Comparative analysis of the complete plastid genomes of desert trees Neltuma and Strombocarpa genera

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Carevic, Liesbeth van den Brink, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3422232/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Neltuma alba (Algarrobo blanco), Neltuma chilensis (Algarrobo Chileno) and Strombocarpa strombulifera (Fortuna) are some of the few trees found in small highly fragmented populations, throughout the Atacama Desert, indicating their drought resistance. We found that the complete chloroplast genomes of N. alba and N. chilensis are larger in size compared to species of the Strombocarpa genus. However, the Strombocarpa species presented slightly more GC content than the Neltuma species. Therefore, we assume that Strombocarpa species have been exposed to stronger evolution than Neltuma species. We observed high variation values in the number of cpSSRs (chloroplast simple sequence repeats) and repeated elements among Neltuma and Strombocarpa species. Very low nucleotide diversity values were found in Neltuma , while ten highly variable regions found in Strombocarpa , can likely be used to resolve uncertainties in phylogeny, and for DNA barcoding. Although in general our study supports the phylogeny of other studies, the biggest inconsistency was the nesting of Prosopis cineraria within the Neltuma clade and showed a divergence time of 1.85 Mya. With this study we provide valuable information about isolated populations of tree species that provide important ecosystem services in hostile environments before they disappear, due to an ongoing fragmentation of their populations. Biological sciences/Genetics/Plant genetics Biological sciences/Ecology Biological sciences/Genetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Legumes have a cosmopolitan distribution and are ecologically important in almost all biomes of the world as their fruits are a food source or their root biology nourishes soils 1,2 , even in ecosystems as extreme as the Atacama Desert. Leguminosae ( Fabaceae ) is one of the largest angiosperm family in terms of species numbers and one of the most diverse family and is classified into three subfamilies ( Caesalpinioideae , Mimosoideae , and Papilionoideae ), which have close to 770 genera and over 19,500 species 1,3 . Burkart (1976) 4 taxonomic monograph of the genus Prosopis L. (Mesquite) recognized 44 species, which are distributed across Southwest Asia, Africa, and (predominantly) America 5,6 . Prosopis species from the Strombocarpa Bentham and Algarobia DC. Emend. Burk sections are trees and normally inhabitants of arid and semiarid regions 6,7 . These species inhabit the Atacama Desert in Chile, which extends for over 1000 km between latitudes 19°S and 30°S and is bordered by the Coastal Cordillera to the west and the Andean Cordillera to the east 8 . Surviving in a place as hostile as the Atacama Desert, where radiation is high, and so is water stress 9,10 is already an accomplishment, but these trees also provide local people with important resources such as fruits, juice, futter and wood 7 . Three species from the formerly known Strombocarpa section ( Prosopis strombulifera (Argentine screwbean) and the endemics Prosopis burkartii and Prosopis tamarugo (Tamarugo)) 7,11 and individuals belonging to different species from the formerly know Algarobia section ( Prosopis chilensis , Prosopis flexuosa , and Prosopis alba ) can be found in the Atacama Desert 6 . The scientific names of these species and the concept of Prosopis established by Bentham (1875) 12 and Burkart (1976) 4 has only currently been disintegrated, because Prosopis was found to be polyphyletic based on both chloroplast (cpDNA) and nuclear DNA (nDNA) 5,13,14 . As a consequence, the old Prosopis cluster was divided in six genera – Anonychium , Prosopis , Neltuma , Strombocarpa , Xerocladia and Indopiptadenia . The species of the above mentioned Algarobia section were renamed as Neltuma alba , Neltuma chilensis , Neltuma flexuosa , and the species of the above mentioned Strombocarpa section as Strombocarpa tamarugo , Strombocarpa burkartii and Strombocarpa strombulifera 5 . This division was based on short DNA sequences, as is common practice in taxonomy. However, compared to short DNA sequences, a complete chloroplast genome of approximately 160,000 bp can offer more information about the phylogenetic relationships and gives a full overview of the specific genes and the structure of its genome. Chloroplast and mitochondria are the powerhouse of a plant cell, responsible for the majority of the energy produced, through photosynthesis and photorespiration, respectively; these organelles have originated endosymbiotically from eubacterial cells, where most genes were either lost or transferred to the host nucleus 15 . The chloroplast genome is a valuable taxonomic resource with rich genetic information 16 , as it is highly conserved and maternally inherited 17 . In angiosperms, most chloroplast genomes are composed of circular DNA molecules and have a quadripartite organization consisting of two copies of inverted repeats (IRs), which divide the rest of chloroplast genome into a large single copy (LSC) region and small single copy (SSC) region 17 . There are approximately 110–130 genes included in chloroplast DNA, consisting of rRNA-coding genes, protein-coding genes, and tRNA-coding genes 16 . Because the chloroplast genome can provide valuable information to support the conservation of threatened trees 18 , gaining insights in chloroplast DNA of the legume tree populations from Atacama Desert could help their conservation. Chloroplast genome sequences are commonly used in plant phylogeny, phylogeographic and genome evolution studies 16,17 . Lately, the use of complete chloroplast genome as a “super-barcoding” method has become an excellent approach allowing for the increase of the phylogenetic resolution at lower taxonomic levels in plants 19,20 . However, in the Atacama Desert only a few plastomes of the native and endemic herbaceous plants 21,22 , shrubs 23,24 and leguminous trees 25,26 have been characterized so far. Unfortunately, several species of trees of the genera Neltuma and Strombocarpa are in vulnerable and endangered conservation status in Chile, e.g. Neltuma chilensis and Strombocarpa tamarugo . Neltuma chilensis and Neltuma alba are restricted to southern Peru, northern and central Chile, southwestern Bolivia and northwestern, western and central Argentina 27–29 . The not threatened Strombocarpa strombulifera is widely distributed from the Arizona desert (U.S.A.) to Patagonia (Argentina) 30 . However, in the Atacama Desert, Neltuma alba, Neltuma chilensis , as well as Strombocarpa strombulifera populations, are fragmented and restricted to oases or valley (forming populations of only a few individuals), and geographically isolated from each other by large areas of land 6,7,31 . Although Neltuma alba and Strombocarpa strombulifera are in the conservation status of “Least Concern” it is urgently necessary to identify the plastomes of their genera now, before their more endangered cousins go extinct. Until now, there is no complete chloroplast genome available for any N. alba , N. chilensis and S. strombulifera , but they are needed to confirm phylogenetic relationships between them, and with closely related species. Therefore, in this study we provide and analyze the complete chloroplast genomes of N. alba , N. chilensis and S. strombulifera , in terms of structure, gene composition, divergence time and phylogeny. Methods Plant material and DNA isolation Fresh leaves of Neltuma alba (Griseb.) C.E. Hughes & G.P. Lewis, Neltuma chilensis (Molina) C.E. Hughes & G.P. Lewis and Strombocarpa strombulifera (Lam.) A. Gray were collected in Copiapó (27°21'39.3"S 70°20'33.8"W), Chacabuco (33°05'24.9"S 70°39'07.3"W) and Pampa del Tamarugal (20°27'59.9"S 69°33'23.5"W) in Chile, respectively. Identification of samples was done according to the taxonomic criteria described by Burkart (1976) 4 . Additionally, the samples were verified by the forester Boris Burgos of the Corporación Nacional Forestal (CONAF) from the Atacama Region. The specimens were deposited in the Departamento de Silvicultura y Conservación de la Naturaleza herbarium of Universidad de Chile (under the names that were correct at the time of deposition: Prosopis alba , EIF13329; Prosopis chilensis , EIF13328; and Prosopis strombulifera , EIF13350). DNA was isolated from the leaves using the modified cetyl-trimethylammonium bromide (CTAB) protocol 7 . The DNA was quantified with a Qubit™ 3.0 fluorometer and a Qubit™ dsDNA HS Assay Kit, according to the protocol supplied by the manufacturer. DNA integrity was verified with an Agilent 2100 Bioanalyzer prior to sequencing. Genome sequencing, assembling and annotation Sequencing libraries were generated by a TruSeq Nano DNA LT Kit (Illumina, San Diego, CA). The final libraries were run on an Agilent 2100 Bioanalyzer to verify the fragment size distribution and concentration. Sequencing was performed with an Illumina sequencing platform, at Genoma Mayor (Universidad Mayor, Chile). Paired-end sequences of 150 bp were generated for each read (R1 and R2). The filtered reads were assembled using SPAdes 4 software version 3.13.0 32 , using three k-mers parameters: - k 33, 55 and 77. The plastid was annotated with PGA software 33 and CPGAVAS2 34 , after which it was manually corrected when needed. The graphical map of the plastid was generated by Organellar Genome DRAW (OGDRAW) 35 , and the complete nucleotide sequences were deposited in the NCBI GenBank database (OP672364, OP672365 and OP672366, under the names Prosopis alba , Prosopis chilensis and Prosopis strombulifera , respectively). Genome comparison, repeat and phylogenetic analysis The plastid structures (LSC/IR, IR/SSC) of N. alba , N. chilensis and S. strombulifera and of five closely related species, i.e. Neltuma juliflora (Sw.) Raf., Strombocarpa tamarugo (Phil.) C.E. Hughes & G.P. Lewis, Prosopis farcta (Banks & Sol.) J.F. Macbr. and Prosopis cineraria (L.) Druce, of the Mimoseae tribe were visualized and compared using IRScope 36 . We used sequence data of whole plastome (obained from GenBank) of species from the genera Neltuma , Strombocarpa and Prosopis for the identification of the simple sequence repeats (SSRs). These SSRs were identified using MISA software 37 with the following search parameters: ten for mononucleotide, eight for dinucleotide, four for trinucleotide and tetranucleotide, and three for pentanucleotide and hexanucleotide. To identify the tandem repeats (forward, palindromic, reverse, and complement) of these species we used REPuter 38 with the following parameters: hamming distance equal to 3, minimal repeat size set to 30 bp, and maximum computed repeats set to 300 bp. A sliding window analysis (window length: 600 pb, step size: 200 bp) was performed to assess the variability (Pi) between plastid of N. alba and N. chilensis , S. tamarugo and S. strombulifera , the genera Strombocarpa and Neltuma , and the tribe Mimoseae with DnaSP v. 5 software 39 . The complete plastid genome sequence of N. alba (OP672364), N. chilensis (OP672365), S. strombulifera (OP672366), S. tamarugo (MW582314), N. glandulosa (NC_026683), N. juliflora (MN104889), P. farcta (MZ073639), P. cineraria (MN104890) and Leucaena trichandra (NC_028733) as outgroup species were used in the phylogenetic analysis. Eighty protein-coding genes (PCG) sequences were aligned separately using MAFFT v7 40 and any gaps in the alignment were trimmed using TrimAL v1.4 41 . Afterwards, the sequences were concatenated with Mesquite 3.81 software 42 . The analyses of the plastid genomes´ 80 PCG sequences were conducted using the maximum likelihood (ML) method. The complete plastid genome sequence of the nine species were analyzed using the Bayesian inference (BI) methods. The best-fitting nucleotide substitution model of sequence evolution, model TVM + I + G, was determined based on the Akaike Information Criterion (AIC) using the MrModeltest v2.3 43 . The ML analyses were performed using RAxML-HPC BlackBox v.8.1.24 44 with 1,000 bootstrap replicates. The BI analysis was conducted using MrBayes v.3.2 45 with the CIPRES Science Gateway v3.3 46 . The Markov Chain Monte Carlo (MCMC) algorithm was calculated for 5,000,000 generations, and the sampling tree for every 1,000 generations. The first 25% of generations were discarded as burn-in. In the analysis, bootstrap support (BS) values were estimated in the ML, and the reliability of clades in the Bayesian analysis was evaluated by means of posterior probability (PP). The trees were visualized with FigTree 47 . Divergence time estimate To estimate the divergence time of the species we used BEAST v2.6.0 48 based on the complete plastid genome sequence of eight species of the Prosopis , Neltuma and Strombocarpa genera, and Leucaena trichandra as an outgroup species. All genome sequences were aligned with MAFFT, and then the file was imported to BEAUTi interface to generate a file for BEAST, after applying HKY + Γ substitution model, “Empirical” frequency, strict molecular clock model and “Yule” model speciation. Divergence times were estimated combining two calibration points. The TimeTree tool ( http://www.timetree.org 49 ) was used to fix the node age of Leucaena trichandra and the Prosopis genus, which is known to have diverged 35 Mya (33.2–40.3 Mya) 13,50–52 . We considered a second calibration as well, fixing the node age of Mezquite clade ( N. alba , N. chilensis , N. juliflora , among others) at 3.65 Mya (3.31–3.99 Mya) after the study of 13 . The Markov Chain Monte Carlo (MCMC) was run for 6 million generations, sampling