Biogeographical origin of Andrographis Wall. ex Nees (Acanthaceae) in eastern peninsular India based on molecular phylogenetics

Biogeographical origin of Andrographis Wall. ex Nees (Acanthaceae) in eastern peninsular India based on molecular phylogenetics | 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 Research Article Biogeographical origin of Andrographis Wall. ex Nees (Acanthaceae) in eastern peninsular India based on molecular phylogenetics Siddharthan Surveswaran, Yuvasri Kalaimani This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4934034/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 genus Andrographis is predominantly peninsular Indian distribution after the resurrection of Haplanthus which is predominantly southeast Asian in distribution. Previous study have shown its close phylogenetic affinity with Haplanthodes and Haplanthus . In this study we constructed a most comprehensive phylogeny of the tribe Andrographideae using five plastid regions, rbcL , matK , trnLF , psbA - trnH and trnGR . The results show polyphyly between Andrographis , Haplanthodes and Haplanthus , resulting in a Andrographis sensu lato (s.l.) clade. Within this earliest splitting clade consisted of Andrographis and Haplanthus ovatus . The next split was with Haplanthodes followed by Andrographis sensu stricto (s.s) clade. Within the Andrographis clade, we observed two clades one was western in distribution and the other was eastern in distribution in peninsular India. Biogeographical analysis suggested an eastern peninsular Indian origin of Andrographis and followed by dispersal into the relative wetter western peninsular India. We deduced the time of diversification of the clades of Andrographis . We discuss the role of south Asian winter monsoon in the diversification of Andrographis which has adapted to aridity. Acanthaceae Biogeography phylogeny Deccan plateau Eastern Ghats Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The family Acanthaceae is pantropical in distribution with about 4900 species and 191 accepted genera (McDade et al. 2008 ; Manzitto-Tripp et al. 2022 ). Most are herbs or shrubs, including some twining forms, and some spiny forms. According to the most recent revised classification, Acanthaceae is classified into four subfamilies: Nelsonioideae, Thunbergioideae, Acanthoideae, and Avicennioideae. Acanthoideae, the most diverse subfamily, comprises eight recognised tribes: Acantheae, Physacantheae, Barlerieae, Andrographideae, Whitfieldieae, Neuracantheae, Ruellieae, and Justicieae. The subfamily Acanthoideae is found only in the Old World, and its tribe Andrographideae is exclusively Asian. Pollen with apertural margins and or surfaces conspicuously thickened intricate ornamentation with conical spines is the unique character of the tribe. Other synapomorphies are ascending cochlear aestivation (imbricate aestivation: one outer petal, three imbricate and one inner), two stamens with or without staminodes and usually bilabiate corollas. They share these characters Justiceae. However, the number of seeds per capsule is numerous, 6–20 whereas it is 2–4 in Justiceae (Manzitto-Tripp et al. 2022 ). There are currently 8 recognized genera: Andrographis Wall. ex Nees, Diotacanthus Benth., Graphandra J. B. Imlay, Gymnostachyum Nees, Haplanthodes Kuntze, Haplanthus Nees, Phlogacanthus Nees and Sphinctacanthus Benth. (Manzitto-Tripp et al. 2022 ). Of these, Haplanthodes with 4 species is endemic to peninsular India (Deshmukh et al. 2021 ). Haplanthus also with 4 species was reinstated from Andrographis based on morphological analysis (Gnanasekaran et al. 2016 ). One species of Haplanthus occurs in peninsular India and three are in southeast (SE) Asia including Andaman islands. Gymnostachyum with 51 species is mostly distributed in SE Asia with few species in peninsular India. Phlogacanthus with 41 species is also mostly SE Asian. Graphandra and Sphinctacanthus are distributed in SE Asia with 1 species each. Sphinctacanthus is distributed in northeast India and Myanmar with only one species, S. griffthii which has close morphological as well as geographical affinities with Phlogacanthus and Gymnostachyum (Hansen 1985 ). Diotacanthus with two species from southern India is sometimes treated with Phlogacanthus ( POWO 2024 ) . Cystacanthus has been merged under Phlogacanthus as a section (Deng et al. 2020b ). The genus Andrographis Wall. ex Nees is composed of annual herbaceous plants native to Asia with 25 species (POWO 2024 ). Almost all are native to peninsular India except of the medicinally important species Andrographis paniculata and Andrographis echioides (syn. Indoneesiela echioides ) which are spread in SE Asia and Sri Lanka (POWO 2024 ). A. paniculata is distributed all over the world and it may be cultivated or naturalised due to its medicinal value. Most of the other Andrographis species are rare and endemic, and they are especially distributed in the eastern parts of the Deccan plateau and the Eastern Ghats (Neeraja et al. 2015 ). Andrographis paniculata has been used in ethnomedicine by various cultures (Samy et al. 2008 ; Hossain et al. 2014 ). Its most active compound, a diterpenoid, Andrographolide and is called the “King of Bitters”. It has been used in treating snake bites (Premendran et al. 2011 ; Nayak et al. 2020 ), treatment for dengue (Panraksa et al. 2017 ) and also implicated in the cure of cancer (Attri et al. 2024 ). During the COVID-19 pandemic, a decoction of Andrographis was used as a cure for the systems and recovery of patients. There are various reports, a few showing some efficacy (Sa-Ngiamsuntorn et al. 2021 ) and many others showing no efficacy (Kanokkangsadal et al. 2023 ; Siripongboonsitti et al. 2023 ). Similarly, Andrographis echiodes has been used as a medicinal plant by various cultures (Kumar et al. 2007 ; Samy et al. 2008 ; Chinnappan 2012 ) and it has been implicated to have anticancer and antibacterial activities (Elangovan et al. 2015 ). Molecular phylogenetic analysis of the tropical Asian Andrographideae is sparse and the monophyly of the genera is still in a state of flux (Deng et al. 2020a ; Manzitto-Tripp et al. 2022 ). Arolla et al. (Arolla et al. 2015 ) attempted a phylogeny of Andrographis and their collection was mostly from Andhra Pradesh, ie., the Eastern Ghats and associated areas. Surveswaran et al., ((Surveswaran et al. 2022 ) showed a close relationship between Haplanthodes and Haplanthus with Andrographis . However, a comprehensive phylogeny Andrographis and analysis of its biogeography within India are lacking. Therefore, in this study, we aimed to, 1) establish the monophyly of Andrographis , 2) to prove the Indian origin of Andrographis and 3) to test whether there are separate lineages in the western and eastern parts of peninsular India associated with the Western Ghats and Eastern Ghats. To this end, we used five plastid markers, rbcL , matK , trnLF , psbA - trnH and trnGR , mostly from the GenBank and a few newly generated sequences. Using this data we performed the most comprehensive phylogeny of Andrographideae followed by a dated phylogeny and performed a biogeographic analysis using Bayesian and Likelihood methods respectively. Materials and Methods Plant material, DNA extraction and PCR amplification Samples of A. echioides were collected from the University of Hyderabad campus in Telangana and Kalburgi (Gulbarga) in Karnataka. The sequences of the rest of the Andrographideae, including several other genera of Acanthaceae, were downloaded from the GenBank database (Table 1 ). Total genomic DNA was isolated using the CTAB method. The study utilized plastid markers: two coding regions, matK and rbcL and three non-coding regions, trnG - trnR , trnL - trnF , and psbA - trnH . The primers used were the same as in Surveswaran et al., ( 2022 ). Each PCR reaction had the following components: 10 µl of the 2x Taq DNA Polymerase Master Mix RED (Ampliqon, Odense, Denmark), 1 µl of forward and reverse primers (10 pmol/µl) each, and 1–2 µl (> 10ng to < 200ng of the extracted DNA) of the template DNA and made upto to 20 µl with PCR grade water. The PCR steps were as follows: Initial denaturation: 94°C, 2 mins; cyclic denaturation: 94°C, 30 secs; cyclic annealing: 58°C, 30 secs; cyclic extension: 72°C, 50 secs; 35 cycles; and final extension: 72°C, 3 mins. The PCR products were verified on agarose gels before being sent for Sanger sequencing to Barcode Biosciences Pvt. Ltd. (Bangalore, India). Table 1 Species names, voucher information and accession numbers of sequences obtained from the GenBank and generated in this study. No. Species Voucher matK rbcL tnrL-F psbA-trnH trnG-R Andrographis affinis SK1723 MH142855 Andrographis alata HJCB2250,SK1721B, CNS06AA2013 MT445715 MH142897 MH142862 MT445682 MT445664 Andrographis atropurpurea SK1716D MH142866 Andrographis beddomei Cherukupalli, N, Osmania University, India MN857164 MN857163 Andrographis echioides DSD02F, SK1682A KX378015 (Prashanth hande), Daniel.s.n,, CNS07AE2013 MW601018 MW601009 MH142868 KC118378 (Tripp) Andrographis echioides UOHxx, University of Hyderabad XXXXXXX XXXXXXX XXXXXXX XXXXXXX Andrographis echioides UOHxx, Gulbarga India XXXXXXX XXXXXXX XXXXXXX XXXXXXX Andrographis elongata SK1708B MH142871 Andrographis glandulosa HJCBE265, CNS04AG2013 KP455309 MT445702 MT445686 MT445667 Andrographis gracilis SK1724C MH142876 Andrographis lawsonii Samydurai,P, Bharathiyar University, India MH428005 MH428004 Andrographis lineata var. lawii DSD08E MW601019 MW601010 Andrographis lobelioides Samydurai, P, Bharathiyar University, India MH423499 MH388772 Andrographis longipedunculata SK1694C MH142880 Andrographis macrobotrys SK1715B, DSD03B MW601021 MW601012 MH142885 Andrographis megamalayana HJCB1454, HJCB1190 MT445716 MT445700 MT445683 MT445665 Andrographis nallamalyana CNS01AN2013 KP455311 KP455305 Andrographis paniculata CNS05AP2013, HJCB0572 KF521888 KF521878 KT074995 LC743551 MT445666 Andrographis producta DSD04C MW601024 MW601015 Andrograophyis serpyllifolia DSD06A MW601025 MW601016 MH142891 Haplanthodes neilgherryensis MML-532 MT445703 MT445690 MT445668 MT445650 Haplanthodes plumosa MML-546 MT445708 MT445691 MT445673 MT445655 Haplanthodes tentaculata MML-531 MT445712 MT445695 MT445677 MT445659 Haplanthodes verticillatus MML-553 MT445710 MT445693 MT445675 MT445657 Haplanthodes.sp. HSP1 MML-544 MT445713 MT445696 MT445678 MT445660 Haplanthus ovatus 1 DSD05A, HJCB5432, HJCB1962 MW601022 MW601013 MT445679 MT445662 Haplanthus ovatus 2 HJCB1962 MT445714 MT445698 Gymnostachyum canescens Dev et al., KFRI, Peechi, India KM217081 KM217085 Gymnostachyum febrifugum Dev et al., KFRI, Peechi, India KM217082 KM217086 Gymnostachyum warrierianum SAD-2014 KM217083 KM217087 Cystacanthus paniculatus CPG25312 KX526467 Cystacanthus turgidus RBG Kew 1996 − 479 K EU528919 KJ140526 Phlogacanthus curviflorus CPG151, BB0613 KX526475 KX527201 MW722784 KR533704 Phlogacanthus parviflorus Boro,N. Guwahati University, India MW810858 Phlogacanthus thyrsiflorus Boro,N. Guwahati University, India MW722783 KC118401 Barleria lupulina BKF189453 KT161362 Barleria prionitis CMPR8789, BKF189452 OL580741 MZ461574 KT075002 KT161339 Sequence assembly and phylogenetic analysis Forward and reverse sequences were assembled using the pregap4 and gap4 modules in the Staden package ver. 2.0.0 beta 11 (Staden et al. 2000 ). Sequences were aligned using the CLUSTAL W algorithm in MEGA version 11 (Tamura et al. 2021 ). Individual