Western Ghats Endemic Euphaea fraseri Forms the Basal Lineage of the Euphaea Clade, Supporting an Out-of-India Origin for the Genus

Western Ghats Endemic Euphaea fraseri Forms the Basal Lineage of the Euphaea Clade, Supporting an Out-of-India Origin for the Genus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Western Ghats Endemic Euphaea fraseri Forms the Basal Lineage of the Euphaea Clade, Supporting an Out-of-India Origin for the Genus Joel Philip, Archita Sharma, Arajush Payra, Arati Joshi, Pankaj Koparde This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8452342/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The Euphaea damselflies, highly remarkable for their red–black coloration, represent specialized inhabitants of fast-flowing montane streams and are distributed across the Indo-Malayan biogeographical region. However, the evolutionary history and spatial diversification of the genus are not well resolved, especially with respect to the origins of Western Ghats endemics relative to the Southeast Asian taxa. Herein, we present the first complete mitochondrial genome of the Western Ghats endemic Euphaea fraseri . The assembled mitogenome is 15,267 bp in length and comprises 13 protein-coding genes (PCGs), 22 tRNA genes, and two ribosomal RNA genes (12S and 16S rRNA), following the canonical mitochondrial organization of Zygoptera. Phylogenetic analyses based on (i) the whole mitogenome and (ii) a concatenated dataset of COI and 16S rRNA consistently recovered a well-supported Western Ghats Euphaea clade. E. fraseri is a basal relative to all sampled congeners and thus represents a deeply divergent lineage distinct from Southeast Asian Euphaea species. Divergence time estimation places the split between E. fraseri and the remaining Euphaea species around 15–20 Ma (Miocene), indicating an ancient lineage that predates the diversification of Southeast Asian taxa. The basal positioning and Miocene divergence of E. fraseri thus provide strong support for an "Out of India" hypothesis, wherein the Western Ghats lineage represents an early-branching remnant from which later dispersal and radiation into Southeast Asia may have occurred. These results add new genomic and temporal data that help elucidate the deep evolutionary history of Euphaea and emphasize the biogeographic importance of the Western Ghats as a refuge for ancient odonate lineages. Further studies using a larger genomic dataset, including missing taxa, will provide insights into the evolutionary relationships between other endemic and endangered groups from the Western Ghats. Biological sciences/Ecology Earth and environmental sciences/Ecology Biological sciences/Evolution Biological sciences/Genetics Biological sciences/Molecular biology Biological sciences/Zoology Phylomitogenomics Biogeography Damselfly Montane Streams Miocene Figures Figure 1 Figure 2 Figure 3 Introduction Odonates (dragonflies & damselflies) are an ancient order of insects whose origin dates back to around 300 million years ago during the Carboniferous period (Grimaldi and Engel, 2005 ). Considered as one of the first few ancient insect orders to take flight, odonates today contribute to multitudes of ecosystem services, such as pest control and nutrient cycling, and inspire artists and storytellers around the world as a wetland-associated symbol of freedom and power (Samways, 2008 ). During the Carboniferous period, odonate-like ancestors, collectively referred to as Protodonata, dominated the aerial predatory niche; these forms, though lacking in several defining characteristics of modern odonates, represent an important stem in the odonate evolution. Recent studies using the mitochondrial data along with fossil calibrations of dragonflies may have originated in the Triassic period, with the Cretaceous period emerging as the critical period for the initial radiation of the main Odonata lineages (Huang et al. 2025 ). Family Euphaediae, one of the most unique zygoptera families, with nine genera and 80 species, is widely distributed in the Palaarctic, Australasia, and Asia (Paulson et al., 2025 ). The members of this family are known for their habit of occupying fast-flowing streams in montane areas (Kalkman et al., 2020 ). The origin of the family Euphaeidae can be traced back to the early Eocene, supported by the fossil records of individuals from its sister groups Eodichromatinae and Epallaginae, which indicates that diversification of the damselflies within the superfamily Calopterygoidea occurred during this period, highlighting that the Euphaid lineage already existed during this period (Bechly, 1998 ; Archibald and Cannings, 2021; Archibald et al., 2024 ). The Indo-Malayan region, one of the eight major biogeographic realms of the world, hosts 36 species of the family (Paulson et al., 2025 ). The region is dominated by tropical and subtropical moist forests. The majority of the species of the Euphaea genus are distributed throughout Southeast Asian countries. India is home for eight Euphaea species, of which five species namely, Euphaea cardinalis (Fraser, 1924); Euphaea dispar Rambur, 1842; Euphaea fraseri (Laidlaw, 1920); Euphaea pseudodispar Sadasivan & Bhakare, 2021 