Mango (Mangifera indica L), an evergreen dicotyledonous angiosperm often referred as <king of fruits= is adapted to grow in tropical and sub-tropical regions of the world (Mukherjee 1949a; Rabah et al. 2017; Singh et al. 2016; Vasanthaiah et al. 2007) . It is considered as one of the most economically successful fresh fruits cultivated in more than 100 countries, with 55 million tonnes produced in 2020. India has produced approximately 24.7 million tonnes as the world9s largest mango producer accounting for 45% total mango production followed by Indonesia (6.6%), Mexico (4.3%), China (4.3%), and Pakistan (4.3%) (FAOSTAT 2022). Besides being consumed fresh, ripe and unripe mangoes are used to produce pickles, chutney, juices, cereal flakes, sauce, and jam building high demand for mangoes on the international market (Saúco 2016).

The taxonomic history of the genus Mangifera (Anacardiaceae) reveals consistent recognition of two major groups with the number of species reported varying between 45 -69 (Hou 1978; Kostermans and Bompard 1993; Mukherjee 1949a) . The most accepted classification described by (Kostermans and Bompard 1993) defines 69 species mainly based on morphological descriptors of reproductive tissues. Of the 69 species, 58 are divided into two subgenera, Mangifera and Limus, with the remaining 11 species placed in an uncertain position due to insufficient voucher material. The subgenus Mangifera is characterized by free stamen filaments and a four/five lobed cushion-shaped papillose disc broader than the base of the ovary while and the subgenus Limus is characterized by united stamen filaments and a stalk-like disc narrower than the base of the ovary. The sub genus Mangifera includes 47 species further divided into four sections: Marchandora Pierre, Euantherae Pierre, Rawa Kosterm, and Mangifera Ding. In the subgenus Mangifera, section Marchandora Pierre includes only M. gedebe which has the unique character of labyrinthine seed. The three species (M. pentandra Hooker, M. cochinchinensis Engler, M. caloneura Kurz) belonging to section Euantherae Pierre are characterized by five fertile stamens. The section Rawa Kosterm includes nine species which are not well delimited. Mangifera Ding Hou is the largest section in the genus with more than 30 species including domesticated mango (M. indica) (Bompard 2009; Kostermans and Bompard 1993) . The 11 species in sub genus Limus are further divided into two sections:

A total of 13 samples belonging to 11 Mangifera species were selected for sequencing (Table 1). Leaf tissue of all M. indica varieties and Mangifera species, except M. pajang and M. caesia, are sourced from trees grafted onto M. indica cv. Kensington Pride rootstock at the Walkamin Research Station, Mareeba, (17°08̍ 02″S and 145°25 37″E), North Queensland, Australia. M. pajang and M. caesia were sourced from trees at Treefarm, El Arish (-17° 47'59.99"S and 146°00'0.00"E) and Fruit Forest Farm (www.fruitforestfarm.com.au, East Feluga, (17°53'46.0"S and 145°59'38.0"E), Queensland, Australia, respectively. Leaves after harvest were snap-frozen in liquid nitrogen, transported under dry ice and stored at -70℃ until processed for DNA extraction. Frozen leaf tissue was first coarsely ground using a mortar and pestle and then finely ground using the Qiagen Tissue lyser (Qiagen, USA). DNA extraction was carried out from fine pulverized mango leaf tissue samples using a cetyltrimethylammonium bromide method (Furtado 2014). The quality and quantity of DNA were assessed for acceptable absorbance ratios (ideal 1.8-2.0 at A260/280 and over 2.0 at 260/230) using Nanodrop Spectrophotometer (Thermo Fisher Scientific). DNA degradation and quantity were assessed by resolving sample and standard DNA by agarose gel electrophoresis (Thermo Fisher Scientific). The isolated DNA was subjected to next-generation short read sequencing (NGS) on an Illumina HiSeq 2000 platform at the Ramaciotti Centre for Genomics, University of New South Wales (UNSW), Australia to obtain sequence data with coverage of the genome that was not less than 20X (Table S1). Sub Genera

Ploidy Level (2n) Unknown Unknown Unknown Unknown Unknown 40 40 40 40 40 40 40 Unknown

Smooth fibrous or non-fibrous fruit: edible Prominent fruit/horticultural characteristics Juicy, very acid and fibrous fruit: edible Resistance to anthracnose (fruit skin) Used as a rootstock for M. indica in Malaysia Fruit flesh: deep-orange-yellow, fibrous, acid to acid sweet, mildly fragrant: edible Largest fruit in the genus Fruit with good aroma, sour taste: edible Drought tolerance/ resistance to anthracnose (fruit skin) and gummosis Firm, fibrous, sweet to acid-sweet, juicy fruit with a strong smell and turpentine flavour: edible Resistance to anthracnose Sweet flesh with strong fragrance in the fruit: edible Very juicy fruit, fibrous, pleasant, sweet taste pulp: edible Small glossy yellowish fruit with acid-sweet taste:edible Juicy, very acid fruit with turpentine and lemon taste: edible Small fruits, with an attractive purple color and a distinctive aroma: edible Soft and juicy flesh with moderate to little fibre, sweet with a characteristic flavour excellent eating quality Firm to soft flesh, low in fibre content, sweet with characteristic aroma, very pleasant taste: edible Fruit with firm, medium juicy, medium amount of fibre of good eating quality Highly resistant to anthracnose disease

