Md Rasedujjaman

Guillaume Maire

Philippe Robert

3D optical computational microscopy in reflection configuration

come

Introduction

Data acquisition and Results

Acknowledgment

The tomographic diffractive microscope (TDM) can be implemented in either transmission configuration or reflection configuration. TDM in reflection configuration has higher Fourier spatial frequency data along the optical-axis of the microscope in comparison to the transmission configuration and also reflective samples can be imaged. We have recently exploited the specific features of such a configuration [START_REF] Maire | High-resolution tomographic diffractive microscopy in reflection configuration[END_REF]. This optical tomographic microscope coupled to sophisticated inversion schemes could be a good candidate for detecting the immunological synapse of T lymphocyte activation. Presently, no technique permits to perform a fast detection of T lymphocyte activation at an early stage which is very promising in medical diagnosis applications.

In doing so we have first considered polystyrene bead (comparable to the size of Tcell) in water medium and detected the interface. This same experiment could be used for detecting the immunological synapse.

Keywords: Reflection tomography, T lymphocyte, Synapse

I sincerely express my gratitude to the summer school on Intelligent signal processing for FrontIEr Research and Industry (INFIERI 2019), PETLAB, HUST for giving me the opportunity to participate this wonderful event. I also would like to acknowledge Marie Skłodowska-Curie Actions -European Commission, AMidex foundation and RPACA for funding this research project.

For data acquisition we use a custom made Linux based software (OpticsbenchUI). With this software the galvanic mirror is controlled for illuminating the sample at desired angle and the image sequences are recorded by the camera accordingly. Few of the recorded holograms are shown in Fig. 2(b). The images are recorded as HD5 format and then the necessary data are extracted by MATLAB. IFFT algorithm is then used to go to k-space from image space as shown in Fig 2(c). This allows us to filter the data. Once this process is done we go back again in image space by using FFT and combine all the images to reconstruct the interface of the sample as in Fig 2(e).

Experimental setup

The tomographic microscope is presented in Fig. 1. The light source is a low coherence supercontinuum laser (NKT Photonics SuperK Extreme EXW-12), that can be filtered from 450 nm to 800 nm. The laser beam is linearly polarized and then divided into a reference beam and a beam directed towards the sample called object beam. The object beam and the reference beam are interfered off-axially. Due to the low coherence any parasitic reflection from the optical components in the object beam path does not interfere with the measurement. A galvanic mirror (M, Newport FSM-300) permits to control the deflection of the sample beam, while a beam expander and diaphragm D generate a wide collimated beam with near homogeneous power density. This beam illuminates the sample after transmission through the microscope objective (can be varied to have different NA) and the associated tube lens (L). The field scattered by the object is collected by the microscope objective (Nikon Apo TIRF oil 100×, NA =1.49) and imaged on a sCMOS camera with pixel size of 6.5μm(Andor Zyla) after passing through relay lenses L2 and L3 to obtain a global magnification of about 300.

Future work

We have successfully reconstructed a polystyrene bead with dimension comparable to that of T-cell. Of course the bead is well defined in geometry and therefor mathematical model for reconstruction is greatly simplified. For T lymphocyte as shown in Fig. 3, the shape is not regular and requires more advanced algorithm for detecting the synapses [START_REF] Robert | Kinetics and mechanics of two-dimensional interactions between T cell receptors and different activating ligands[END_REF] with the same experimental setup and data acquisition method as used for the beads.

Theory and Design Theory

Tomographic diffraction microscopy is based on the same principle of the off-axis digital holography. Lets consider the incident field in space domain as follows:

But we do not observe the filed rather intensity is recorded and consequently phase information is lost.

When the E inc (r) is incident on an object, the object scatters the incident field, this is called scattered field E d (r). Then the total field is given by E(r) = E inc (r) + E d (r). If the total field E(r) is interfered with another field called reference field E ref whose phase is known, the field detected E intf (interference) will be as follows, E intf (r) = E(r) + E ref (r). Then the resulting intensity from the above equation has the desired phase term φ(r) as shown below, where, ∆φ(r) represents the phase difference. Now we can extract both the phase and intensity information.

Fig. 1 .

 1 Fig. 1. Block diagram of the TDM in reflection configuration : M, rotating mirror; BE, beam expander; D, diaphragm; L1 ,tube lens; L2,...,5 lenses; BS1,...,3 , beamsplitters.

Fig. 2 .

 2 Fig. 2. (a) Bead (dia of 6μm) on substrate, (b) Hologram at different illumination angle(74 0 , 61 0 , 23 0 , 67 0 ). (c) The hologram in Fourier space (d) The desired order as filtered form (c). (e) The bead interface (Note: for images (b-e) both x and y axis indicates No. of pixel)

Fig. 3 .

 3 Fig. 3. Antigen presenting cell (APC) sitting on top of glass substrate: plane wave illumination at various angle

E

  inc =E 0 e i φ ( r )I inc (r )= | E inc (r ) | 2

+ | E ref (r ) | 2 +2|E (r ) |. | E ref (r ) | . cos [ Δ φ (r ) ]


Aubriot

; Knapp

MM; Poczai S ; Syfert

P ; Buerki

Xavier Aubriot
email: xavier.aubriot@univ-rennes1.fr

Sandra Knapp

Mindy Syfert

Péter Poczai

Sven Buerki
email: svenbuerki@boisestate.edu

Shedding new light on the origin and spread of the brinjal eggplant (Solanum melongena L.) and its wild relatives

Keywords: Eggplant (aubergine), crop wild relatives (CWRs), phylogenomics, chloroplast genome, biogeography, molecular dating, dispersion, mammalian herbivores

PREMISE OF THE STUDY:

While brinjal eggplant (Solanum melongena L.) is the second most important solanaceaous vegetable crop, we lack firm knowledge of its evolutionary relationships. This in turn limits efficient use of crop wild relatives in eggplant improvement. Here, we examine the hypothesis of linear step-wise expansion of the eggplant group from Africa to Asia.

METHODS:

We use museum collections to generate nuclear and full-plastome data for all species of the eggplant clade. We combine a phylogenomic approach with distribution data to infer a biogeographic scenario for the clade.

KEY RESULTS:

The eggplant clade has Pleistocene origins in northern Africa. Dispersions to tropical Asia gave rise to Solanum insanum, the wild progenitor of the eggplant, and to Africa distinct lineages of widespread and southern-African species. Results suggest that spread of species to southern Africa is recent and was likely facilitated by large mammalian herbivores feeding on Solanum fruits (African elephant and impala).

CONCLUSIONS:

Rather than a linear 'Out Of Africa' sequence, our results are more consistent with an initial dispersion event into Asia, and subsequent wide dispersion and differentiation across Africa 1 1 2 3 4 5 7 8 9 1

driven by large mammalian herbivores. Our evolutionary results will impact future work on eggplant domestication and use of wild relatives in breeding of this increasingly important solanaceous crop.

INTRODUCTION

Crop wild relatives (CWRs) are likely to play a significant role in securing 21 st century food security [START_REF] Dempewolf | Adapting agriculture to climate change: a global initiative to collect, conserve, and use crop wild relatives[END_REF], due to their potential for use in breeding to withstand adverse impacts of climate change, increasing scarcity of nutrients, water and other inputs, and new pests and diseases [START_REF] Hopkins | Crop wild relatives: plant conservation for food security[END_REF]. Understanding the phylogenetic relationships, geographic origins and spread of CWRs is critical for improving their use in future plant breeding in the face of environmental and climate change.

The brinjal eggplant, Solanum melongena L. (also known as the aubergine; Solanaceae) is the second most important economically important solanaceaous vegetable crop after tomato (FAO, 2017). The brinjal eggplant has a long history of cultivation (see [START_REF] Daunay | History and iconography of eggplant[END_REF][START_REF] Wang | Ancient Chinese literature reveals pathways of eggplant domestication[END_REF], and is thought to have been domesticated in Asia several times [START_REF] Daunay | Eggplant[END_REF][START_REF] Meyer | Phylogeographic relationships among Asian eggplants and new perspectives on eggplant domestication[END_REF]. Increasing genetic diversity in the cultivated crop is a major target in brinjal eggplant breeding [START_REF] Muñoz-Falcón | Diversity in commercial varieties and landraces of black eggplants and implications for broadening the breeders' gene pool[END_REF]. Improvements in yield, fruit and postharvest quality [START_REF] Vilanova | Development of a linkage map of eggplant based on a S. incanum x S. melongena backcross generation[END_REF], resistance to plant pathogens [START_REF] Pautasso | Impacts of climate change on plant diseases -opinions and trends[END_REF][START_REF] Bebber | The global spread of crop pests and pathogens[END_REF], and introduction of abiotic stress traits such as tolerance to increased drought, temperatures, and extreme precipitation are all important, especially in the face of climate change model predictions of higher temperatures and extreme weather patterns worldwide [START_REF] Cattivelli | Drought tolerance improvement in crop plants: an integrated view from breeding to genomics[END_REF]. Crop wild relatives are an increasingly important source for traits and genes for brinjal eggplant improvement [START_REF] Daunay | The use of wild species for the genetic improvement of brinjal (Solanum melongena L.) and tomato (Lycopersicon esculentum Miller)[END_REF]Muñóz-Falcón et al., 2009;[START_REF] Hurtado | Diversity and relationships of eggplants from three geographically distant secondary centers of diversity[END_REF][START_REF] Kaushik | Phenotyping of eggplant wild relatives and interspecific hybrids with conventional and phenomics descriptors provides insight for their potential utilization in breeding[END_REF], and knowledge about their distribution, traits and threat status has been the focus of recent studies [START_REF] Syfert | Crop wild relatives of the brinjal eggplant (Solanum melongena; Solanaceae): poorly represented in genebanks and many species at risk of extinction[END_REF]Kinsell et al., unpublished).

Solanum L., with c. 1,400 species, is one of the largest genera of flowering plants [START_REF] Frodin | History and concepts of big plant genera[END_REF] and has been the subject of much recent taxonomic and phylogenetic work (e.g., see references in [START_REF] Särkinen | A phylogenetic framework for evolutionary study of the nightshades (Solanaceae): a dated 1000-tip tree[END_REF][START_REF] Vorontsova | A revision of the spiny solanums, Solanum subgenus Leptostemonum (Solanaceae) in Africa and Madagascar[END_REF]. The species-level taxonomy of S. melongena and its wild relatives has long been recognised as complex (Lester and Hasan, 1991;[START_REF] Daunay | Eggplant[END_REF], but recent revision and clarification of taxonomic limits in the group (Knapp et al., 2013;[START_REF] Vorontsova | A revision of the spiny solanums, Solanum subgenus Leptostemonum (Solanaceae) in Africa and Madagascar[END_REF] has provided a set of species identities for use in further analyses.