and Euphaea thosegharensis Sadasivan & Bhakare, 2021 and the recently discovered Euphaea wayanadensis Anooj, Susanth & Sadasivan, 2025 are restricted to the Western Ghats region (Anooj et al. 2025 ) and rest two species, Euphaea masoni Selys, 1879 and Euphaea ochracea Selys, 1859 are confined in the Northeastern states of India (Bhakare et al. 2021 ). The Malabar Torrent Dart Euphaea fraseri is endemic to the Western Ghats biodiversity hotspot distributed in the states of Tamil Nadu, Kerala, Karnataka, Goa, and Maharashtra (Mitra 2002 ; Ranganekar et al. 2010; Koparde et al. 2015 ; Subramanian et al. 2018 ; Mujumdar et al. 2020 ; Sawant et al.2022). The Euphaea species of the Western Ghats all are similar in appearance and are characterized by the broadly opaque black coloration with metallic iridescence on the apices of the hindwings of the males, shorter and rounded forewings; the 10th abdominal segment has a distinct keel; discoidal cell is transversed; the anal appendages is present with a forcipate cerci and tiny paraprocts (Fraser, 1934 ). The Malabar Torrent Dart Euphaea fraseri is a habitat specialist occupying areas of fast-flowing streams in the montane wet forests of the Western Ghats (Koparde et al., 2015 ; Subramanian et al., 2018 ). It is a red and black-colored damselfly having characteristic frons and thoracic patterns. The species is classified as Least Concern by the IUCN Red-List; however, there is little or no information available on its population size and trend, total geographical range, and area of occupancy (Kakkasery, 2011 ). Habitat modification and degradation are reported to be common threats to the endemic Odonata fauna of the Western Ghats (Koparde et al., 2015 ; Subramanian et al., 2018 ). Literature on the evolutionary relationships of endemic odonates of India and Indo-Malaya is scarce. To investigate the evolutionary origins of the unique Western Ghats endemic damselfly, here, we generated the complete mitochondrial genome of Euphaea fraseri . The phylogenomic study based on mitogenomes provided insights into the evolutionary history and phylogenetic positioning (Huang et al., 2025 ) of the species of the Euphaea genus. Further, we performed a phylogenetic analysis (COI & 16s rRNA) with a larger set of species belonging to the Euphaea genus to uncover patterns in diversification. The information provided here will be useful in future studies on population genetics, evolutionary biology, and phylogenomics. Materials and Methods Taxon Sampling, DNA Extraction & Next Generation Sequencing Specimens were collected from a field site in Maharashtra State, India. Specimens were stored in 90% alcohol and transported to the lab. Collected specimens were identified with the help of taxonomic keys of Fraser (1934). Necessary collection permits were obtained from the State Forest Department and the Maharashtra State Biodiversity Board. Total genomic DNA was extracted from the specimens using DNease Blood and Tissue Kit (Qiagen, Hilden, Germany) using the standard protocol. For library preparation, we used the TruSeq DNA Nano kit. NGS was performed using the Illumina Novaseq6000 platform (150 bp paired reads). For the library, 10 GB of raw data were obtained. NGS Data Quality Assessment and Processing FastQC was performed on the raw R1 and R2 reads obtained for the sample. The data obtained after the NGS run were filtered and curated. A total of 23,385,231 R1 and 23,385,231 R2 raw reads were generated, of which 44,536,502 good-quality reads were used for further analysis. We used MitoZ for assembly, annotation, and visualization of the mitogenome (Meng et al. 2019). A contig of 15267 bases was assembled, having a minimum depth of 600x. To visualize the entire mitochondrial genome with annotated features, a circos plot was created using Circos (Krzywinski et al. 2009). We further used the MITOS webserver to create a linear plot (http://mitos.bioinf.uni-leipzig.de/). Briefly, fastp version 0.22.0 was used to clean raw reads. Cleaned reads were assembled using Megahit. The HMMER program was used to extract candidate mitochondrial sequences and further filtered them by taxonomic assignment. Sequences belonging to non-target taxon were discarded. The BWA program was used to create reference genome index files. The assembled genome using the Megahit program from the MitoZ toolkit was used as a reference genome in this case, as no closely related genomes were available. The BWA tool was used to map pair-end reads on the reference genome. Resulting alignment metrics were converted into BAM files using Samtools. Paired-end reads were assembled using the PEAR software package (https://cme.h-its.org/exelixis/web/software/pear/). PEAR evaluates all possible paired-end read overlaps without requiring the target fragment size as input. In addition, it implements a statistical test for minimizing false-positive results. The quality of assembled reads was again checked by FastQC. Clean reads were then filtered based on the quality of reads and length of the reads using the fastp package (Chen et al. 2018). COI and 16s rRNA Sequencing COI and 16s gene amplification was done for four Euphaea species ( E. fraseri, E. pseudodispar, E. cardinalis, E. dispar )collected from the Western Ghats for the construction of the concatenated tree. The COI gene amplification of the specimens was done using LCO primer (Forward: 5'-GGTCAACAAATCATAAAGATATTGG-3') and HCO primer (Reverse: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3') (Folmer et al., 1994). The 16s rRNA gene amplification was done using 16sf primer (Forward: 5′-CGGCCTGTTTAACAAAAACAT-3′) and 16sr (Reverse: 5′-CCCGGTTTGAACTCAGATCATGT-3′) (Hasegawa & Kasuya, 2006). The cycle conditions for both the samples were 95 ◦ C for 2 min followed by 40 cycles of 95 ◦ C for 30sec, 50 ◦ C for 30sec, and 72 ◦ C for one min. Mitophylogenomic and Phylogenetic Analyses The generated mitochondrial genome of Euphaea fraseri was aligned with 14 other complete mitochondrial genomes from various odonate species obtained from the GenBank database (Supplementary Table 1, Huang et al., 2025). The alignment was performed using MAFFT, tree construction based on the Maximum Likelihood approach (1000 bootstraps), and visualization was carried out in IQ-TREE v2. For fossil calibration, we used three secondary calibration points as used in earlier studies (Huang et al., 2025). A dated mitogenome phylogeny was constructed using treePL (Stephen et al., 2012). Individuals of the sub-order Anisoptera were set as outgroups ( Polycanthagyna melanictera, Periaeschna flinti, Davidius lunatus, Ictinogomphus pertinax ). The lab-generated sequences of COI and 16S rRNA genes from four Euphaea species were examined and manually curated using Chromas v2.6.6 (Technelysium Pty Ltd), and consensus sequences were generated in MEGA v11.0.13 (Kumar et al., 2024). Sequences for other species were retrieved from the GenBank database (Supplementary Table 2). All sequences were translated into protein-coding format to check for the presence of stop codons. The analysis was based on the concatenation of the COI and 16S rRNA genes. A total of eighteen sequences (four lab-generated and fourteen from GenBank) were aligned using the ClustalW tool with default settings for the concatenated (COI + 16S rRNA) dataset. The phylogenetic tree was constructed using the maximum likelihood method with the ML + rapid bootstrap option (1000 replicates) implemented in RAxMLGUI v2.0.15 (Stamatakis, 2014), with two species from the family Libellulidae designated as outgroups ( Trithemis aurora, Crocothemis servelia ). A model test was performed before tree construction, and the model with the best BIC value was selected. The resulting tree was visualized and edited using FigTree v1.4.4. Results The length of the assembled mitogenome of Euphaea fraseri is 15267 bp. The mitogenome consists of 37 genes, 13 protein-coding genes (PCGs), two rRNA genes (small and large ribosomal RNA), and 22 tRNA genes. Total GC content is 41.71%. The genome structure, gene order, and total gene number resemble E. formosa and E. ochracea (Figure 1). The phylogenomic analysis revealed that the Western Ghats endemic E. fraseri is basal to the Euphaea clade (Figure 2). The divergence time estimates reveal that E. fraseri diversified between 15 Ma to 20 Ma (~16 Ma), indicating the oldest divergence amongst the studied Euphaea members. Phylogenetic analysis integrating more species revealed a similar pattern, whereas the Euphaea species were separated into two well-defined clades: the Western Ghats and Southeast Asian (Figure 3). Discussion The study provided the first-ever complete mitochondrial genome of the Western Ghats endemic E. fraseri. The phylogenomic analysis revealed that E. fraseri is basal to the Southeast Asian species, diversifying around 16 Ma in the Miocene epoch of the Neogene period, indicative of Out of India diversification of the Euphaea Genus. The superfamily Calopterygoidea started diversifying in the middle stages of the Paleogene period (Huang et al., 2025), with the origin of the family Euphaidae occurring between the Eocene or Oligocene epochs of the Paleogene period. The collision of India with the Eurasian plate happened before or around the same time in 50-55 Ma, which is in the early Eocene epoch; this might have created opportunities for the introduction of the Euphaeid lineages within the Indian Subcontinent. The origin of E. fraseri is intriguing as its older (Dysphaea & Bayadera genera) and younger (Southeast Asian Euphaea species) evolutionary relatives are from South & Southeast Asia, suggesting plausible gaps in data and knowledge. The mitophylogenomic analysis suggested that E. fraseri might have evolved and colonized the Western Ghats, followed by its congeners radiating into Southeast Asia, corroborating the Out of India hypothesis (Loria & Prendini, 2020). In our study, the positioning of E. masoni, which is basal to its Southeast Asian congeners, along with the positioning of the rest of the Euphaea species, appears identical to the previous mitophylogenomic reconstructions (Huang et al., 2025). This validates our positioning of E. fraseri and is, in fact, basal to all the Southeast Asian congeners and has an out-of-India origin. The diversification of E. formosa, E. yayeyamana, E. decorata, and E. ornata isseen within the last ~2.5 Ma, in the middle to lower