In addition to generating sequence data for the 11 Mangifera species, publicly available Illumina sequencing paired-end reads were downloaded from the National Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) for five Mangifera species namely M. sylvatica, M. odorata, M. percisiformis, M. hiemalis and M. indica cv. Tommy Atkins (Table S2). Chloroplast genomes for each species were assembled using two methods: a chloroplast assembly pipeline (CAP) described by (Moner et al. 2018) in CLC Genomic

Genome annotations for assembled chloroplast genomes were performed using GeSeq online tool (https://chlorobox.mpimp-golm.mpg.de/geseq.html) and M. indica cv. Tommy Atkins (Accession: NC_035239.1) was used as the reference genome. Based on the evolutionary relationships observed in chloroplast genome-based phylogenetic analysis, closely related species within the main clades and subclades were compared to determine their evolutionary relationships. Chloroplast genomes of the species were subjected to pairwise alignment in Geneious using the MAFFT alignment tool and the number INDELs, substitutions and SNPs present between the sequences were identified.

Here, we used a list of single copy nuclear genes. Details of the genes were not available for Mangifera species. Therefore, Citrus sinensis, the closest relative of M. indica for which the details of single copy nuclear genes were available (Li et al. 2017) was used as the reference, to extract corresponding single-copy genes in mango. Single copy genes (107) in C. sinensis were mapped against the coding DNA sequences/gene models of M. indica cv. Alphonso (Wang et al. 2020) in CLC-GWB. Out of 107, 47 were identified as single-copy genes in M. indica. Then, trimmed paired-end illumina reads of each species were mapped against 47 single-copy genes of M. indica, and consensus gene sequences were extracted. All 47 genes were identified as single-copy genes in Mangifera species. Also, the same 47 gene sequences were extracted from A. occidentale. Details of the single copy genes in mango are indicated in Table S3.

Phylogenetic analysis of Mangifera species was undertaken using the chloroplast genome sequences and also for the single-copy nuclear gene sequences. Corresponding sequences of A. occidentale were used as an outgroup. Apart from the 13 samples sequenced in this study, five species for which chloroplast genomes and nuclear gene sequences generated by downloading sequence data from NCBI were also included.

For chloroplast genomes-based phylogenetic analysis, Sequences were imported to the Geneious 2022.2.2 software (www.geneious.com) and aligned by multiple sequence alignment using MAFFT (MAFFT v7.490) alignment tool (Katoh and Standley 2013) . Two methods were used for phylogenetic analysis: Maximum likelihood (ML) method and Bayesian inference (BI) method. jMfodelTest v2.1.4 (Darriba et al. 2012) was used to select the best fitting nucleotide substitution model using Cyberinfrastructure for Phylogenetic Research (CIPRES) Science Gateway (http://www.phylo.org/) (Table S4). ML analysis was performed in RaXML GUI 2.0 (v 2.0.10) (Stamatakis 2014) with 1000 bootstrap replicates under Akaike information criterion. Bayesian analysis was carried out in Geneious software using MrBayes v. 3.2 (Ronquist et al. 2012) under the Bayesian information criterion (Table S4). iTOL v.6 tool (https://itol.embl.de/) (Letunic and Bork, 2021) was used to visualize and edit the phylogenies. Using posterior probability (PP) and bootstrap support (BS) to evaluate the supports of the phylogenetic tree implemented under BI and ML methods respectively, final trees obtained from both approaches were compared and tree topologies were validated.

For nuclear gene sequences, phylogenetic trees were generated using two approaches: gene concatenation and coalescent approach to analyse any topological incongruence and for a better understanding of evolutionary relationships among species.

All 47 genes of were concatenated in the same order to get a one long sequence per species. Sequences for all the species were imported to the Geneious 2022.2.2 software (www.geneious.com) and aligned by MAFFT alignment. Phylogenetic trees were constructed using ML method and BI methods after selecting the best fitting nucleotide substitution model by running jMfodelTest v2.1.4 (Darriba et al. 2012) (Table S4). ML analysis was performed in RAxML (version 8) (Stamatakis 2014) with 1000 bootstrap replicates, and Bayesian analysis was carried out in Geneious software using MrBayes v. 3.2 (Ronquist et al. 2012) (Table S4). iTOL v.6 tool (https://itol.embl.de/) (Letunic and Bork 2021) was used to visualize and edit the phylogenetic trees. By using PP and BS values to evaluate the supports of the phylogenetic tree implemented under BI and ML methods respectively, final trees obtained from both approaches were compared and tree topologies were validated.