alignments were checked and manually corrected using AliView ver. 1.28 (Larsson 2014 ). The alignments were concatenated using SequenceMatrix ver. 1.9 (Vaidya et al. 2011 ). Sequence alignment characteristics such as parsimony informative sites were estimated using PAUP ver. 4.0 beta 10 (Swofford 2003 ). The best-fit model of sequence evolution was deduced using jModeltest ver. 2.1.10 (Darriba et al. 2012 ). The selection of the best model was based on the Akaike Information Criterion with correction (AICc). Bayesian phylogenetic analysis was performed using MrBayes ver. 3.2.7 (Ronquist and Huelsenbeck 2003 ; Ronquist et al. 2012 ). A partitioned dataset with the best model selected according to jModteltest2 was used. Ten million runs of Metropolis-coupled Markov chains (MCMC) were run as two independent runs with 4 chains each. The convergence of the runs was checked using the average standard deviation of split frequencies being less than 0.01 and also by Tracer ver.1.7.2 as described below for BEAST analysis. Maximum-likelihood analysis was performed in IQTREE ver. 2.0.7 with model selection of the partitioned dataset followed by tree-building using the merged partitions and ultrafast bootstrapping with 1000 replicates (Minh et al. 2020 ). For Bayesian analysis, BEAST ver. 2.7.6 (Bouckaert et al. 2019 ) was used. An XML file for input into BEAST was prepared using BEAUTi. The best-fit model for each partition was set up. Molecular dating analysis was performed using secondary data calibration from (Tripp and McDade 2014 ; Surveswaran et al. 2022 ). The age of Andrographideae was between 18.44 and 11.86 million years ago (mya) with a mean of 9.44 mya. These dates were modelled using a uniform distribution with upper, lower and offset values as 12, 18 and 5 respectively. Ten million MCMC iterations were performed, with sampling every 10,000 runs. The convergence of the runs was checked using Tracer ver. 1.7.2 (Rambaut et al. 2018 ) as effective sample sizes (ESS) values for all parameters greater than 200. After the run was complete, the consensus tree was computed using Treeannotator ver. 2.7.6 with a burnin of 25% of the initial trees. Trees were visualised in FigTree ver. 1.4.4 (Rambaut 2018 ). Since Andrographideae is tropical Asian in distribution and the diversity of Andrographis is high in peninsular India is high, we separated peninsular India to the east and west, in accordance to the Eastern Ghats and Western Ghats, the mountain stretches which harbour high biodiversity in peninsular India. The regions taken for analysis were South East Asia (including, south China, northeast India and peninsular Malaysia ) designated as A, Eastern Ghats denoted as E, Africa denoted as F, Sri Lanka denoted as L and Western Ghats denoted as W. The distribution data of the species were collected from GBIF (GBIF.org 2024 ), India Flora Online (India Flora Online 2024 ) and POWO (POWO 2024 ). Biogeographic analysis was performed using the BioGeoBEARS (Matzke 2014 , 2018 ), R package in R ver. 4.4.1 (R Core Team 2024 ) using RStudio ver. 2024.04.1 Build 748 (RStudio Team 2020 ). All models of biogeography, DEC, Divalike and Bayarealike along with jump dispersal (+ J) were analysed. The max areas were set to FALSE in the DEC model, and the rest of the models were set to TRUE. The best-fit model for biogeography was selected based on the AICc values after the likelihood ratio test implemented in BioGeoBears. Results In this work, most of the sequence data were obtained from the GenBank database except accessions of Andrographis echioides , one from Hyderabad, Telangana and another from Kalburgi, Karnataka, were collected (Fig. 1 ). Their sequences were generated for the study despite the availability of data for one A. echioides accession in the public databases as a comparison. The combined molecular sequence dataset including matK , rbcL , psbA - trnH , trnL - trnF , and trnG - trnR consisted of 2510 nucleotides of which 204 (8%) were parsimoniously informative. The nucleotide statistics and best-fit models for each dataset and the combined dataset are summarised (Table 2 ). The dataset included all the genera of Andrographideae except Diatacanthus , Graphandra and Sphinctacanthus sensu Manzitto-Tripp et al. ( 2022 ). Table 2 Sequence information plastid markers used Dataset Taxa Length Conserved sites Parsimony informative characters Variable characters Best model according to AICc (calculated using jModeltest2) Combined genes 37 2510 2137 204 373 GTR + G matK 27 689 546 69 142 GTR + I psbA - trnH 14 549 492 35 56 HKY + G rbcL 14 329 233 37 96 GTR trnG - trnR 17 365 310 49 54 HKY trnL - trnF 29 558 525 14 25 HKY The Bayesian phylogenetic analysis tree showed a polyphyly of Andrographis with respect to Haplanthodes and Haplanthus (Fig. 2 ) considered here as Andrographis sensu lato (s.l). The Bayesian Posterior Probability (BPP) support > 0.95 was considered very high support, > 0.9 to 0.94 was considered good support and 90% was considered very high support, > 50–90% was considered good support and anything below 50% was considered unsupported. Phlogacanthus (incl. Cystacanthus ) was monophyletic with good MLBS support but without BPP support. Gymnostachyum with three species was highly supported. Eighteen of the 25 currently recognised Andrographis (POWO 2024 ) were used in this study. Andrographis s.l. could be further divided into 4 clades in our analysis (Fig. 2 ). The first, well supported clade consisted for Haplanthus ovatus along with A. elongata , A. alata and A. affinis ( Haplanthus - Andrographis clade, clade no. 1, Fig. 2 ). The second, well supported clade was the Haplanthodes clade (clade 2, Fig. 2 ) which included all the 4 species of Haplanthodes . The next fully supported clade contained only Andrographis species i.e., Andrographis sensu stricto clade. This Andrographis s.s clade was divided into two sister sub-clades which are designated as Andrographis clade I and Andrographis clade II (clades 3,4, Fig. 2 ). These sister clades are different in their biogeography Andrographis clade I is found in the eastern part of peninsular India whereas the Andrographis clade II is found in the western part of peninsular India. The Andrographis clade I (clade 3, Fig. 2 ) was not well supported and contained eastern peninsular Indian species: A. nallamalayana , A. serpyllifolia , A. glandulosa , A. echioides , A. beddomei and A. longipedunculata . In this clade, except for A. nallamalayana , which is the earliest divergent species, the rest of the nested clades were highly supported. The position of different accessions was variable and did not show congruence with dating analysis (Fig. 3 ). The Andrographis clade II (clade 4, Fig. 2 ) was significantly supported and included A. atropurpurea , A. gracilis , A. lobelioides , A. lineata , A. lawsonii , A. megamalyana , A. macrobotrys , A. paniculata and A. producta . The relationship among the species in this clade was not well resolved. All species in this clade were in the western peninsular India. The dated BEAST phylogram of the same partitioned dataset described above resolved the same topology as the Bayesian analysis. However, some minor differences in the not supported clades were different between the ML analysis and BEAST analysis. The major clades were supported with > 0.95 BPP support in BEAST analysis (Fig. 3 ). The median age of the nodes as inferred from the BEAST analysis are indicated along with the 95% highest posterior density in square brackets (95% HPD). The age of the tribe Andrographideae clade and the Andrographis s.l . clade was found to be 18.17 MYA and 9.09 MYA respectively. The Andrographis clade I, (clade 3, in Fig. 2 ) with the inclusion with A. nallamalayana was not supported. With the exclusion of A. nallamalayana , the remaining species were highly supported as monophyletic in BPP. In the BEAST analysis, A. echioides accessions 1 and 2, collected by us, were monophyletic without support. A. echiodes from the GenBank was closest in relationship to A. beddomei also without support. The rest of the clades, clades, 1,2,4 were well supported. The monophyly of Gymnostachyum and Phlogacanthus was moderately supported ( 0.95 BPP). The locality information of Andrographis and other taxa was collected from GBIF and POWO and summarised (Supplementary data, Table S1 ). The best model of biogeographical distribution according to the AICc was found to be DEC without jump (+ J parameter). The alternative distribution models, such as DIVALIKE, and BAYAREA-like with and without J parameter, were tested in BioGeoBEARS (Supplementary data, Fig. S2 . Table S2 ). According to the DEC model of biogeographical distribution as inferred in BioGeoBEARS, the ancestry of the tribe Andrographideae was found to be western region of peninsular India. The Gymnostachyum clade and the Phlogacanthus clade have an ancestry in western peninsular India whereas the Andrographis s.l. clade has its ancestry in the eastern peninsular India. There have been two dispersals into the western part of India, that is in the Haplanthodes clade (clade 2) and in the Andrographis clade II (clade 4). Discussion The molecular phylogenetics of the tribe Andrographideae is not well understood (Manzitto-Tripp et al. 2022 ) and therefore this study is the most comprehensive work on the tribe. The sampling of the genus is more than 70% of the currently accepted species according to POWO. The largest genus of Andrographideae is Gymnostachyum with 51 species spreading from India in the west to the Philippines in the east. The second most speciose genus is Phlogacanthus with 41 which spreads eastwards up to Sulawesi from India. Phlogacanthus now includes Cystacanthus (Deng et al. 2020b ) but we have retained the old name in the figures in this work for easy recognition. The next largest genus is Andrographis with 25 species that are exclusively distributed in India. The distribution extends up to Myanmar which is adjacent to India but these species are now treated under Haplanthus (Gnanasekaran et al. 2016 ). We have sampled only Haplanthus ovatus which is distributed in the eastern parts of India, in the Mahendragiri mountains, which is a part of the Eastern Ghats. The other species of Haplanthus , H . hygrophiloides and H . laxiflorus show a distribution eastwards from India such as Myanmar, Bhutan, Thailand, Vietnam, Cambodia, Malaysia and parts of the Andaman Islands (Gnanasekaran et al. 2016 ). H. hygrophiloides (Pegu, Myanmar), H. laxiflorus var. tenuiflora (Andaman Islands, India), H. laxiflorus var. recedens (Tenasserim, Mooleeyit, Myanmar) and H. rosulatus (Thailand) need to be added to the phylogeny to understand the status of Haplanthus in relation to Andrographis . Additionally, the morphological synapomorphies uniting the three species Andrographis , A. affinis , A. alata and A. elongata with Haplanthus requires investigation. These three species, though originated in India they have a distribution eastwards and southwards, i.e., southeast Asia and Sri Lanka. The ancestral region of the entire Andrographis is the eastern peninsular India. Within this, A ndrographis-Haplanthodes clade (clade 1) is also eastern peninsular Indian in origin. The Indian endemic, Haplanthodes , though western Indian in distribution, is eastern Indian in origin and the speciation might have happened after dispersal into the western peninsular Indian region. Similarly in ancestry of the Andgrograhis