Pleistocene epoch, in accordance with Huang et al. (2025). This time is marked by a high amount of climatic oscillations, causing glacial land bridges between the Asian mainland and continental islands, which significantly affected the species distribution and induced speciation (Huang and Lin, 2011). Diversification within the Western Ghats may also have been induced due such similar historical climatic fluctuations and habitat formation and fragmentation processes. The concatenated tree (COI & 16s rRNA) also showed a similar pattern, grouping all the Western Ghat species of Euphaea in a separate clade from their Southeast Asian counterparts originating from the same ancestral node. This pattern implies that the eventual isolation of the Western Ghats from the Northeastern and other regions of India likely contributed to the genetic differentiation between the species that are isolated within the Western Ghats from the other Euphaea species distributed outside the Western Ghats. Our study revealed that endemic and missing species such as E. fraseri might hold the key to furthering our understanding of the evolution and biogeography of specialist aquatic insects. Adding more mitogenomes in phylogenomic reconstructions will leverage taxonomic and biogeographical studies and resolve ambiguities in future studies. Declarations Author Contributions: Conceptualization, methodology, project administration, funding, analysis, and writing: PK Data collection, curation, methodology, analysis, and writing: JP, AS Field work and writing: AP Analysis: AJ All authors (except AJ) reviewed the manuscript Funding: Dr. Pankaj Koparde: Department of Science and Technology, Ministry of Science and Technology, India, DST-SERB/2020/0019 Conflict of Interest: The authors have no conflict of interest to declare. References Grimaldi, D., & Engel, M. S. (2005). Evolution of the Insects. Cambridge University Press. Samways, M.J., 2008. Dragonflies as focal organisms in contemporary conservation biology. (ed.Cordoba-Aguilar, A.), Dragonflies and Damselflies. Oxford University Press, Oxford, New York, pp. 97–108. Huang et al. Phylogenetic relationships and divergence times of Odonata inferred from mitochondrial genome. iScience , 28, https://doi.org/10.1016/j.isci.2025.111806 (2025). Bechly, G. 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Stamatakis: "RAxML Version 8: A tool for Phylogenetic Analysis and Post-Analysis of Large Phylogenies". In Bioinformatics, 2014, open access. Stephen A. Smith, Brian C. O’Meara, treePL: divergence time estimation using penalized likelihood for large phylogenies, Bioinformatics , Volume 28, Issue 20, October 2012, Pages 2689–2690, https://doi.org/10.1093/bioinformatics/bts492 Loria, S.F., Prendini, L. Out of India, thrice: diversification of Asian forest scorpions reveals three colonizations of Southeast Asia. Sci Rep 10, 22301 (2020). https://doi.org/10.1038/s41598-020-78183-8 Additional Declarations No competing interests reported. Supplementary Files Supplementarytables.docx Supplementary Files Supplementary Table 1. List of sequences used to construct phylogenomic tree Supplementary Table 2. List of sequences used to construct phylogenetic (COI & 16s rRNA genes) tree Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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16s rRNA) sequences using maximum likelihood of 18 Odonata sequences.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8452342/v1/5defa2f193c3868d0a4ac5fc.png"},{"id":105441927,"identity":"2f7ef749-c227-4f06-b3c6-993e5371d4db","added_by":"auto","created_at":"2026-03-26 06:12:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1065498,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8452342/v1/540bcbf4-d5be-4210-86bc-643c42c69cf1.pdf"},{"id":101320772,"identity":"5994b5d7-9de1-4f16-b188-76e35ca302ee","added_by":"auto","created_at":"2026-01-28 12:57:35","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12191,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Files\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 1. \u003c/strong\u003eList of sequences used to construct phylogenomic tree\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 2.\u003c/strong\u003e List of sequences used to construct phylogenetic (COI \u0026amp; 16s rRNA genes) tree\u003c/p\u003e","description":"","filename":"Supplementarytables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8452342/v1/d79ea7ddf840fbec494145ad.