For coalescent approach, single ML gene trees were constructed by ML method using RAxML (version 8) (Stamatakis 2014). We searched for the best-scoring ML tree using a GTR+GAMMA model with 1000 bootstrap replicates. Low support branches (BS<10%) in gene trees were collapsed to minimize potential impacts of gene tree error for species tree reconstruction. Then, these gene trees were used to construct a coalescent-based species tree using ASTRAL-III (Zhang et al. 2018)

Illumina sequencing conducted for the 13 samples belonging to 11 Mangifera species in this study resulted in 60,699,616 to 181,601,786 raw reads with 150bp mean read length. Trimmed paired-end reads at 0.01 quality limits (Phred score > 20) ranged between 59,763,897 and 171,303,402 reads. The data size of the trimmed reads of all 13 samples corresponded to over 20x of the genome size, therefore all were selected for the chloroplast assembly (Table S1). Raw reads downloaded from NCBI for five Mangifera species (M. odorata, M. sylvatica, M. percisiformis, M. hiemalis and M. indica cv. Tommy Atkins) had a total of 99,649,506 to 127,708,722 reads which ranged from 94,450,606 to 117,445,811 after trimming at 0.01 quality limits. For all the species, the mean coverage was higher than 20x genome size, which enabled them to be included in the analysis (Table S2). The chloroplast genome and respective raw reads were also available for the species M. longipes (synonym: M. laurina), but the average coverage after quality trimming was less than 20x, and therefore this was not included for the analysis. The Get Organelle pipeline resulted in two output files for the chloroplast genome for each species/ genotype, due to possibility of the SSC occurring in both orientations in the chloroplast genomes in plants. Therefore, the two chloroplast sequences for each species were aligned with the reference (M. indica; Accession: NC_035239.1) in Clone Manager Professional 9 to select the sequence with the widely accepted SSC orientation (59LSC39:59IR139:5SSC39:39IR259). The size of the chloroplast genomes of 13 wild Mangifera species and three cultivars of M. indica ranged from 151,752bp to158,965bp of which the smallest and the largest genomes were recorded for M. caesia and M. laurina Lombok, respectively (Table 2). The typical quadripartite structure of the chloroplast genome was recorded in all 14 Mangifera species and the lengths of LSC, SSC, and IR regions ranged between 86,507 to 98,334 bp, 18,319 to 19,064 bp and 17,177 to 26,412 bp, respectively where overall guanine – cytosine content (GC content) ranged from 37.6 to 37.9 %. The chloroplast genomes for all species had the same number of total genes (115), rRNA (4) and tRNA (31) and protein encoding genes (80) (Table 2). Although the size of the chloroplast genomes varies across the Mangifera species, three cultivars of M. indica had identical chloroplast genomes. Two M. odorata accessions had slightly different chloroplast genome sizes, where the accession we sequenced had a genome size of 158,889bp while for the sample for which the data was downloaded from NCBI (M. odorata*) had a genome size of 158,883 bp, representing a 5 bp difference. The length difference was due to two deletions revealed in M. odorata*, one located in a non- coding region of LSC while the other located in the intron1 region of the PetD gene of LSC. The chloroplast sequence of M. indica cultivar Kensington Pride (Fig. 1) is a representation of the chloroplast sequence of the 14 Mangifera species which have the same number of genes although there are differences in total chloroplast size and the sizes of the LSC, IR1 and IR2 and

A multiple chloroplast sequence alignment conducted using A. occidentale as the outgroup followed by phylogenetic tree construction resulted in a ML tree and a Bayesian tree with same tree topology. BS and PP values of the final tree are presented in Fig. 2. The model of nucleotide substitutions for ML analysis was GTR+G whereas for the Bayesian analysis, it was TPM1uf+G. The tree developed with the ML approach showed a BS of 100 at most of the nodes and PP of one in all the nodes. In the whole plastome tree, three main clades were identified. First, 14 Mangifera species were clustered into two distinct clades in which only M. caesia belonging to section Dissidue in the subgenus Limus was placed in first clade (Clade A). Other 13 species were grouped into a separate clade indicating their evolutionary distinct relationship to M. caesia, which were then clustered into two sub clades (clade B and clade C). Clade B included a total of six species which belong to different categories in the classification. M. pajang and M. odorata belong to sub genus Limus while M. casturi and M. laurina belong to subgenus Mangifera. M. percisiformis and M. hiemalis are two species placed under uncertain position in the classification. Within clade B, species in subgenera Mangifera (Clade BI), Limus (Clade BII), and species being classified in an uncertain position (Clade BIII) have localized into well supported distinct clades (BS=100, PP=1). The species belong to subgenera Mangifera and Limus and were sister to each other and both together have become a sister clade to species placed in uncertain position in the classification. Clade C had species belonging only to the sub genus Mangifera which was further divided into three subclades (clades CI, CII and CIII). Interestingly, four wild species (M. lalijiwa, M. applanata, M. altissima and M. caloneura) were clustered with three cultivars of domesticated mango (M. indica) (Clade CI). Although species belong to section Mangifera and Euantherae are characterised by the presence of one and multiple fertile stamens respectively, M. caloneura in section Euantherae was clustered with species belonging to section Mangifera. Furthermore, M. sylvatica and M. zeylanica; two species within the clade C were separately clustered into distinct clades, CII and CIII respectively. M. sylvatica is the sister to domesticated clade (clade CI) and M. zeylanica (Clade CIII) has become sister to the clade includes CI and CII. Therefore, the phylogeny based on whole chloroplast genome clustered species belong to different groups inferring the close genetic and evolutionary relationships of their chloroplast genomes (Fig. 2).