every 1,000 generations. We ran the program using the input file XML generated by BEAUTi in BEAST. Final log files were checked in Tracer 1.7.1 53 . We used the TreeAnnotator program 48 with a 10% burn-in. Figtree software was used to visualize phylogenetic trees, using the extent of the 95% highest posterior density (HPD) intervals for each divergence time. Results The chloroplast genome lengths of N. alba , N. chilensis and S. strombulifera comprise 162,980 bp, 163,047 bp and 160,569 bp respectively and its structure contains a typical quadripartite structure with two inverted repeat regions (IR; 25,919 bp, 25,919 bp and 26,026 bp respectively) separated by a large single copy region (LSC; 92,300 bp, 92,356 bp and 89,569 bp respectively) and a small single copy region (SSC; 18,842 bp, 18,853 bp and 18,623 bp respectively) (Figure 1, Table 1). Its length and structure are similar to those of the other species of the Neltuma , Strombocarpa and Prosopis genera, which vary in the IRs between 25,919 bp and 25,935 bp, in the LSC between 91,062 bp and 92,937 bp and in the SSC between 18,643 bp and 18,880 bp (Table 1). The cp genomes of the Strombocarpa species ( S. strombulifera 160,569 bp; and S. tamarugo 161,575) are smaller (~2,000 bp) than those of the Neltuma species ( N. alba 162,980; and N. chilensis 163,047) (Table 1). The GC content in the chloroplast of N. alba and N. chilensis (35.9%) is slightly less than in S. strombulifera (36.2%), but the overall GC content was similar to other species of the Neltuma , Strombocarpa and Prosopis genera (Table 1). In total, 128 genes were found in cp genomes of N. alba , N. chilensis and S. strombulifera , which included 83 coding genes, 8 rRNA genes, and 37 tRNA genes (Table 1). Of these genes, six coding genes (ndhB, rpl2 , rpl23 , rps12 , rps7 and ycf2 ), four rRNA genes and seven tRNA genes located in the IR regions contained duplicated genes (Table 2). Eighteen genes had introns, including nine PCGs ( atpF , ndhA , ndhB , petB , petD , rpl2 , rpl16 , rpoC1 and rps16 ), except for S. strombulifera , which had ten PCGs, additionally including ycf2 to the ones mentioned before. Six tRNAs ( trnA UGC , trnG GCC , trnI GAU , trnK UUU , trnL UAA and trnV UAC ) contained one intron, while three PCGs ( ycf3 , rps12 and clpP ) had two introns (Table 2). We compared the simple sequence repeats (SSRs) from the cp genomes of the species of the Neltuma , Strombocarpa and Prosopis genera. The maximum number was 100 SSRs in N. juliflora , 95 SSRs in P. cineraria , 92 SSRs in N. glandulosa and S. strombulifera , 90 SSRs in N. chilensis , 88 SSRs in N. alba and, whereas S. tamarugo and P. farcta only had 70 SSRs (Figure 2A). Mononucleotide repeats (A/T) were the most common repeats and the highest in number, ranging from 61 ( S. tamarugo ) to 80 ( S. strombulifera ); mononucleotide repeats C/G were found in all species except in S. tamarugo and S. strombulifera (Figure 2B). The number of dinucleotide SSRs (AT/AT) was similar in all species except in S. tamarugo and S. strombulifera in which no dinucleotide SSRs were found. In all species, 10-12 trinucleotide repeats AAT/ATT were found, but in S. tamarugo and S. strombulifera only 4 and 5 respectively, were present. Moreover, only S. tamarugo had trinucleotide repeats AAG/CTT (Figure 2B). In general, four pentanucleotide SSRs type (AAAAT/ATTTT, AAATT/AATTT, AATAT/ATATT and AATGG/ATTCC) were found in all species, but only one pentanucleotide SSR was present in P. farcta (AACTT/AAGTT), S. strombulifera and S. tamarugo (AATAG/ATTCT), and S. strombulifera (AATTC/AATTG) (Figure 2B). Only two hexanucleotide SSRs, AATATT/AATATT and AAATAG/ATTTCT, were observed in S. strombulifera and P. farcta , respectively (Figure 2B). The amounts of repeats varied between 57 and 80 in the cp genomes of S. tamarugo (88), P. farcta (74), N. juliflora (73), P. cineraria (72), N. glandulosa (67), N. alba (60), N. chilensis (60) and S. strombulifera (57) (Figure 3A). The number of complement repeat in S. tamarugo was higher (5 repeats) than in the rest of the species (Figure 3A). The total number of palindromic repeats was less in Strombocarpa species ( S. strombulifera , 22; and S. tamarugo , 24) than in species of the Neltuma ( N. alba , 28; N. chilensis , 27; N. glandulosa , 29; and N. juliflora , 30) and Prosopis ( P. cineraria , 33; P. farcta , 27) genera (Figure 3B). On the other hand, the total number of forward repeats was less in the cp genome of N. alba and N. chilensis (25 each), than in the rest of the species, where they varied between 30 to 40 (Figure 3C). Palindromic and forward repeats with lengths of 30-39 bp were the most common and abundant repeats in the species of the Neltuma , Strombocarpa and Prosopis genera (Figure 3B-C). The number of reverse repeat (range of 30-39 bp) in S. tamarugo was higher (with 25 repeats) than in the rest of the species (Figure 3D). The expansion and contraction of the IR and SC regions contributes to the differentiation in chloroplast genome size in some genera and families. For that reason, we compared of SSC, LSC, IRA, and IRB border regions of the species of the Neltuma , Strombocarpa and Prosopis genera. In all species, rps19 genes were located in the junction between LSC and IRb region (JLB), of which 176 to 188 bp were located at the LSC region and 91 to 103 bp located at the IRb region (Figure 4). In all species, the rpl2 gene was entirely located in the IR regions (Figure 4). The ndhF gene of Neltuma and Prosopis species were located in the SSC region, 137-156 bp away from the IRb-SSC border, while in the two Strombocarpa species this gene was located approx. 67 bp away from the IRb-SSC border. At the SSC-IRA border, the ycf1 gene extended into the SSC region, at varying lengths ranging from 4,760 bp in S. tamarugo to 4,794 bp in N. juliflora and P. cineraria , however, in N. alba and S. strombulifera the gene was entirely located in the SSC region, 963-973 bp away from the IRA-SSC border. In general, the truncated copy of ycf1 was located in the IRB region (except in N. alba and S. strombulifera ), while one end extended into the SSC region for 17 bp only in N. juliflora and P. cineraria . The distance between rps19 and the LSC/IRA border was only 2 bp in N. glandulosa and N. juliflora . In most species, the trnH gene was located in the LSC region, 2-16 bp away from the IRA-LSC border, but in N. juliflora and P. cineraria it was much more distant. In general, the structure of the cp genomes of Neltuma , Strombocarpa and Prosopis were similar in arrangement (Figure 4). In the genus Neltuma , the mean estimated value for nucleotide diversity (Pi) between N. alba and N. chilensis was Pi= 0,00037 (ranging from 0 to 0.0416); and the regions with the highest values of Pi were rps16-trnQ UUG (Pi = 0.005), accD-psaI (Pi = 0.0416) and ycf2- trnI CAU (Pi = 0.005) (Figure 5A). In the genus Strombocarpa , the mean estimated Pi value between S. strombulifera and S. tamarugo was 0,00522 (ranging from 0 to 0.0433) (Figure 5A); and the regions with the highest values of Pi were matK-rps16 (Pi = 0.0433), trnK-psbI (Pi = 0.0366), trnS GCU -trnG GCC (Pi = 0.0233), petN-psbM (Pi = 0.0216), psaB-psaA (Pi = 0.0216), rbcL-accD (Pi = 0.0350), psbE-petG (Pi = 0.0383), rpoA-rpl36 (Pi = 0.0250), rps7-ndhB (Pi = 0.0200) and ycf2 (Pi = 0.0416) (Figure 5B). Among Neltuma and Strombocarpa species, the mean estimated Pi value was 0,00759 (ranging from 0 to 0.0627); and the regions with the highest values of Pi were matK-rps16 (Pi = 0.0563), trnQ UUG -trnS GCU (Pi = 0.0472), trnS GCU -atpA (Pi = 0.0627), petN-trnD GUC (Pi = 0.0288), psaB-psaA (Pi = 0.0444), atpB-rbcL (Pi = 0.0541), psbL-petL (Pi = 0.0361), rps18-rps12 (Pi = 0.0341), ndhE-rrn23S (Pi = 0.0277), ndhB (Pi = 0.0255) and ycf2 (Pi = 0.0347) (Figure 5C). For all Mimoseae species used in this study, the mean estimated Pi value was 0,01236 (ranging from 0 to 0.1171); and ~31 regions with Pi values greater than 0.02 were observed (Figure 5D). The results of the ML and IB trees had similar topologies when we compared 80 protein-coding genes of the plastid genomes of the nine Mimosacea species (Figure 6). The ML phylogenetic analysis revealed four clades, one joined S. tamarugo and S. strombulifera (BP=100), the second clade contained N. glandulosa , N. juliflora , P. cineraria , N. chilensis and N. alba (BP=100), the third clade was formed by P. farcta , and the fourth clade was formed by the outgroup L. trichandra (Figure 6A). The BI phylogenetic analysis with the nine Mimosaceae species showed high support (PP=1.00) and their topology was identical to ML analysis (Figure 6B). The second clade showed three subclades: one containing N. glandulosa which was separated from the rest of species with high support (BP=100; PP=1.00), while the second contained N. juliflora and P. cineraria (ML, BP=100; BI, PP=1.00), and the third contained N. chilensis and N. alba with high support (BP=100; PP=1.00) (Figure 6). Divergence time for the Neltuma , Strombocarpa and Prosopis species based on the sequence of the chloroplast genomes is shown in Figure 7, and suggests that Netuma , Strombocarpa and Prosopis species shared a common ancestor around 43.11 Mya (95% highest posterior density (HPD): 37.72-48.07 Mya) in the Eocene. The age estimate for the split between Prosopis in the Old World and the new world species was 33.52 Mya (95% HPD: 29.48-37.70 Mya) in the early Oligocene. Strombocarpa and Neltuma genera diverged in the New World around 22.32 Mya (95% HPD: 19.55-25.08 Mya) in the early Miocene (Figure 7). Within of the genus Strombocarpa , S. strombulifera and S. tamarugo diverged around 8.70 Mya (95% HPD: 7.52-9.89 Mya) in the late Miocene (Figure 7). While the species of the genus Neltuma diverged into two clades much later, around 2.96 Mya (95% HPD: 2.62-3.33 Mya) in the Pliocene (Figure 7). One clade containing N. alba , N. chilensis and N. glandulosa diverged 1.12 Mya (95% HPD: 0.85-1.39 Mya) when a subclade formed between N. alba and N. chilensis that diverged 0.66 Mya (95% HPD: 0.49-0.84 Mya). The other clade containing P. cineraria and N. juliflora diverged 1.85 Mya (95% HPD: 1.56-2.12 Mya) (Figure 7). Table 1. General features of the Neltuma , Strombocarpa and Prosopis plastid genomes. Species Accession Size (bp) GC (%) LSC (bp) SSC (bp) IR (bp) N° Genes Protein-coding genes tRNA genes rRNA genes N. alba OP672364 162,980 35.9 92,300 18,842 25,919 128 83 37 8 N. chilensis OP672365 163,047 35.9 92,356 18,853 25,919 128 83 37 8 N. glandulosa NC_026683 163,040 35.9 92,322 18,880 25,919 128 83 37 8 N. juliflora * MN104889 163,237 35.9 92,495 18,880 25,931 132 85 39 8 S. strombulifera OP672366 160,569 36.2 89,569 18,623 26,026 128 83 37 8 S. tamarugo MW582314 161,575 36.0 91,062 18,643 25,935 127 82 37 8 P. cineraria * MN104890 163,677 35.9 92,937 18,878 25,931 131 85 38 8 P. farcta MZ073639 162,900 35.9 92,156 18,880 25,932 127 82 37 8 (*) values of features of the species are described by Asaf et al (2020) Table 2 . Gene composition of the plastid genome of N. alba, N. chilensis and S. strombulifera . Category of genes Group of genes Names of genes N° Photosynthesis Photosystem I psaA, psaB, psaC, psaI, psaJ 5 Photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ 15 ATP synthase atpA, atpB, atpE, atpF b , atpH, atpI 6 NADH-dehydrogenase ndhA b , ndhB ab , ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK 12 cytochrome b/f complex petA, petB b , petD b , petG, petL, petN 6 Large subunit RUBISCO rbcL 1 Protein synthesis and DNA replication Transfer RNAs trnA-UGC ab , trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-UCC, trnG-GCC b , trnH-GUG, trnI-GAU ab , trnI-CAU a , trnK-UUU b , trnL-UAA b , trnL-CAA a , trnL-UAG, trnM-CAU, trnN-GUU a , trnP-UGG, trnQ-UUG, trnR-ACG a , trnR-UCU, trnS-GGA, trnS-UGA, trnS-GCU, trnT-GGU, trnT-UGU, trnV-UAC b , trnV-GAC a , trnW-CCA, trnY-GUA 37 Ribosomal RNAs rrn16S a , rrn23S a , rrn4.5S a , rrn5S a 8 Ribosomal Protein large-subunit rpl14, rpl2 ab , rpl16 b , rpl20, rpl23 a , rpl32, rpl33, rpl36 10 DNA dependent RNA polymerase rpoA, rpoB, rpoC1 b , rpoC2 4 Ribosomal Protein Small-subunit rps11, rps12 ac , rps14, rps15, rps16 b , rps18, rps19, rps2, rps3, rps4, rps7 a , rps8 14 Other functions Subunit of Acetyl-CoA-carboxylase accD 1 c-type cytochrome synthesis gene ccsA 1 Envelop membrane protein cemA 1 Protease clpP c 1 Maturase matK 1 Initiation Factor infA 0 Unknown function Conserved open reading frames ycf1, ycf2 a , ycf3 c , ycf4 5 Total 128 a Duplicated genes; b Genes containing introns; c Genes containing two introns Discussion Genomic research with NGS technology has developed rapidly, allowing efficient sequencing of complete plastid genomes 54 . Molecular differences in the complete chloroplast genome between species and individuals provide a good means of comparison 55 . The cp genome offers several advantages over the nuclear genome, such as unique haploid structure, structural conservation, maternal inheritance, and moderate rate of evolution 55,56 . In our comparative study of the plastid genomes of N. alba , N. chilensis , and S. strombulifera analyzing gene content, structure, divergence time, and phylogeny we found that the complete chloroplast genomes of N. alba and N. chilensis are conserved in size compared to species of the Strombocarpa genus. The chloroplast of N. alba and N. chilensis showed similar values for genome size and the number of genes compared to Neltuma juliflora and Neltuma glandulosa described by Asaf 57 , ~ 163.000 bp for both, while the number of genes varied between 131 and 128. The number of genes was similar between the Neltuma and Strombocarpa genera, although S. tamarugo lost the gene psbL (remaining with 127 genes only) 25 . The absence of the psbL gene has been