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Western Ghats Endemic Euphaea fraseri Forms the Basal Lineage of the Euphaea Clade, Supporting an Out-of-India Origin for the Genus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOdonates (dragonflies \u0026amp; damselflies) are an ancient order of insects whose origin dates back to around 300\u0026nbsp;million years ago during the Carboniferous period (Grimaldi and Engel, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Considered as one of the first few ancient insect orders to take flight, odonates today contribute to multitudes of ecosystem services, such as pest control and nutrient cycling, and inspire artists and storytellers around the world as a wetland-associated symbol of freedom and power (Samways, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). During the Carboniferous period, odonate-like ancestors, collectively referred to as Protodonata, dominated the aerial predatory niche; these forms, though lacking in several defining characteristics of modern odonates, represent an important stem in the odonate evolution. Recent studies using the mitochondrial data along with fossil calibrations of dragonflies may have originated in the Triassic period, with the Cretaceous period emerging as the critical period for the initial radiation of the main Odonata lineages (Huang et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFamily Euphaediae, one of the most unique zygoptera families, with nine genera and 80 species, is widely distributed in the Palaarctic, Australasia, and Asia (Paulson et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The members of this family are known for their habit of occupying fast-flowing streams in montane areas (Kalkman et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The origin of the family Euphaeidae can be traced back to the early Eocene, supported by the fossil records of individuals from its sister groups Eodichromatinae and Epallaginae, which indicates that diversification of the damselflies within the superfamily Calopterygoidea occurred during this period, highlighting that the Euphaid lineage already existed during this period (Bechly, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Archibald and Cannings, 2021; Archibald et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Indo-Malayan region, one of the eight major biogeographic realms of the world, hosts 36 species of the family (Paulson et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The region is dominated by tropical and subtropical moist forests. The majority of the species of the \u003cem\u003eEuphaea\u003c/em\u003e genus are distributed throughout Southeast Asian countries. India is home for eight \u003cem\u003eEuphaea\u003c/em\u003e species, of which five species namely, \u003cem\u003eEuphaea cardinalis\u003c/em\u003e (Fraser, 1924); \u003cem\u003eEuphaea dispar\u003c/em\u003e Rambur, 1842; \u003cem\u003eEuphaea fraseri\u003c/em\u003e (Laidlaw, 1920); \u003cem\u003eEuphaea pseudodispar\u003c/em\u003e Sadasivan \u0026amp; Bhakare, 2021 and \u003cem\u003eEuphaea thosegharensis\u003c/em\u003e Sadasivan \u0026amp; Bhakare, 2021 and the recently discovered \u003cem\u003eEuphaea wayanadensis Anooj, Susanth \u0026amp; Sadasivan, 2025\u003c/em\u003e are restricted to the Western Ghats region (Anooj et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and rest two species, \u003cem\u003eEuphaea masoni\u003c/em\u003e Selys, 1879 and \u003cem\u003eEuphaea ochracea\u003c/em\u003e Selys, 1859 are confined in the Northeastern states of India (Bhakare et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The Malabar Torrent Dart \u003cem\u003eEuphaea fraseri\u003c/em\u003e is endemic to the Western Ghats biodiversity hotspot distributed in the states of Tamil Nadu, Kerala, Karnataka, Goa, and Maharashtra (Mitra \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Ranganekar et al. 2010; Koparde et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Subramanian et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mujumdar et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sawant et al.2022).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eEuphaea\u003c/em\u003e species of the Western Ghats all are similar in appearance and are characterized by the broadly opaque black coloration with metallic iridescence on the apices of the hindwings of the males, shorter and rounded forewings; the 10th abdominal segment has a distinct keel; discoidal cell is transversed; the anal appendages is present with a forcipate cerci and tiny paraprocts (Fraser, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1934\u003c/span\u003e). The Malabar Torrent Dart \u003cem\u003eEuphaea fraseri\u003c/em\u003e is a habitat specialist occupying areas of fast-flowing streams in the montane wet forests of the Western Ghats (Koparde et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Subramanian et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It is a red and black-colored damselfly having characteristic frons and thoracic patterns. The species is classified as Least Concern by the IUCN Red-List; however, there is little or no information available on its population size and trend, total geographical range, and area of occupancy (Kakkasery, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Habitat modification and degradation are reported to be common threats to the endemic Odonata fauna of the Western Ghats (Koparde et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Subramanian et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLiterature on the evolutionary relationships of endemic odonates of India and Indo-Malaya is scarce. To investigate the evolutionary origins of the unique Western Ghats endemic damselfly, here, we generated the complete mitochondrial genome of \u003cem\u003eEuphaea fraseri\u003c/em\u003e. The phylogenomic study based on mitogenomes provided insights into the evolutionary history and phylogenetic positioning (Huang et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) of the species of the \u003cem\u003eEuphaea\u003c/em\u003e genus. Further, we performed a phylogenetic analysis (COI \u0026amp; 16s rRNA) with a larger set of species belonging to the Euphaea genus to uncover patterns in diversification. The information provided here will be useful in future studies on population genetics, evolutionary biology, and phylogenomics.