Assembled chloroplast genomes were imported to Geneious software to conduct pairwise alignment to identify number and types of variants present between the species clustered as sister taxa in the two main clades (clade B and C) of the chloroplast phylogeny. A total of 116 variants were observed between M. laurina and M. casturi while 75 variants were found between M. odorata and M. pajang (Table 3). The two species, M. percisiformis and M. hiemalis, for which we assembled the chloroplast genomes from raw read data available in NCBI, differed by 45 variants revealing the close evolutionary relationships among these sister taxa in clade BIII. In clade CI, pairwise comparison of wild species with M. indica cv Kensington Pride showed that, M. altissima and M. indica had identical chloroplast sequences. Moreover, M. lalijiwa and M. applanata also had an identical chloroplast genome which only differed from M. indica by having a single nucleotide deletion located in a non-coding region in LSC. Furthermore, despite reporting distinct morphological characteristics from M. indica, M. caloneura only had one single nucleotide insertion and one single nucleotide deletion in non-coding regions compared to M. indica. Diversity within the chloroplast genomes clustered in clade CI was very low. Clade Name in the phylogenetic tree BI BII BIII CI CII CIII

Species/ genotypes in comparison M. laurina M. casturi M.odorata M. odorata * M. pajang M. persiciformis* M. hiemalis* M altissima M. lalijiwa M. applanata M. caloneura (Xoai queo) M. indica cv. Kensington Pride M. indica cv.Tommy Atkins M. indica cv. Alphonso M. sylvatica* M. indica cv. Kensington Pride M. zeylanica M. indica cv. Kensington Pride * Species for which chloroplast genomes were assembled using raw data downloaded from NCBI

Total number of variations between A vs B 116 2 75 45 0 0 0 0 0 1 1 2 0 3 96 175 30 17 9 1 2 22 18

The same approach was used to construct a nuclear phylogeny with concatenation-based approach as was applied in constructing the chloroplast phylogeny. A. occidentale was used as the outgroup. A total of 47 common single copy nuclear genes out of 107 (Li et al. 2017) were identified and selected for Mangifera species. The multiple sequence alignment was 71,881 bp in length and ML and Bayesian trees resulted in the same tree topology. The final tree with BS values and PP values is presented in Fig. 3a. Although some of the nodes showed less BS support values, all the nodes were supported with high PP values. The model of nucleotide substitutions for ML analysis was GTR+I+G whereas TPM1+I+G was used for the Bayesian analysis.

Except for M. sylvatica, the other eight species belonging to subgenus Mangifera were clustered into one main distinct clade. M. lalijiwa, M. applanata and M. caloneura with M. casturi were clustered into one clade and M. altissima, M. zeylanica, and M. indica cultivars were clustered into another clade within the main clade. M. applanata and M. lalijiwa were sister taxa to each other. Furthermore, two M. indica cultivars (Kensington Pride and Tommy Atkins) showed closer genetic relationship to M. zeylanica and M. altissima revealing close evolutionary relationship of the two wild species to domesticated mango. Moreover, although M. sylvatica was closely related to species in domesticated clade in chloroplast phylogeny, it was clustered with the two species placed in uncertain position in the classification in the nuclear phylogeny where it was more closely related to M. hiemalis than to M. percisiformis.

Furthermore, both chloroplast and concatenation-based nuclear phylogenies revealed that M. caesia is evolutionarily distant from the rest of the Mangifera species (Fig. 2 and 3a). Grouping of species in both chloroplast genome and nuclear genes-based analysis does not completely concur with the accepted classification described by (Kostermans and Bompard 1993) for genus Mangifera. Incongruence in tree topologies could be seen between the phylogenies developed based on the whole plastome genome and the nuclear genes.