Previous analyses recognised only two species in the Eggplant clade (e.g., Lester and Hasan, 1991;[START_REF] Weese | Eggplant origins: out of Africa, into the Orient[END_REF], but further work with African solanums suggested the clade comprised ten species characterized in part by their andromonoecious breeding system (see Table 1 in Knapp et al., 2013). Even though the number of taxa recognised increased, several extremely widespread species (e.g., S. campylacanthum A.Rich. across eastern Africa) include much geographical variation, suggesting a role for ecological structuring in these taxa [START_REF] Ranil | Solanum insanum L. (subgenus Leptostemonum Bitter, Solanaceae), the neglected wild progenitor of eggplant (S. melongena L): a review of taxonomy, characteristics and uses aimed at its enhancement for improved eggplant breeding[END_REF][START_REF] Vorontsova | A revision of the spiny solanums, Solanum subgenus Leptostemonum (Solanaceae) in Africa and Madagascar[END_REF].

Solanum melongena and its wild relatives are members of the largest clade of the genus, the Leptostemonum Clade (the 'spiny' solanums; [START_REF] Bohs | Major clades in Solanum based on ndhF sequences[END_REF][START_REF] Weese | Eggplant origins: out of Africa, into the Orient[END_REF][START_REF] Särkinen | A phylogenetic framework for evolutionary study of the nightshades (Solanaceae): a dated 1000-tip tree[END_REF], and within that, a monophyletic group comprising almost all Old World spiny solanums [START_REF] Stern | Molecular delimitation of clades within New World species of the "spiny solanums" (Solanum subgenus Leptostemonum)[END_REF][START_REF] Vorontsova | African spiny Solanum (subgenus Leptostemonum, Solanaceae): a thorny phylogenetic tangle[END_REF][START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF]. The Eggplant clade has been recognised as monophyletic in previous analyses (e.g., [START_REF] Stern | Molecular delimitation of clades within New World species of the "spiny solanums" (Solanum subgenus Leptostemonum)[END_REF][START_REF] Weese | Eggplant origins: out of Africa, into the Orient[END_REF], but sampling of Old World taxa was limited. Using the same set of genebank accessions [START_REF] Weese | Eggplant origins: out of Africa, into the Orient[END_REF] tested Lester's (Lester and Hasan, 1991) evolutionary scenario of brinjal eggplant evolution, but showed that the weedy species S. linnaeanum Hepper & P.M.-L.Jaeger also belonged to the group. Their analysis [START_REF] Weese | Eggplant origins: out of Africa, into the Orient[END_REF] supported the scenario of an African origin for the group with step-wise expansion to tropical Asia and migration back to the Middle East as feral forms. [START_REF] Vorontsova | African spiny Solanum (subgenus Leptostemonum, Solanaceae): a thorny phylogenetic tangle[END_REF] expanded the dataset by including many African taxa and still recovered a monophyletic Eggplant clade, but with little internal resolution. Their results placed the narrow Kenyan endemic S. agnewiorum Voronts. within the monophyletic Eggplant clade, a surprising result given its small fruit and hermaphroditic flowers. Inclusion of more species from Africa and southeast Asia [START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF] revealed that two additional African species with hermaphroditic flowers were members of the monophyletic group (S. lanzae J.-P.Lebrun & Stork and S. usambarense Bitter & Dammer). These studies used both plastid and nuclear molecular markers, and although the circumscription of the Eggplant clade improved, resolution within it was still poor. Although the brinjal eggplant and its wild relatives have clear morphological, molecular and eco-geographical differences (see [START_REF] Meyer | Phylogeographic relationships among Asian eggplants and new perspectives on eggplant domestication[END_REF]Knapp et al., 2013;[START_REF] Ranil | Solanum insanum L. (subgenus Leptostemonum Bitter, Solanaceae), the neglected wild progenitor of eggplant (S. melongena L): a review of taxonomy, characteristics and uses aimed at its enhancement for improved eggplant breeding[END_REF], they have been difficult to resolve using traditional molecular markers [START_REF] Vorontsova | African spiny Solanum (subgenus Leptostemonum, Solanaceae): a thorny phylogenetic tangle[END_REF][START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF].

High-throughput sequencing and DNA library preparation improvements have made obtaining whole plastid genomes from total DNA extraction affordable for many non-model organisms driving a rapid increase in the number of genomes in public sequence repositories (see [START_REF] Tonti-Filippini | What can we do with 1000 plastid genomes?[END_REF]. It has been suggested that complete plastid genomes can be used as 'superbarcodes' thus providing an abundance of characters to increase resolution in poorly resolved phylogenies [START_REF] Williams | Integration of complete chloroplast genome sequences with small amplicon datasets improves phylogenetic resolution in Acacia[END_REF].

Here we use sequences of whole chloroplast genomes to improve circumscription of and resolution in the Eggplant clade, and to test hypotheses concerning the origin of the close relatives of the cultivated eggplant, Solanum melongena. To account for morphological and geographical variability of the species, we designed a two-fold approach. First, we used Sanger data to test species circumscriptions for four widespread species of the Eggplant clade; we investigated two nuclear loci, ITS and the waxy (GBSSI) gene, and one plastid intergenic spacer, ndhF-rpL32, to check for topological incongruences. Second, based on this first analysis, we generated a plastome phylogeny of the Eggplant clade that we used to re-assess Lester and Hasan's (1991) dispersion of the Eggplant clade as currently circumscribed. We relate dispersion and spread in widespread eastern African species to large mammalian herbivores that feed on Solanum fruits [START_REF] Pringle | Low functional redundancy among mammalian browsers in regulating an encroaching shrub (Solanum campylacanthum) in African savannah[END_REF][START_REF] Kartzinel | DNA metabarcoding illuminates dietary niche partitioning be African large herbivores[END_REF]. Our results have implications for future research on wild sources of genetic variability for eggplant improvement, particularly in the context of global climate change and efforts to develop the genetic basis for sustainable pest control.

MATERIALS AND METHODS

Taxon sampling-Sampling for preliminary Sanger analysis-For Sanger analysis, we sampled across the distribution range of four widespread species (S. campylacanthum, S. cerasiferum Dunal, S. incanum L. and S. insanum L.) in order to test species monophyly and reveal any geographic patterns. For the widespread and weedy eastern African species S. campylacanthum, we sampled three specimens from the northernmost part of its distribution area (Ethiopia), one from Tanzania and two from the southernmost part (Namibia and South-Africa). Both S. cerasiferum and S. incanum are distributed across northern sub-Saharan Africa, with S. incanum also extending into regions of Middle East and central Asia as far as Pakistan [START_REF] Vorontsova | A revision of the spiny solanums, Solanum subgenus Leptostemonum (Solanaceae) in Africa and Madagascar[END_REF]. For S. cerasiferum we used specimens from Ethiopia and the Central African Republic, and for S. incanum one specimen from the westernmost part of the distribution range (Burkina Faso), two from the centre (Kenya and Oman), as well as one from the easternmost part (Pakistan). Solanum insanum, the wild progenitor of the cultivated eggplant [START_REF] Meyer | Phylogeographic relationships among Asian eggplants and new perspectives on eggplant domestication[END_REF][START_REF] Ranil | Solanum insanum L. (subgenus Leptostemonum Bitter, Solanaceae), the neglected wild progenitor of eggplant (S. melongena L): a review of taxonomy, characteristics and uses aimed at its enhancement for improved eggplant breeding[END_REF], is distributed across tropical Asia, but also occurs in Madagascar; it does not occur in Africa with no populations in East Asia and Africa [START_REF] Ranil | Solanum insanum L. (subgenus Leptostemonum Bitter, Solanaceae), the neglected wild progenitor of eggplant (S. melongena L): a review of taxonomy, characteristics and uses aimed at its enhancement for improved eggplant breeding[END_REF]; we sampled two specimens from tropical Asia (China and India) and one individual from Madagascar. Our sampling is not designed to explore the structure of relationships between the populations of S. insanum and S. melongena; this will require population level sampling across the range of both taxa (e.g., [START_REF] Meyer | Phylogeographic relationships among Asian eggplants and new perspectives on eggplant domestication[END_REF]W. Jinxiu, pers. comm.).

To place our study within the wider framework of Old World spiny solanums, we also sampled closely related species (sensu [START_REF] Vorontsova | African spiny Solanum (subgenus Leptostemonum, Solanaceae): a thorny phylogenetic tangle[END_REF], Aubriot et al., 2016). A clade of four tropical Asian species ("Solanum violaceum and relatives" sensu [START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF], as well as three African species (S. nigriviolaceum Bitter, S. polhillii Voronts. and S. supinum Dunal) were found to be closely related to the Eggplant clade [START_REF] Vorontsova | African spiny Solanum (subgenus Leptostemonum, Solanaceae): a thorny phylogenetic tangle[END_REF][START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF]; therefore we included in our sampling one accession of S. violaceum Ortega and one accession for each of these three African species. Two informal groupings of a large number of African and Asian species, the 'Anguivi grade' and the 'Climbing clade' [START_REF] Vorontsova | African spiny Solanum (subgenus Leptostemonum, Solanaceae): a thorny phylogenetic tangle[END_REF][START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF] were also sampled in order to better test the circumscription of the Eggplant clade. We selected six species of the 'Anguivi grade', including the two cultivated African solanums, the Gboma eggplant (S. macrocarpon L.) and the Scarlet eggplant (S. aethiopicum L.), as well as their wild progenitors (S. dasyphyllum Schumach. & Thonn. and S. anguivi Lam. respectively). Most species of the 'Anguivi grade' have geographic distributions centred in continental eastern Africa, but several species occupy disjunct regions such as the Seychelles and India (see [START_REF] Vorontsova | African spiny Solanum (subgenus Leptostemonum, Solanaceae): a thorny phylogenetic tangle[END_REF][START_REF] Vorontsova | A revision of the spiny solanums, Solanum subgenus Leptostemonum (Solanaceae) in Africa and Madagascar[END_REF][START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF] for details on geographic distributions). To identify potential long-distance dispersal events, we sampled one species with a tropical Asian distribution (S. trilobatum L.) and another that occurs within the Middle Eastern region (S. glabratum Dunal). We used an eastern African scandent shrub from the 'Climbing clade', S. richardii Dunal, to root all the trees. Sampling for this analysis includes 36 accessions (24 species), accounting for all 13 species of the Eggplant clade and for 11 additional spiny solanum species from Africa and Asia.