observed in some other eudicots, magnoliids, and monocots as well 58 . The genome sizes of S. strombulifera (160,569 bp) and S. tamarugo (161,575 bp) were smaller compared to the Neltuma species (~ 163.000 bp) 25 . However, the Strombocarpa species presented slightly more GC content (36.0%-36.2%) compared to the Neltuma species (35.9%). These GC values fall within the limit of variation registered in others studies 25,57 . Furthermore, a study about several orchid species, showed that the species with a smallest chloroplast size ( Pholidota cantonensis , 158,786 bp), had a highest GC content (37.47%) 59 , similar to our observations. The chloroplast genome tends to reduce its size during evolution 60 , and gene length might be affected by selection during the evolution of spermatophytes 61 . The variations in chloroplast genome size among closely related species can be attributed to IRs, LSC, SSC, intergenic regions, and gene numbers 61 . In this study, very little variation in IRs and intergenic regions was observed between N. alba and N. chilensis , resulting in very few differences in genome size, while there is a large variation in these regions in the genomes of S. strombulifera and S. tamarugo . Therefore, we assume that Strombocarpa species have been exposed to stronger evolution than Neltuma species. A total of 70 to 100 chloroplast simple sequence repeats (cpSSRs) were founded in the cp genomes of the species of the Neltuma , Strombocarpa and Prosopis genera. Our results showed high variation values in the number of cpSSRs among Neltuma and Strombocarpa species, being the highest for N. juliflora (100) and the lowest for S. tamarugo ( 70 ). The most abundant cpSSR motif types in Neltuma , Strombocarpa and Prosopis were mono-nucleotides, which is the most abundant repeat type in angiosperms cp genomes 62 . Only Strombocarpa species did not show mononucleotide C/G motifs, nor dinucleotide motifs and additionally, they had a lower number of trinucleotide AAT/ATT motifs. However, the Strombocarpa species were the only species that presented the pentanucleotide AATAG/ATTCT motifs. It has been shown in Cyatheaceae , that the characteristics of cpSSRs can provide useful phylogenetic information at the genus level, such as phylogenetic relationships, but also about the number, relative abundance, motif type and relative density of cpSSRs 63 . In a similar way, our results demonstrate that the cpSSRs among Neltuma and Strombocarpa , both in number and cpSSR motifs, are likely genus specific. Repeat sequences are considered to play an important role in genome recombination, rearrangements and contain fundamental phylogenetic information 64,65 . We found differences in the repeated elements of the cp genome between Neltuma and Strombocarpa species. The highest total number of repeat elements (palindrome, forward, reverse and complement) was found in S. tamarugo (88) and the lowest in S. strombulifera ( 57 ). In general, the total number of palindromic repeats was less in Strombocarpa species than in Neltuma species. However, the total number of forward repeats was less in N. alba and N. chilensis than in the Strombocarpa species. On the other hand, the number of complement and reverse (range of 30–39 bp) repeats in S. tamarugo was higher than in the Neltuma species. In the majority of the species in this study, the most abundant repeat elements detected were, in order, forward, palindromic and reverse. This corresponds to other studies about cp genomes of mimosoid species 66,67 , although S. tamarugo is an exception in terms of reverse and complements repeats numbers. Throughout of the evolution of plastid genomes, structural rearrangements occur, for example in the IRs, which are frequently subject to expansion, contraction or even complete loss 68 . An increased length of IR-SSC boundaries plays an important role in mimosoid plastome size variation 69 . For example, eight mimosoid plastomes of the tribe Acacia and Inga exhibited an unusual 13 kb IR-SSC boundary shift into the SSC region 67,69 , and the size of these plastomes was found significantly affected by a IR-SC boundary shift, as well as by repeat content 67 . We observed a slight IR expansion into SSC in S. strombulifera (26.026 bp) and S. tamarugo (25.935 bp) in comparison to the Netuma species. Therefore, the SSC regions of the Strombocarpa species showed contraction, and were the shortest SSC regions compared with those of the Neltuma and Prosopis genera. Asaf et al 57 did not detect IR expansion in Neltuma and Prosopis species, however, they detected a slight expansion in the outgroup species of the genus Adenanthera (with a length of 26,028 bp), similar to what we found the in Strombocarpa species. The study of Asaf Asaf et al 57 did not, however, include Strombocarpa species to compare to the Neltuma and Prosopis species. Similar to Asaf 57 , we found a partially duplicated rps19 gene at the beginnings and ends of the IR regions in N. alba , N. chilensis , S. strombulifera and S. tamarugo (including 91 bp in IR). In of most Mimosoideae species, the rps19 is located in the LSC/IRB junction (JLB), with 98–109 bp of the 5′ end of this gene into the IR region 67 . The ndhF gene was located closer to the IRB-SSC border (JSB) in Strombocarpa species (up to 67 bp) than in Neltuma and Prosopis species (137 to 156 bp). Likewise, the ndhF gene in the species of the genera Adenanthera , Parkia , Piptadenia , Leucaena and Dichrostachys ( Mimosoideae ) was found entirely within the SSC region (ranging 11 to 150 away from the JSB junction), however, in species of the tribe Acacia and Inga ( Mimosoideae ) it was found within the JSB junction, resulting in the duplication of this gene 67 . Several models concerning the expansion and contraction of IR regions have been proposed to explain the possible mechanisms that result in shifts in the IR-LSC junctions 70 . In our case, we detected that Strombocarpa species had a larger contraction of the LSC region then Neltuma and Prosopis species. The structural differences presented among the plastomes of the Neltuma and Strombocarpa species reinforce the idea and necessity to disintegrate the Prosopis cluster, as proposed by Hughes et al 5 . However, for the new genera it would have been recommendable to have kept the names of the sections ( Algarobia and Strombocarpa , as proposed by Burkart 4 for the new genera. The nucleotide diversity (Pi) analysis of Neltuma and Strombocarpa plastomes showed more variations in the LSC and SSC regions than the IR regions. In addition, strong differences of nucleotide diversity value were found between Neltuma and Strombocarpa species. The Pi values between Neltuma species were so low that we found only three variable regions ( rps16-trnQ UUG , accD-psaI and ycf2- trnI CAU ), whereas in Strombocarpa species we found ten regions with high Pi values ( matK-rps16 , trnK-psbI , trnS GCU - trnG GCC , petN-psbM , psaB-psaA , rbcL-accD , psbE-petG , rpoA-rpl36 , rps7-ndhB and ycf2 ). We believe that these ten highly variable regions found in Strombocarpa species, can be of use to resolve uncertainties in phylogenetic analysis of the genus, as well as for DNA barcoding. However, as a very low number of variable regions was found in the species of the Neltuma genus, it will be necessary for further studies to include a sufficient number of samples in order to identify the best regions for identification within the genus Neltuma . The phylogenetic results (ML and BI) based on 80 protein-coding genes of the plastid genome of nine Mimosoideae species showed that S. strombulifera formed a strongly supported group with S. tamarugo (BP = 100; PP = 1.00), and the Neltuma group appeared as paraphyletic because P. cineraria was part of a well-supported clade (BP = 62; PP = 1.00) with N. juliflora , N. alba and N. chilensis . P. farcta , however appeared as sister group of Neltuma and Strombocarpa clade, as expected. Within the Neltuma clade, N. alba formed a highly supported clade with N. chilensis (BP = 100; PP = 1.00), and so did N. juliflora with P. cineraria (BP = 100; PP = 1.00), whereas N. glandulosa appeared as a strongly supported sister group to both (BP = 100; PP = 1.00). With the exception of P. cineraria (further discussed in the next paragraph), the Neltuma group was monophyletic with Strombocarpa group as its sister clade. Although S. strombulifera and S. tamarugo formed a well-supported group, these two species showed important differences in genome size, number of genes and nucleotide diversity with high degree of variation. These genetic differences in the chloroplast correspond to the findings of Burkart 4 who separated S. tamarugo and S. strombulifera into the Cavernicarpae and Strombocarpa e series, respectively. The same was observed by Catalano et al 13 through a three-marker analysis ( trnS-psbC , G3pdh , NIA ), who found two well supported groups, one of them corresponding to the Cavenicarpae series (including Prosopis ferox and P. tamarugo ) and the other formed by North American species of the Strombocarpa e series (including Prosopis pubescens and Prosopis palmeri ). Undoubtedly, the biggest inconsistency observed in our phylogenetic analysis was the nesting of P. cineraria within the Neltuma clade. According to the results of Asaf et al 57 , P. cineraria forms a group with high support with N. juliflora . It is interesting and unexpected that P. cineraria did not form a group with P. farcta , both of them being Old World species, but nested with N. juliflora , N. glandulosa , N. alba and N. chilensis , which are New World species. However, according to the phylogenetic analysis performed by Catalano et al 13 , there are more distant relationships among species from the Old World sections and closer relationships among species of the American sections ( Strombocarpa , Algarobia , and Monilicarpa sections). Prosopis cineraria is one of the most common trees of the Indian desert, Arabian Peninsula and, in general, is abundant throughout the middle east 57,71 , whereas N. juliflora is native to the Caribbean, Central and northern South America 72 . However, Neltuma juliflora was introduced to Ethiopia and the Middle East around 1970 and over the years this species has spread outside the plantation areas, adversely affecting natural habitats and rangelands 73 . This invasive plant is characterized by vigorous growth which helps it to outcompete indigenous plant species 74 . Neltuma juliflora seeds survive in livestock and warthogs’ droppings, which serve as a vehicle for the plant to reach distant areas and to expand their distribution throughout the region 74,75 . We hypothesize that N. juliflora might have crossed with some individuals of P. cineraria in a natural way, giving offspring to a hybrid with a phenotype resembling P. cineraria but, when N. juliflora acted as the maternal part, with the cp genome of N. juliflora . This could be a logic explanation for the nesting of P. cineraria within the Neltuma clade, if the samples used by Asaf et al. 57 were obtained from a P. cineraria resembling hybrid. Estimate of divergence time in plant groups have been important in order to understand their phylogeographic history and evolutionary biology 76 . Our molecular dating analysis suggests that Leucaena trichandra as root species diverged in the Middle Eocene (mean = 43.11 Mya; 95% HPD = 37.72–48.07 Mya). Later, P. farcta diverged in the Early Oligocene (mean = 33.52 Mya; 95% HPD = 29.48–37.70 Mya), while P. cineraria diverged together with the Neltuma species in the Pleistocene. Sudalaimuthuasari et al 71 , using whole genome sequencing with 76,554 genes, estimated that P. cineraria and P. alba diverged ~ 23Mya. Undoubtedly, this divergence time is closer to what would be expected for species of the genus Prosopis that belongs to the Old World, but not for P. cineraria , whose complete chloroplast genome data show a divergence time of 1.85 Mya (95% HPD: 1.56–2.12 Mya), strengthening our suspicion of this sample being a natural hybrid. Although a previous study indicates that the divergence between Strombocarpa and Neltuma genera occurred in the Oligocene 13 , our results show that these genera diverged in the early Miocene (mean = 22.32 Mya; 95% HPD = 19.55–25.08 Mya). The molecular divergence time found in Neltuma and Strombocarpa genera is close to the diversification of the major clades in the subfamily Mimosoideae , which occurred in the Late Miocene 13,50 . Our results showed that Strombocarpa diverged in the Late Miocene (mean = 8.70 Mya; 95% HPD = 7.52–9.89 Mya), which is supported by the fossil Prosopisinoxylon anciborae , a Mimosoideae species with a high similarity to genus Prosopis L. (currently re-delimitated), reported to have occurred during the Late Miocene in the Catamarca Province, Argentina 77 . Additionally, a similar divergence time, around 9.21 Mya (8.35–10.07), for the genus Strombocarpa was found Catalano et al 13 . Our results also showed that the Neltuma genus started diverging in the Pliocene (mean = 2.96 Mya; 95% HPD = 2.62–3.33 Mya) and continued in the Pleistocene. This corresponds to the Mesquite species (e.g. N. alba , N. juliflora , N. glandulosa , N. chilensis , N. alpataco and N. nigra ) whose divergence time started in the Pliocene and continued in the Pleistocene, (mean = 3.65 Mya; 95% HPD = 3.31–3.99 Mya) 13 . Tree species such as Neltuma and Strombocarpa are subject to a number of ecological selective pressures due to the hostile conditions of the Atacama Desert. Chloroplast genes are involved in regulatory responses to various abiotic stresses, including heat, chilling, salinity, drought and radiation 78,79 . Therefore, the here presented chloroplast genomes of the Neltuma and Strombocarpa species can play an