Previously proposed hybrids were included in our dataset and possibility of hybridization events also were observed for some species when compared chloroplast and concatenation-based nuclear phylogenies. Therefore, to further analyse the phylogenetic relationships among Mangifera species with respect to nuclear genes, coalescence approach was utilised to develop individual nuclear gene trees thereby to develop a species tree. Individual gene trees were analysed to see close evolutionary relationships among species. In coalescence-based species tree, local posterior probability support (LPP) values are indicated in the branches (/1). In both concatenation and coalescence based nuclear phylogenies, species belonging to subgenus Mangifera, and species placed in uncertain position in the classification were clustered in one clade with high support values (BS=99, PP=1, LPP=0.93) (Fig. 3). Within this clade, pattern of clustering into sub-clades was different for some species between the two nuclear phylogenies but BS and LPP support values also were low for some sub-clades. In both concatenation and coalescence-based phylogenies, M.casturi and M.lalijiwa are clustered together as sister taxa (BS=100, PP1, LPP= 0.92) and M. hiemalis, M. sylvatica and M. percisiformis were clustered in to one subclade (BS=100, PP=1, LPP=0.96). But in coalescence-based tree, M. hiemalis, M. sylvatica and M. percisiformis were closely related to six other species belong to subgenus Mangifera (M. altissima, M. applanata, M.indica, M. caloneura, M.zeylanica and M. laurina) than M. casturi and M. lalijiwa while these eight species in subgenus Mangifera were clustered together into one clade in concatenation-based tree.

M.odorata is a proposed hybrid between M. indica and M. foetida and M. casturi is a proposed hybrid between M. indica and M. quadrifida. Within our dataset, only one of the parents are available for these hybrids. Although it is not possible to validate the hybridity due to absence of one of the parents, we analysed individual gene trees to support the hybridity by recording the number of gene trees where the hybrids were clustered with the parent available in our dataset (M. indica). Out of 47 gene trees, M. odorata was clustered with M. indica as sister taxa in only four gene trees and they were not supported with high BS values (Table 4, Figure S1). Similarly, M. casturi was also clustered with M. indica as sister taxa in four gene trees only in which BS support were weak in three gene trees for this clade (Table 4, Figure S1). We observed close evolutionary relationship between M. zeylanica and M. indica and also between M. hiemalis and M. sylvatica in nuclear phylogenies. Therefore, we assumed that M. zeylanica might have undergone domestication and M. sylvatica may have cross hybridised with M. hiemalis during evolution of these species. Analysing individual gene trees revealed that M. zeylanica was clustered with M. indica as sister taxa in 14 gene trees and M. sylvatica was clustered with and M. hiemalis as sister taxa in 12 gene trees. Some individual gene trees for M. zeylanica and M. sylvatica showed less BS support when clustering with M. indica and M. hiemalis respectively (Table 4, Figure S1). × × × × × × × × × × × √ × × × × × × × × × √ √ × √ √ × × × × × × × × × × × √ × √ √ × × × × × × × × × × × × × √ × × × × √ √ √ × × × × √ × × × × √ × √ √ × √ × × × × × ×

Determination of phylogenetic relationships among crop species provides basic information for predicting their evolutionary history, taxonomical classification, evaluating their diversity and importance in plant breeding (Zhang et al. 2012) . Although genetic analysis of plants has improved rapidly with advanced sequencing technology, many phylogenetic studies in the genus Mangifera have relied on a set of molecular markers such as amplified fragment length polymorphisms (AFLP), rapid amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR) and the sequencing of limited numbers of targeted regions in the chloroplast genome (maturase K, trnL-F spacer regions) and nuclear ribosomal DNA (internal transcribed spacer/ITS region) (Eiadthong et al. 1999; Fitmawati et al. 2017; Fitmawati 2016; Hartana 2010; Hidayat et al. 2011; Schnell and Knight Jr 1992; Yonemori et al. 2002) . This is the first comparative analysis of phylogenetic relationship within the genus using both whole chloroplast genomes and multiple single-copy nuclear genes.