Sampling for whole chloroplast genomes analysis-After using Sanger sequencing for initial assessment of species delimitation in widespread species we generated full plastome data for all species of the Eggplant clade, with the exception of the cultivated brinjal eggplant for which we had two sets of Illumina reads at our disposal. If a species was not shown to be monophyletic based on preliminary Sanger analyses, one accession per lineage was included. All outgroup species used in the preliminary analysis were included in the full-plastome analysis, with the exception of S. nigriviolaceum for which we had insufficient DNA extract available.

DNA extraction, amplification, sequencing and data assembly-Silica gel dried or herbarium material was ground using a Mixer Mill MM 300 with glass beads (Qiagen Inc., Valencia, CA, USA) and with molecular grade sand. Total genomic DNA was extracted using the same two-step protocol than the one described by [START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF]. We have also extracted chloroplast DNA from fresh leaves of Solanum melongena 'Black Beauty' following [START_REF] Shi | An improved chloroplast DNA extraction procedure for whole plastid genome sequencing[END_REF].

Amplified DNA regions used in the preliminary Sanger analysis were selected based on previous broad-scope studies on Solanum phylogenetics [START_REF] Särkinen | A phylogenetic framework for evolutionary study of the nightshades (Solanaceae): a dated 1000-tip tree[END_REF][START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF]. These latter have shown that the plastid intergenic spacer ndhF-rpL32, the internal transcribed spacer (ITS) and the granule-bound starch synthase I (GBSSI) or waxy gene are particularly fitted for resolving phylogenetic relationships as the species level. Considering the abundant literature on DNA barcoding of landplants and on phylogenetics of Solanum, the use of these three rapidly evolving and a priori unlinked DNA regions should be reliable for testing current species boundaries. We downloaded 62 sequences from GenBank, and generated 61 for this study (see Appendix). For each additional accession, the three DNA regions were amplified and sequenced following the same procedures and techniques than those described in [START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF]. Sanger reads were assembled and edited in Geneious v.7.1.9 (Biomatters Ltd., Auckland, New Zealand). All sequences were automatically aligned with MAFFT v.7.305 [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: Improvements in performance and usability[END_REF], using the L-INS-i algorithm.

6

Newly generated sequences are deposited in GenBank and listed in the Appendix.

Starting quantities of total genomic DNA for the 25 samples used in the phylogenomic study were determined by measurement with a Qubit 2.0 Fluorometer (Life Technologies Corp., Carlsbad, CA). DNAs were diluted to approximately 3.8 ng/μl and μl and l l and libraries were prepared using the TruSeq Nano DNA Library Prep Kit (Illumina, San Diego, CA, USA) following manufacturer instructions.

Sequencing was performed at the Natural History Museum (London) sequencing facility using the Illumina MiSeq platform (2 × 250 bp) and loading concentration of 6 pM. Illumina adapters were removed, and all reads were quality-trimmed and quality-filtered using Trimmomatic 0.35 [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF] with default settings.

Plastome assembly, annotation and alignment-All paired reads were de-novo assembled with IDBA-UD 1.1.1 [START_REF] Peng | IDBA-UD: A de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth[END_REF], using maximum k-value = 250 bp and minimum k-value = 50 bp. Resulting contigs were uploaded into Geneious v.7.1.9 [START_REF] Kearse | Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data[END_REF] where they were quality-checked and length-filtered. Segments of the de novo plastomes were then circularized by matching end points. A high coverage plastid genome (800×) was also assembled de novo for Solanum melongena 'Black Beauty' from the collected reads using the built-in Geneious assembler platform with zero mismatches and gaps allowed among the reads. A similar procedure was conducted using Velvet v1. 2.10 (Zerbino and Birney, 2008) with k-mer length 37, minimum contig length 74 and default settings by applying 800× upper coverage limit. The results of the two de novo methods were compared and inspected. Sanger-based gap closure and IR junction verification was carried out following [START_REF] Moore | Using plastid genome-scale data to resolve enigmatic relationships among basal angiosperms[END_REF].

Sequence inconstancies were checked by mapping each set of reads to their respective plastome as described in [START_REF] Wysocki | Evolution of the bamboos (Bambusoideae; Poaceae): a full plastome phylogenomic analysis[END_REF]. Assembled plastomes were aligned to the completely annotated bittersweet (Solanum dulcamara L.) plastome available on GenBank (NC035724; Amiryousefi et al., unpublished). Annotations were transferred from the references to the assembled plastomes using a similarity threshold of 80%, and were then checked and refined manually. All 25 plastomes were aligned with MAFFT v.7.305 using the FFT-NS-2 algorithm, and the alignment was inspected for structural mutations. The pairwise distances between each pair of plastomes were calculated and analysed using the Kimura Maximum Composite Likelihood model implemented in MEGA 7.0 [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets[END_REF].

Phylogenetic analyses and dating-Sanger and genomic alignments were subjected to both maximum likelihood (ML) and Bayesian inference (BI) analyses. All analyses were run via the CIPRES Science Gateway [START_REF] Miller | Creating the CIPRES science gateway for inference of large phylogenetic trees[END_REF]) using RAxML-HPC v.8.1.24 (Stamatakis, 2014) for ML and MrBayes 3.2.6 [START_REF] Huelsenbeck | MRBAYES: Bayesian inference of phylogenetic trees[END_REF] for BI. In all analyses gaps were treated as missing data (N) and Solanum richardii served as the outgroup. Sanger alignments were analysed both separately and as combined datasets. RAxML analyses were run by using the rapid bootstrap algorithm with 1,000 bootstrap iterations. The GTR + G rate substitution model was applied to each matrix in order to fit the substitution models implemented in BI (see below); the proportion of invariable sites was not estimated as it is not recommended by RAxML developer [START_REF] Stamatakis | The RAxML v8[END_REF].

Bootstrap replicate trees were used to draw bipartitions on the optimal ML tree; nodes with bootstrap support values (BS) below 50% were pruned to obtain the ML 50% majority-rule tree. For BI analyses, the best-fitting nucleotide substitution model inferred with MrModeltest v.2.3 [START_REF] Nylander | MrModeltest v2. Computer program and documentation distributed by the author[END_REF] via estimation of the Akaike information criterion was assigned to each of DNA region. The regions ndhF-rpL32 and ITS followed a GTR + I + G substitution model, whereas the GTR + I model better fitted for the waxy region. MrBayes analyses constituted of two independent parallel runs of four Markov chains each, run for 10 million generations and trees were sampled every 1000 generations. Adequate mixing of Markov chains and convergence of the two runs were confirmed with Tracer v1.6 (Rambaut et al., 2014); after a 10% burn-in, remaining trees were used to generate a 50% Bayesian majority-rule consensus tree. Congruence between the topologies obtained for each of the three markers was visually inspected before concatenating them into a combined matrix that was divided into three partitions, to which the best-fitted substitution models were applied. The combined matrix was run under the same conditions detailed above for the single marker matrices. We used the same BI and ML approaches to analyse the phylogenomic dataset. The plastome matrix was analysed with MrModeltest, and the GTR + I + G nucleotide substitution model was selected as best-fitting the data. For same reasons than stated above, this model was implemented in MrBayes whereas the GTR + G rate substitution model was preferred for RAxML analyses. All conditions of MrBayes and RAxML runs were identical to those implemented for analyses of Sanger alignments.

Relative divergence times, together with the topology were estimated using BEAST v.1.8.4 [START_REF] Drummond | Bayesian phylogenetics with BEAUti and the BEAST 1.7[END_REF], with a GTR + I + G rate substitution model applied to the plastome matrix.

A Yule speciation tree prior and a relaxed uncorrelated clock-model that allows rates to vary independently along branches [START_REF] Drummond | Relaxed phylogenetics and dating with confidence[END_REF] were used with all other parameters set to default. We used two temporal constraints to calibrate the BEAST analyses: (1) age of the Eggplant clade (mean = 1.54 Myr; standard deviation = 0.5), ( 2) age of the root (mean = 3.38 Myr; standard deviation = 0.5). These were derived from the Solanaceae-wide phylogeny of [START_REF] Särkinen | A phylogenetic framework for evolutionary study of the nightshades (Solanaceae): a dated 1000-tip tree[END_REF], with uncertainty regarding these dates incorporated by assigning normal prior distributions to the two calibration points [START_REF] Couvreur | Molecular phylogenetics reveal multiple tertiary vicariance origins of the African rain forest trees[END_REF][START_REF] Evans | The evolution of succulence: insights from a remarkable radiation in Madagascar[END_REF]. Four independent BEAST analyses were run, each 100 million generations, sampling every 10,000 generations. Convergence of all parameters was assessed using Tracer 1.5 (Rambaut et al., 2014), the first 10% of each of the four Markov chains 8 was removed as burn-in, and chains were combined with LogCombiner 1.7.2. [START_REF] Drummond | Bayesian phylogenetics with BEAUti and the BEAST 1.7[END_REF] to calculate the maximum clade credibility tree. 2012). Linder's regions are statistically defined using patterns from groups of vascular plants and vertebrates, and were shown to be robust between groups. This biogeographic framework is well suited for these solanums with 10 of 23 taxa included in our analysis restricted to one region, and several clades that are characteristic of a set of connected biogeographic regions (Southern African clade; Fig. 1B).