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003eTaxon Sampling, DNA Extraction \u0026amp; Next Generation Sequencing\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSpecimens were collected from a field site in Maharashtra State, India. Specimens were stored in 90% alcohol and transported to the lab. Collected specimens were identified with the help of taxonomic keys of Fraser (1934). Necessary collection permits were obtained from the State Forest Department and the Maharashtra State Biodiversity Board. Total genomic DNA was extracted from the specimens using DNease Blood and Tissue Kit (Qiagen, Hilden, Germany) using the standard protocol. For library preparation, we used the TruSeq DNA Nano kit. NGS was performed using the Illumina Novaseq6000 platform (150 bp paired reads). For the library, 10 GB of raw data were obtained. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNGS Data Quality Assessment and Processing\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFastQC was performed on the raw R1 and R2 reads obtained for the sample. The data obtained after the NGS run were filtered and curated. A total of 23,385,231 R1 and 23,385,231 R2 raw reads were generated, of which 44,536,502 good-quality reads were used for further analysis. We used MitoZ for assembly, annotation, and visualization of the mitogenome (Meng et al. 2019). A contig of 15267 bases was assembled, having a minimum depth of 600x. To visualize the entire mitochondrial genome with annotated features, a circos plot was created using Circos (Krzywinski et al. 2009). We further used the MITOS webserver to create a linear plot (http://mitos.bioinf.uni-leipzig.de/). Briefly, fastp version 0.22.0 was used to clean raw reads. Cleaned reads were assembled using Megahit. The HMMER program was used to extract candidate mitochondrial sequences and further filtered them by taxonomic assignment. Sequences belonging to non-target taxon were discarded. The BWA program was used to create reference genome index files. The assembled genome using the Megahit program from the MitoZ toolkit was used as a reference genome in this case, as no closely related genomes were available. The BWA tool was used to map pair-end reads on the reference genome. Resulting alignment metrics were converted into BAM files using Samtools. Paired-end reads were assembled using the PEAR software package (https://cme.h-its.org/exelixis/web/software/pear/). PEAR evaluates all possible paired-end read overlaps without requiring the target fragment size as input. In addition, it implements a statistical test for minimizing false-positive results. The quality of assembled reads was again checked by FastQC. Clean reads were then filtered based on the quality of reads and length of the reads using the fastp package (Chen et al. 2018).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCOI and 16s rRNA Sequencing\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCOI and 16s gene amplification was done for four \u003cem\u003eEuphaea\u003c/em\u003e species (\u003cem\u003eE. fraseri, E. pseudodispar, E. cardinalis, E. dispar\u003c/em\u003e)collected from the Western Ghats for the construction of the concatenated tree. The COI gene amplification of the specimens was done using LCO primer (Forward: 5'-GGTCAACAAATCATAAAGATATTGG-3') and HCO primer (Reverse: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3') (Folmer et al., 1994). The 16s rRNA gene amplification was done using 16sf primer (Forward: 5′-CGGCCTGTTTAACAAAAACAT-3′) and 16sr (Reverse: 5′-CCCGGTTTGAACTCAGATCATGT-3′) (Hasegawa \u0026amp; Kasuya, 2006). The cycle conditions for both the samples were 95\u003csup\u003e◦\u003c/sup\u003eC for 2 min followed by 40 cycles of 95\u003csup\u003e◦\u003c/sup\u003eC for 30sec, 50\u003csup\u003e◦\u003c/sup\u003eC for 30sec, and 72\u003csup\u003e◦\u003c/sup\u003eC for one min.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMitophylogenomic and Phylogenetic Analyses\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe generated mitochondrial genome of \u003cem\u003eEuphaea fraseri\u003c/em\u003e was aligned with 14 other complete mitochondrial genomes from various odonate species obtained from the GenBank database (Supplementary Table 1, Huang et al., 2025). The alignment was performed using MAFFT, tree construction based on the Maximum Likelihood approach (1000 bootstraps), and visualization was carried out in IQ-TREE v2. For fossil calibration, we used three secondary calibration points as used in earlier studies (Huang et al., 2025). A dated mitogenome phylogeny was constructed using treePL (Stephen et al., 2012). Individuals of the sub-order Anisoptera were set as outgroups (\u003cem\u003ePolycanthagyna melanictera, Periaeschna