Chloroplast genomes for seven species were assembled for the first time in this study for M. pajang, M. altissima, M. caesia, M. lalijiwa, M. zeylanica, M. appalanta and M. casturi. Different pipelines and programs such as SPAdes (Bankevich et al. 2012) , SOAPdenovo2 (Luo et al. 2012) , ORG.Asm (Coissac et al. 2016), IOGA (Bakker et al. 2016) , Fast-Plast (Afinit 2017), Organelle_PBA (Soorni et al. 2017), NOVOPlasty (Dierckxsens et al. 2017), chloroExtractor (Ankenbrand et al. 2018) , CAP (Moner et al. 2018) and Get Organelle toolkit (Jin et al. 2020) are available to assemble organelle genomes. Here, we have used CAP and Get Organelle pipeline to assemble plastome genomes independently for each species. The two approaches used in CAP (reference-guided mapping and de-novo assembly) eliminate many errors in genomes developed from each approach giving a highly accurate final chloroplast genome. The Get Organelle pipeline; a fast and versatile toolkit used to assemble organelle genomes via de novo approach is also capable of generating all possible arrangements of the chloroplast genome present because of flip-flop configurations or other isomers mediated by repeats (Jin et al. 2020) . Therefore, a comparison of chloroplast genomes generated from CAP and the Get Organelle pipeline validated the development of highly accurate final chloroplast genomes for all the species. Annotation of chloroplast genome with Ge Seq software discovered a total of 115 genes including 80 protein-coding genes, 31 tRNA genes, and four rRNA genes for all the species used in this study. More genes have been annotated in this analysis compared to previous studies, in which (Zhang et al. 2020) reported a total of 112 genes (78 protein-coding genes, 30tRNA genes, 4 rRNA genes) for M. sylvatica while 113 genes (79 protein-coding genes, 30tRNA genes, 4 rRNA genes) for M. sylvatica, M. odorata, M. longipes, M. percisiformis, M. hiemalis and M. indica were reported by (Niu et al. 2021) .

Phylogenetic relationships within genus Mangifera showed topological incongruence for some species with respect to whole chloroplast and nuclear genes trees which may be caused by introgressive hybridization, allopolyploidy or incomplete lineage sorting. Reproductive compatibility between different species allows native cytoplasm of a species to be easily replaced by another through hybridization which has been detected both in animals (mitochondrial capture) (Liu et al. 2016; Rebbeck et al. 2011) and plants (chloroplast capture) (Rieseberg 1995; Rieseberg and Soltis 1991). In plants, chloroplast capture events have been reported in many plant families including Maleae, Rosaceae (Liu et al. 2020) , Nothofagaceae (Acosta and Premoli 2010) Scrophulariaceae (Wolfe and Elisens 1995) , Apiaceae (Yi et al. 2015) , Poaceae (Ananda et al. 2021; Moner et al. 2020) , Rubiaceae (Charr et al. 2020; Guyeux et al. 2019) and Myrtaceae (Healey et al. 2018). Hybridization followed by recurrent backcrossing have explained discrepancies between chloroplast and nuclear gene-based phylogenies in diverse families of plants (Liu et al. 2017; Smith and Sytsma 1990; Stegemann et al. 2012; Tsitrone et al. 2003) . In mango, evidence for inter-specific reproductive compatibility was reported for M. indica and M. laurina. A cross between M. indica and M. laurina have produced 60 successful hybrids (Bally et al. 2010) . Hybrid origins were reported for M. odorata (cross-hybrid between M. indica and M. foetida) (Teo et al. 2002) and M. casturi (Matra et al. 2021) .

Close genetic relationship between M. applanata and M. altissima has been reported in a phylogenetic analysis conducted based on one chloroplast gene (Maturase K) while M. indica and M. caloneura have nested into two distinct clades (Hidayat et al. 2011). In our study, based on whole chloroplast genomes, M. laijiwa, M. applanata, M. altissima and M. caloneura were clustered with M. indica sharing 99.9% sequence similarity. These four wild relatives clustered with domesticated mango into a distinct clade even in concatenation-based nuclear phylogeny showing their close evolutionary relationship to M. indica whereas only M. laijiwa out of the above four species clustered separately in the coalescent approach. Furthermore, two of the M. indica cultivars (Kensington Pride and Tommy Atkins) were more closely related to M. altissima than M. indica cv. Alphonso failing to resolve M. indica from M. altissima based on nuclear gene sequences. A close evolutionary relationship between M. altissima and M. indica was also observed in the coalescence approach. Therefore, due to remarkably close evolutionary relationships observed among these species in chloroplast and nuclear phylogenies, we suggest these four wild relatives and cultivated mango are very closely related and might have shared or descended from the same common ancestor.

Considering domestication of M. indica, although a single domestication event has been reported based on historical records (Mukherjee 1972; Singh et al. 2016) , two independent domestication events have also proposed for M. indica in India and Indochina (Bompard 2009) . Based on a population genomics study, Warschefsky and von Wettberg (2019) suggested that mango domestication is a complex process and it may involve multiple domestication events and interspecific hybridization; two common phenomena observed in perennial fruit crop domestication. Their results also have indicated a high genetic diversity among M. indica cultivars distributed outside from the region where the mango was originated and a unique genetic diversity in Southeast Asian cultivars compared to other populations. Warschefsky and von Wettberg (2019) suggest that the origin and initial cultivation of mango may have taken place in Southeast Asia and further improvement and domestication may have occurred in India. In addition, cross-hybridization was highly likely to occur between wild relatives and M. indica at the early stages of domestication because of the presence of a high number of species and presence of evidence for crossbreeding. Thus, apart from descending from a common ancestor, cross-hybridization between M. indica and the four wild relatives is also a possible phenomenon that may have further contributed to the close evolutionary relationships observed in our study. However, this close evolutionary relationship could be further supported by including multiple replicates per species which is a limitation in this study.