Biogeographic inference and dispersion analysis-Species

Boundaries of the biogeographic regions used in this study are subject to some uncertainty due to contrasting climatic patterns (e.g. the wet aseasonal Congolian region vs the drier and more seasonal Sudanian region). Areas around the Tanzania/μl and Kenya border in east Africa correspond to a zone of high mountainous complexity (i.e. Albertine Rift and Kilimanjaro Massif), and have been previously assigned to either the Somalian or the Zambesian regions, depending on the data types used [START_REF] White | The vegetation of Africa: a descriptive memoir to accompany the UNESCO/μl and AET-FAT/μl and UNSO vegetation map of Africa[END_REF][START_REF] White | The AETFAT chorological classification of Africa: history, methods and applications[END_REF][START_REF] Linder | The partitioning of Africa: Statistically defined biogeographical regions in sub-Saharan Africa[END_REF]. This region has also been identified as a centre of diversity for African spiny solanums [START_REF] Syfert | Crop wild relatives of the brinjal eggplant (Solanum melongena; Solanaceae): poorly represented in genebanks and many species at risk of extinction[END_REF][START_REF] Vorontsova | A revision of the spiny solanums, Solanum subgenus Leptostemonum (Solanaceae) in Africa and Madagascar[END_REF]. In addition, two of our sampled species, S. polhillii and S. usambarense, are restricted to this transitional region [START_REF] Vorontsova | A revision of the spiny solanums, Solanum subgenus Leptostemonum (Solanaceae) in Africa and Madagascar[END_REF] and assigning species to particular biogeographic region was challenging; therefore we accounted for this complexity by assigning these two species to both the Somalian and the Zambesian regions.

Handling of the Middle East area presents another challenge as this region does not account for any endemic species of spiny solanums in our sampling [START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF]. Only S. glabratum and S. incanum are present in the Middle East, both have distribution ranges centred on northeastern Africa (i.e., Sudanian region). The Red Sea is not a strong dispersal barrier, being only about 25 km wide at its southernmost and narrowest (natural) point, and we combined the Middle East with the Sudanian region, hence delimiting our region B. Similarly, it is difficult to draw the eastern boundary of region B as the Middle East acts as a biogeographic crossroads between Asia and Africa, its biota being of composite origin [START_REF] Buerki | Contrasting biogeographic and diversification patterns in two mediterranean-type ecosystems[END_REF]. Among all spiny solanums native of the Middle East region, two are distributed from eastern tropical India to the Arabian Peninsula (S. pubescens Willd., S. virginianum L.) while others are not present in tropical regions as their distributions are restricted to dry regions between northeastern Africa and northwestern India (S. cordatum Forssk., S. forskalii Dunal, S. glabratum, S. incanum, S. platacanthum Dunal). Given such distribution patterns, we used the Thar Desert of India as the eastern boundary of zone B (Sudanian region and Middle East). This delimitation also corresponds to the western limit of the Asian monsoon climatic regime, acting as a proxy of the limit between the tropical areas of region A (Tropical Asia) and the mostly dry areas of region B [START_REF] Holt | An update of Wallace's zoogeographic regions of the world[END_REF].

The history of Madagascar and of its neighbouring archipelagos (Mascarenes, Granitic Seychelles and Comoros) is characterized by complex geological and climatic events that accompanied their isolation from Africa and the Indian subcontinent. The Malagasy endemic flora has a composite origin, with elements from Africa, the Indian subcontinent and South-East Asia [START_REF] Buerki | Spatio-temporal history of the endemic genera of Madagascar[END_REF]. The spiny solanums endemic to Madagascar (not sampled here) are not closely related to the Eggplant clade and form a monophyletic clade whose phylogenetic placement is still unresolved [START_REF] Vorontsova | African spiny Solanum (subgenus Leptostemonum, Solanaceae): a thorny phylogenetic tangle[END_REF][START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF] within the larger Old World lineage of spiny solanums.

We sampled four species that are native to Madagascar and the Mascarenes; two are present in Africa and in Madagascar (S. anguivi and S. richardii), while the two others have a distribution range centred on tropical Asia, with S. insanum (the wild progenitor of the eggplant) present in Madagascar (Knapp et al., 2013;[START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF][START_REF] Ranil | Solanum insanum L. (subgenus Leptostemonum Bitter, Solanaceae), the neglected wild progenitor of eggplant (S. melongena L): a review of taxonomy, characteristics and uses aimed at its enhancement for improved eggplant breeding[END_REF] and S. violaceum recorded from Mauritius [START_REF] Vorontsova | A revision of the spiny solanums, Solanum subgenus Leptostemonum (Solanaceae) in Africa and Madagascar[END_REF]. Hence, we treated Madagascar and the neighbouring archipelagos as a distinct biogeographic area; the Malagasy region (region G).

The dispersal-extinction-cladogenesis (DEC) likelihood model implemented in Lagrange v.

20130526 [START_REF] Ree | Maximum likelihood inference of geographic range evolution by dispersal, local extinction, and cladogenesis[END_REF] was used to investigate the biogeographic history of the Eggplant clade. The analysis was performed on the BEAST maximum clade credibility tree using the following set of parameters: (1) species ranges were coded using presence in the seven biogeographic regions;

(2) maximum range size at nodes was constrained to 3; (3) areas not formed of adjacent regions were excluded from the analysis (adjacency matrix); (4) the dispersal constraints matrix was used to set the dispersal rates between the seven biogeographic regions with a value of 1 for adjacent regions (e.g., E and F), 0.5 between sea-separated regions (e.g., G) and 0.01 for nonadjacent regions (e.g., A and F).

Ancestral area reconstruction for each node was plotted on the BEAST maximum clade credibility tree using pie charts (see [START_REF] Buerki | An evaluation of new parsimony-based versus parametric inference methods in biogeography: a case study using the globally distributed plant family Sapindaceae[END_REF] for details on the R script).

To assess the potential influence of fruit-eating migratory mammals in the dispersion of the African widespread Eggplant clade species (Solanum campylacanthum, S. cerasiferum and S. 10 incanum), a distribution map of these species together with two of their known large-mammalian seed dispersers (African elephant, Loxodonta africana (Blumenbach) and impala, Aepyceros melampus (Lichentenstein); see [START_REF] Pringle | Low functional redundancy among mammalian browsers in regulating an encroaching shrub (Solanum campylacanthum) in African savannah[END_REF][START_REF] Kartzinel | DNA metabarcoding illuminates dietary niche partitioning be African large herbivores[END_REF] was reconstructed using the R packages maps and dismo. Shapefiles of the current distributions of the African elephant and impala were downloaded from the IUCN Red List of Threatened Species website (IUCN SSC Antelope Specialist Group, 2016;[START_REF] Blanc | Loxodonta africana. The IUCN Red List of Threatened Species[END_REF]. Using georeferenced herbarium specimens, we gathered data on 1,150 populations of S. campylacanthum, S. cerasiferum and S. incanum. A Venn diagram showing the overlap between the distributions of the widespread Eggplant clade species and the African elephant and impala was inferred using the R package VennDiagram.

RESULTS

Sanger reconstructions and species delimitation-Visual comparison of 50% majority-rule

trees obtained from single marker matrices with BI and ML showed no strongly supported topological conflict (BS > 75% and PP > 0.9) (only the BI consensus trees shown; see Appendix S1a,b,c in the Supplemental Data with the online version of this article). Topologies are overall all poorly resolved with the exception of few strongly supported nodes; they only differed in the degree of resolution, with the ITS consensus trees exhibiting higher proportions of supported nodes than those inferred from waxy and ndhF-rpL32.

The combined Sanger dataset yielded a total of 3,121 base pairs (bp) of which 256 bp were variable. There were no supported conflicts between the BI and ML 50% majority-rule trees, with the BI tree showing a much greater resolution than the ML one, albeit often with poorly supported nodes.

The combined topology (Appendix S1d) shows that the Eggplant clade as previously defined [START_REF] Vorontsova | African spiny Solanum (subgenus Leptostemonum, Solanaceae): a thorny phylogenetic tangle[END_REF][START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF] is monophyletic (PP = 0.99) and it includes the Cape Verde endemic Solanum rigidum Lam. Within the Eggplant clade, two of the widespread species are resolved as monophyletic (S. campylacanthum, S. cerasiferum) and one as paraphyletic (S. incanum). The three accessions of S. incanum from the eastern part of the species distribution range (Kenya, Oman and Pakistan) form a strongly supported lineage (BS = 99%; PP = 1) that excludes the accession from Burkina Faso; the latter is sister to the clade formed by the eggplant (S. melongena) and its wild progenitor (S. insanum). Solanum melongena is part of a polytomy that includes all three accessions of S. insanum; this confirms previous phylogenetic results [START_REF] Vorontsova | African spiny Solanum (subgenus Leptostemonum, Solanaceae): a thorny phylogenetic tangle[END_REF][START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF].

Affinities of the species of the Eggplant clade remain largely unresolved using these markers, with the exception of a group of species from southern Africa (S. aureitomentosum Bitter, S. lichtensteinii Willd., S. linnaeanum, S. umtuma Voronts. & S.Knapp) forming a strongly supported group (BS = 90%; PP = 1) that had been recognised in previous analyses [START_REF] Vorontsova | African spiny Solanum (subgenus Leptostemonum, Solanaceae): a thorny phylogenetic tangle[END_REF][START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF].