s.s. Clade is also eastern and Andrographis (clade 4) might have diversified after dispersal into the western peninsular Indian region. In summary, within the eastern peninsular India originated Andrographis , there are two dispersals into the western peninsular region associated with the Western Ghats, one, Haplanthodes and another, the Andrographis s.s. clade II Though Andrographis is endemic to the Indian peninsula, the medicinally important A. paniculata and A. echioides are only widespread. A. paniculata is probably under cultivation worldwide due its medicinal properties (GBIF.org 2024 ). A. echioides is found in Sri Lanka and also probably cultivated in other parts of the world as a potential medicinal plant. However, these occurrences may be due to human mediated distribution due to their use as medicinal plants. Some other species such as A. macrobotrys , A. elongata and A. alata also extend their distribution towards Sri Lanka and southeast Asia. These are most probably recent range expansions. The Eastern Ghats is a stretch of mountains which extends throughout the eastern part of peninsular India like its western counterpart, Western Ghats. The Eastern Ghats, though not much studied as the Western Ghats, host several congeners with Western Ghats and would have probably been used as a connecting bridge between the Western Ghats and southeast Asia. However, the Eastern Ghats mountain ranges, though disjunct, is larger in area and harbours a high level of biodiversity due to its diverse forest types in moist and cooler altitudes (Banerji 1990 ; Ramachandran et al. 2018 ; Thulsi Rao et al. 2019 ; Panda et al. 2020 ). One of the greatest plant geographers after Humboldt was the botanist Joseph Dalton Hooker (Egerton 2019 ) and the author of the 7 volume treatise, Flora of British India has discussed about the botanical regions and provinces of British India in his “Sketch of the flora of British India''. He divides the former British India into 9 provinces: 1. Eastern Himalaya, 2. Western Himalaya, 3. Indus Plain, 4. Gangetic Plain, 5. Malabar, 6. The Deccan, 7. Ceylon, 8. Burma, and 9. The Malayan Peninsula (Hooker 1904 ). The Western Ghats species belong to the Malabar region and the Eastern Ghats species belong to the Deccan region of Hooker’s division. The Deccan plateau is an elevated region which encompasses the major part of peninsular India which adjoins the Western Ghats in the west and harbours the Eastern Ghats mountains in the east. The Deccan region is mostly semi-arid because the high mountains of the Western Ghats break the rain clouds and reduce the rainfall towards the eastern regions. The Deccan region is therefore dominated by grasslands and deciduous vegetation (Simkins 1926 ; Khadkikar et al. 1999 ; Dash et al. 2019 ; Panda et al. 2020 ; Bhunia and Patra 2024 ). The age of diversification of Andrographis s.l. as inferred in our study is around 9.09 with a 95% HPD of 7.01 to 11.65 MYA. This corresponds to the age of diversification of other plant genera of plants and animals in the Indian peninsula (Deepak and Karanth 2018 ; Surveswaran et al. 2021 ) which has been most probably due to the evolution of the South Asian Monsoon. The diversification of Andrographis matches with the onset and intensification of the Indian and Asian monsoon as a result of enhanced aridity in the Asian interior around 9 − 8 MYA (Zhisheng et al. 2001 ). The Eastern Ghats clade started diversification around 4.02 MYA with a 95% HPD of 2.56 to 5.37 MYA. Within this period, around 3.6–2.6 MYA, due to various geological events the South Asian summer monsoon and East Asian summer monsoons might have weakened and the winter monsoons have become prominent (Zhisheng et al. 2001 ). The biota of peninsular India, most of which is the Deccan plateau, is dependent on the monsoonal rainfall for their survival. In India, South West or summer monsoons bring rain to the windward side of the Western Ghats from June to September. As the monsoon clouds weaken, the leeward side of the western ghats adjoining the Deccan plateau receives less rainfall but life depends on these seasonal rains for their survival. As the weakened winds reach north towards the Himalayas and retreat, they bring moisture for the Himalayan region. This is the North East monsoon or the winter monsoon which happens during October to December and brings rain to the eastern coast of India which is relatively drier during the summer monsoon period. The Deccan plateau and the Eastern Ghats forms a part of the region fed by the Northeast monsoon (Sankalia and Joshi 1957 ; Rajeevan et al. 2022 ). Therefore the eastern part of the Indian peninsula is generally arid and receives less rainfall and therefore considered as a semiarid region. Andrographis of the eastern peninsular Indian region are probably adapted to this semi-arid condition as shown by some xeric characters such as smaller and narrower leaves and the presence of spines in some of the Acanthaceae members. Several dry adapted plants and animals of the peninsular India are adapted to semi-arid conditions of the Deccan plateau and depend on the monsoon rains for their survival (Surveswaran et al. 2021 ). In conclusion, we find that Andrographis s.l. is almost entirely Indian in origin with a few recent dispersals into southeast Asia and Sri Lanka. Andrographis has originated and diversified in the semi-arid conditions of peninsular India which is majorly represented by the Deccan plateau. They might have originated from wet-adapted forms of Andrographideae in the Western Ghats region. The age of diversification matches with the age of intensification of the summer monsoon climate or South West monsoon in peninsular India. Further, diversification within the eastern clades of Andrographis correlates with the rise of the winter monsoon or North East monsoon which is much pronounced in the eastern region of peninsular India. Declarations Acknowledgements SS acknowledges University of Hyderabad, Institution of Eminence (IoE) grant, UoH-IoE-RC5-22-042 for experimental work. Author contributions Conceptualization: S.S; Methodology: S.S, Y.K; Formal analysis: S.S. Y.K; Resources: S.S, Y.K; Data Curation: S.S,Y.K; Writing - Original Draft: S.S,Y.K; Writing - Review & Editing: S.S; Visualization: S.S,Y.K; Project administration: S.S,Y.K; Funding acquisition: S.S. Conflict of interest: The authors have no relevant financial or non-financial interests to disclose. References Arolla RG, Cherukupalli N, Khareedu VR, Vudem DR (2015) DNA barcoding and haplotyping in different species of Andrographis . Biochem Syst Ecol 62:91–97. https://doi.org/10.1016/j.bse.2015.08.001 Attri DC, Dhyani P, Trivedi VL, et al (2024) Current evidence on molecular mechanisms of andrographolide in cancer. Curr Med Chem. https://doi.org/10.2174/0109298673295496240530100728 Banerji PK (1990) On the geology of the eastern ghats of orissa and andhra pradesh, india. In: Precambrian continental crust and its economic resources. 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Environ Monit Assess 191:784. https://doi.org/10.1007/s10661-019-7686-7 Panraksa P, Ramphan S, Khongwichit S, Smith DR (2017) Activity of andrographolide against dengue virus. Antiviral Res 139:69–78. https://doi.org/10.1016/j.antiviral.2016.12.014 POWO (2024) Plants of the World Online. Facilitated by the Royal Botanic Gardens, Kew. In: Plants of the World Online. Kew Science. http://www.plantsoftheworldonline.org/. Accessed 22 Jun 2024 Premendran SJ, Salwe KJ, Pathak S, et al (2011) Anti-cobra venom activity of plant Andrographis paniculata and its comparison with polyvalent anti-snake venom. J Nat Sci Biol Med 2:198–204. https://doi.org/10.4103/0976-9668.92326 Rajeevan M, Mohapatra M, Unnikrishnan CK, et al (2022) Northeast Monsoon of South Asia, Meteorological Monograph Ramachandran RM, Roy PS, Chakravarthi V, et al (2018) Long-term land use and land cover changes (1920–2015) in Eastern Ghats, India: Pattern of dynamics and challenges in plant species conservation. Ecological Indicators 85:21–36. https://doi.org/10.1016/j.ecolind.2017.10.012 Rambaut A, Drummond AJ, Xie D, et al (2018) Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Syst Biol 67:901–904. https://doi.org/10.1093/sysbio/syy032 Rambaut A (2018) FigTree. Version 1.4.4. Institute of Evolutionary Biology, University of Edinburgh Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574. https://doi.org/10.1093/bioinformatics/btg180 Ronquist F, Teslenko M, van der Mark P, et al (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. 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The Geographical Teacher Supplement no. 2:1–92 Siripongboonsitti T, Ungtrakul T, Tawinprai K, et al (2023) Efficacy of Andrographis paniculata extract treatment in mild to moderate COVID-19 patients being treated with favipiravir: A double-blind, randomized, placebo-controlled study (APFaVi trial). Phytomedicine 119:155018. https://doi.org/10.1016/j.phymed.2023.155018 Staden R, Beal KF, Bonfield JK (2000) The Staden package, 1998. Methods Mol Biol 132:115–130. https://doi.org/10.1385/1-59259-192-2:115 Surveswaran S, Kambale SS, Srivatsav M, et al (2021) Origin and diversification of Indian Ceropegieae (Apocynaceae) and possible relation to the Indian monsoon. J Syst Evol 59:93–112. https://doi.org/10.1111/jse.12578 Surveswaran S, Tiwari N, Karanth PK, et al (2022) Molecular phylogenetics and character evolution in Haplanthodes (Acanthaceae), an endemic genus from peninsular India. Nord J Bot. https://doi.org/10.1111/njb.03238 Swofford DL (2003) PAUP*. Phylogenetic analysis using parsimony (* and other methods). Version 4. . Sinauer Associates, Sunderland., USA Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis version 11. Mol Biol Evol 38:3022–3027. https://doi.org/10.1093/molbev/msab120 Thulsi Rao K, Sai Bhaskar Reddy N, Umamaheswar Reddy C (2019) Eastern Ghats Environmental Outlook. Greens’ Alliance For Conservation of Eastern Ghats, Hyderabad, India Tripp EA, McDade LA (2014) A rich fossil record yields calibrated phylogeny for Acanthaceae (Lamiales) and evidence for marked biases in timing and directionality of intercontinental disjunctions. Syst Biol 63:660–684. https://doi.org/10.1093/sysbio/syu029 Vaidya G, Lohman DJ, Meier R (2011) SequenceMatrix: concatenation software for the fast assembly of multi-gene datasets with character set and codon information. Cladistics 27:171–180. https://doi.org/10.1111/j.1096-0031.2010.00329.x Zhisheng A, Kutzbach JE, Prell WL, Porter SC (2001) Evolution of Asian monsoons and phased uplift of the Himalaya-Tibetan plateau since Late Miocene times. Nature 411:62–66. https://doi.org/10.1038/35075035 Additional Declarations No competing interests reported. Supplementary Files FigS1.AndrographisDECvsDECJM0unconstrainedv2nullfalsemerged.pdf TableS1.AndrographisBiogeographytable.docx TableS2.BioGeoBEARSrestable.xlsx 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4934034","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":349890369,"identity":"855e036f-e7b1-4b4a-ab1a-1db64141c3dc","order_by":0,"name":"Siddharthan Surveswaran","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYDACZgbDBwwMB8CIgcGAOC3GBiRqYWAwk0BoIQbItzNvq+apuJPYd4D54QeGgjuEtRgcZiu7zXPmWeLMA2zGEgwGz4jQwsxjdpu37XDihgMMZiATiHBYM49ZMUQL+zfitDAc5jFjhmjhIdIWoF+KJeecOWw88zBPsUQCUQ7rP7zxw5uKw7J9x9s3fvjwhxiHQYFjAzOQTCBeAwODPSmKR8EoGAWjYIQBAObXO1vToGwVAAAAAElFTkSuQmCC","orcid":"","institution":"University of Hyderabad","correspondingAuthor":true,"prefix":"","firstName":"Siddharthan","middleName":"","lastName":"Surveswaran","suffix":""},{"id":349890370,"identity":"4fa3158b-99c5-437d-a7f0-056d79e8b351","order_by":1,"name":"Yuvasri Kalaimani","email":"","orcid":"","institution":"University of Hyderabad","correspondingAuthor":false,"prefix":"","firstName":"Yuvasri","middleName":"","lastName":"Kalaimani","suffix":""}],"badges":[],"createdAt":"2024-08-18 15:29:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4934034/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4934034/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64591642,"identity":"12bdb7ce-e423-4699-b8ef-3691b7554730","added_by":"auto","created_at":"2024-09-16 09:33:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1821204,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eAndrographis echioides\u003c/em\u003e. a-c: Samples from University of Hyderabad campus. D: habit from Gulbarga fort, Kalburgi.