M. zeylanica, is an endemic species to Sri Lanka which is only discovered in forests of wet and intermediate zones, showing scattered distribution. Close evolutionary relationship was observed in the concatenation-based nuclear phylogeny between M. zeylanica and M. indica in spite of having a distinct chloroplast genome when compared the variations between two species (with 44 INDELs, 122 SNPs and nine substitutions). Therefore, we hypothesized that cross hybridization might have occurred between an early lineage of M. zeylanica and M. indica or its close wild relative. Since the species have a distinct chloroplast genome, we assumed that during the cross, M. indica may have most likely acted as the pollen donor/ or paternal parent, resulting hybrids which carry the chloroplast genome M. zeylanica and nuclear genes of both M. zeylanica and M. indica/it9s close relative. The nuclear phylogeny/species tree based on the coalescence approach also showed a close relationship between M. indica and M. zeylanica. Clustering of M. zeylanica with M. indica in 14 individual gene trees suggested that M. zeylanica might have a hybrid origin. But as the BS/PP and LPP values are relatively low for this clade in individual gene trees as well as in both the consensus trees, it is also possible that the set of genes are not sufficiently variable to give a better resolution in the phylogeny. Therefore, it is difficult to make a conclusion about the hybrid origin for M. zeylanica.

Among six species (M. casturi, M. laurina, M. pajang, M. odorata, M. percisiformis and M. hiemalis) nested together in the chloroplast phylogeny, M. casturi is a cultivated species in Indonesia. This endemic species is only found in cultivation (Rhodes and Maxted 2016) and was proposed to be a natural hybrid between M. indica and M. quadrifida according to a SNP analysis (Warschefsky 2018) . Since M. casturi has shown higher affinity to M. indica than to M. quadrifida instead of being direct intermediate between two species, it was further suggested that M. casturi is most likely a result of an F1 hybrid backcrossed with M. indica (Warschefsky 2018; (Warschefsky and von Wettberg 2023) . Microsatellite marker based analysis showed broad genetic variation among four M. casturi accessions (Kasturi, Cuban, Pinari and Pelipisan) and DNA barcording based phylogenetic analysis suggested several species as ancesters for M. casturi (Matra et al. 2021) . Genetic variation has also been confirmed between 16 accessions of M. casturi using SNP markers (N. Dillon, pers. comm.). Therefore a combination of microsatellite and DNA barcoading data support that M. quadrifida and M. indica was hybridised to result in M. casturi and F1 hybrids may have further hybridized with the ancestors of the parental species or multiple other Mangifera species to generate hybrid cultivars which have high genetic diversity (Matra et al. 2021) . In our study, a close genetic relationship was observed between M. casturi and species in the domesticated clade of the concatenation-based nuclear phylogeny despite having distinct chloroplast genomes. In contrast, in the coalescence approach, M.casturi showed a relatively distant evolutionary relationship with M. indica both in species tree and in individual gene trees. Therefore, according to our results, coalescence-based nuclear phylogenies don9t strongly support the parentage of M. indica for M. casturi. Since a very low number of genes are shared between M. indica and M. casturi, if M. indica is one of the parents, it is not possible that M. casturi is a first-generation hybrid. Also, the absence of the data for other proposed parent (M. quadrifida) and other wild relatives limits analysing the input of M. quadrifuda and any other species for the hybrid origin of M. casturi. M. laurina is a cultivated species in Indonesia where its wild distribution ranges from Myanmar, Cambodia, Vietnam and Malesia, Thailand to New Guinea. Analysis of ITS genomic region (Yonemori. et al. 2002) have revealed close evolutionary relationship between M. laurina and M. indica. Analysis of Maturase K chloroplast genomic region has differentiated Indonesia and Thailand specimens collected for M. laurina. Since common interspecific hybridization has been suggested for this species (Hartana 2010), it is possible that M. laurina may have cross hybridized with other species after intoroduction to the regions where it is widely cultivated. Due to the relatively close evolutionary relationship observed between M. laurina and M. indica in nuclear gene analysis despite the chloroplast genome being distinct, it might be possible to occur hybridization between early lineage of M. laurina and M. indica. Current data and results only support the close evolutionary relationship between the two species, but further analysis should be conducted with multiple samples for both species and also including more species in the genus.