Plastome phylogenomic analyses-Plastome lengths ranged from 155,017 to 155,820 bp, with a mean coverage between 10.2× and 254.8×; see Table 1 and Appendix S2 for details on the plastomes. The plastome alignment was 159,227 bp in length, of which 3,890 bp were variable. There were no supported conflicts between the BI topologies obtained with BEAST and MrBayes, and the RAxML 50% majority-rule tree (see Appendix S3). Phylogenetic trees are all fully resolved with the exception of a polytomy in the MrBayes and RAxML 50% majority-rule trees. For the MrBayes topology all other nodes have PP ≥ 0.99 (Appendix S3b). For the BEAST and RAxML trees, 13 out of 15 nodes within the Eggplant clade are strongly supported (i.e., BS ≥ 87% and PP ≥ 0.97; Fig. 1B and Appendix S3a,c). Plastome trees confirm results from the Sanger analyses, but provide much better resolution of relationships in the clade (Fig. 1B). The Eggplant clade (sensu [START_REF] Vorontsova | African spiny Solanum (subgenus Leptostemonum, Solanaceae): a thorny phylogenetic tangle[END_REF][START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF]) is recovered as monophyletic; it is sister (BS = 82%; PP = 0.94) to a lineage formed by two African (Solanum polhillii and S. supinum) and one tropical Asian species (S. trilobatum), partly confirming previous phylogenetic results [START_REF] Vorontsova | African spiny Solanum (subgenus Leptostemonum, Solanaceae): a thorny phylogenetic tangle[END_REF]; but see [START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF] for an alternative position of S. trilobatum). The first-branching lineage, here called 'Kilimanjaro clade' (Fig. 1B), groups three species from the eastern African region (S. agnewiorum, S. lanzae, S. usambarense). Affinities within this clade are still unresolved; in the BEAST analysis S. agnewiorum branches first but this result is not supported (PP = 0.41). The next branching lineage is formed by the eggplant and its wild progenitor, S. insanum, the two accessions of the cultivated eggplant being grouped together (Fig. 1B). This lineage is sister to a monophyletic group that includes all remaining species of the Eggplant clade and is composed of two sister clades: (1) the Southern African clade (Fig. 1B) with four species from southern Africa (S. aureitomentosum Bitter, S. lichtensteinii, S. linnaeanum and S. umtuma), and (2) the Widespread clade (Fig. 1B) that includes three species with large distribution ranges (S. campylacanthum, S. cerasiferum and S. incanum) and the Cape Verde islands endemic S. rigidum. Within the Southern African clade, S. aureitomentosum branches first, followed by S. linnaeanum that is sister to a moderately supported grouping formed by S. lichtensteinii and S. umtuma (BS = 83%; PP = 1). Solanum campylacanthum is the earliest diverging species of the Widespread clade; it is sister to a lineage in which S. cerasiferum branches first, followed by one of the two accession of S. incanum (from Kenya) that is in turn sister to a grouping formed by the Cape Verdean S. rigidum and the second accession of S. incanum (from Burkina Faso). its distribution range into area E (Zambesian region). There is a high level of biogeographic uncertainty on the node separating the cultivated eggplant plus its progenitor (S. insanum) from the rest of the species in the clade. This is due to a disjunct distribution between these two clades (Fig. 1B). The most parsimonious scenario invokes two dispersal events leading to the most common ancestor of the whole clade therefore achieving a widespread distribution across regions BE and A (Tropical Asian region). The disjunct distribution of S. insanum in A and G (Malagasy region) most likely results from a long-distance dispersal event from A to G during the last million years. The majority of inferred dispersal events took place between the Southern African and Widespread clades. Southward and westward dispersal from B has been active in these two clades over the last 2 Myrs, and F (Southern African region) was colonized during the past 1 million years. In addition to having acted as a cradle, area B has been a pivotal region in shaping the current distribution of the eggplant clade. The inferred dispersion scenarios are illustrated in Fig. 1A.

Historical biogeography of the Eggplant clade and dispersion analysis-The

The distribution of Solanum campylacanthum, S. cerasiferum and S. incanum together with their large mammalian seed dispersers is displayed in Fig. 2A. The distribution of African elephants and impalas covers 53.7% (617 in total) of the populations of these three widespread species (Fig. 2B).

The elephant range currently only covers 24.3% (150) of the three Solanum species populations, while the impala distribution covers 94.4% (583); most Solanum populations only overlap the impala range (467 populations) (Fig. 2B).

DISCUSSION

While studies focusing on the morpho-ecological and genetic diversity of the eggplant and its wild relatives are increasing (e.g., [START_REF] Gramazio | Transcriptome analysis and molecular marker discovery in Solanum incanum and S. aethiopicum, two close relatives of the common eggplant (Solanum melongena) with interest for breeding[END_REF][START_REF] Kaushik | Phenotyping of eggplant wild relatives and interspecific hybrids with conventional and phenomics descriptors provides insight for their potential utilization in breeding[END_REF][START_REF] Ranil | Solanum insanum L. (subgenus Leptostemonum Bitter, Solanaceae), the neglected wild progenitor of eggplant (S. melongena L): a review of taxonomy, characteristics and uses aimed at its enhancement for improved eggplant breeding[END_REF][START_REF] Syfert | Crop wild relatives of the brinjal eggplant (Solanum melongena; Solanaceae): poorly represented in genebanks and many species at risk of extinction[END_REF][START_REF] Acquadro | Coding SNPs analysis highlights genetic relationships and evolution pattern in eggplant complexes[END_REF] our study is the first designed to test phylogenetic and biogeographic scenarios in the Eggplant clade (sensu [START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF]. Our sampling for Sanger and genomic studies allowed us to test recent species delimitations and build a phylogeny for the Eggplant clade that is well resolved internally. This study strongly supports Solanum insanum, the only wild Eggplant clade species currently distributed within the original centre of domestication of S. melongena [START_REF] Meyer | Phylogeographic relationships among Asian eggplants and new perspectives on eggplant domestication[END_REF], as its most likely progenitor (Fig. 1A,B). The Somalian and Middle East region is identified as the cradle of the Eggplant clade with recent dispersions to southern/μl and western Africa and tropical Asia; distribution data suggest that large savannah herbivores contributed to the geographical expansion of several of the species (Fig. 2A,B). The results do not, however, provide a definitive biogeographic scenario for the disjunct distribution between S. insanum + S. melongena and the rest of the species of the Eggplant clade that are all restricted to Africa and western Asia (see node 10 in Appendix S4a).

13 Phylogenetic relationships and species concepts in the eggplant clade-Within the Eggplant clade, four species concepts are particularly challenging: Solanum campylacanthum, S. cerasiferum, S. incanum and S. insanum (the three first species belong to the Widespread clade, the last is sister to S. melongena; Fig. 1B). Widespread across Africa and Asia, these species are also extremely polymorphic, with morphological characters that vary within and between populations (Knapp et al., 2013;[START_REF] Ranil | Solanum insanum L. (subgenus Leptostemonum Bitter, Solanaceae), the neglected wild progenitor of eggplant (S. melongena L): a review of taxonomy, characteristics and uses aimed at its enhancement for improved eggplant breeding[END_REF]. [START_REF] Olet | Variation within the Solanum incanum complex in Uganda and its relationship with Solanum cerasiferum[END_REF] suggested that S. campylacanthum (as "S.

incanum") and S. cerasiferum belong to the same biological species; our phylogenetic results support the monophyly of these taxa and confirm they represent two distinct lineages. Sanger and fullplastome data unambiguously confirm S. insanum as the wild progenitor of the cultivated brinjal eggplant. While the two eggplant accessions are sister in the plastome topology, pairwise distances between the two plastomes of the eggplant and plastome of S. insanum are identical (1E-4) and c. 25 times smaller than the mean interspecific pairwise distances of the dataset (2.8E-3). Strong plastome identity between the brinjal eggplant and its wild progenitor is not surprising as they are known to interbreed relatively freely when in sympatry (e.g., [START_REF] Davidar | The potential for crop to wild hybridization in eggplant (Solanum melongena; Solanaceae) in southern India[END_REF][START_REF] Mutegi | Genetic diversity and population structure of wild/μl and weedy eggplant (Solanum insanum, Solanaceae) in southern India: Implication for conservation[END_REF]. Our results also illustrate the fact that, while the cultivated eggplant and S. insanum present distinctive morphological features [START_REF] Ranil | Solanum insanum L. (subgenus Leptostemonum Bitter, Solanaceae), the neglected wild progenitor of eggplant (S. melongena L): a review of taxonomy, characteristics and uses aimed at its enhancement for improved eggplant breeding[END_REF], diagnostic molecular data are still lacking. Solanum incanum is recovered as paraphyletic, with the sample from the westernmost part of the distribution range (Burkina Faso) that does not group unambiguously with the other S. incanum accessions.

Genomic data resolve the Cape Verde endemic S. rigidum nested within S. incanum, strongly supported as sister to the sample from Burkina Faso. The plastid genome of S. rigidum is very similar to that of the two accessions of S. incanum; mean pairwise distance (MPD) between these three plastomes is 3.5E-4. The phylogeny does not reflect morphological characters; S. rigidum is morphologically similar to S. cerasiferum and readily distinguishable from S. incanum by its deeply lobed leaves and sparser indumentum. This result can be interpreted in two ways. Solanum rigidum may be a recent morpho-geographical segregate of the continental S. incanum, both taxa having conserved their ancestral chloroplast genome. Internal transcribed spacer (ITS) data (Appendix S1a) resolve S. rigidum as sister to S. macrocarpon (gboma eggplant) and S. dasyphyllum, albeit this result is poorly supported (PP = 0.82); this would suggest a hybrid origin via chloroplast capture with contributions from S. incanum (as the maternal parent of the cross) and arguably from S. macrocarpon or its wild progenitor, S. dasyphyllum (see also [START_REF] Knapp | Wild relatives of the eggplant (Solanum melongena L.: Solanaceae): New understanding of species names in a complex group[END_REF]. Chloroplast capture revealed by whole plastid genome sequences has been reported in the tomato clade (Solanum sect.

Lycopersicon; Poczai et al., unpublished) as well as among species of Gossypium [START_REF] Chen | Molecular evolution of the plastid genome during diversification of the cotton genus[END_REF] and Gaultheria [START_REF] Zhang | Plastid phylogenomics and adaptive evolution of Gaultheria series Trichophyllae (Ericaceae), a clade from sky islands of the Himalaya-Hengduan Mountains[END_REF]. Obtaining broader geographic sampling of Eggplant clade species, particularly S. rigidum and accessions from western Africa, using data suited to this taxonomic depth, such as nuclear SSRs or SNPs (e.g., [START_REF] Van Loo | Intervarietal and intravarietal genetic structure in Douglas-fir: nuclear SSRs bring novel insights into past population demographic processes, phylogeography, and intervarietal hybridization[END_REF][START_REF] Acquadro | Coding SNPs analysis highlights genetic relationships and evolution pattern in eggplant complexes[END_REF] may help better resolve circumscription of S. incanum and S. rigidum.