\u003c/p\u003e","description":"","filename":"Fig.1echioides.tiff.png","url":"https://assets-eu.researchsquare.com/files/rs-4934034/v1/aef1c16180064c04158bcb6f.png"},{"id":64591639,"identity":"12433962-d9a8-4421-ad0e-b32ed8dd26e2","added_by":"auto","created_at":"2024-09-16 09:33:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":261690,"visible":true,"origin":"","legend":"\u003cp\u003eBayesian phylogenetic analysis tree generated by MrBayes using five combined plastid markers, \u003cem\u003ematK\u003c/em\u003e, \u003cem\u003erbcL\u003c/em\u003e,\u003cem\u003etnrL-F\u003c/em\u003e, \u003cem\u003epsbA\u003c/em\u003e-\u003cem\u003etrnH\u003c/em\u003e and \u003cem\u003etrnG-R\u003c/em\u003e. Numbers on branches indicate Bayesian Posterior Probability (BPP) and Maximum-likelihood bootstrap support (MLBS) support respectively. Scale indicates number of substitutions along the branches.\u003c/p\u003e","description":"","filename":"Fig.2combined37.nex.con.treedit1.png","url":"https://assets-eu.researchsquare.com/files/rs-4934034/v1/65895bd219a70a1da6e9c605.png"},{"id":64592296,"identity":"4a84e937-46f9-4581-9059-87b8117803f3","added_by":"auto","created_at":"2024-09-16 09:41:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":462187,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular dating chronogram generated by BEAST using five combined plastid markers, \u003cem\u003ematK\u003c/em\u003e, \u003cem\u003erbcL\u003c/em\u003e,\u003cem\u003etnrL-F\u003c/em\u003e, \u003cem\u003epsbA\u003c/em\u003e-\u003cem\u003etrnH\u003c/em\u003e and \u003cem\u003etrnG-R\u003c/em\u003e. Numbers below branches indicate mean ages of the nodes in millions of years ago (MYA). Numbers in parentheses above branches indicate 95% highest posterior density ranges. Asterisk in branches indicate high posterior probability support, i.e., 0.95.\u003c/p\u003e","description":"","filename":"Fig.3combine37beastdatingmatk.con95HPD.tre2.png","url":"https://assets-eu.researchsquare.com/files/rs-4934034/v1/034104b59962259abcb86913.png"},{"id":64592648,"identity":"b5901c1b-0803-4f81-b5f2-91721763ad6a","added_by":"auto","created_at":"2024-09-16 09:49:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1330218,"visible":true,"origin":"","legend":"\u003cp\u003eBiogeographical analysis tree inferred from BioGeoBEARS analysis according to the DEC model. Distribution ranges are indicated in the tips. Ancestral areas of the nodes are inferred by BioGeoBears. Biogeographical ranges: W: western peninsular India, AF: Africa, A: Asia, E: eastern peninsular India and L: Sri Lanka.\u003c/p\u003e","description":"","filename":"Fig.4BioGeoBEARSedited18824.png","url":"https://assets-eu.researchsquare.com/files/rs-4934034/v1/fff3fe1182528c9c7d4bd074.png"},{"id":109759693,"identity":"1d38b582-f206-4b60-b965-ac64e9bc7f7b","added_by":"auto","created_at":"2026-05-22 07:27:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4892706,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4934034/v1/34b7c7d1-263b-4c29-b6d3-d566a59c250f.pdf"},{"id":64592297,"identity":"be9b9740-b6d7-4ec0-9e61-0ed4abdfc100","added_by":"auto","created_at":"2024-09-16 09:41:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":365957,"visible":true,"origin":"","legend":"","description":"","filename":"FigS1.AndrographisDECvsDECJM0unconstrainedv2nullfalsemerged.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4934034/v1/8f908fbe1337e5bf64073efa.pdf"},{"id":64591641,"identity":"5d06917d-d1fe-4b47-9391-b2eb9afed554","added_by":"auto","created_at":"2024-09-16 09:33:37","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":306258,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.AndrographisBiogeographytable.docx","url":"https://assets-eu.researchsquare.com/files/rs-4934034/v1/71568d4daae964f6bfae37c6.docx"},{"id":64591646,"identity":"7250b7ce-7247-4892-9191-921fb839bb74","added_by":"auto","created_at":"2024-09-16 09:33:38","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10468,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.BioGeoBEARSrestable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4934034/v1/34200b150d5da8fe17d73e1e.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biogeographical origin of Andrographis Wall. ex Nees (Acanthaceae) in eastern peninsular India based on molecular phylogenetics","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe family Acanthaceae is pantropical in distribution with about 4900 species and 191 accepted genera (McDade et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Manzitto-Tripp et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Most are herbs or shrubs, including some twining forms, and some spiny forms. According to the most recent revised classification, Acanthaceae is classified into four subfamilies: Nelsonioideae, Thunbergioideae, Acanthoideae, and Avicennioideae. Acanthoideae, the most diverse subfamily, comprises eight recognised tribes: Acantheae, Physacantheae, Barlerieae, Andrographideae, Whitfieldieae, Neuracantheae, Ruellieae, and Justicieae. The subfamily Acanthoideae is found only in the Old World, and its tribe Andrographideae is exclusively Asian.\u003c/p\u003e \u003cp\u003ePollen with apertural margins and or surfaces conspicuously thickened intricate ornamentation with conical spines is the unique character of the tribe. Other synapomorphies are ascending cochlear aestivation (imbricate aestivation: one outer petal, three imbricate and one inner), two stamens with or without staminodes and usually bilabiate corollas. They share these characters Justiceae. However, the number of seeds per capsule is numerous, 6\u0026ndash;20 whereas it is 2\u0026ndash;4 in Justiceae (Manzitto-Tripp et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are currently 8 recognized genera: \u003cem\u003eAndrographis\u003c/em\u003e Wall. ex Nees, \u003cem\u003eDiotacanthus\u003c/em\u003e Benth., \u003cem\u003eGraphandra\u003c/em\u003e J. B. Imlay, \u003cem\u003eGymnostachyum\u003c/em\u003e Nees, \u003cem\u003eHaplanthodes\u003c/em\u003e Kuntze, \u003cem\u003eHaplanthus\u003c/em\u003e Nees, \u003cem\u003ePhlogacanthus\u003c/em\u003e Nees and \u003cem\u003eSphinctacanthus\u003c/em\u003e Benth. (Manzitto-Tripp et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOf these, \u003cem\u003eHaplanthodes\u003c/em\u003e with 4 species is endemic to peninsular India (Deshmukh et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eHaplanthus\u003c/em\u003e also with 4 species was reinstated from \u003cem\u003eAndrographis\u003c/em\u003e based on morphological analysis (Gnanasekaran et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). One species of \u003cem\u003eHaplanthus\u003c/em\u003e occurs in peninsular India and three are in southeast (SE) Asia including Andaman islands. \u003cem\u003eGymnostachyum\u003c/em\u003e with 51 species is mostly distributed in SE Asia with few species in peninsular India. \u003cem\u003ePhlogacanthus\u003c/em\u003e with 41 species is also mostly SE Asian. \u003cem\u003eGraphandra\u003c/em\u003e and \u003cem\u003eSphinctacanthus\u003c/em\u003e are distributed in SE Asia with 1 species each. \u003cem\u003eSphinctacanthus\u003c/em\u003e is distributed in northeast India and Myanmar with only one species, \u003cem\u003eS. griffthii\u003c/em\u003e which has close morphological as well as geographical affinities with \u003cem\u003ePhlogacanthus\u003c/em\u003e and \u003cem\u003eGymnostachyum\u003c/em\u003e (Hansen \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). \u003cem\u003eDiotacanthus\u003c/em\u003e with two species from southern India is sometimes treated with \u003cem\u003ePhlogacanthus (\u003c/em\u003ePOWO \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e\u003cem\u003e)\u003c/em\u003e. \u003cem\u003eCystacanthus\u003c/em\u003e has been merged under \u003cem\u003ePhlogacanthus\u003c/em\u003e as a section (Deng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003eAndrographis\u003c/em\u003e Wall. ex Nees is composed of annual herbaceous plants native to Asia with 25 species (POWO \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Almost all are native to peninsular India except of the medicinally important species \u003cem\u003eAndrographis paniculata\u003c/em\u003e and \u003cem\u003eAndrographis echioides\u003c/em\u003e (syn. \u003cem\u003eIndoneesiela echioides\u003c/em\u003e) which are spread in SE Asia and Sri Lanka (POWO \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A. paniculata is distributed all over the world and it may be cultivated or naturalised due to its medicinal value. Most of the other \u003cem\u003eAndrographis\u003c/em\u003e species are rare and endemic, and they are especially distributed in the eastern parts of the Deccan plateau and the Eastern Ghats (Neeraja et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eAndrographis paniculata\u003c/em\u003e has been used in ethnomedicine by various cultures (Samy et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Hossain et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Its most active compound, a diterpenoid, Andrographolide and is called the \u0026ldquo;King of Bitters\u0026rdquo;. It has been used in treating snake bites (Premendran et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Nayak et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), treatment for dengue (Panraksa et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and also implicated in the cure of cancer (Attri et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). During the COVID-19 pandemic, a decoction of \u003cem\u003eAndrographis\u003c/em\u003e was used as a cure for the systems and recovery of patients. There are various reports, a few showing some efficacy (Sa-Ngiamsuntorn et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and many others showing no efficacy (Kanokkangsadal et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Siripongboonsitti et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Similarly, \u003cem\u003eAndrographis echiodes\u003c/em\u003e has been used as a medicinal plant by various cultures (Kumar et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Samy et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Chinnappan \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and it has been implicated to have anticancer and antibacterial activities (Elangovan et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMolecular phylogenetic analysis of the tropical Asian Andrographideae is sparse and the monophyly of the genera is still in a state of flux (Deng et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e; Manzitto-Tripp et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Arolla et al. (Arolla et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) attempted a phylogeny of \u003cem\u003eAndrographis\u003c/em\u003e and their collection was mostly from Andhra Pradesh, ie., the Eastern Ghats and associated areas. Surveswaran et al., ((Surveswaran et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) showed a close relationship between \u003cem\u003eHaplanthodes\u003c/em\u003e and \u003cem\u003eHaplanthus\u003c/em\u003e with \u003cem\u003eAndrographis\u003c/em\u003e. However, a comprehensive phylogeny \u003cem\u003eAndrographis\u003c/em\u003e and analysis of its biogeography within India are lacking. Therefore, in this study, we aimed to, 1) establish the monophyly of \u003cem\u003eAndrographis\u003c/em\u003e, 2) to prove the Indian origin of \u003cem\u003eAndrographis\u003c/em\u003e and 3) to test whether there are separate lineages in the western and eastern parts of peninsular India associated with the Western Ghats and Eastern Ghats. To this end, we used five plastid markers, \u003cem\u003erbcL\u003c/em\u003e, \u003cem\u003ematK\u003c/em\u003e, \u003cem\u003etrnLF\u003c/em\u003e, \u003cem\u003epsbA\u003c/em\u003e-\u003cem\u003etrnH\u003c/em\u003e and \u003cem\u003etrnGR\u003c/em\u003e, mostly from the GenBank and a few newly generated sequences. Using this data we performed the most comprehensive phylogeny of Andrographideae followed by a dated phylogeny and performed a biogeographic analysis using Bayesian and Likelihood methods respectively.