flinti, Davidius lunatus, Ictinogomphus pertinax\u003c/em\u003e). The lab-generated sequences of COI and 16S rRNA genes from four \u003cem\u003eEuphaea\u003c/em\u003e species were examined and manually curated using Chromas v2.6.6 (Technelysium Pty Ltd), and consensus sequences were generated in MEGA v11.0.13 (Kumar et al., 2024). Sequences for other species were retrieved from the GenBank database (Supplementary Table 2). All sequences were translated into protein-coding format to check for the presence of stop codons. The analysis was based on the concatenation of the COI and 16S rRNA genes. A total of eighteen sequences (four lab-generated and fourteen from GenBank) were aligned using the ClustalW tool with default settings for the concatenated (COI + 16S rRNA) dataset. The phylogenetic tree was constructed using the maximum likelihood method with the ML + rapid bootstrap option (1000 replicates) implemented in RAxMLGUI v2.0.15 (Stamatakis, 2014), with two species from the family \u003cem\u003eLibellulidae\u003c/em\u003e designated as outgroups (\u003cem\u003eTrithemis aurora, Crocothemis servelia\u003c/em\u003e). A model test was performed before tree construction, and the model with the best BIC value was selected. The resulting tree was visualized and edited using FigTree v1.4.4.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe length of the assembled mitogenome of \u003cem\u003eEuphaea fraseri\u003c/em\u003e is 15267 bp. The mitogenome consists of 37 genes, 13 protein-coding genes (PCGs), two rRNA genes (small and large ribosomal RNA), and 22 tRNA genes. Total GC content is 41.71%. The genome structure, gene order, and total gene number resemble \u003cem\u003eE. formosa\u003c/em\u003e and \u003cem\u003eE. ochracea\u003c/em\u003e (Figure 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe phylogenomic analysis revealed that the Western Ghats endemic \u003cem\u003eE. fraseri\u003c/em\u003e is basal to the Euphaea clade (Figure 2). The divergence time estimates reveal that \u003cem\u003eE. fraseri\u003c/em\u003e diversified between 15 Ma to 20 Ma (~16 Ma), indicating the oldest divergence amongst the studied \u003cem\u003eEuphaea\u003c/em\u003e members. Phylogenetic analysis integrating more species revealed a similar pattern, whereas the Euphaea species were separated into two well-defined clades: the Western Ghats and Southeast Asian (Figure 3).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe study provided the first-ever complete mitochondrial genome of the Western Ghats endemic \u003cem\u003eE. fraseri.\u0026nbsp;\u003c/em\u003eThe phylogenomic analysis revealed that \u003cem\u003eE. fraseri\u0026nbsp;\u003c/em\u003eis basal to the Southeast Asian species, diversifying around 16 Ma in the Miocene epoch of the Neogene period, indicative of Out of India diversification of the Euphaea Genus. The superfamily Calopterygoidea started diversifying in the middle stages of the Paleogene period (Huang et al., 2025), with the origin of the family Euphaidae occurring between the Eocene or Oligocene epochs of the Paleogene period. The collision of India with the Eurasian plate happened before or around the same time in 50-55 Ma, which is in the early Eocene epoch; this might have created opportunities for the introduction of the Euphaeid lineages within the Indian Subcontinent. The origin of \u003cem\u003eE. fraseri\u003c/em\u003e is intriguing as its older (Dysphaea \u0026amp; Bayadera genera) and younger (Southeast Asian Euphaea species) evolutionary relatives are from South \u0026amp; Southeast Asia, suggesting plausible gaps in data and knowledge. The mitophylogenomic analysis suggested that \u003cem\u003eE. fraseri\u0026nbsp;\u003c/em\u003emight have evolved and colonized the Western Ghats, followed by its congeners radiating into Southeast Asia, corroborating the Out of India hypothesis (Loria \u0026amp; Prendini, 2020). In our study, the positioning of \u003cem\u003eE. masoni,\u0026nbsp;\u003c/em\u003ewhich is basal to its Southeast Asian congeners, along with the positioning of the rest of the Euphaea species, appears identical to the previous mitophylogenomic reconstructions (Huang et al., 2025). This validates our positioning of \u003cem\u003eE. fraseri\u0026nbsp;\u003c/em\u003eand is, in fact, basal to all the Southeast Asian congeners and has an out-of-India origin. The diversification of \u003cem\u003eE. formosa, E. yayeyamana, E. decorata,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eE. ornata\u0026nbsp;\u003c/em\u003eisseen within the last ~2.5 Ma, in the middle to lower Pleistocene epoch, in accordance with Huang et al. (2025). This time is marked by a high amount of climatic oscillations, causing glacial land bridges between the Asian mainland and continental islands, which significantly affected the species distribution and induced speciation (Huang and Lin, 2011). Diversification within the Western Ghats may also have been induced due such similar historical climatic fluctuations and habitat formation and fragmentation processes.