Our inferred plastome phylogeny provides much better resolution at the interspecific level within the Eggplant clade than previous phylogenetic investigations that either lacked taxonomic representativeness [START_REF] Meyer | Phylogeographic relationships among Asian eggplants and new perspectives on eggplant domestication[END_REF][START_REF] Acquadro | Coding SNPs analysis highlights genetic relationships and evolution pattern in eggplant complexes[END_REF] or phylogenetic resolution [START_REF] Vorontsova | African spiny Solanum (subgenus Leptostemonum, Solanaceae): a thorny phylogenetic tangle[END_REF][START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF] or both [START_REF] Weese | Eggplant origins: out of Africa, into the Orient[END_REF]. The topology we obtained, with the exception of one internal nodes (Fig. 1B), is fully resolved with strongly supported nodes. This plastome phylogeny confirms several phylogenetic hypotheses while it contradicts others. Taxonomic composition and phylogenetic structure of the Eggplant clade as found by [START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF] is confirmed but with the addition of Solanum rigidum and much improved resolution and support. Three species that were found in polytomy at the base of the Eggplant clade (S. agnewiorum, S. lanzae and S. usambarense) here are resolved as a basal Kilimanjaro clade; the Southern African clade and Widespread clade, whose compositions globally overlap clades that were previously poorly supported with Sanger data (see [START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF][START_REF] Vorontsova | African spiny Solanum (subgenus Leptostemonum, Solanaceae): a thorny phylogenetic tangle[END_REF]) are here resolved with highly supported nodes. Phenetic results obtained by [START_REF] Acquadro | Coding SNPs analysis highlights genetic relationships and evolution pattern in eggplant complexes[END_REF] with a data set of 12,859 nuclear SNPs, but with limited taxonomic sampling (6 Eggplant clade spp.) are not fully congruent with our plastome topology; comparisons are difficult due to differences in sampling and methodological approaches.

Effect of savannahs and mammalian dispersers on biogeography of the Eggplant clade-

Our biogeographic results show that the Eggplant clade originated recently, during the last c. 3 Myr, in the Somalian and Middle East region (Fig. 1A,B). Spread then occurred mostly within Africa; the progenitor of the eggplant, Solanum insanum, is the only wild species that is not present in continental Africa, but that has a Malagasy-Southeast Asian distribution. Dispersion to tropical Asia that gave rise to S. insanum is likely to have happened during the Early Pleistocene via the Middle East region; we suggest dispersion to Madagascar was a later event, possibly linked to recent human migration (see [START_REF] Crowther | Ancient crops provide first archaeological signature of the westward Austronesian expansion[END_REF]. Our results challenge Lester and Hasan's (1991) hypothesis of an eastern African origin for the 'ancestors of S. melongena' with subsequent spread to tropical Asia via a stepwise expansion through Middle East. Our biogeographic analysis instead shows that the origin of the Eggplant clade lies in northeastern Africa and the Middle East, with spread both south (Widespread and Southern African clades) and east (S. insanum + S. melongena). Importantly, it shows that the tropical Asian lineage of S. insanum did not proceed from a step-wise expansion through Middle East, but instead from an early dispersion from Africa, unrelated to the southwards spread of the Widespread and Southern African clades. This first attempt to provide a testable biogeographic framework for the Eggplant clade shows that its history consists in a wide dispersion across the African continent rather than an 'Out of Africa' phenomenon. Nesting of Asian species such as S.

15

insanum in otherwise African lineages, suggesting dispersal events in African lineages are not rare in Old World spiny solanums; S. trilobatum and S. violaceum also show this pattern in our plastome phylogeny (see also [START_REF] Aubriot | Tropical Asian species show the Old World clade of "spiny solanums" (the Leptostemonum Clade: Solanaceae) is not monophyletic[END_REF]. Species of the Eggplant clade have a recent biogeographic history and several species have large and complex distribution ranges, which makes it difficult to untangle biotic and abiotic factors that could explain evolutionary dynamics in the clade. Many spiny solanums are plants of disturbed areas, and most species of the Eggplant clade are weedy and occur in nitrogen-rich habitats of aridareas or gaps in tropical forests, in a wide range of altitudes (Vorontosva and Knapp, 2016;[START_REF] Knapp | Wild relatives of the eggplant (Solanum melongena L.: Solanaceae): New understanding of species names in a complex group[END_REF]. Only the three species of the basal Kilimanjaro clade are habitat-restricted; they are found in higher elevation (> 1,200 m) montane woodlands (Vorontosva and Knapp, 2016). These are also either hermaphroditic (Solanum lanzae, S. usambarense) or only weakly andromonoecious (S. agnewiorum) with small fruits, while strong andromonecy characterizes all other species of the Eggplant clade (Vorontosva and Knapp, 2016;[START_REF] Vorontsova | African spiny Solanum (subgenus Leptostemonum, Solanaceae): a thorny phylogenetic tangle[END_REF], with larger fruits. Our data suggest that the combination of breeding system and ecology (see below) in this lineage have been important factors in the dramatic expansion of its distribution range over the last 2 Myrs.

The increased geographical spread of members of the Eggplant clade coincides with profound habitat changes in Africa. Decrease in atmospheric CO2 concentrations and aridification beginning in the late Miocene led to global increase in the biomass of plants using C4 photosynthesis. This resulted in the extension of savannahs and fire-prone environments, first in northeastern Africa and then in South Africa [START_REF] Cerling | Global vegetation change through the Miocene/μl and Pliocene boundary[END_REF][START_REF] Uno | Neogene biomarker record of vegetation change in eastern Africa[END_REF]. Recent studies on hominin environments [START_REF] Cerling | Woody cover and hominin environments in the past 6 million years[END_REF][START_REF] Uno | Neogene biomarker record of vegetation change in eastern Africa[END_REF] found that woody grassland savannahs became persistent in northeastern Africa at least 6 Myr BP, with major increases in the abundance of savannah grazing mammalian s over this period [START_REF] Uno | Late Miocene to Pliocene carbon isotope record of differential diet change among East African herbivores[END_REF]. Open grassland and wooded grassland savannahs are typical habitats for members of both the South-African and Widespread clades, and large savannah mammalians such as African elephants and impalas have been recorded as important herbivores and seed dispersers of Eggplant clade species [START_REF] Pringle | Low functional redundancy among mammalian browsers in regulating an encroaching shrub (Solanum campylacanthum) in African savannah[END_REF][START_REF] Kartzinel | DNA metabarcoding illuminates dietary niche partitioning be African large herbivores[END_REF]. In Africa elephants and impalas have native distributions that overlap those of members of the Eggplant clade, and particularly species of the Widespread clade (see Fig. 2A,B). Although the historical range of African elephants fully encompassed the current distribution of the Eggplant clade species (see [START_REF] Ripple | Collapse of the world's largest herbivores[END_REF], it appears that today the impala is likely to be more important for the seed dispersal of widespread species. We suggest that the extensive geographical expansion over the last 2-3 Myr and the impressive ranges of some of the extant species (e.g., Solanum campylacanthum, S. cerasiferum, S. incanum) is related to the dispersal of seeds by these large, migratory mammalian herbivores.

Contraction of ranges of these large mammalian seed dispersers could ultimately contribute to isolation of populations previously more intermixed, leading to differentiation and speciation. Human use of these species could have also contributed to their recent wide dispersal and spread; many species of the clade are used medicinally and for tanning hides [START_REF] Bitter | Solana Africana Part IV. Repertorium specierum novarum regni vegetabilis[END_REF][START_REF] Ranil | Solanum insanum L. (subgenus Leptostemonum Bitter, Solanaceae), the neglected wild progenitor of eggplant (S. melongena L): a review of taxonomy, characteristics and uses aimed at its enhancement for improved eggplant breeding[END_REF][START_REF] Vorontsova | A revision of the spiny solanums, Solanum subgenus Leptostemonum (Solanaceae) in Africa and Madagascar[END_REF]. Fragments of Solanum seeds have been recorded from a 780,000 yr old hominin site in Israel [START_REF] Melamed | The plant component of an Acheulian diet at Gesher Benot Ya'aqov, Israel[END_REF], and archaeological remains may provide additional information about early interactions between hominins and Eggplant clade species.

Disentangling the relationship between complex changes in ecology and differentiation in the Eggplant clade, and in spiny solanums more broadly, is challenging, especially in the light of recent profound environmental change; new types of data will be required to understand the evolutionary success of this cosmopolitan and very diverse group.

Eggplant domestication, still a biogeographic puzzle-Our understanding of the evolutionary history and biogeography of the eggplant and its wild relatives is now better understood following the analyses conducted here using whole plastome and nuclear sequence data. The two nuclear regions we analysed here do not contradict plastome data, but nuclear genomic data for all the species of the Eggplant clade will be necessary to assess phylogenetic discordance and detect historical gene flow.

Combination of plastome and denser nuclear genomic data would also open the way to in-depth investigation of the genomic basis of environment-specific and lineage-specific adaptations in eggplant CWR (e.g., [START_REF] Pease | Phylogenomics reveals three sources of adaptive variation during a rapid radiation[END_REF]. This will ultimately provide plant breeders and physiologists with an estimate of the potential for selection from standing variation across eggplant CWR, potentially revolutionizing genetic improvement of eggplants.

Our study also shows that even with sampling across the full diversity of the Eggplant clade and a robust phylogenomic framework, our understanding of the biogeographic history of the Eggplant clade can still be improved; in particular dispersion of Solanum insanum lineages to tropical Asia and emergence and domestication of the brinjal eggplant. [START_REF] Meyer | Phylogeographic relationships among Asian eggplants and new perspectives on eggplant domestication[END_REF] suggested that S. melongena was domesticated several times from S. insanum (as S. incanum); use of S. insanum as a medicine [START_REF] Ranil | Solanum insanum L. (subgenus Leptostemonum Bitter, Solanaceae), the neglected wild progenitor of eggplant (S. melongena L): a review of taxonomy, characteristics and uses aimed at its enhancement for improved eggplant breeding[END_REF] and extensive movement of peoples across southeastern Asia support this idea.

Combining sampling for S. insanum through its whole geographical range with the use of recently developed targeted high-throughput sequencing [START_REF] Kadlec | Targeted NGS for species level phylogenomics: 'made to measure' or 'one size fits all[END_REF] will provide a promising approach for reconstructing reliable scenarios of evolution of brinjal eggplant from its wild progenitor, S. insanum.

TABLE 1 Summary of GenBank accession numbers, species name, collection information and plastome composition and coverage for taxa used in the NextGen analyses. Herbarium codes in column 4 follow Index Herbariorum [START_REF] Thiers | μl and[END_REF]. Abbreviation in columns 7 to 10 are as follows: herb. = herbarium material; sil. = silica gel-dried material; LSC = large single copy region; SSC = Small single copy region; IRs = inverted repeat regions. [Information intended for the review process only: The sequences generated for the present study are in the process of being submitted to GenBank.