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003ePlant material, DNA extraction and PCR amplification\u003c/p\u003e \u003cp\u003eSamples of \u003cem\u003eA. echioides\u003c/em\u003e were collected from the University of Hyderabad campus in Telangana and Kalburgi (Gulbarga) in Karnataka. The sequences of the rest of the Andrographideae, including several other genera of Acanthaceae, were downloaded from the GenBank database (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Total genomic DNA was isolated using the CTAB method. The study utilized plastid markers: two coding regions, \u003cem\u003ematK\u003c/em\u003e and \u003cem\u003erbcL\u003c/em\u003e and three non-coding regions, \u003cem\u003etrnG\u003c/em\u003e-\u003cem\u003etrnR\u003c/em\u003e, \u003cem\u003etrnL\u003c/em\u003e-\u003cem\u003etrnF\u003c/em\u003e, and \u003cem\u003epsbA\u003c/em\u003e-\u003cem\u003etrnH\u003c/em\u003e. The primers used were the same as in Surveswaran et al., (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Each PCR reaction had the following components: 10 \u0026micro;l of the 2x Taq DNA Polymerase Master Mix RED (Ampliqon, Odense, Denmark), 1 \u0026micro;l of forward and reverse primers (10 pmol/\u0026micro;l) each, and 1\u0026ndash;2 \u0026micro;l (\u0026gt;\u0026thinsp;10ng to \u0026lt;\u0026thinsp;200ng of the extracted DNA) of the template DNA and made upto to 20 \u0026micro;l with PCR grade water. The PCR steps were as follows: Initial denaturation: 94\u0026deg;C, 2 mins; cyclic denaturation: 94\u0026deg;C, 30 secs; cyclic annealing: 58\u0026deg;C, 30 secs; cyclic extension: 72\u0026deg;C, 50 secs; 35 cycles; and final extension: 72\u0026deg;C, 3 mins. The PCR products were verified on agarose gels before being sent for Sanger sequencing to Barcode Biosciences Pvt. Ltd. (Bangalore, India).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSpecies names, voucher information and accession numbers of sequences obtained from the GenBank and generated in this study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVoucher\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ematK\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003erbcL\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003etnrL-F\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003epsbA-trnH\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003etrnG-R\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis affinis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSK1723\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMH142855\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis alata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHJCB2250,SK1721B, CNS06AA2013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMT445715\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMH142897\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMH142862\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMT445682\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMT445664\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis atropurpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSK1716D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMH142866\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis beddomei\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCherukupalli, N, Osmania University, India\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMN857164\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMN857163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis echioides\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDSD02F, SK1682A KX378015 (Prashanth hande), Daniel.s.n,, CNS07AE2013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMW601018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMW601009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMH142868\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eKC118378\u003c/p\u003e \u003cp\u003e(Tripp)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis echioides\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUOHxx, University of Hyderabad\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eXXXXXXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eXXXXXXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eXXXXXXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eXXXXXXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis echioides\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUOHxx, Gulbarga India\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eXXXXXXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eXXXXXXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eXXXXXXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eXXXXXXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis elongata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSK1708B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMH142871\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis glandulosa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHJCBE265,\u003c/p\u003e \u003cp\u003eCNS04AG2013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKP455309\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT445702\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMT445686\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMT445667\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis gracilis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSK1724C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMH142876\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis lawsonii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSamydurai,P, Bharathiyar University, India\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMH428005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMH428004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis lineata\u003c/em\u003e var. \u003cem\u003elawii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDSD08E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMW601019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMW601010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis lobelioides\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSamydurai, P, Bharathiyar University, India\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMH423499\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMH388772\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis longipedunculata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSK1694C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMH142880\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis macrobotrys\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSK1715B, DSD03B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMW601021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMW601012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMH142885\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis megamalayana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHJCB1454, HJCB1190\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMT445716\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT445700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMT445683\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMT445665\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis nallamalyana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCNS01AN2013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKP455311\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKP455305\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis paniculata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCNS05AP2013, HJCB0572\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKF521888\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKF521878\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eKT074995\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLC743551\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMT445666\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrographis producta\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDSD04C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMW601024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMW601015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAndrograophyis serpyllifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDSD06A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMW601025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMW601016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMH142891\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eHaplanthodes neilgherryensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMML-532\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMT445703\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT445690\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMT445668\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMT445650\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eHaplanthodes plumosa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMML-546\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMT445708\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT445691\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMT445673\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMT445655\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eHaplanthodes tentaculata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMML-531\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMT445712\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT445695\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMT445677\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMT445659\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eHaplanthodes verticillatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMML-553\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMT445710\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT445693\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMT445675\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMT445657\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eHaplanthodes.sp. HSP1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMML-544\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMT445713\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT445696\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMT445678\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMT445660\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eHaplanthus ovatus 1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDSD05A, HJCB5432, HJCB1962\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMW601022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMW601013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMT445679\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMT445662\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eHaplanthus ovatus 2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHJCB1962\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMT445714\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT445698\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eGymnostachyum canescens\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDev et al., KFRI, Peechi, India\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKM217081\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKM217085\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eGymnostachyum febrifugum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDev et al., KFRI, Peechi, India\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKM217082\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKM217086\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eGymnostachyum warrierianum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSAD-2014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKM217083\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKM217087\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCystacanthus paniculatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCPG25312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKX526467\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCystacanthus turgidus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRBG Kew 1996\u0026thinsp;\u0026minus;\u0026thinsp;479 K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEU528919\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eKJ140526\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePhlogacanthus curviflorus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCPG151, BB0613\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKX526475\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKX527201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMW722784\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eKR533704\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePhlogacanthus parviflorus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBoro,N. Guwahati University, India\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMW810858\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePhlogacanthus thyrsiflorus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBoro,N. Guwahati University, India\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMW722783\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eKC118401\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBarleria lupulina\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBKF189453\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eKT161362\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBarleria prionitis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCMPR8789, BKF189452\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOL580741\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMZ461574\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eKT075002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eKT161339\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSequence assembly and phylogenetic analysis\u003c/p\u003e \u003cp\u003eForward and reverse sequences were assembled using the pregap4 and gap4 modules in the Staden package ver. 2.0.0 beta 11 (Staden et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Sequences were aligned using the CLUSTAL W algorithm in MEGA version 11 (Tamura et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Individual alignments were checked and manually corrected using AliView ver. 1.28 (Larsson \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The alignments were concatenated using SequenceMatrix ver. 1.9 (Vaidya et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Sequence alignment characteristics such as parsimony informative sites were estimated using PAUP ver. 4.0 beta 10 (Swofford \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The best-fit model of sequence evolution was deduced using jModeltest ver. 2.1.10 (Darriba et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The selection of the best model was based on the Akaike Information Criterion with correction (AICc).\u003c/p\u003e \u003cp\u003eBayesian phylogenetic analysis was performed using MrBayes ver. 3.2.7 (Ronquist and Huelsenbeck \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Ronquist et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). A partitioned dataset with the best model selected according to jModteltest2 was used. Ten million runs of Metropolis-coupled Markov chains (MCMC) were run as two independent runs with 4 chains each. The convergence of the runs was checked using the average standard deviation of split frequencies being less than 0.01 and also by Tracer ver.1.7.2 as described below for BEAST analysis. Maximum-likelihood analysis was performed in IQTREE ver. 2.0.7 with model selection of the partitioned dataset followed by tree-building using the merged partitions and ultrafast bootstrapping with 1000 replicates (Minh et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For Bayesian analysis, BEAST ver. 2.7.6 (Bouckaert et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) was used. An XML file for input into BEAST was prepared using BEAUTi. The best-fit model for each partition was set up. Molecular dating analysis was performed using secondary data calibration from (Tripp and McDade \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Surveswaran et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The age of Andrographideae was between 18.44 and 11.86\u0026nbsp;million years ago (mya) with a mean of 9.44 mya. These dates were modelled using a uniform distribution with upper, lower and offset values as 12, 18 and 5 respectively. Ten million MCMC iterations were performed, with sampling every 10,000 runs. The convergence of the runs was checked using Tracer ver. 1.7.2 (Rambaut et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) as effective sample sizes (ESS) values for all parameters greater than 200. After the run was complete, the consensus tree was computed using Treeannotator ver. 2.7.6 with a burnin of 25% of the initial trees. Trees were visualised in FigTree ver. 1.4.4 (Rambaut \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince Andrographideae is tropical Asian in distribution and the diversity of \u003cem\u003eAndrographis\u003c/em\u003e is high in peninsular India is high, we separated peninsular India to the east and west, in accordance to the Eastern Ghats and Western Ghats, the mountain stretches which harbour high biodiversity in peninsular India. The regions taken for analysis were South East Asia (including, south China, northeast India and peninsular Malaysia ) designated as A, Eastern Ghats denoted as E, Africa denoted as F, Sri Lanka denoted as L and Western Ghats denoted as W. The distribution data of the species were collected from GBIF (GBIF.org \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), India Flora Online (India Flora Online \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and POWO (POWO \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Biogeographic analysis was performed using the BioGeoBEARS (Matzke \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), R package in R ver. 4.4.1 (R Core Team \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) using RStudio ver. 2024.04.1 Build 748 (RStudio Team \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). All models of biogeography, DEC, Divalike and Bayarealike along with jump dispersal (+\u0026thinsp;J) were analysed. The max areas were set to FALSE in the DEC model, and the rest of the models were set to TRUE. The best-fit model for biogeography was selected based on the AICc values after the likelihood ratio test implemented in BioGeoBears.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn this work, most of the sequence data were obtained from the GenBank database except accessions of \u003cem\u003eAndrographis echioides\u003c/em\u003e, one from Hyderabad, Telangana and another from Kalburgi, Karnataka, were collected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Their sequences were generated for the study despite the availability of data for one \u003cem\u003eA. echioides\u003c/em\u003e accession in the public databases as a comparison.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe combined molecular sequence dataset including \u003cem\u003ematK\u003c/em\u003e, \u003cem\u003erbcL\u003c/em\u003e, \u003cem\u003epsbA\u003c/em\u003e-\u003cem\u003etrnH\u003c/em\u003e, \u003cem\u003etrnL\u003c/em\u003e-\u003cem\u003etrnF\u003c/em\u003e, and \u003cem\u003etrnG\u003c/em\u003e-\u003cem\u003etrnR\u003c/em\u003e consisted of 2510 nucleotides of which 204 (8%) were parsimoniously informative. The nucleotide statistics and best-fit models for each dataset and the combined dataset are summarised (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The dataset included all the genera of Andrographideae except \u003cem\u003eDiatacanthus\u003c/em\u003e, \u003cem\u003eGraphandra\u003c/em\u003e and \u003cem\u003eSphinctacanthus\u003c/em\u003e sensu Manzitto-Tripp et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSequence information plastid markers used\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDataset\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTaxa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLength\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConserved sites\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eParsimony informative characters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVariable characters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBest model according to AICc (calculated using jModeltest2)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCombined genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2510\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e373\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGTR\u0026thinsp;+\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ematK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e689\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e546\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGTR\u0026thinsp;+\u0026thinsp;I\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003epsbA\u003c/em\u003e-\u003cem\u003etrnH\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e549\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e492\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHKY\u0026thinsp;+\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erbcL\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e329\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e233\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGTR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003etrnG\u003c/em\u003e-\u003cem\u003etrnR\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e365\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e310\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHKY\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003etrnL\u003c/em\u003e-\u003cem\u003etrnF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e558\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e525\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHKY\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe Bayesian phylogenetic analysis tree showed a polyphyly of \u003cem\u003eAndrographis\u003c/em\u003e with respect to \u003cem\u003eHaplanthodes\u003c/em\u003e and \u003cem\u003eHaplanthus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) considered here as \u003cem\u003eAndrographis sensu lato\u003c/em\u003e (s.l). The Bayesian Posterior Probability (BPP) support\u0026thinsp;\u0026gt;\u0026thinsp;0.95 was considered very high support, \u0026gt;\u0026thinsp;0.9 to 0.94 was considered good support and \u0026lt;\u0026thinsp;0.9 was considered as not supported. Similarly, Maximum-likelihood bootstrap (MLBS) support\u0026thinsp;\u0026gt;\u0026thinsp;90% was considered very high support, \u0026gt;\u0026thinsp;50\u0026ndash;90% was considered good support and anything below 50% was considered unsupported. \u003cem\u003ePhlogacanthus\u003c/em\u003e (incl. \u003cem\u003eCystacanthus\u003c/em\u003e) was monophyletic with good MLBS support but without BPP support. \u003cem\u003eGymnostachyum\u003c/em\u003e with three species was highly supported.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEighteen of the 25 currently recognised \u003cem\u003eAndrographis\u003c/em\u003e (POWO \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) were used in this study. \u003cem\u003eAndrographis\u003c/em\u003e s.l. could be further divided into 4 clades in our analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The first, well supported clade consisted for \u003cem\u003eHaplanthus ovatus\u003c/em\u003e along with \u003cem\u003eA. elongata\u003c/em\u003e, \u003cem\u003eA. alata\u003c/em\u003e and \u003cem\u003eA. affinis\u003c/em\u003e (\u003cem\u003eHaplanthus\u003c/em\u003e-\u003cem\u003eAndrographis\u003c/em\u003e clade, clade no. 1, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The second, well supported clade was the \u003cem\u003eHaplanthodes\u003c/em\u003e clade (clade 2, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) which included all the 4 species of \u003cem\u003eHaplanthodes\u003c/em\u003e. The next fully supported clade contained only \u003cem\u003eAndrographis\u003c/em\u003e species i.e., \u003cem\u003eAndrographis sensu stricto\u003c/em\u003e clade. This \u003cem\u003eAndrographis s.s\u003c/em\u003e clade was divided into two sister sub-clades which are designated as \u003cem\u003eAndrographis\u003c/em\u003e clade I and \u003cem\u003eAndrographis\u003c/em\u003e clade II (clades 3,4, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These sister clades are different in their biogeography \u003cem\u003eAndrographis\u003c/em\u003e clade I is found in the eastern part of peninsular India whereas the \u003cem\u003eAndrographis\u003c/em\u003e clade II is found in the western part of peninsular India. The \u003cem\u003eAndrographis\u003c/em\u003e clade I (clade 3, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) was not well supported and contained eastern peninsular Indian species: \u003cem\u003eA. nallamalayana\u003c/em\u003e, \u003cem\u003eA. serpyllifolia\u003c/em\u003e, \u003cem\u003eA. glandulosa\u003c/em\u003e, \u003cem\u003eA. echioides\u003c/em\u003e, \u003cem\u003eA. beddomei\u003c/em\u003e and \u003cem\u003eA. longipedunculata\u003c/em\u003e. In this clade, except for \u003cem\u003eA. nallamalayana\u003c/em\u003e, which is the earliest divergent species, the rest of the nested clades were highly supported. The position of different accessions was variable and did not show congruence with dating analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Andrographis clade II (clade 4, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) was significantly supported and included \u003cem\u003eA. atropurpurea\u003c/em\u003e, \u003cem\u003eA. gracilis\u003c/em\u003e, \u003cem\u003eA. lobelioides\u003c/em\u003e, \u003cem\u003eA. lineata\u003c/em\u003e, \u003cem\u003eA. lawsonii\u003c/em\u003e, \u003cem\u003eA. megamalyana\u003c/em\u003e, \u003cem\u003eA. macrobotrys\u003c/em\u003e, \u003cem\u003eA. paniculata\u003c/em\u003e and \u003cem\u003eA. producta\u003c/em\u003e. The relationship among the species in this clade was not well resolved. All species in this clade were in the western peninsular India.\u003c/p\u003e \u003cp\u003eThe dated BEAST phylogram of the same partitioned dataset described above resolved the same topology as the Bayesian analysis. However, some minor differences in the not supported clades were different between the ML analysis and BEAST analysis. The major clades were supported with \u0026gt;\u0026thinsp;0.95 BPP support in BEAST analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The median age of the nodes as inferred from the BEAST analysis are indicated along with the 95% highest posterior density in square brackets (95% HPD). The age of the tribe Andrographideae clade and the \u003cem\u003eAndrographis s.l\u003c/em\u003e. clade was found to be 18.17 MYA and 9.09 MYA respectively. The \u003cem\u003eAndrographis\u003c/em\u003e clade I, (clade 3, in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) with the inclusion with \u003cem\u003eA. nallamalayana\u003c/em\u003e was not supported. With the exclusion of \u003cem\u003eA. nallamalayana\u003c/em\u003e, the remaining species were highly supported as monophyletic in BPP. In the BEAST analysis, \u003cem\u003eA. echioides\u003c/em\u003e accessions 1 and 2, collected by us, were monophyletic without support. \u003cem\u003eA. echiodes\u003c/em\u003e from the GenBank was closest in relationship to \u003cem\u003eA. beddomei\u003c/em\u003e also without support. The rest of the clades, clades, 1,2,4 were well supported. The monophyly of \u003cem\u003eGymnostachyum\u003c/em\u003e and \u003cem\u003ePhlogacanthus\u003c/em\u003e was moderately supported (\u0026lt;\u0026thinsp;0.9\u0026thinsp;\u0026gt;\u0026thinsp;0.95 BPP).\u003c/p\u003e \u003cp\u003eThe locality information of Andrographis and other taxa was collected from GBIF and POWO and summarised (Supplementary data, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The best model of biogeographical distribution according to the AICc was found to be DEC without jump (+\u0026thinsp;J parameter). The alternative distribution models, such as DIVALIKE, and BAYAREA-like with and without J parameter, were tested in BioGeoBEARS (Supplementary data, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). According to the DEC model of biogeographical distribution as inferred in BioGeoBEARS, the ancestry of the tribe Andrographideae was found to be western region of peninsular India. The \u003cem\u003eGymnostachyum\u003c/em\u003e clade and the \u003cem\u003ePhlogacanthus\u003c/em\u003e clade have an ancestry in western peninsular India whereas the \u003cem\u003eAndrographis\u003c/em\u003e s.l. clade has its ancestry in the eastern peninsular India. There have been two dispersals into the western part of India, that is in the \u003cem\u003eHaplanthodes\u003c/em\u003e clade (clade 2) and in the \u003cem\u003eAndrographis\u003c/em\u003e clade II (clade 4).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe molecular phylogenetics of the tribe Andrographideae is not well understood (Manzitto-Tripp et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and therefore this study is the most comprehensive work on the tribe. The sampling of the genus is more than 70% of the currently accepted species according to POWO. The largest genus of Andrographideae is \u003cem\u003eGymnostachyum\u003c/em\u003e with 51 species spreading from India in the west to the Philippines in the east. The second most speciose genus is \u003cem\u003ePhlogacanthus\u003c/em\u003e with 41 which spreads eastwards up to Sulawesi from India. \u003cem\u003ePhlogacanthus\u003c/em\u003e now includes \u003cem\u003eCystacanthus\u003c/em\u003e (Deng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e) but we have retained the old name in the figures in this work for easy recognition. The next largest genus is \u003cem\u003eAndrographis\u003c/em\u003e with 25 species that are exclusively distributed in India. The distribution extends up to Myanmar which is adjacent to India but these species are now treated under \u003cem\u003eHaplanthus\u003c/em\u003e (Gnanasekaran et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). We have sampled only \u003cem\u003eHaplanthus ovatus\u003c/em\u003e which is distributed in the eastern parts of India, in the Mahendragiri mountains, which is a part of the Eastern Ghats. The other species of \u003cem\u003eHaplanthus\u003c/em\u003e, \u003cem\u003eH\u003c/em\u003e. \u003cem\u003ehygrophiloides\u003c/em\u003e and \u003cem\u003eH\u003c/em\u003e. \u003cem\u003elaxiflorus\u003c/em\u003e show a distribution eastwards from India such as Myanmar, Bhutan, Thailand, Vietnam, Cambodia, Malaysia and parts of the Andaman Islands (Gnanasekaran et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). \u003cem\u003eH. hygrophiloides\u003c/em\u003e (Pegu, Myanmar), \u003cem\u003eH. laxiflorus\u003c/em\u003e var. \u003cem\u003etenuiflora\u003c/em\u003e (Andaman Islands, India), \u003cem\u003eH. laxiflorus\u003c/em\u003e var. \u003cem\u003erecedens\u003c/em\u003e (Tenasserim, Mooleeyit, Myanmar) and \u003cem\u003eH. rosulatus\u003c/em\u003e (Thailand) need to be added to the phylogeny to understand the status of \u003cem\u003eHaplanthus\u003c/em\u003e in relation to \u003cem\u003eAndrographis\u003c/em\u003e. Additionally, the morphological synapomorphies uniting the three species \u003cem\u003eAndrographis\u003c/em\u003e, \u003cem\u003eA. affinis\u003c/em\u003e, \u003cem\u003eA. alata\u003c/em\u003e and \u003cem\u003eA. elongata\u003c/em\u003e with \u003cem\u003eHaplanthus\u003c/em\u003e requires investigation. These three species, though originated in India they have a distribution eastwards and southwards, i.e., southeast Asia and Sri Lanka.