important role in understanding the plants adaptations to these hostile environments. The chloroplast genome structure of legumes is particularly interesting, because it contains multiple rearrangements, expansions, contractions, and loss of genetic content, which are all very useful for phylogenetic studies 79 . Phylogenetic analysis can aid conservation of species through the confirmation of taxonomic status, clarification of evolutionary relationships and consequently the determination fo conservation priorities 80 . Additionally, phylogeographic studies offer valuable information for conservation purposes as they describe the geographical distribution of genetic variability among species populations 81 . With this study, we discovered differences in chloroplast genomes of Neltuma and Strombocarpa , species improving our understanding of its phylogeny and evolution, in hope to aid the conservation of these valuable species before it is too late and they disappear. Conclusion In this work, we present for the first time the assembly and characterization of the chloroplast genomes of Neltuma alba , Neltuma chilensis and Strombocarpa strombulifera . We found enough variation in genome size, GC content, repetitive elements and nucleotide diversity to support the disintegration of the former genus Prosopis L. The chloroplasts presented in this study provide a better understanding of the diversification of Neltuma , Strombocarpa and Prosopis as well as important information for evolutionary, phylogenomic and biogeographic studies for other species of the Fabaceae family. Declarations Acknowledgements RC thanks projects ANID-FONDECYT initiation 11230668. RC and FC thanks AFOREST, a Millenium Nucleus supported by ANID – MILENIO – NCS2022_024. WH thanks "Convenio ESR UTA2295". RC thanks Neri Contreras-Ascencio and Ana Díaz-Sanchez for their sampling support. Funding This work was supported by the ANID-FONDECYT Initiation into Research grant 11230668 and by the Universidad de Atacama (DIUDA 22423 project). PJ was funded by the German Research Foundation (DFG) with the grant number JU 3228/1-1. LvdB was funded by ANID PIA/ACT 210038. Authors' contributions . R.C., L.vdB., F.C.: data analyses, writing manuscript. R.C., L.vdB., F.C., W.H., P.J.: data interpretation, writing and editing manuscript. R.C., F.C., L.vdB: experimental analysis. R.C., L.vdB., W.H.: Analysis and interpretation of result. R.C., L.vdB., P.J.: editing-review original draft. All the authors have approved the final manuscript. Conflicts of interest/Competing interests . There is no conflict of interest between the authors of this manuscript. Ethics approval . This article does not contain any studies with human participants or animals performed by any of the authors. Research Permit . This research complies with the corresponding research permits according to national and international standards, for the collection of material from Neltuma alba, Neltuma chilensis and Strombocarpa strombulifera, and the care of flora and fauna. The research permit was granted by CONAF (National Forestry Corporation) N° N00024/08-11-2019 (JBH/FAP/JVO) and N° N00003-2023/27-01-2023 (NOO/FAP/JVO). Consent to participate . All the authors of this manuscript declare that we participated in the design and preparation of this manuscript. Consent for publication . All authors authorize the publication of this manuscript. Availability of data and material . The datasets generated and analyzed during the current study are available in the Genome Database on National Center for Biotechnology Information (NCBI) repository under the accession number OP672364 for Neltuma alba, OP672365 for Neltuma chilensis and OP672366 for Strombocarpa strombulifera. The BioProject and BioSample accession numbers on NCBI for Neltuma alba are PRJNA1026123 and SAMN37734720, for Neltuma chilensis are PRJNA1026131 and SAMN37735133, and for Strombocarpa strombulifera are PRJNA1026137 and SAMN37735326. 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Some biological characteristics that foster the invasion of Prosopis juliflora (Sw.) DC. at Middle Awash Rift Valley Area, north-eastern Ethiopia. J. Arid Environ. 58, 135–154 (2004). Premoli, A. C., Mathiasen, P., Cristina Acosta, M. & Ramos, V. A. Phylogeographically concordant chloroplast DNA divergence in sympatric Nothofagus s.s. How deep can it be? New Phytol. 193, 261–275 (2012). Martínez, L. C. A. Prosopisinoxylon anciborae nov. gen. et sp. (Leguminosae, Mimosoideae) from the Late Miocene Chiquimil Formation (Santa María Group), Catamarca, Argentina. Rev. Palaeobot. Palynol. 158, 262–271 (2010). Song, Y., Feng, L., Alyafei, M. A. M., Jaleel, A. & Ren, M. Function of chloroplasts in plant stress responses. Int. J. Mol. Sci. 22, (2021). Daniell, H., Lin, C. S., Yu, M. & Chang, W. J. Chloroplast genomes: Diversity, evolution, and applications in genetic engineering. Genome Biol. 17, (2016). Byrne, M. Phylogenetics and the conservation of a diverse and ancient flora. Comptes Rendus - Biol. 326, (2003). Bobo-Pinilla, J., Salmerón-Sánchez, E., Mendoza-Fernández, A. J., Mota, J. F. & Peñas, J. Conservation and Phylogeography of Plants: From the Mediterranean to the Rest of the World. Diversity 14, (2022). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 25 Jan, 2024 Reviews received at journal 19 Jan, 2024 Reviewers agreed at journal 09 Jan, 2024 Reviewers agreed at journal 22 Dec, 2023 Reviewers invited by journal 21 Dec, 2023 Editor assigned by journal 31 Oct, 2023 Editor invited by journal 23 Oct, 2023 Submission checks completed at journal 23 Oct, 2023 First submitted to journal 08 Oct, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3422232","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":242078740,"identity":"188b3715-1fde-439a-8c79-61bda1d4a652","order_by":0,"name":"Roberto Contreras-Díaz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYFACHnQB9gYGCSAlg19LApjF2AAROADWgmEUHi0SCfi1mLf3Hnxc+cOGgb+99/jjiop78vIz3xje+MFgh1OLzJlzyYZnEtIYJM6cS2w8c6bYcMPtHGPLHoZknFokJHLMJBsSDjMYSOQYNja2JTBukM4xk+BhOIBPi/lPhJZ/CfbzZ54xk/yDX4sZI0JLQ0Jiww0eM2m8tvCcS5ZsSEvjkThzxnBmw7GE5A1n0oqtZQzw+IW99+DHBhsbOf72HoOPDTUJtvPbD2+8+abCTg6XFhhAN9OAkIZRMApGwSgYBfgAALsoT8LL8bSSAAAAAElFTkSuQmCC","orcid":"","institution":"Universidad de Atacama","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Roberto","middleName":"","lastName":"Contreras-Díaz","suffix":""},{"id":242078741,"identity":"394fedb2-1c93-4a5a-81eb-2d289a7ce510","order_by":1,"name":"Felipe S. Carevic","email":"","orcid":"","institution":"Universidad Arturo Prat","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Felipe","middleName":"S.","lastName":"Carevic","suffix":""},{"id":242078743,"identity":"55b6c3d2-b78b-4d46-b899-902f7a6e1cb2","order_by":2,"name":"Liesbeth van den Brink","email":"","orcid":"","institution":"ECOBIOSIS, Universidad de Concepción","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Liesbeth","middleName":"van den","lastName":"Brink","suffix":""},{"id":242078746,"identity":"5e9dcdb0-fda5-4254-af6e-92ec60d8ae7f","order_by":3,"name":"Wilson Huanca-Mamani","email":"","orcid":"","institution":"Universidad de Tarapacá","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wilson","middleName":"","lastName":"Huanca-Mamani","suffix":""},{"id":242078748,"identity":"a696ca43-bc02-4cef-b497-e65c358faa99","order_by":4,"name":"Patrick Jung","email":"","orcid":"","institution":"University of Applied Sciences Kaiserslautern","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"","lastName":"Jung","suffix":""}],"badges":[],"createdAt":"2023-10-08 22:29:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3422232/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3422232/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":45340665,"identity":"7c276a9b-9745-44f6-bdf9-f986f550e91b","added_by":"auto","created_at":"2023-10-27 20:21:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1345682,"visible":true,"origin":"","legend":"\u003cp\u003eCircular gene map of the chloroplast genomes of \u003cem\u003eNeltuma alba\u003c/em\u003e, \u003cem\u003eNeltuma chilensis\u003c/em\u003e and \u003cem\u003eStrombocarpa strombulifera\u003c/em\u003e. Genes were colored according to their functional group. Small single copy (SSC), large single copy (LSC), and inverted repeats (IRA, IRB) were indicated.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3422232/v1/76e9a446707090efe9b8a2dc.png"},{"id":45340662,"identity":"b7ad1b1b-869e-41ba-8f16-c0779b9f2d22","added_by":"auto","created_at":"2023-10-27 20:21:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":97792,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of simple sequence repeats (SSRs) of the \u003cem\u003eN. alba\u003c/em\u003e, \u003cem\u003eN. chilensis\u003c/em\u003e, \u003cem\u003eS. strombulifera\u003c/em\u003e, \u003cem\u003eS. tamarugo\u003c/em\u003e, \u003cem\u003eN. glandulosa\u003c/em\u003e, \u003cem\u003eN. juliflora\u003c/em\u003e, \u003cem\u003eP. cineraria\u003c/em\u003eand \u003cem\u003eP. farcta\u003c/em\u003e chloroplast genomes. Total numbers of SSRs of each motif unit (A) and frequency of SSR motifs in different repeat class types (B).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3422232/v1/79ff0db1a23f4ffcb525e388.png"},{"id":45340660,"identity":"5ba87ec6-be48-46f1-9f21-df3504db5a58","added_by":"auto","created_at":"2023-10-27 20:21:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":123673,"visible":true,"origin":"","legend":"\u003cp\u003eRepeat structure analysis of the \u003cem\u003eN. alba\u003c/em\u003e, \u003cem\u003eN. chilensis\u003c/em\u003e, \u003cem\u003eS. strombulifera\u003c/em\u003e, \u003cem\u003eS. tamarugo\u003c/em\u003e, \u003cem\u003eN. glandulosa\u003c/em\u003e, \u003cem\u003eN. juliflora\u003c/em\u003e, \u003cem\u003eP. cineraria\u003c/em\u003e and \u003cem\u003eP. farcta\u003c/em\u003echloroplast genomes. Total numbers long repeat types: Palindrome, Forward, Reverse and Complement (A), number of palindrome repeats (B), number of forward repeats (C) and number of reverse repeats (D) by length.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3422232/v1/9be5b1c6bbe5d979cb5ac409.png"},{"id":45341465,"identity":"1a5cc07d-78b0-4f4e-bfcb-7d6d772b7a84","added_by":"auto","created_at":"2023-10-27 20:29:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2146719,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of chloroplast genomes between the Long Single Copy (LSC), Short Single Copy (SSC) and Inverted Repeat (IRa and IRb) junction regions among \u003cem\u003eNeltuma\u003c/em\u003e, \u003cem\u003eStrombocarpa\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003especies.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3422232/v1/33dc6c12b030f4770978cae8.png"},{"id":45341464,"identity":"5bc90608-0054-41f1-ade9-30edcd43d5ba","added_by":"auto","created_at":"2023-10-27 20:29:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1463466,"visible":true,"origin":"","legend":"\u003cp\u003eSliding window analysis of the whole plastid of the two \u003cem\u003eNeltuma\u003c/em\u003especies (a), of the two \u003cem\u003eStrombocarpa\u003c/em\u003especies (b), of the genera \u003cem\u003eStrombocarpa\u003c/em\u003eand \u003cem\u003eNeltuma\u003c/em\u003e (c), and of the tribe \u003cem\u003eMimoseae\u003c/em\u003e (d). X-axis: Nucleotide position, Y-axis: Nucleotide diversity (Pi). The regions with highest Pi values were plotted considering 2× the median value as the cutoff point indicated by the green dashed line.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3422232/v1/6a12c833bbf0fbe1d18e6fba.png"},{"id":45340666,"identity":"9e9a32ab-46c3-4042-9a62-22cd7f380bf0","added_by":"auto","created_at":"2023-10-27 20:21:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":203488,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular phylogenetic analysis based on 80 protein-coding genes of the plastid genome of nine \u003cem\u003eMimoseae\u003c/em\u003e species inferred by maximum likelihood (a) and Bayesian inference (b) methods. Numbers in the nodes are ML bootstrap values (a) and Bayesian posterior probabilities values (b).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3422232/v1/c055c06225f41353b7731c6a.png"},{"id":45340663,"identity":"9f56ea27-0e21-43ac-b2ba-2d4e7e967398","added_by":"auto","created_at":"2023-10-27 20:21:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":178458,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eNeltuma, Strombocarpa and Prosopis\u003c/em\u003e chronogram showing divergence times estimated using BEAST program based on data from nine whole plastid genomes. The divergence times of each clade are displayed near each node. Blue bars represent 95% highest posterior density values for the estimated mean dates. The nodes 1 and 2 correspond to calibration points.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3422232/v1/c67ce4da33bed42e33cd9d61.png"},{"id":45342248,"identity":"fd266678-ea9f-41ca-8b34-055e5b1813f2","added_by":"auto","created_at":"2023-10-27 20:37:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1796625,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3422232/v1/48c02a4d-6c54-49a8-b245-47d427bab102.