Once the GenBank accession numbers for all these sequences have been generated, we will update the table].

  hypothesis of linear step-wise migration from Africa to Asia and use ancestral areas reconstruction to investigate biogeography and

  distributions were determined from collection data stored in Solanaceae Source Database (2017, http:/μl and /μl and www.solanaceaesource.org/μl and ); cultivated species (Solanum melongena, S. aethiopicum, S. macrocarpon) were excluded from the biogeographic analysis. Because several species are widespread, with distribution areas that encompass all eastern Africa (S. campylacanthum) or that extend from western Africa to Pakistan (S. incanum), circumscription of biogeographic areas that are meaningful in terms of patterns of species distribution and that are coherent climatically and biogeographically speaking is challenging. We defined seven biogeographic areas based on climatic and biogeographic evidence (see below): (A) Tropical Asian region; (B) Somalian and Middle East region; (C) Sudanian region; (D) Congolian region; (E) Zambesian region; (F) Southern African region; (G) Malagasy region (see Fig. 1A). Four of these (C, D, E and F) directly correspond to those of Linder et al. (

  most likely biogeographic scenario for the Eggplant clade is displayed in Figure 1b. The Eggplant clade originated in area B (Somalian and Middle East region) sometimes during the early Pleistocene. The Kilimanjaro clade maintained this distribution range with the exception of Solanum usambarense, which expanded 12

FIGURE 1 FIGURE 2

 12 FIGURE 1 Phylogeny and biogeography of the Eggplant clade based on whole chloroplast genome sequences. (A) Map showing the seven biogeographic areas used to infer the biogeographic history of the Eggplant clade. Dotted lines indicate instances of complex biome transitions. Arrows illustrate the dispersion events inferred from the biogeographic analysis. (B) Full-plastome dated phylogeny of the Eggplant Clade (consensus of 4 BEAST analyses; 159,227 bp matrix). All nodes have PP ≥ 0.97 and BS ≥ 87% except for the nodes designated with *. Green bars correspond to 95% HDP intervals for the dates of the nodes (Myr). Geographical distribution for each terminal is indicated using the biogeographic regions subdivision. The most probable ancestral area is figured at each node of the Eggplant clade; high level of biogeographic uncertainty is indicated with dotted lines. Pie-charts with relative probabilities of ancestral states at each node are provided in Appendix S4a,b. Hypothesised dispersion (D) and extinction (E) events are figured above the branches of the tree. Within the Eggplant clade, clades are given provisional names based on their distributions. Names of cultivated species are in bold. Pictures from top to bottom and left to right are fruits of the cultivated eggplant ( Solanum melongena), S. incanum, S. linnaeanum, S. insanum (eggplant wild relative) and S. agnewiorum. Photograph credits: (S. melongena, S. insanum) X. Aubriot; (S. incanum, S. agnewiorum) M.S. Vorontsova; (S. linnaeanum) S. Knapp.

ACKNOWLEDGEMENTS

We thank the curators of A, BM, K, LISC and MH for permission to sample DNA from specimens held in their collections; Pr. H. Balslev (Aarhus University), the Bangkok Forest Herbarium (BKF), Pr. P. Chantaranothai and the Botanical team of the Applied Taxonomic Research Center of Khon Kaen University for field assistance in Thailand; Dr. D. Narasimhan (Madras Christian College), Dr. V. Sampath Kumar, Dr. G.V.S Murthy, Dr. P. Lakshminarasimhan, Dr. A. Benniamin, G. Gnanasekaran (Botanical Survey of India), P. Kale and R. Singh (BLAT) for their invaluable assistance during visits of Indian herbaria. We thank M.S. Vorontsova, M.-C. Daunay, M.C. Duarte and J. Prohens for their comments that significantly improved the manuscript; we thank the NHM sequencing facility team, particularly A. Briscoe and K. Hopkins, for assistance with molecular work. This work was funded in part by the Natural History Museum's Natural Resources and Hazards Initiative and by the Natural History Museum's Departmental Investment Fund.

APPENDIX

Summary of species, country, voucher (collector and number), herbarium acronym (codes follow [START_REF] Thiers | μl and[END_REF]) and GenBank accession numbers for taxa used in the preliminary Sanger study, provided in the following order: ITS, waxy and ndhF-rpL32. Dashed lines indicate that the region was not sampled for this accession. Newly generated sequences are indicated with an asterisk following the accession number. [Information intended for the review process only: GenBank numbers for sequences already published are provided. The sequences generated for the present study are in the process of being submitted to GenBank and we have used our laboratory extraction ID (XA plus three digits) to refer to them. Once the GenBank accession numbers for all these sequences have been generated, we will update the table where appropriate]. L.,Tanzania,Vorontsova et al. 156 (BM), XAS232*, -, XAS232*. S. aethiopicum L., Olmstead S-74 (WTU), -, AY996378, -. S. agnewiorum Voronts.,Kenya,Vorontsova et al. 195 (BM)

Solanum aethiopicum


Xin Zhao
email: zhaoxin@hzu.edu.cn

Jing Wang

Yang Jiao

Haijiao Yu

Yao Qin

Quanming Li

Guogang Zhao

Principal Component Clustering Analysis Apply to the Amino Acid Content in Antler Based on Matlab

Keywords: amino, Amino acid, principal component cluster analysis, Cluster analysis method, Matlab

The antler is a very high nutritional value of supplements, but also a very important medicine, antler occupies a very important position in Chinese medicine. Antler is rich in amino acids, which contains more than seven kinds of essential amino acids. In the organic component of antler, amino acids are the topped content of nutrients, and in them, the highest is glycine; amino acids are the basic components of living organism tissue cells, and play a pivotal role for life events. If the body lacks any kind of essential amino acids, it can cause physiological dysfunction, affecting the normal antibody metabolism, leading to disease. In the paper, it accord to the data of amino acids which contain different specifications antler herbs, analyze and compare the relationship of amino acids between sika deer antler and red deer antler using the principal component cluster analysis .The results showed that sika deer antler with red deer antler have the similar medicine effect and different essential amino acids nutrients.

Introduction

Antler is not ossification of sika deer and red deer bucks dense hairs, and it is common health tonic medicine, it mainly has Warming impotence, Yijing blood, gluten bone health and other effects [START_REF]Chinese Pharmacopoeia Commission. Pharmacopoeia of the people's Republic of China[M[END_REF][START_REF] Ni Jiakui | Summary of clinical application of deer products[J][END_REF][START_REF] Chen Daixian | Character identification of 12 kinds of pilose antler medicinal materials[J][END_REF][START_REF] Bernhard | The chemical composition and structure of Deer antlers[END_REF][START_REF] Silave | Amino acid and mineral compsition of antlers and pantocrine[J]. Sbornik nauchnykh rabot nauchno-Issledovatel'skoi laboratot -ii Pantovogo ollenevodstva[END_REF] . Antler has been used as medicine two thousand years, and is recorded in Han Dynasty "Shen Nong's Herbal Classic" [START_REF] Zhang Jinghua | Determination of trace elements in elk antler of sika deer and red deer[J][END_REF][START_REF] Ding Yuhua | Determination of the chemical constituents of deer antler[J]. special wild economic animal and plant research[END_REF][START_REF] Dan | Sun xiaoqiu. Sika deer and different parts of amino acids, total phospholipids, calcium and phosphorus content of research[J][END_REF] . So far, antler medicinal ingredient is still not very clear, in the works of process, quality assessment, identifying the authenticity, there is no scientific chemical composition as the standard indicators, and it restricts further development and utilization of the antler [START_REF] Yulin | Research Progress on chemical composition of pilose antler[END_REF][START_REF] Li Zehong | Comparison of amino acid content in two branched antler and antler three branched[J][END_REF][START_REF] Ma Shengliang | Analysis of several chemical constituents in Xingkai Lake Sika Deer[END_REF] . Therefore, it is in urgent need to carry in-depth study, it should systematically study the antler active ingredients and pharmacological effects using modern analytical methods, this work not only has theoretical significance, but also important practical value, which can found the clear antler quality standars, and provide a scientific basis for the further development and utilization of the antler. This paper analyze and compare the relationship of amino acids between sika deer antler and red deer antler using the principal component cluster analysis.

The Data Extraction and Related Work

In this paper, the data is from the article by Zhang Song [START_REF] Zhang Song | The analysis of amino acid content of different specifications of goods in Antler[J][END_REF] which name is Determination of amino acids in Cornu Cervi Pantotrichum of different specifications, and then extract the content of essential.

Mathematical Model Theory

Principal Component Cluster Analysis

In the principal component cluster analysis, when the variance contribution of first principal component 1

F is high ( Generally more than 85% ), it can be considered that the principal component can reflect the information provided by the original P variables, at this time can be based on the first principal component score to sort the assessment. 1. The erroneous zone of comprehensive score of principal component: In many cases, the variance contribution rate of first principal component is not high enough, it should to thought a number of main components which are named as

r F F F , , , 2 1
 , in order to sort the sample, the common method is that we make the variance contribution rate i a of principal components i F as weight, and construct the composite score about main component:

r r F a F a F a F a H       3 3 2 2 1 1 , (1)
Then, according to the formula, the main component of the sample is calculated and sorted. The comprehensive score calculation looks very reasonable, it seems to increase the information content, that is, to increase the variance contribution rate, in fact, this is just an illusion, by calculating the variance of H we can found, it is possible to get completely opposite conclusions.

          p i i i i r i i i i r i i a a F Var a H Var 1 2 1 2 1 2 ) ( ) (    , (2)
From this derivation, we found that the variance of composite score is smaller than the variance of the first principal component, indicating that the information contained in comprehensive principal component scores than the first principal component contains even less information. 2. Principal component clustering: For the multi-index evaluation system sort problem,if the variance contribution rate of the first principal component is not enough, that is, the raw data that is expressed in the first principal component information is not enough, only by the first principal component score of the samples will have sidedness about sort. At this time, the principal component analysis and cluster analysis can be combined, that is principal component cluster analysis.