\u003c/p\u003e \u003cp\u003eThe ancestral region of the entire \u003cem\u003eAndrographis\u003c/em\u003e is the eastern peninsular India. Within this, A\u003cem\u003endrographis-Haplanthodes\u003c/em\u003e clade (clade 1) is also eastern peninsular Indian in origin. The Indian endemic, \u003cem\u003eHaplanthodes\u003c/em\u003e, though western Indian in distribution, is eastern Indian in origin and the speciation might have happened after dispersal into the western peninsular Indian region. Similarly in ancestry of the Andgrograhis s.s. Clade is also eastern and Andrographis (clade 4) might have diversified after dispersal into the western peninsular Indian region. In summary, within the eastern peninsular India originated \u003cem\u003eAndrographis\u003c/em\u003e, there are two dispersals into the western peninsular region associated with the Western Ghats, one, \u003cem\u003eHaplanthodes\u003c/em\u003e and another, the \u003cem\u003eAndrographis\u003c/em\u003e s.s. clade II\u003c/p\u003e \u003cp\u003eThough \u003cem\u003eAndrographis\u003c/em\u003e is endemic to the Indian peninsula, the medicinally important \u003cem\u003eA. paniculata\u003c/em\u003e and \u003cem\u003eA. echioides\u003c/em\u003e are only widespread. \u003cem\u003eA. paniculata\u003c/em\u003e is probably under cultivation worldwide due its medicinal properties (GBIF.org \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eA. echioides\u003c/em\u003e is found in Sri Lanka and also probably cultivated in other parts of the world as a potential medicinal plant. However, these occurrences may be due to human mediated distribution due to their use as medicinal plants. Some other species such as \u003cem\u003eA. macrobotrys\u003c/em\u003e, \u003cem\u003eA. elongata\u003c/em\u003e and \u003cem\u003eA. alata\u003c/em\u003e also extend their distribution towards Sri Lanka and southeast Asia. These are most probably recent range expansions.\u003c/p\u003e \u003cp\u003eThe Eastern Ghats is a stretch of mountains which extends throughout the eastern part of peninsular India like its western counterpart, Western Ghats. The Eastern Ghats, though not much studied as the Western Ghats, host several congeners with Western Ghats and would have probably been used as a connecting bridge between the Western Ghats and southeast Asia. However, the Eastern Ghats mountain ranges, though disjunct, is larger in area and harbours a high level of biodiversity due to its diverse forest types in moist and cooler altitudes (Banerji \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Ramachandran et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Thulsi Rao et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Panda et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). One of the greatest plant geographers after Humboldt was the botanist Joseph Dalton Hooker (Egerton \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and the author of the 7 volume treatise, Flora of British India has discussed about the botanical regions and provinces of British India in his \u0026ldquo;Sketch of the flora of British India''. He divides the former British India into 9 provinces: 1. Eastern Himalaya, 2. Western Himalaya, 3. Indus Plain, 4. Gangetic Plain, 5. Malabar, 6. The Deccan, 7. Ceylon, 8. Burma, and 9. The Malayan Peninsula (Hooker \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1904\u003c/span\u003e). The Western Ghats species belong to the Malabar region and the Eastern Ghats species belong to the Deccan region of Hooker\u0026rsquo;s division. The Deccan plateau is an elevated region which encompasses the major part of peninsular India which adjoins the Western Ghats in the west and harbours the Eastern Ghats mountains in the east. The Deccan region is mostly semi-arid because the high mountains of the Western Ghats break the rain clouds and reduce the rainfall towards the eastern regions. The Deccan region is therefore dominated by grasslands and deciduous vegetation (Simkins \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1926\u003c/span\u003e; Khadkikar et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Dash et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Panda et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Bhunia and Patra \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe age of diversification of \u003cem\u003eAndrographis\u003c/em\u003e s.l. as inferred in our study is around 9.09 with a 95% HPD of 7.01 to 11.65 MYA. This corresponds to the age of diversification of other plant genera of plants and animals in the Indian peninsula (Deepak and Karanth \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Surveswaran et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) which has been most probably due to the evolution of the South Asian Monsoon. The diversification of \u003cem\u003eAndrographis\u003c/em\u003e matches with the onset and intensification of the Indian and Asian monsoon as a result of enhanced aridity in the Asian interior around 9\u0026thinsp;\u0026minus;\u0026thinsp;8 MYA (Zhisheng et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The Eastern Ghats clade started diversification around 4.02 MYA with a 95% HPD of 2.56 to 5.37 MYA. Within this period, around 3.6\u0026ndash;2.6 MYA, due to various geological events the South Asian summer monsoon and East Asian summer monsoons might have weakened and the winter monsoons have become prominent (Zhisheng et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The biota of peninsular India, most of which is the Deccan plateau, is dependent on the monsoonal rainfall for their survival. In India, South West or summer monsoons bring rain to the windward side of the Western Ghats from June to September. As the monsoon clouds weaken, the leeward side of the western ghats adjoining the Deccan plateau receives less rainfall but life depends on these seasonal rains for their survival. As the weakened winds reach north towards the Himalayas and retreat, they bring moisture for the Himalayan region. This is the North East monsoon or the winter monsoon which happens during October to December and brings rain to the eastern coast of India which is relatively drier during the summer monsoon period. The Deccan plateau and the Eastern Ghats forms a part of the region fed by the Northeast monsoon (Sankalia and Joshi \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1957\u003c/span\u003e; Rajeevan et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore the eastern part of the Indian peninsula is generally arid and receives less rainfall and therefore considered as a semiarid region. \u003cem\u003eAndrographis\u003c/em\u003e of the eastern peninsular Indian region are probably adapted to this semi-arid condition as shown by some xeric characters such as smaller and narrower leaves and the presence of spines in some of the Acanthaceae members. Several dry adapted plants and animals of the peninsular India are adapted to semi-arid conditions of the Deccan plateau and depend on the monsoon rains for their survival (Surveswaran et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn conclusion, we find that \u003cem\u003eAndrographis\u003c/em\u003e s.l. is almost entirely Indian in origin with a few recent dispersals into southeast Asia and Sri Lanka. \u003cem\u003eAndrographis\u003c/em\u003e has originated and diversified in the semi-arid conditions of peninsular India which is majorly represented by the Deccan plateau. They might have originated from wet-adapted forms of Andrographideae in the Western Ghats region. The age of diversification matches with the age of intensification of the summer monsoon climate or South West monsoon in peninsular India. Further, diversification within the eastern clades of \u003cem\u003eAndrographis\u003c/em\u003e correlates with the rise of the winter monsoon or North East monsoon which is much pronounced in the eastern region of peninsular India.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eSS acknowledges University of Hyderabad, Institution of Eminence (IoE) grant, UoH-IoE-RC5-22-042 for experimental work.\u0026nbsp;\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eConceptualization: S.S; Methodology: S.S, Y.K; Formal analysis: S.S. Y.K; Resources: S.S, Y.K; Data Curation: S.S,Y.K; Writing - Original Draft: S.S,Y.K; Writing - Review \u0026amp; Editing: S.S; Visualization: S.S,Y.K; Project administration: S.S,Y.K; Funding acquisition: S.S.\u0026nbsp;\u003c/p\u003e\n\n\n\n\u003cp\u003eConflict of interest: The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArolla RG, Cherukupalli N, Khareedu VR, Vudem DR (2015) DNA barcoding and haplotyping in different species of \u003cem\u003eAndrographis\u003c/em\u003e. Biochem Syst Ecol 62:91\u0026ndash;97. https://doi.org/10.1016/j.bse.2015.08.001\u003c/li\u003e\n\u003cli\u003eAttri DC, Dhyani P, Trivedi VL, et al (2024) Current evidence on molecular mechanisms of andrographolide in cancer. 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Cladistics 27:171\u0026ndash;180. https://doi.org/10.1111/j.1096-0031.2010.00329.x\u003c/li\u003e\n\u003cli\u003eZhisheng A, Kutzbach JE, Prell WL, Porter SC (2001) Evolution of Asian monsoons and phased uplift of the Himalaya-Tibetan plateau since Late Miocene times. Nature 411:62\u0026ndash;66. https://doi.org/10.1038/35075035\u003c/li\u003e\n\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":"Acanthaceae, Biogeography, phylogeny, Deccan plateau, Eastern Ghats","lastPublishedDoi":"10.21203/rs.3.rs-4934034/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4934034/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe genus \u003cem\u003eAndrographis\u003c/em\u003e is predominantly peninsular Indian distribution after the resurrection of \u003cem\u003eHaplanthus\u003c/em\u003e which is predominantly southeast Asian in distribution. Previous study have shown its close phylogenetic affinity with \u003cem\u003eHaplanthodes\u003c/em\u003e and \u003cem\u003eHaplanthus\u003c/em\u003e. In this study we constructed a most comprehensive phylogeny of the tribe Andrographideae using five plastid regions, \u003cem\u003erbcL\u003c/em\u003e, \u003cem\u003ematK\u003c/em\u003e, \u003cem\u003etrnLF\u003c/em\u003e, \u003cem\u003epsbA\u003c/em\u003e-\u003cem\u003etrnH\u003c/em\u003e and \u003cem\u003etrnGR\u003c/em\u003e. The results show polyphyly between \u003cem\u003eAndrographis\u003c/em\u003e, \u003cem\u003eHaplanthodes\u003c/em\u003e and \u003cem\u003eHaplanthus\u003c/em\u003e, resulting in a \u003cem\u003eAndrographis sensu lato\u003c/em\u003e (s.l.) clade. Within this earliest splitting clade consisted of \u003cem\u003eAndrographis\u003c/em\u003e and \u003cem\u003eHaplanthus ovatus\u003c/em\u003e. The next split was with Haplanthodes followed by \u003cem\u003eAndrographis sensu stricto\u003c/em\u003e (s.s) clade. Within the \u003cem\u003eAndrographis\u003c/em\u003e clade, we observed two clades one was western in distribution and the other was eastern in distribution in peninsular India. Biogeographical analysis suggested an eastern peninsular Indian origin of \u003cem\u003eAndrographis\u003c/em\u003e and followed by dispersal into the relative wetter western peninsular India. We deduced the time of diversification of the clades of \u003cem\u003eAndrographis\u003c/em\u003e. We discuss the role of south Asian winter monsoon in the diversification of \u003cem\u003eAndrographis\u003c/em\u003e which has adapted to aridity.\u003c/p\u003e","manuscriptTitle":"Biogeographical origin of Andrographis Wall. ex Nees (Acanthaceae) in eastern peninsular India based on molecular phylogenetics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-16 09:33:32","doi":"10.21203/rs.3.rs-4934034/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":"a6191886-61cd-4556-ba15-9ef56e7c0538","owner":[],"postedDate":"September 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T05:40:05+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-16 09:33:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4934034","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4934034","identity":"rs-4934034","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}