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative analysis of the complete plastid genomes of desert trees Neltuma and Strombocarpa genera","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLegumes have a cosmopolitan distribution and are ecologically important in almost all biomes of the world as their fruits are a food source or their root biology nourishes soils \u003csup\u003e1,2\u003c/sup\u003e, even in ecosystems as extreme as the Atacama Desert. \u003cem\u003eLeguminosae\u003c/em\u003e (\u003cem\u003eFabaceae\u003c/em\u003e) is one of the largest angiosperm family in terms of species numbers and one of the most diverse family and is classified into three subfamilies (\u003cem\u003eCaesalpinioideae\u003c/em\u003e, \u003cem\u003eMimosoideae\u003c/em\u003e, and \u003cem\u003ePapilionoideae\u003c/em\u003e), which have close to 770 genera and over 19,500 species \u003csup\u003e1,3\u003c/sup\u003e. Burkart (1976)\u003csup\u003e4\u003c/sup\u003e taxonomic monograph of the genus \u003cem\u003eProsopis\u003c/em\u003e L. (Mesquite) recognized 44 species, which are distributed across Southwest Asia, Africa, and (predominantly) America \u003csup\u003e5,6\u003c/sup\u003e. \u003cem\u003eProsopis\u003c/em\u003e species from the \u003cem\u003eStrombocarpa\u003c/em\u003e Bentham and \u003cem\u003eAlgarobia\u003c/em\u003e DC. Emend. Burk sections are trees and normally inhabitants of arid and semiarid regions \u003csup\u003e6,7\u003c/sup\u003e. These species inhabit the Atacama Desert in Chile, which extends for over 1000 km between latitudes 19\u0026deg;S and 30\u0026deg;S and is bordered by the Coastal Cordillera to the west and the Andean Cordillera to the east \u003csup\u003e8\u003c/sup\u003e. Surviving in a place as hostile as the Atacama Desert, where radiation is high, and so is water stress \u003csup\u003e9,10\u003c/sup\u003e is already an accomplishment, but these trees also provide local people with important resources such as fruits, juice, futter and wood \u003csup\u003e7\u003c/sup\u003e. Three species from the formerly known \u003cem\u003eStrombocarpa\u003c/em\u003e section (\u003cem\u003eProsopis strombulifera\u003c/em\u003e (Argentine screwbean) and the endemics \u003cem\u003eProsopis burkartii\u003c/em\u003e and \u003cem\u003eProsopis tamarugo\u003c/em\u003e (Tamarugo)) \u003csup\u003e7,11\u003c/sup\u003e and individuals belonging to different species from the formerly know \u003cem\u003eAlgarobia\u003c/em\u003e section (\u003cem\u003eProsopis chilensis\u003c/em\u003e, \u003cem\u003eProsopis flexuosa\u003c/em\u003e, and \u003cem\u003eProsopis alba\u003c/em\u003e) can be found in the Atacama Desert \u003csup\u003e6\u003c/sup\u003e. The scientific names of these species and the concept of \u003cem\u003eProsopis\u003c/em\u003e established by Bentham (1875)\u003csup\u003e12\u003c/sup\u003e and Burkart (1976)\u003csup\u003e4\u003c/sup\u003e has only currently been disintegrated, because \u003cem\u003eProsopis\u003c/em\u003e was found to be polyphyletic based on both chloroplast (cpDNA) and nuclear DNA (nDNA) \u003csup\u003e5,13,14\u003c/sup\u003e. As a consequence, the old \u003cem\u003eProsopis\u003c/em\u003e cluster was divided in six genera \u0026ndash; \u003cem\u003eAnonychium\u003c/em\u003e, \u003cem\u003eProsopis\u003c/em\u003e, \u003cem\u003eNeltuma\u003c/em\u003e, \u003cem\u003eStrombocarpa\u003c/em\u003e, \u003cem\u003eXerocladia\u003c/em\u003e and \u003cem\u003eIndopiptadenia\u003c/em\u003e. The species of the above mentioned \u003cem\u003eAlgarobia\u003c/em\u003e section were renamed as \u003cem\u003eNeltuma alba\u003c/em\u003e, \u003cem\u003eNeltuma chilensis\u003c/em\u003e, \u003cem\u003eNeltuma flexuosa\u003c/em\u003e, and the species of the above mentioned \u003cem\u003eStrombocarpa\u003c/em\u003e section as \u003cem\u003eStrombocarpa tamarugo\u003c/em\u003e, \u003cem\u003eStrombocarpa burkartii\u003c/em\u003e and \u003cem\u003eStrombocarpa strombulifera\u003c/em\u003e \u003csup\u003e5\u003c/sup\u003e. This division was based on short DNA sequences, as is common practice in taxonomy. However, compared to short DNA sequences, a complete chloroplast genome of approximately 160,000 bp can offer more information about the phylogenetic relationships and gives a full overview of the specific genes and the structure of its genome.\u003c/p\u003e \u003cp\u003eChloroplast and mitochondria are the powerhouse of a plant cell, responsible for the majority of the energy produced, through photosynthesis and photorespiration, respectively; these organelles have originated endosymbiotically from eubacterial cells, where most genes were either lost or transferred to the host nucleus \u003csup\u003e15\u003c/sup\u003e. The chloroplast genome is a valuable taxonomic resource with rich genetic information \u003csup\u003e16\u003c/sup\u003e, as it is highly conserved and maternally inherited \u003csup\u003e17\u003c/sup\u003e. In angiosperms, most chloroplast genomes are composed of circular DNA molecules and have a quadripartite organization consisting of two copies of inverted repeats (IRs), which divide the rest of chloroplast genome into a large single copy (LSC) region and small single copy (SSC) region \u003csup\u003e17\u003c/sup\u003e. There are approximately 110\u0026ndash;130 genes included in chloroplast DNA, consisting of rRNA-coding genes, protein-coding genes, and tRNA-coding genes \u003csup\u003e16\u003c/sup\u003e. Because the chloroplast genome can provide valuable information to support the conservation of threatened trees \u003csup\u003e18\u003c/sup\u003e, gaining insights in chloroplast DNA of the legume tree populations from Atacama Desert could help their conservation. Chloroplast genome sequences are commonly used in plant phylogeny, phylogeographic and genome evolution studies \u003csup\u003e16,17\u003c/sup\u003e. Lately, the use of complete chloroplast genome as a \u0026ldquo;super-barcoding\u0026rdquo; method has become an excellent approach allowing for the increase of the phylogenetic resolution at lower taxonomic levels in plants \u003csup\u003e19,20\u003c/sup\u003e. However, in the Atacama Desert only a few plastomes of the native and endemic herbaceous plants \u003csup\u003e21,22\u003c/sup\u003e, shrubs \u003csup\u003e23,24\u003c/sup\u003e and leguminous trees \u003csup\u003e25,26\u003c/sup\u003e have been characterized so far.\u003c/p\u003e \u003cp\u003eUnfortunately, several species of trees of the genera \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003e are in vulnerable and endangered conservation status in Chile, e.g. \u003cem\u003eNeltuma chilensis\u003c/em\u003e and \u003cem\u003eStrombocarpa tamarugo\u003c/em\u003e. \u003cem\u003eNeltuma chilensis\u003c/em\u003e and \u003cem\u003eNeltuma alba\u003c/em\u003e are restricted to southern Peru, northern and central Chile, southwestern Bolivia and northwestern, western and central Argentina \u003csup\u003e27\u0026ndash;29\u003c/sup\u003e. The not threatened \u003cem\u003eStrombocarpa strombulifera\u003c/em\u003e is widely distributed from the Arizona desert (U.S.A.) to Patagonia (Argentina) \u003csup\u003e30\u003c/sup\u003e. However, in the Atacama Desert, \u003cem\u003eNeltuma alba, Neltuma chilensis\u003c/em\u003e, as well as \u003cem\u003eStrombocarpa strombulifera\u003c/em\u003e populations, are fragmented and restricted to oases or valley (forming populations of only a few individuals), and geographically isolated from each other by large areas of land \u003csup\u003e6,7,31\u003c/sup\u003e. Although \u003cem\u003eNeltuma alba\u003c/em\u003e and \u003cem\u003eStrombocarpa strombulifera\u003c/em\u003e are in the conservation status of \u0026ldquo;Least Concern\u0026rdquo; it is urgently necessary to identify the plastomes of their genera now, before their more endangered cousins go extinct. Until now, there is no complete chloroplast genome available for any \u003cem\u003eN. alba\u003c/em\u003e, \u003cem\u003eN. chilensis\u003c/em\u003e and \u003cem\u003eS. strombulifera\u003c/em\u003e, but they are needed to confirm phylogenetic relationships between them, and with closely related species. Therefore, in this study we provide and analyze the complete chloroplast genomes of \u003cem\u003eN. alba\u003c/em\u003e, \u003cem\u003eN. chilensis\u003c/em\u003e and \u003cem\u003eS. strombulifera\u003c/em\u003e, in terms of structure, gene composition, divergence time and phylogeny.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material and DNA isolation\u003c/h2\u003e \u003cp\u003eFresh leaves of \u003cem\u003eNeltuma alba\u003c/em\u003e (Griseb.) C.E. Hughes \u0026amp; G.P. Lewis, \u003cem\u003eNeltuma chilensis\u003c/em\u003e (Molina) C.E. Hughes \u0026amp; G.P. Lewis and \u003cem\u003eStrombocarpa strombulifera\u003c/em\u003e (Lam.) A. Gray were collected in Copiap\u0026oacute; (27\u0026deg;21'39.3\"S 70\u0026deg;20'33.8\"W), Chacabuco (33\u0026deg;05'24.9\"S 70\u0026deg;39'07.3\"W) and Pampa del Tamarugal (20\u0026deg;27'59.9\"S 69\u0026deg;33'23.5\"W) in Chile, respectively. Identification of samples was done according to the taxonomic criteria described by Burkart (1976)\u003csup\u003e4\u003c/sup\u003e. Additionally, the samples were verified by the forester Boris Burgos of the Corporaci\u0026oacute;n Nacional Forestal (CONAF) from the Atacama Region. The specimens were deposited in the Departamento de Silvicultura y Conservaci\u0026oacute;n de la Naturaleza herbarium of Universidad de Chile (under the names that were correct at the time of deposition: \u003cem\u003eProsopis alba\u003c/em\u003e, EIF13329; \u003cem\u003eProsopis chilensis\u003c/em\u003e, EIF13328; and \u003cem\u003eProsopis strombulifera\u003c/em\u003e, EIF13350). DNA was isolated from the leaves using the modified cetyl-trimethylammonium bromide (CTAB) protocol \u003csup\u003e7\u003c/sup\u003e. The DNA was quantified with a Qubit\u0026trade; 3.0 fluorometer and a Qubit\u0026trade; dsDNA HS Assay Kit, according to the protocol supplied by the manufacturer. DNA integrity was verified with an Agilent 2100 Bioanalyzer prior to sequencing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eGenome sequencing, assembling and annotation\u003c/h2\u003e \u003cp\u003eSequencing libraries were generated by a TruSeq Nano DNA LT Kit (Illumina, San Diego, CA). The final libraries were run on an Agilent 2100 Bioanalyzer to verify the fragment size distribution and concentration. Sequencing was performed with an Illumina sequencing platform, at Genoma Mayor (Universidad Mayor, Chile). Paired-end sequences of 150 bp were generated for each read (R1 and R2). The filtered reads were assembled using SPAdes 4 software version 3.13.0 \u003csup\u003e32\u003c/sup\u003e, using three k-mers parameters: - k 33, 55 and 77. The plastid was annotated with PGA software \u003csup\u003e33\u003c/sup\u003e and CPGAVAS2 \u003csup\u003e34\u003c/sup\u003e, after which it was manually corrected when needed. The graphical map of the plastid was generated by Organellar Genome DRAW (OGDRAW) \u003csup\u003e35\u003c/sup\u003e, and the complete nucleotide sequences were deposited in the NCBI GenBank database (OP672364, OP672365 and OP672366, under the names \u003cem\u003eProsopis alba\u003c/em\u003e, \u003cem\u003eProsopis chilensis\u003c/em\u003e and \u003cem\u003eProsopis strombulifera\u003c/em\u003e, respectively).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGenome comparison, repeat and phylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe plastid structures (LSC/IR, IR/SSC) of \u003cem\u003eN. alba\u003c/em\u003e, \u003cem\u003eN. chilensis\u003c/em\u003e and \u003cem\u003eS. strombulifera\u003c/em\u003e and of five closely related species, i.e. \u003cem\u003eNeltuma juliflora\u003c/em\u003e (Sw.) Raf., \u003cem\u003eStrombocarpa tamarugo\u003c/em\u003e (Phil.) C.E. Hughes \u0026amp; G.P. Lewis, \u003cem\u003eProsopis farcta\u003c/em\u003e (Banks \u0026amp; Sol.) J.F. Macbr. and \u003cem\u003eProsopis cineraria\u003c/em\u003e (L.) Druce, of the \u003cem\u003eMimoseae\u003c/em\u003e tribe were visualized and compared using IRScope \u003csup\u003e36\u003c/sup\u003e. We used sequence data of whole plastome (obained from GenBank) of species from the genera \u003cem\u003eNeltuma\u003c/em\u003e, \u003cem\u003eStrombocarpa\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e for the identification of the simple sequence repeats (SSRs). These SSRs were identified using MISA software \u003csup\u003e37\u003c/sup\u003e with the following search parameters: ten for mononucleotide, eight for dinucleotide, four for trinucleotide and tetranucleotide, and three for pentanucleotide and hexanucleotide. To identify the tandem repeats (forward, palindromic, reverse, and complement) of these species we used REPuter \u003csup\u003e38\u003c/sup\u003e with the following parameters: hamming distance equal to 3, minimal repeat size set to 30 bp, and maximum computed repeats set to 300 bp. A sliding window analysis (window length: 600 pb, step size: 200 bp) was performed to assess the variability (Pi) between plastid of \u003cem\u003eN. alba\u003c/em\u003e and \u003cem\u003eN. chilensis\u003c/em\u003e, \u003cem\u003eS. tamarugo\u003c/em\u003e and \u003cem\u003eS. strombulifera\u003c/em\u003e, the genera \u003cem\u003eStrombocarpa\u003c/em\u003e and \u003cem\u003eNeltuma\u003c/em\u003e, and the tribe \u003cem\u003eMimoseae\u003c/em\u003e with DnaSP v. 5 software \u003csup\u003e39\u003c/sup\u003e. The complete plastid genome sequence of \u003cem\u003eN. alba\u003c/em\u003e (OP672364), \u003cem\u003eN. chilensis\u003c/em\u003e (OP672365), \u003cem\u003eS. strombulifera\u003c/em\u003e (OP672366), \u003cem\u003eS. tamarugo\u003c/em\u003e (MW582314), \u003cem\u003eN. glandulosa\u003c/em\u003e (NC_026683), \u003cem\u003eN. juliflora\u003c/em\u003e (MN104889), \u003cem\u003eP. farcta\u003c/em\u003e (MZ073639), \u003cem\u003eP. cineraria\u003c/em\u003e (MN104890) and \u003cem\u003eLeucaena trichandra\u003c/em\u003e (NC_028733) as outgroup species were used in the phylogenetic analysis. Eighty protein-coding genes (PCG) sequences were aligned separately using MAFFT v7 \u003csup\u003e40\u003c/sup\u003e and any gaps in the alignment were trimmed using TrimAL v1.4 \u003csup\u003e41\u003c/sup\u003e. Afterwards, the sequences were concatenated with Mesquite 3.81 software \u003csup\u003e42\u003c/sup\u003e. The analyses of the plastid genomes\u0026acute; 80 PCG sequences were conducted