\u003c/p\u003e\n\u003cp\u003eThe concatenated tree (COI \u0026amp; 16s rRNA) also showed a similar pattern, grouping all the Western Ghat species of \u003cem\u003eEuphaea\u003c/em\u003e in a separate clade from their Southeast Asian counterparts originating from the same ancestral node. This pattern implies that the eventual isolation of the Western Ghats from the Northeastern and other regions of India likely contributed to the genetic differentiation between the species that are isolated within the Western Ghats from the other Euphaea species distributed outside the Western Ghats.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur study revealed that endemic and missing species such as \u003cem\u003eE. fraseri\u003c/em\u003e might hold the key to furthering our understanding of the evolution and biogeography of specialist aquatic insects. Adding more mitogenomes in phylogenomic reconstructions will leverage taxonomic and biogeographical studies and resolve ambiguities in future studies.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, methodology, project administration, funding, analysis, and writing: PK\u003c/p\u003e\n\u003cp\u003eData collection, curation, methodology, analysis, and writing: JP, AS\u003c/p\u003e\n\u003cp\u003eField work and writing: AP\u003c/p\u003e\n\u003cp\u003eAnalysis: AJ\u003c/p\u003e\n\u003cp\u003eAll authors (except AJ) reviewed the manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. Pankaj Koparde: Department of Science and Technology, Ministry of Science and Technology, India, DST-SERB/2020/0019\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGrimaldi, D., \u0026amp; Engel, M. 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Out of India, thrice: diversification of Asian forest scorpions reveals three colonizations of Southeast Asia. \u003cem\u003eSci Rep\u003c/em\u003e 10, 22301 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-020-78183-8\u003c/span\u003e\u003cspan address=\"10.1038/s41598-020-78183-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Phylomitogenomics, Biogeography, Damselfly, Montane Streams, Miocene","lastPublishedDoi":"10.21203/rs.3.rs-8452342/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8452342/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Euphaea damselflies, highly remarkable for their red\u0026ndash;black coloration, represent specialized inhabitants of fast-flowing montane streams and are distributed across the Indo-Malayan biogeographical region. However, the evolutionary history and spatial diversification of the genus are not well resolved, especially with respect to the origins of Western Ghats endemics relative to the Southeast Asian taxa. Herein, we present the first complete mitochondrial genome of the Western Ghats endemic \u003cem\u003eEuphaea fraseri\u003c/em\u003e. The assembled mitogenome is 15,267 bp in length and comprises 13 protein-coding genes (PCGs), 22 tRNA genes, and two ribosomal RNA genes (12S and 16S rRNA), following the canonical mitochondrial organization of Zygoptera. Phylogenetic analyses based on (i) the whole mitogenome and (ii) a concatenated dataset of COI and 16S rRNA consistently recovered a well-supported Western Ghats Euphaea clade. \u003cem\u003eE. fraseri\u003c/em\u003e is a basal relative to all sampled congeners and thus represents a deeply divergent lineage distinct from Southeast Asian Euphaea species. Divergence time estimation places the split between E. fraseri and the remaining Euphaea species around 15\u0026ndash;20 Ma (Miocene), indicating an ancient lineage that predates the diversification of Southeast Asian taxa. The basal positioning and Miocene divergence of \u003cem\u003eE. fraseri\u003c/em\u003e thus provide strong support for an \"Out of India\" hypothesis, wherein the Western Ghats lineage represents an early-branching remnant from which later dispersal and radiation into Southeast Asia may have occurred. These results add new genomic and temporal data that help elucidate the deep evolutionary history of Euphaea and emphasize the biogeographic importance of the Western Ghats as a refuge for ancient odonate lineages. Further studies using a larger genomic dataset, including missing taxa, will provide insights into the evolutionary relationships between other endemic and endangered groups from the Western Ghats.\u003c/p\u003e","manuscriptTitle":"Western Ghats Endemic Euphaea fraseri Forms the Basal Lineage of the Euphaea Clade, Supporting an Out-of-India Origin for the Genus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 12:55:42","doi":"10.21203/rs.3.rs-8452342/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9bfd5577-4fd9-40d6-bbdb-258d949b300f","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61800468,"name":"Biological sciences/Ecology"},{"id":61800469,"name":"Earth and environmental sciences/Ecology"},{"id":61800470,"name":"Biological sciences/Evolution"},{"id":61800471,"name":"Biological sciences/Genetics"},{"id":61800472,"name":"Biological sciences/Molecular biology"},{"id":61800473,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2026-03-26T06:11:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-28 12:55:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8452342","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8452342","identity":"rs-8452342","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}