Cluster analysis is a method which classify the sample or variable according closeness degree of properties, although the cluster analysis can classify well the data which contain many indexes, but it can not get the evaluation result of the degree of excellence. The thought of principal component cluster analysis is to analysis principal components firstly, and then take a number of the main components of the sample to do cluster analysis, last combine with the first principal component score sort to classify the sample ranked. Thus we get a new comprehensive evaluation method, the specific work is following: selecting the R principal components accoding to the cumulative contribution rate calculate the principal component scores:

r k X a X a X a F p pk k k k , , 2 , 1 , 2 2 1 1        (3)
The selected new data matrix (

r F F F , , , 2 1 
) can be analysed by system clustering. Then using the calculated mean score of the samples determine the order between different classes; Finally, according to the first principal component score of samples determine the order of samples, and give the comprehensive evaluation. The result by Matlab of Seven kinds of essential amino acid content using principal component cluster analysis is shown in Figure 1, the waxes of (5, 9, 19, 28) are clustered into a large class, and the other three waxes are clustered into a class, the result of cluster analysis about sika deer antler and red deer antler is not clear. Sika Deer Velvet Deer Antler wax and Red Deer antler wax tablets can be regarded as the same level, and its amino acid content is similar, indicating that Sika Deer Velvet Deer Antler wax and Red Deer antler have similar medicine effect.

Results and Discussion

with the Principal Component Cluster Analysis Methods for Data Processing

Each essential amino acid content reflects the quality of antler, we divide it into seven indicators, they are (Met) methionine 1 X , (Ile) isoleucine 2 X , (Leu) leucine 3 X , (Phe) phenylalanine 4 X , (Val) valine 5 X , (Lys) lysine 6 X , (Thr) threonine 7 X . For the principal component analysis of essential amino acid about Red Deer, we calculate a linear combination of each main component represented by the variables,

0 3554 . 0 X X X X X X X Z        (4) , - - - -
0 4808 . 0 X X X X X X X Z    (5)
The score for each factor in the amino acid content of Red deer: The result of pedigree cluster analysis about principal component scores using matlab is following :

0 3651 . 0 X X X X X X X Z        (6) , - - - -
0 5572 . 0 X X X X X X X Z    (7)
The score for each factor in the amino acid content of Sika deer: In the figure 3, the number 4 and the number 13 get together. Table 1 shows that Figure 3 make the waxes get together.

Compared Sika Deer antler and Red Deer antler principal component analysis of essential amino acids, from the linear combination of the first principal component shows that when the quality of Red Deer antler and Sika Deer antler are the same, the methionine and isoleucine content of the Sika Deer antler are higher than Red Deer antler, and the rest of essential amino acids are lower than the Red Deer antler antler. Among them, the isoleucine content of sika deer antler is 0.05 times bigger than the red deer antler, the methionine content of sika deer antler is 0.01 times bigger than the red deer antler, the rest of the essential amino acids of sika deer antler are 1% less than red deer antler. This explains the differences about the amino acids content of sika deer antler and red deer antler.

Conclusions

In this paper, the amino acid content of sika deer antler and red deer antler was analyzed using principal component cluster analysis. The principal component cluster analysis explained the similar medicine effect and different active ingredient between the sika deer antler and red deer. From the charts of article, we can see that the different amino acid content can be clustered into a pile, this shows the amino acid content is very close, so red deer antler and sika deer antler have similar medicine effect. By the principal component analysis shows the difference of essential amino acids nutrients between the sika deer antler and red deer antler, and provides a theoretical basis for evaluation of the quality characteristics of the antler.

Fig. 1 .

 1 Fig. 1. Velvet antler human body essential amino acids principal components clustering

,

Figure 2 .

 2 Figure 2. Ma pilose antler human body essential amino acids principal components clustering For the principal component analysis of essential amino acid about Sika Deer, we calculate a linear combination of each main component represented by the variables,

,

Figure 3 .

 3 Figure 3. Plum velvet antler human body essential amino acids principal component cluster

Table 1 .

 1 Red deer antler amino acids score

	Red deer	The principal component score 1	The principal component score 2
	1	4.9780496	-1.0985852
	2	6.2697454	-1.2895272
	3	6.991833	-1.70675
	4	5.9332288	-1.0979714
	5	7.353369	-0.451135
	6	5.4390096	-1.1220619
	7	5.7467428	-1.160236
	8	4.400715	-0.7897087
	9	7.166991	-0.598925
	10	2.7852456	-0.4334046
	11	3.898019	-0.4907397
	12	5.0588716	-1.666578
	13	5.854442	-0.959294
	14	6.830866	-1.49329

Table 2 . Sika deer antler amino acids score

 2 The result of pedigree cluster analysis about principal component scores using matlab is following:

	Sika deer	The principal component score 1	The principal component score 2
	16	5.7469903	-1.5991154
	17	5.2282053	-1.1448584
	18	5.2473394	-1.1999092
	19	7.423939	-1.014214
	20	6.95486	-1.70147
	21	5.6532929	-1.2921322
	22	5.624774	-1.093257
	23	10.072079	-1.679304
	24	6.739466	-1.934784
	25	6.144351	-1.527741
	26	8.511762	-1.862528
	27	5.6281678	-1.4326174
	28	5.854442	-0.068999133
	29	6.830866	-0.070970537
	30	5.9528372	-0.064596331


P M François

B Sandoz

P Decq

S Laporte

Quantification of the stress generated by the endoscopic movement in the brain parenchyma during intra ventricular surgical procedure

Keywords: Brain, hydrocephalus, endoscope, Finite element analysis

Introduction

Endoscopy is increasingly used for intra ventricular surgeries such as hydrocephalus and tumor removal [START_REF] Miwa | Neuroendoscopic biopsy and the treatment of tumor-associated hydrocephalus of the ventricular and paraventricular region in pediatric patients: a nationwide study in Japan[END_REF].

Robotic based surgeries are more and more developed to control the trajectory and the movement of the endoscope and then to enhance the accuracy of surgical procedures and to reduce post-operative issues. The use of computer-aided robot is then well-suited to accurately reach ventricles cavities through the brain parenchyma and to minimize cerebral damage caused by the movement of the endoscope. On a mechanical point of view, moderate and severe traumatic brain injury (TBI) had a 50% risk to locally occur when Von Mises stress respectively exceeds 18 and 38 kPa [START_REF] Willinger | Human head tolerance limits to specific injury mechanisms[END_REF].

The purpose of this study was to assess the Von Mises stress generated in the brain by the endoscopic movement.

Methods

FE model: geometry and material properties

The geometry and the mesh of both the endoscope and the surrounding cerebral layer were designed with the Hypermesh ® software (Altair Engineering, 1985). Endoscope and brain were represented by two cylindrical parts, respectively 5.2 mm and 80 mm of external diameters. Brain model was a hollow at its center along its principal axis, of 5.6 mm of diameter; the endoscope part was placed along its center (Figure 1).

The thickness of the cerebral layer of one patient and the center of rotation of the endoscope were personalized using MRI images.

Incompressible hyper-elastic material constitutive law was implemented for the brain model (Table 1), with a Poisson coefficient of 0,495 [START_REF] Mihai | A comparison of hyperelastic constitutive models applicable to brain and fat tissues[END_REF].

A linear perfectly elastic material was used for the endoscope model (E = 69 GPa and ν = 0.33). An interface TYPE 19 (a node to surface combined with a surface to surface interface) was set to simulate the contact between the endoscope and the brain model. It was assumed that the movement between the endoscope and the brain is nearly frictionless. Thus, a friction coefficient of 0.01 was set. Brain and endoscope models were meshed with H8C Standard 8-nodes elements, with 2*2*2 integration points, no hourglass, and a Lagrangian solid full integration formulation.

The size of the elements in the radial direction was linearly interpolated in order to have smaller elements close to the contact.

Boundary conditions

On the upper face of the model, the nodes with a radial position higher than 20 mm were fully constrained in order to simulate the contact between the brain and the inner surface of the skull.

The node at the center of the upper surface of the endoscope (the center of rotation CR) was constrained in translation in the three directions and free to rotate. The movement was imposed to the node set at the center of the bottom surface of the endoscope.

A real trajectory measured by a robot (Renishaw ® ) during an actual hydrocephalus surgery (3300 s of surgery)

W 1 , 2 , 3 = ∑ p C p m p ( m p 1 + m p 2 + m p 3 -3)
CONTACT pm. François pierre-marc.Francois@ensam.eu

The two thresholds to evaluate the brain lesions after the surgery (18 kPa and 38 kPa) were determined for external impact tests [START_REF] Willinger | Human head tolerance limits to specific injury mechanisms[END_REF]. In a first step, it gives good estimation of brain damage, but in the future, further work should be performed to use a more suitable criterion for the present clinical procedure. Even if this model can be personalized regarding the thickness of the cerebral layer and the position of the center of rotation of the endoscope, it still remains to take into account the variation of mechanical properties between a patient and another. Finally, this model might be evaluated using the visible injury scares on MRI after the surgery.

Conclusions

A finite element model simulating an actual endoscopic movement in the cerebral parenchyma had been designed in order to quantify the Von Mises stress generated into the brain. It could be used downstream hydrocephalus surgeries to evaluate the physiological state of the brain and to better plan the procedure in term of lesion.

Results and discussion

The generated Von Mises stress was mapped on the 3D geometry of the model until the 70th second. Due to numerical convergence problem, the simulation had to be stopped. Results were presented Figure 2.

The maximal Von Mises stress locally reached at this step of the simulation was 23 kPa, which corresponds to about 90% of risk of moderate neurological lesions and less than 10% to get a severe TBI [START_REF] Willinger | Human head tolerance limits to specific injury mechanisms[END_REF].

Several improvements are planned. First, convergence problems have to be solved in order to simulate the 3300s of surgery. The shape of the mesh was designed to avoid too highly distorted elements creation during the simulation. More realistic boundary conditions can be implemented. Due to numerical optimization, the brain is not in contact with the endoscope at the initial time step, whereas it completely surrounds it in real context. The viscosity was neglected because the speed of the endoscope was very low (0.417 mm/s). Nevertheless, a visco hyper-elastic law will be implemented to improve the model. Table 1. Fourth order ogden law for hyper-elastic properties.

Figure 1 .

 1 Figure 1. personalized position of the model: mesh of the endoscope (blue) and the surrounding cerebral layer (red). the center of rotation of the endoscope (Cr) corresponded to the enter point in the head. (adapted from netter 2002).

Figure 2 .

 2 Figure 2. example of Von mises stress (mpa) generated by the endoscope path, slice view, after 7 s of surgery (18 kpa: 50% of risk of moderate tBi).