using the maximum likelihood (ML) method. The complete plastid genome sequence of the nine species were analyzed using the Bayesian inference (BI) methods. The best-fitting nucleotide substitution model of sequence evolution, model TVM\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G, was determined based on the Akaike Information Criterion (AIC) using the MrModeltest v2.3 \u003csup\u003e43\u003c/sup\u003e. The ML analyses were performed using RAxML-HPC BlackBox v.8.1.24 \u003csup\u003e44\u003c/sup\u003e with 1,000 bootstrap replicates. The BI analysis was conducted using MrBayes v.3.2 \u003csup\u003e45\u003c/sup\u003e with the CIPRES Science Gateway v3.3 \u003csup\u003e46\u003c/sup\u003e. The Markov Chain Monte Carlo (MCMC) algorithm was calculated for 5,000,000 generations, and the sampling tree for every 1,000 generations. The first 25% of generations were discarded as burn-in. In the analysis, bootstrap support (BS) values were estimated in the ML, and the reliability of clades in the Bayesian analysis was evaluated by means of posterior probability (PP). The trees were visualized with FigTree \u003csup\u003e47\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDivergence time estimate\u003c/h2\u003e \u003cp\u003eTo estimate the divergence time of the species we used BEAST v2.6.0 \u003csup\u003e48\u003c/sup\u003e based on the complete plastid genome sequence of eight species of the \u003cem\u003eProsopis\u003c/em\u003e, \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003e genera, and \u003cem\u003eLeucaena trichandra\u003c/em\u003e as an outgroup species. All genome sequences were aligned with MAFFT, and then the file was imported to BEAUTi interface to generate a file for BEAST, after applying HKY\u0026thinsp;+\u0026thinsp;Γ substitution model, \u0026ldquo;Empirical\u0026rdquo; frequency, strict molecular clock model and \u0026ldquo;Yule\u0026rdquo; model speciation. Divergence times were estimated combining two calibration points. The TimeTree tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.timetree.org\u003c/span\u003e\u003cspan address=\"http://www.timetree.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e \u003csup\u003e49\u003c/sup\u003e) was used to fix the node age of \u003cem\u003eLeucaena trichandra\u003c/em\u003e and the \u003cem\u003eProsopis\u003c/em\u003e genus, which is known to have diverged 35 Mya (33.2\u0026ndash;40.3 Mya) \u003csup\u003e13,50\u0026ndash;52\u003c/sup\u003e. We considered a second calibration as well, fixing the node age of \u003cem\u003eMezquite\u003c/em\u003e clade (\u003cem\u003eN. alba\u003c/em\u003e, \u003cem\u003eN. chilensis\u003c/em\u003e, \u003cem\u003eN. juliflora\u003c/em\u003e, among others) at 3.65 Mya (3.31\u0026ndash;3.99 Mya) after the study of \u003csup\u003e13\u003c/sup\u003e. The Markov Chain Monte Carlo (MCMC) was run for 6\u0026nbsp;million generations, sampling every 1,000 generations. We ran the program using the input file XML generated by BEAUTi in BEAST. Final log files were checked in Tracer 1.7.1 \u003csup\u003e53\u003c/sup\u003e. We used the TreeAnnotator program \u003csup\u003e48\u003c/sup\u003e with a 10% burn-in. Figtree software was used to visualize phylogenetic trees, using the extent of the 95% highest posterior density (HPD) intervals for each divergence time.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe chloroplast genome lengths of \u003cem\u003eN. alba\u003c/em\u003e, \u003cem\u003eN. chilensis\u003c/em\u003e and \u003cem\u003eS. strombulifera\u003c/em\u003e comprise 162,980 bp, 163,047 bp and 160,569 bp respectively and its structure contains a typical quadripartite structure with two inverted repeat regions (IR; 25,919 bp, 25,919 bp and 26,026 bp respectively) separated by a large single copy region (LSC; 92,300 bp, 92,356 bp and 89,569 bp respectively) and a small single copy region (SSC; 18,842 bp, 18,853 bp and 18,623 bp respectively) (Figure 1, Table 1). Its length and structure are similar to those of the other species of the \u003cem\u003eNeltuma\u003c/em\u003e, \u003cem\u003eStrombocarpa\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e genera, which vary in the IRs between 25,919 bp and 25,935 bp, in the LSC between 91,062 bp and 92,937 bp and in the SSC between 18,643 bp and 18,880 bp (Table 1). The cp genomes of the \u003cem\u003eStrombocarpa\u003c/em\u003e species (\u003cem\u003eS. strombulifera\u003c/em\u003e 160,569 bp; and \u003cem\u003eS. tamarugo\u003c/em\u003e 161,575) are smaller (~2,000 bp) than those of the \u003cem\u003eNeltuma\u003c/em\u003e species (\u003cem\u003eN. alba\u003c/em\u003e 162,980; and \u003cem\u003eN. chilensis\u003c/em\u003e 163,047) (Table 1).\u0026nbsp;The GC content in the chloroplast of \u003cem\u003eN. alba\u003c/em\u003e and \u003cem\u003eN. chilensis\u003c/em\u003e (35.9%) is slightly less than in \u003cem\u003eS. strombulifera\u003c/em\u003e (36.2%), but the overall GC content was similar to other species of the \u003cem\u003eNeltuma\u003c/em\u003e, \u003cem\u003eStrombocarpa\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e genera (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn total, 128 genes were found in cp genomes of \u003cem\u003eN. alba\u003c/em\u003e, \u003cem\u003eN. chilensis\u003c/em\u003e and \u003cem\u003eS. strombulifera\u003c/em\u003e, which included 83 coding genes, 8 rRNA genes, and 37 tRNA genes (Table 1). Of these genes, six coding genes (ndhB, \u003cem\u003erpl2\u003c/em\u003e, \u003cem\u003erpl23\u003c/em\u003e, \u003cem\u003erps12\u003c/em\u003e, \u003cem\u003erps7\u003c/em\u003e and \u003cem\u003eycf2\u003c/em\u003e), four rRNA genes and seven tRNA genes located in the IR regions contained duplicated genes (Table 2). Eighteen genes had introns, including nine PCGs (\u003cem\u003eatpF\u003c/em\u003e, \u003cem\u003endhA\u003c/em\u003e, \u003cem\u003endhB\u003c/em\u003e, \u003cem\u003epetB\u003c/em\u003e, \u003cem\u003epetD\u003c/em\u003e, \u003cem\u003erpl2\u003c/em\u003e, \u003cem\u003erpl16\u003c/em\u003e, \u003cem\u003erpoC1\u003c/em\u003e and \u003cem\u003erps16\u003c/em\u003e), except for \u003cem\u003eS. strombulifera\u003c/em\u003e, which had ten PCGs, additionally including \u003cem\u003eycf2\u0026nbsp;\u003c/em\u003eto the ones mentioned before. Six tRNAs (\u003cem\u003etrnA\u003csup\u003eUGC\u003c/sup\u003e\u003c/em\u003e, \u003cem\u003etrnG\u003csup\u003eGCC\u003c/sup\u003e\u003c/em\u003e, \u003cem\u003etrnI\u003csup\u003eGAU\u003c/sup\u003e\u003c/em\u003e, \u003cem\u003etrnK\u003csup\u003eUUU\u003c/sup\u003e\u003c/em\u003e, \u003cem\u003etrnL\u003csup\u003eUAA\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003etrnV\u003csup\u003eUAC\u003c/sup\u003e\u003c/em\u003e) contained one intron, while three PCGs (\u003cem\u003eycf3\u003c/em\u003e, \u003cem\u003erps12\u003c/em\u003e and \u003cem\u003eclpP\u003c/em\u003e) had two introns (Table 2).\u003c/p\u003e\n\u003cp\u003eWe compared the simple sequence repeats (SSRs) from the cp genomes of the species of the \u003cem\u003eNeltuma\u003c/em\u003e, \u003cem\u003eStrombocarpa\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e genera. The maximum number was 100 SSRs in \u003cem\u003eN. juliflora\u003c/em\u003e, 95 SSRs in \u003cem\u003eP. cineraria\u003c/em\u003e, 92 SSRs in \u003cem\u003eN.\u003c/em\u003e \u003cem\u003eglandulosa\u003c/em\u003e and \u003cem\u003eS. strombulifera\u003c/em\u003e, 90 SSRs in \u003cem\u003eN. chilensis\u003c/em\u003e, 88 SSRs in \u003cem\u003eN. alba\u003c/em\u003e and, whereas S. \u003cem\u003etamarugo\u003c/em\u003e and \u003cem\u003eP. farcta\u003c/em\u003e only had 70 SSRs (Figure 2A). Mononucleotide repeats (A/T) were the most common repeats and the highest in number, ranging from 61 (\u003cem\u003eS. tamarugo\u003c/em\u003e) to 80 (\u003cem\u003eS. strombulifera\u003c/em\u003e); mononucleotide repeats C/G were found in all species except in \u003cem\u003eS. tamarugo\u003c/em\u003e and \u003cem\u003eS. strombulifera\u003c/em\u003e (Figure 2B). The number of dinucleotide SSRs (AT/AT) was similar in all species except in \u003cem\u003eS. tamarugo\u003c/em\u003e and \u003cem\u003eS. strombulifera\u003c/em\u003e in which no dinucleotide SSRs were found. In all species, 10-12 trinucleotide repeats AAT/ATT were found, but in \u003cem\u003eS. tamarugo\u003c/em\u003e and \u003cem\u003eS.\u003c/em\u003e \u003cem\u003estrombulifera\u003c/em\u003e only 4 and 5 respectively, were present. Moreover, only \u003cem\u003eS. tamarugo\u003c/em\u003e had trinucleotide repeats AAG/CTT (Figure 2B). In general, four pentanucleotide SSRs type (AAAAT/ATTTT, AAATT/AATTT, AATAT/ATATT and AATGG/ATTCC) were found in all species, but only one pentanucleotide SSR was present in \u003cem\u003eP. farcta\u0026nbsp;\u003c/em\u003e(AACTT/AAGTT), \u003cem\u003eS. strombulifera\u003c/em\u003e and \u003cem\u003eS. tamarugo\u003c/em\u003e (AATAG/ATTCT), and \u003cem\u003eS. strombulifera\u003c/em\u003e (AATTC/AATTG) (Figure 2B). Only two hexanucleotide SSRs, AATATT/AATATT and AAATAG/ATTTCT, were observed in \u003cem\u003eS. strombulifera\u003c/em\u003e and \u003cem\u003eP. farcta\u003c/em\u003e, respectively (Figure 2B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe amounts of repeats varied between 57 and 80 in the cp genomes of \u003cem\u003eS. tamarugo\u003c/em\u003e (88), \u003cem\u003eP. farcta\u003c/em\u003e (74), \u003cem\u003eN. juliflora\u003c/em\u003e (73), \u003cem\u003eP. cineraria\u003c/em\u003e (72), \u003cem\u003eN. glandulosa\u003c/em\u003e (67), \u003cem\u003eN. alba\u003c/em\u003e (60), \u003cem\u003eN. chilensis\u003c/em\u003e (60) and \u003cem\u003eS. strombulifera\u003c/em\u003e (57) (Figure 3A). The number of complement repeat in \u003cem\u003eS. tamarugo\u003c/em\u003e was higher (5 repeats) than in the rest of the species (Figure 3A). The total number of palindromic repeats was less in \u003cem\u003eStrombocarpa\u003c/em\u003e species (\u003cem\u003eS. strombulifera\u003c/em\u003e, 22; and \u003cem\u003eS. tamarugo\u003c/em\u003e, 24) than in species of the \u003cem\u003eNeltuma\u003c/em\u003e (\u003cem\u003eN. alba\u003c/em\u003e, 28; \u003cem\u003eN. chilensis\u003c/em\u003e, 27; \u003cem\u003eN. glandulosa\u003c/em\u003e, 29; and \u003cem\u003eN. juliflora\u003c/em\u003e, 30) and \u003cem\u003eProsopis\u003c/em\u003e (\u003cem\u003eP. cineraria\u003c/em\u003e, 33; \u003cem\u003eP.\u003c/em\u003e \u003cem\u003efarcta\u003c/em\u003e, 27) genera (Figure 3B). On the other hand, the total number of forward repeats was less in the cp genome of \u003cem\u003eN. alba\u003c/em\u003e and \u003cem\u003eN. chilensis\u003c/em\u003e (25 each), than in the rest of the species, where they varied between 30 to 40 (Figure 3C). Palindromic and forward repeats with lengths of 30-39 bp were the most common and abundant repeats in the species of the \u003cem\u003eNeltuma\u003c/em\u003e, \u003cem\u003eStrombocarpa\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e genera (Figure 3B-C). The number of reverse repeat (range of 30-39 bp) in \u003cem\u003eS. tamarugo\u003c/em\u003e was higher (with 25 repeats) than in the rest of the species (Figure 3D).\u003c/p\u003e\n\u003cp\u003eThe expansion and contraction of the IR and SC regions contributes to the differentiation in chloroplast genome size in some genera and families. For that reason, we compared of SSC, LSC, IRA, and IRB border regions of the species of the \u003cem\u003eNeltuma\u003c/em\u003e, \u003cem\u003eStrombocarpa\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e genera. In all species, \u003cem\u003erps19\u003c/em\u003e genes were located in the junction between LSC and IRb region (JLB), of which 176 to 188 bp were located at the LSC region and 91 to 103 bp located at the IRb region (Figure 4). In all species, the \u003cem\u003erpl2\u003c/em\u003e gene was entirely located in the IR regions (Figure 4). The \u003cem\u003endhF\u003c/em\u003e gene of \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e species were located in the SSC region, 137-156 bp away from the IRb-SSC border, while in the two \u003cem\u003eStrombocarpa\u003c/em\u003e species this gene was located approx. 67 bp away from the IRb-SSC border. At the SSC-IRA border, the \u003cem\u003eycf1\u003c/em\u003e gene extended into the SSC region, at varying lengths ranging from 4,760 bp in \u003cem\u003eS. tamarugo\u003c/em\u003e to 4,794 bp in \u003cem\u003eN. juliflora\u003c/em\u003e and \u003cem\u003eP. cineraria\u003c/em\u003e, however, in \u003cem\u003eN. alba\u003c/em\u003e and \u003cem\u003eS.\u003c/em\u003e \u003cem\u003estrombulifera\u003c/em\u003e the\u003cem\u003e\u0026nbsp;\u003c/em\u003egene was entirely located in the SSC region, 963-973 bp away from the IRA-SSC border. In general, the truncated copy of \u003cem\u003eycf1\u003c/em\u003e was located in the IRB region (except in \u003cem\u003eN. alba\u003c/em\u003e and \u003cem\u003eS. strombulifera\u003c/em\u003e), while one end extended into the SSC region for 17 bp only in \u003cem\u003eN. juliflora\u003c/em\u003e and \u003cem\u003eP. cineraria\u003c/em\u003e. The distance between \u003cem\u003erps19\u003c/em\u003e and the LSC/IRA border was only 2 bp in \u003cem\u003eN. glandulosa\u003c/em\u003e and \u003cem\u003eN. juliflora\u003c/em\u003e. In most species, the \u003cem\u003etrnH\u003c/em\u003e gene was located in the LSC region, 2-16 bp away from the IRA-LSC border, but in \u003cem\u003eN. juliflora\u003c/em\u003e and \u003cem\u003eP. cineraria\u003c/em\u003e it was much more distant. In general, the structure of the cp genomes of \u003cem\u003eNeltuma\u003c/em\u003e, \u003cem\u003eStrombocarpa\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e were similar in arrangement (Figure 4).