - 3 ] lacked specific details on the identification keys used to identify mosquito species. This lack of detailed methodology in those studies, combined with our phylogenetic findings, suggests that Ae. pseudalbopictus specimens may have been previously classified as a novel cryptic species of Ae. albopictus . On the other hand it may be possible that Wang et al . (2012) or Hide et al . (2024) misidentified their specimens as Ae. pseudalbopictus but their consistent morphological and molecular differentiation of Ae. pseudalbopictus makes this less likely. The misidentification of Ae. pseudalbopictus as a cryptic species of Ae. albopictus has significant implications for vector surveillance and control in dengue- and chikungunya-endemic regions. Ae. albopictus is a well-documented vector, while the vectorial capacity of Ae. pseudalbopictus remains less studied. Misidentification could lead to inaccurate assessments of vector distribution, abundance, and behaviour, potentially skewing epidemiological models and control strategies. For example, differences in insecticide resistance or host-feeding preferences between Ae. albopictus and Ae. pseudalbopictus could affect the efficacy of interventions. Our analysis, by robustly confirming the conspecificity of Ae. nr. albopictus with Ae. pseudalbopictus and clarifying Ae. unalom ’s distinctiveness, underscores the need for integrated taxonomic approaches that combine multi-locus molecular data with rigorous morphological examinations based on established keys, such as those by Huang (1972) [ 4 ] and Barraud (1934) [ 6 ]. While our analysis is robust, it is limited by the small number of Ae. pseudalbopictus sequences available (five COI and three ITS2 sequences); however, the consistent clustering of Ae. nr. albopictus with Ae. pseudalbopictus across both loci provides strong support for our conclusions. Additional sequencing of Ae. pseudalbopictus populations from India and Southeast Asia would strengthen our conclusions. To prevent taxonomic errors, we propose a standardized framework for Stegomyia species delimitation: multi-locus sequencing including mitochondrial and nuclear markers; morphological examinations using established keys; comprehensive sampling of all sympatric Stegomyia species; and, integration of ecological data to understand species boundaries. Special attention is required when analyzing rDNA, particularly ITS2, due to intragenomic variation due to indel or SNPs [ 12 , 16 - 17 ]. Such variation can lead to ambiguous sequence chromatograms or alignment errors, potentially inflating perceived genetic divergence. Hide et al . have also reported mixed bases in ITS2 of Ae. pseudalbopictus which can be explained by presence of indel caused by variation in number of GC-repeats. This framework would enhance taxonomic accuracy and support effective vector control in regions where Stegomyia species are public health threats. Conclusion The evidence presented here indicates that the reported new cryptic species Aedes nr. albopictus is conspecific to Ae. pseudalbopictus . This finding highlights the critical need for rigorous taxonomic studies that integrate morphological examinations with comprehensive molecular analyses, including all known sympatric species, before proposing new species. Accurate species identification is essential for effective vector surveillance and control, particularly for Ae. albopictus and Ae. pseudalbopictus , which are vectors of dengue and chikungunya in endemic regions. Author contributions OPS: data analysis and writing first draft; RM: data analysis; RD: contributed to the manuscript. Competing Interests The authors declare that they have no known competing interests Acknowledgement The authors acknowledge the supportive research environment and resources provided by the Indian Council of Medical Research (ICMR) and the National Institute of Malaria Research (NIMR) that contributed to the development of the ideas presented in this manuscript. The views expressed are those of the authors and do not represent the official stance of ICMR or NIMR. References 1. ↵ Biswas S , Rajkonwar J , Jena SR , Gogoi P , Nirmolia T , Vinayagam S , Hazarika G , Sihag AK , Borah B , Pebam R , Kaur H , Baruah K , Narain K , Subbarao SK , Bhattacharyya DR , Borkakoty B , Bhowmick IP . Detection of a sympatric cryptic species mimicking Aedes albopictus (Diptera: Culicidae) in dengue and Chikungunya endemic forest villages of Tripura, India, posing a daunting challenge for vector research . Sci Rep . 2025 ; 15 ( 1 ): 14237 . doi: 10.1038/s41598-025-96146-9 . 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