Out of the remaining four species (M. pajang, M. odorata, M. percisiformis and M. hiemalis) clustered within the same main clade in chloroplast phylogeny, M. pajang is an endemic species originating from and cultivated in Borneo, Indonesia. M. odorata cultivated in Malaysia is a proposed hybrid between M. indica and M. foetida from which it showed more affinity to M. foetida than to M. indica (Teo et al. 2002; Yonemori. et al. 2002) . Furthermore, closer genetic relationship of M. foetida to M. pajang compared to M indica has been suggested (Schnell and Knight Jr 1992). Having a distinct chloroplast genome and a distant phylogenetic relationship to M. indica in the chloroplast tree suggests that M. odorata might have captured chloroplast genome from M. foetida and ancestor of M. indica might have contributed as the male progenitor. In both concatenation and coalescence approaches for nuclear genes, M. odorata showed a relatively distant evolutionary relationship with M. indica. Since individual gene trees cluster the two species in four gene trees only with weak support, it is less likely that M. odorata is a first-generation hybrid. Though a small M. indica background was suggested for M. odorata, without being the other suggested parent, the hybrid status and parentage of M. odorata is inconclusive. Another discrepancy observed from the chloroplast and nuclear trees is related to the position of M. sylvatica. Previous studies have revealed a close evolutionary relationship between M. indica and M. sylvatica based on restriction fragment length polymorphism (RFLP) (Eiadthong et al. 1999), ITS (Yonemori. et al. 2002) marker analysis and whole chloroplast genome analysis (Niu et al., 2021) . In our study, although M. sylvatica showed a close genetic relationship with M. indica in chloroplast phylogeny, it nested with M. hiemalis in the nuclear phylogeny. M. hiemalis is an endemic species to China and M. sylvatica is also one of the cultivated species in China (Wang et al. 2020; Baul et al. 2016) . Therefore, both species share the same geographical distribution. Since individual gene trees revealed clustering with M. hiemalis as sister taxa in 12 trees, M. sylvatica might have a hybrid origin where the hybridization occurred long ago, but the low BS values in individual gene trees does not strongly support this hypothesis.

Topological incongruence observed by chloroplast genome and single copy nuclear gene-based phylogenies reveal that there is a potential for inter-specific hybridization in the genus. But less BS values and weak resolution in gene trees of coalescence approach and low BS/PP/LPP support values in some of the branches of concatenation-based nuclear phylogeny and species tree are clear evidence that the nuclear genes are not well distinguished/ might not vary across the group of species studied. Less variability of nuclear genes and absence of one of the parents for proposed hybrids limited concluding about possible hybridization event/s occurred in the genus and hybrid of M. odorata and M. casturi. But even if we have hybrids in our dataset with the presence of both parents, phylogenies will show their close evolutionary relationships if it is a recent generation hybrid. Therefore, results of this study suggests that the whole group sufficiently closely related with each other so that we needed large amount of data to get a concatenated/consensus tree. The history of evolution of the species and hybridization is complex in the genus and requires more species to get better understanding. However, is it possible that out of 69 distinct species identified in the genus, some or many of them may have either domestication input or cross-hybridized with other wild relatives.

Our analysis of determining evolutionary relationships within the genus Mangifera, based on whole chloroplast genome and 47 single copy nuclear genes, revealed close genetic relationship among species and discrepancies between whole plastome and nuclear gene-based phylogenies. We suggest that the five species including M. indica, M. altissima, M. applanata, M. caloneura, M. lalijiwa are very closely related and might have descended from the same common ancestor due to their close evolutionary relationship. It was difficult to confirm the hybrid origin of M. odorata and M. casturi as suggested previously due to absence of one of the proposed parents within our dataset and due to clustering of the available proposed parent in only a low number of gene trees. Relatively high number of gene trees showed close evolutionary relationship between M. zeylanica and M. indica, and M. sylvatica and M. hiemalis. However, evidence did not strongly support the possible hybridization due to weak BS/PP and LPP supports in phylogenies. Moreover, it was observed that geographical proximity might have facilitated possible hybridization events. Despite limited number of species used in the study, it seems that evolution of species and hybridization in the genus Mangifera is a complex process. This is the first comparative analysis of evolutionary relationships within the genus with whole chloroplast genome and multiple nuclear genes. These findings provide an understanding about the nature of hybridization within the genus between wild and domesticated mango revealing potential domestication input into some species. Validation of hybridity and accuracy of evolutionary relationships within the genus can be highly supported and improved by adding more species including potential parents and sampling species from different geographical locations.

Robert J. Henry: Conceptualization and design, Methodology, Supervision, Validation, Project administration,

All data supporting the findings of this study are available within the paper and its Supplementary Information. 10. Data Archiving Statement Raw Illumina sequence read data were submitted to NCBI9 Short Read Archive (SRA) database under Bio project ID PRJNA940204 and under Bio sample ID9s SAMN33621737- SAMN33621749. Chloroplast genomes will be submitted to NCBI under Bio project ID PRJNA940204 and accession numbers will be provided. 11. References Acosta MC, Premoli AC (2010). Evidence of chloroplast capture in south American Nothofagus (subgenus Nothofagus, Nothofagaceae). Mol Phylogenet Evol 54(1): 235-242. https://doi.org/10.1016/j.ympev.2009.08.008.