\u003c/p\u003e\n\u003cp\u003eIn the genus \u003cem\u003eNeltuma\u003c/em\u003e, the mean estimated value for nucleotide diversity (Pi) between \u003cem\u003eN. alba\u003c/em\u003e and \u003cem\u003eN. chilensis\u003c/em\u003e was Pi= 0,00037 (ranging from 0 to 0.0416);\u0026nbsp;and the regions with the highest values of Pi were \u003cem\u003erps16-trnQ\u003csup\u003eUUG\u003c/sup\u003e\u003c/em\u003e (Pi = 0.005), \u003cem\u003eaccD-psaI\u003c/em\u003e (Pi = 0.0416) and \u003cem\u003eycf2- trnI\u003csup\u003eCAU\u003c/sup\u003e\u003c/em\u003e (Pi = 0.005) (Figure 5A). In the genus \u003cem\u003eStrombocarpa\u003c/em\u003e, the mean estimated Pi value between \u003cem\u003eS. strombulifera\u003c/em\u003e and \u003cem\u003eS. tamarugo\u003c/em\u003e was 0,00522 (ranging from 0 to 0.0433) (Figure 5A);\u0026nbsp;and the regions with the highest values of Pi were \u003cem\u003ematK-rps16\u003c/em\u003e (Pi = 0.0433), \u003cem\u003etrnK-psbI\u003c/em\u003e (Pi = 0.0366), \u003cem\u003etrnS\u003csup\u003eGCU\u003c/sup\u003e-trnG\u003csup\u003eGCC\u003c/sup\u003e\u003c/em\u003e (Pi = 0.0233), \u003cem\u003epetN-psbM\u003c/em\u003e (Pi = 0.0216), \u003cem\u003epsaB-psaA\u003c/em\u003e (Pi = 0.0216), \u003cem\u003erbcL-accD\u003c/em\u003e (Pi = 0.0350), \u003cem\u003epsbE-petG\u003c/em\u003e (Pi = 0.0383), \u003cem\u003erpoA-rpl36\u003c/em\u003e (Pi = 0.0250), \u003cem\u003erps7-ndhB\u003c/em\u003e (Pi = 0.0200) and \u003cem\u003eycf2\u003c/em\u003e (Pi = 0.0416) (Figure 5B).\u0026nbsp;Among \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003e species, the mean estimated Pi value was 0,00759 (ranging from 0 to 0.0627); and the regions with the highest values of Pi were \u003cem\u003ematK-rps16\u003c/em\u003e (Pi = 0.0563), \u003cem\u003etrnQ\u003csup\u003eUUG\u003c/sup\u003e-trnS\u003csup\u003eGCU\u003c/sup\u003e\u003c/em\u003e (Pi = 0.0472), \u003cem\u003etrnS\u003csup\u003eGCU\u003c/sup\u003e-atpA\u003c/em\u003e (Pi = 0.0627), \u003cem\u003epetN-trnD\u003csup\u003eGUC\u003c/sup\u003e\u003c/em\u003e (Pi = 0.0288), \u003cem\u003epsaB-psaA\u003c/em\u003e (Pi = 0.0444), \u003cem\u003eatpB-rbcL\u003c/em\u003e (Pi = 0.0541),\u0026nbsp;\u003cem\u003epsbL-petL\u003c/em\u003e (Pi = 0.0361),\u0026nbsp;\u003cem\u003erps18-rps12\u003c/em\u003e (Pi = 0.0341), \u003cem\u003endhE-rrn23S\u003c/em\u003e (Pi = 0.0277), \u003cem\u003endhB\u003c/em\u003e (Pi = 0.0255)\u0026nbsp;and \u003cem\u003eycf2\u003c/em\u003e (Pi = 0.0347) (Figure 5C).\u0026nbsp;For all \u003cem\u003eMimoseae\u003c/em\u003e species used in this study, the mean estimated Pi value was 0,01236 (ranging from 0 to 0.1171); and ~31 regions with Pi values greater than 0.02 were observed (Figure 5D).\u003c/p\u003e\n\u003cp\u003eThe results of the ML and IB trees had similar topologies when we compared 80 protein-coding genes of the plastid genomes of the nine \u003cem\u003eMimosacea\u003c/em\u003e species (Figure 6).\u0026nbsp;The ML phylogenetic analysis revealed four clades, one joined \u003cem\u003eS.\u003c/em\u003e \u003cem\u003etamarugo\u003c/em\u003e and \u003cem\u003eS. strombulifera\u003c/em\u003e (BP=100), the second clade contained \u003cem\u003eN. glandulosa\u003c/em\u003e, \u003cem\u003eN. juliflora\u003c/em\u003e, \u003cem\u003eP. cineraria\u003c/em\u003e, \u003cem\u003eN. chilensis\u003c/em\u003e and \u003cem\u003eN. alba\u003c/em\u003e (BP=100), the third clade was formed by \u003cem\u003eP. farcta\u003c/em\u003e, and the fourth clade was formed by the outgroup \u003cem\u003eL.\u003c/em\u003e \u003cem\u003etrichandra\u003c/em\u003e (Figure 6A).\u0026nbsp;The BI phylogenetic analysis with the nine \u003cem\u003eMimosaceae\u003c/em\u003e species showed high support (PP=1.00) and their topology was identical to ML analysis (Figure 6B). The second clade showed three subclades: one containing \u003cem\u003eN. glandulosa\u003c/em\u003e which was separated from the rest of species with high support (BP=100; PP=1.00), while the second contained \u003cem\u003eN. juliflora\u003c/em\u003e and \u003cem\u003eP. cineraria\u003c/em\u003e (ML, BP=100; BI, PP=1.00), and the third contained \u003cem\u003eN. chilensis\u003c/em\u003e and \u003cem\u003eN. alba\u003c/em\u003e with high support (BP=100; PP=1.00) (Figure 6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDivergence time for the \u003cem\u003eNeltuma\u003c/em\u003e, \u003cem\u003eStrombocarpa\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e species based on the sequence of the chloroplast genomes is shown in Figure 7, and suggests that \u003cem\u003eNetuma\u003c/em\u003e, \u003cem\u003eStrombocarpa\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e species shared a common ancestor around 43.11 Mya (95% highest posterior density (HPD): 37.72-48.07 Mya) in the Eocene. The age estimate for the split between \u003cem\u003eProsopis\u003c/em\u003e in the Old World and the new world species was 33.52 Mya (95% HPD: 29.48-37.70 Mya) in the early Oligocene. \u003cem\u003eStrombocarpa\u003c/em\u003e and \u003cem\u003eNeltuma\u003c/em\u003e genera diverged in the New World around 22.32 Mya (95% HPD: 19.55-25.08 Mya) in the early Miocene (Figure 7). Within of the genus \u003cem\u003eStrombocarpa\u003c/em\u003e, \u003cem\u003eS. strombulifera\u003c/em\u003e and \u003cem\u003eS.\u003c/em\u003e \u003cem\u003etamarugo\u003c/em\u003e diverged around 8.70 Mya (95% HPD: 7.52-9.89 Mya) in the late Miocene (Figure 7). While the species of the genus \u003cem\u003eNeltuma\u003c/em\u003e diverged into two clades much later, around 2.96 Mya (95% HPD: 2.62-3.33 Mya) in the Pliocene (Figure 7). One clade containing \u003cem\u003eN. alba\u003c/em\u003e, \u003cem\u003eN. chilensis\u003c/em\u003e and \u003cem\u003eN. glandulosa\u003c/em\u003e diverged 1.12 Mya (95% HPD: 0.85-1.39 Mya) when a subclade formed between \u003cem\u003eN. alba\u003c/em\u003e and \u003cem\u003eN. chilensis\u003c/em\u003e that diverged 0.66 Mya (95% HPD: 0.49-0.84 Mya). The other clade containing \u003cem\u003eP. cineraria\u003c/em\u003e and \u003cem\u003eN.\u003c/em\u003e \u003cem\u003ejuliflora\u003c/em\u003e diverged 1.85 Mya (95% HPD: 1.56-2.12 Mya) (Figure 7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e General features of the \u003cem\u003eNeltuma\u003c/em\u003e, \u003cem\u003eStrombocarpa\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e plastid genomes.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"642\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.683229813664596%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecies\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.267080745341614%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAccession\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSize (bp)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.900621118012422%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGC (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLSC (bp)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSSC (bp)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eIR (bp)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.763975155279503%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eN\u0026deg; Genes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.472049689440993%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein-coding genes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003etRNA genes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003erRNA genes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.683229813664596%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eN. alba\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.267080745341614%\" valign=\"top\"\u003e\n \u003cp\u003eOP672364\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\" valign=\"top\"\u003e\n \u003cp\u003e162,980\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.900621118012422%\" valign=\"top\"\u003e\n \u003cp\u003e35.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e92,300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e18,842\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e25,919\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.763975155279503%\" valign=\"top\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.472049689440993%\" valign=\"top\"\u003e\n \u003cp\u003e83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.683229813664596%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eN. chilensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.267080745341614%\" valign=\"top\"\u003e\n \u003cp\u003eOP672365\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\" valign=\"top\"\u003e\n \u003cp\u003e163,047\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.900621118012422%\" valign=\"top\"\u003e\n \u003cp\u003e35.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e92,356\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e18,853\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e25,919\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.763975155279503%\" valign=\"top\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.472049689440993%\" valign=\"top\"\u003e\n \u003cp\u003e83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.683229813664596%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eN. glandulosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.267080745341614%\" valign=\"top\"\u003e\n \u003cp\u003eNC_026683\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\" valign=\"top\"\u003e\n \u003cp\u003e163,040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.900621118012422%\" valign=\"top\"\u003e\n \u003cp\u003e35.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e92,322\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e18,880\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e25,919\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.763975155279503%\" valign=\"top\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.472049689440993%\" valign=\"top\"\u003e\n \u003cp\u003e83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.683229813664596%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eN. juliflora\u0026nbsp;\u003c/em\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.267080745341614%\" valign=\"top\"\u003e\n \u003cp\u003eMN104889\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\" valign=\"top\"\u003e\n \u003cp\u003e163,237\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.900621118012422%\" valign=\"top\"\u003e\n \u003cp\u003e35.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e92,495\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e18,880\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e25,931\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.763975155279503%\" valign=\"top\"\u003e\n \u003cp\u003e132\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.472049689440993%\" valign=\"top\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.683229813664596%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eS. strombulifera\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.267080745341614%\" valign=\"top\"\u003e\n \u003cp\u003eOP672366\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\" valign=\"top\"\u003e\n \u003cp\u003e160,569\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.900621118012422%\" valign=\"top\"\u003e\n \u003cp\u003e36.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e89,569\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e18,623\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e26,026\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.763975155279503%\" valign=\"top\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.472049689440993%\" valign=\"top\"\u003e\n \u003cp\u003e83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.683229813664596%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eS. tamarugo\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.267080745341614%\" valign=\"top\"\u003e\n \u003cp\u003eMW582314\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\" valign=\"top\"\u003e\n \u003cp\u003e161,575\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.900621118012422%\" valign=\"top\"\u003e\n \u003cp\u003e36.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e91,062\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e18,643\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e25,935\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.763975155279503%\" valign=\"top\"\u003e\n \u003cp\u003e127\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.472049689440993%\" valign=\"top\"\u003e\n \u003cp\u003e82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.683229813664596%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eP. cineraria\u003c/em\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.267080745341614%\" valign=\"top\"\u003e\n \u003cp\u003eMN104890\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\" valign=\"top\"\u003e\n \u003cp\u003e163,677\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.900621118012422%\" valign=\"top\"\u003e\n \u003cp\u003e35.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e92,937\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e18,878\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e25,931\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.763975155279503%\" valign=\"top\"\u003e\n \u003cp\u003e131\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.472049689440993%\" valign=\"top\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.683229813664596%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eP. farcta\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.267080745341614%\" valign=\"top\"\u003e\n \u003cp\u003eMZ073639\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\" valign=\"top\"\u003e\n \u003cp\u003e162,900\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.900621118012422%\" valign=\"top\"\u003e\n \u003cp\u003e35.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e92,156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e18,880\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.919254658385094%\" valign=\"top\"\u003e\n \u003cp\u003e25,932\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.763975155279503%\" valign=\"top\"\u003e\n \u003cp\u003e127\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.472049689440993%\" valign=\"top\"\u003e\n \u003cp\u003e82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.074534161490684%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e(*) values of features of the species are described by Asaf et al (2020)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e. Gene composition of the plastid genome of \u003cem\u003eN. alba, N. chilensis and S. strombulifera\u003c/em\u003e.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"495\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003eCategory of genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003eGroup of genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003eNames of genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003eN\u0026deg;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003ePhotosynthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003ePhotosystem I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003epsaA, psaB, psaC, psaI, psaJ\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003ePhotosystem II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003epsbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003eATP synthase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eatpA, atpB, atpE, atpF\u003csup\u003eb\u003c/sup\u003e, atpH, atpI\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003eNADH-dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003endhA\u003csup\u003eb\u003c/sup\u003e, ndhB\u003csup\u003eab\u003c/sup\u003e, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003ecytochrome b/f complex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003epetA, petB\u003csup\u003eb\u003c/sup\u003e, petD\u003csup\u003eb\u003c/sup\u003e, petG, petL, petN\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003eLarge subunit RUBISCO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003erbcL\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003eProtein synthesis and DNA replication\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003eTransfer RNAs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003etrnA-UGC\u003csup\u003eab\u003c/sup\u003e, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-UCC, trnG-GCC\u003csup\u003eb\u003c/sup\u003e, trnH-GUG, trnI-GAU\u003csup\u003eab\u003c/sup\u003e, trnI-CAU\u003csup\u003ea\u003c/sup\u003e, trnK-UUU\u003csup\u003eb\u003c/sup\u003e, trnL-UAA\u003csup\u003eb\u003c/sup\u003e, trnL-CAA\u003csup\u003ea\u003c/sup\u003e, trnL-UAG, trnM-CAU, trnN-GUU\u003csup\u003ea\u003c/sup\u003e, trnP-UGG, trnQ-UUG, trnR-ACG\u003csup\u003ea\u003c/sup\u003e, trnR-UCU, trnS-GGA, trnS-UGA, trnS-GCU, trnT-GGU, trnT-UGU, trnV-UAC\u003csup\u003eb\u003c/sup\u003e, trnV-GAC\u003csup\u003ea\u003c/sup\u003e, trnW-CCA, trnY-GUA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003eRibosomal RNAs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003errn16S\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e, rrn23S\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e, rrn4.5S\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e, rrn5S\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003eRibosomal Protein large-subunit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003erpl14, rpl2\u003csup\u003eab\u003c/sup\u003e, rpl16\u003csup\u003eb\u003c/sup\u003e, rpl20, rpl23\u003csup\u003ea\u003c/sup\u003e, rpl32, rpl33, rpl36\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003eDNA dependent RNA polymerase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003erpoA, rpoB, rpoC1\u003csup\u003eb\u003c/sup\u003e, rpoC2\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003eRibosomal Protein Small-subunit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003erps11, rps12\u003csup\u003eac\u003c/sup\u003e, rps14, rps15, rps16\u003csup\u003eb\u003c/sup\u003e, rps18, rps19, rps2, rps3, rps4, rps7\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e, rps8\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003eOther functions\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003eSubunit of Acetyl-CoA-carboxylase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eaccD\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003ec-type cytochrome synthesis gene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eccsA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003eEnvelop membrane protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ecemA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003eProtease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eclpP \u003csup\u003ec\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003eMaturase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ematK\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003eInitiation Factor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003einfA\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003eUnknown function\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003eConserved open reading frames\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eycf1, ycf2\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e, ycf3 \u003csup\u003ec\u003c/sup\u003e, ycf4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.22222222222222%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.535353535353536%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eTotal\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.8686868686868685%\" valign=\"top\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Duplicated genes; \u003csup\u003eb\u003c/sup\u003e Genes containing introns; \u003csup\u003ec\u003c/sup\u003e Genes containing two introns\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eGenomic research with NGS technology has developed rapidly, allowing efficient sequencing of complete plastid genomes \u003csup\u003e54\u003c/sup\u003e. Molecular differences in the complete chloroplast genome between species and individuals provide a good means of comparison \u003csup\u003e55\u003c/sup\u003e. The cp genome offers several advantages over the nuclear genome, such as unique haploid structure, structural conservation, maternal inheritance, and moderate rate of evolution \u003csup\u003e55,56\u003c/sup\u003e. In our comparative study of the plastid genomes of \u003cem\u003eN. alba\u003c/em\u003e, \u003cem\u003eN. chilensis\u003c/em\u003e, and \u003cem\u003eS. strombulifera\u003c/em\u003e analyzing gene content, structure, divergence time, and phylogeny we found that the complete chloroplast genomes of \u003cem\u003eN. alba\u003c/em\u003e and \u003cem\u003eN. chilensis\u003c/em\u003e are conserved in size compared to species of the \u003cem\u003eStrombocarpa\u003c/em\u003e genus. The chloroplast of \u003cem\u003eN. alba\u003c/em\u003e and \u003cem\u003eN. chilensis\u003c/em\u003e showed similar values for genome size and the number of genes compared to \u003cem\u003eNeltuma juliflora\u003c/em\u003e and \u003cem\u003eNeltuma glandulosa\u003c/em\u003e described by Asaf\u003csup\u003e57\u003c/sup\u003e, ~\u0026thinsp;163.000 bp for both, while the number of genes varied between 131 and 128. The number of genes was similar between the \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003e genera, although \u003cem\u003eS. tamarugo\u003c/em\u003e lost the gene \u003cem\u003epsbL\u003c/em\u003e (remaining with 127 genes only) \u003csup\u003e25\u003c/sup\u003e. The absence of the \u003cem\u003epsbL\u003c/em\u003e gene has been observed in some other eudicots, magnoliids, and monocots as well \u003csup\u003e58\u003c/sup\u003e. The genome sizes of \u003cem\u003eS. strombulifera\u003c/em\u003e (160,569 bp) and \u003cem\u003eS. tamarugo\u003c/em\u003e (161,575 bp) were smaller compared to the \u003cem\u003eNeltuma\u003c/em\u003e species (~\u0026thinsp;163.000 bp) \u003csup\u003e25\u003c/sup\u003e. However, the \u003cem\u003eStrombocarpa\u003c/em\u003e species presented slightly more GC content (36.0%-36.2%) compared to the \u003cem\u003eNeltuma\u003c/em\u003e species (35.9%). These GC values fall within the limit of variation registered in others studies \u003csup\u003e25,57\u003c/sup\u003e. Furthermore, a study about several orchid species, showed that the species with a smallest chloroplast size (\u003cem\u003ePholidota cantonensis\u003c/em\u003e, 158,786 bp), had a highest GC content (37.47%) \u003csup\u003e59\u003c/sup\u003e, similar to our observations. The chloroplast genome tends to reduce its size during evolution \u003csup\u003e60\u003c/sup\u003e, and gene length might be affected by selection during the evolution of spermatophytes \u003csup\u003e61\u003c/sup\u003e. The variations in chloroplast genome size among closely related species can be attributed to IRs, LSC, SSC, intergenic regions, and gene numbers \u003csup\u003e61\u003c/sup\u003e. In this study, very little variation in IRs and intergenic regions was observed between \u003cem\u003eN. alba\u003c/em\u003e and \u003cem\u003eN. chilensis\u003c/em\u003e, resulting in very few differences in genome size, while there is a large variation in these regions in the genomes of \u003cem\u003eS. strombulifera\u003c/em\u003e and \u003cem\u003eS. tamarugo\u003c/em\u003e. Therefore, we assume that \u003cem\u003eStrombocarpa\u003c/em\u003e species have been exposed to stronger evolution than \u003cem\u003eNeltuma\u003c/em\u003e species.\u003c/p\u003e \u003cp\u003eA total of 70 to 100 chloroplast simple sequence repeats (cpSSRs) were founded in the cp genomes of the species of the \u003cem\u003eNeltuma\u003c/em\u003e, \u003cem\u003eStrombocarpa\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e genera. Our results showed high variation values in the number of cpSSRs among \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003e species, being the highest for \u003cem\u003eN. juliflora\u003c/em\u003e (100) and the lowest for \u003cem\u003eS. tamarugo\u003c/em\u003e (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e). The most abundant cpSSR motif types in \u003cem\u003eNeltuma\u003c/em\u003e, \u003cem\u003eStrombocarpa\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e were mono-nucleotides, which is the most abundant repeat type in angiosperms cp genomes \u003csup\u003e62\u003c/sup\u003e. Only \u003cem\u003eStrombocarpa\u003c/em\u003e species did not show mononucleotide C/G motifs, nor dinucleotide motifs and additionally, they had a lower number of trinucleotide AAT/ATT motifs. However, the \u003cem\u003eStrombocarpa\u003c/em\u003e species were the only species that presented the pentanucleotide AATAG/ATTCT motifs. It has been shown in \u003cem\u003eCyatheaceae\u003c/em\u003e, that the characteristics of cpSSRs can provide useful phylogenetic information at the genus level, such as phylogenetic relationships, but also about the number, relative abundance, motif type and relative density of cpSSRs \u003csup\u003e63\u003c/sup\u003e. In a similar way, our results demonstrate that the cpSSRs among \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003e, both in number and cpSSR motifs, are likely genus specific.\u003c/p\u003e \u003cp\u003eRepeat sequences are considered to play an important role in genome recombination, rearrangements and contain fundamental phylogenetic information \u003csup\u003e64,65\u003c/sup\u003e. We found differences in the repeated elements of the cp genome between \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003e species. The highest total number of repeat elements (palindrome, forward, reverse and complement) was found in \u003cem\u003eS. tamarugo\u003c/em\u003e (88) and the lowest in \u003cem\u003eS. strombulifera\u003c/em\u003e (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). In general, the total number of palindromic repeats was less in \u003cem\u003eStrombocarpa\u003c/em\u003e species than in \u003cem\u003eNeltuma\u003c/em\u003e species. However, the total number of forward repeats was less in \u003cem\u003eN. alba\u003c/em\u003e and \u003cem\u003eN. chilensis\u003c/em\u003e than in the \u003cem\u003eStrombocarpa\u003c/em\u003e species. On the other hand, the number of complement and reverse (range of 30\u0026ndash;39 bp) repeats in \u003cem\u003eS. tamarugo\u003c/em\u003e was higher than in the \u003cem\u003eNeltuma\u003c/em\u003e species. In the majority of the species in this study, the most abundant repeat elements detected were, in order, forward, palindromic and reverse. This corresponds to other studies about cp genomes of mimosoid species \u003csup\u003e66,67\u003c/sup\u003e, although \u003cem\u003eS. tamarugo\u003c/em\u003e is an exception in terms of reverse and complements repeats numbers.\u003c/p\u003e \u003cp\u003eThroughout of the evolution of plastid genomes, structural rearrangements occur, for example in the IRs, which are frequently subject to expansion, contraction or even complete loss \u003csup\u003e68\u003c/sup\u003e. An increased length of IR-SSC boundaries plays an important role in mimosoid plastome size variation \u003csup\u003e69\u003c/sup\u003e. For example, eight mimosoid plastomes of the tribe \u003cem\u003eAcacia\u003c/em\u003e and \u003cem\u003eInga\u003c/em\u003e exhibited an unusual 13 kb IR-SSC boundary shift into the SSC region \u003csup\u003e67,69\u003c/sup\u003e, and the size of these plastomes was found significantly affected by a IR-SC boundary shift, as well as by repeat content \u003csup\u003e67\u003c/sup\u003e. We observed a slight IR expansion into SSC in \u003cem\u003eS. strombulifera\u003c/em\u003e (26.026 bp) and \u003cem\u003eS. tamarugo\u003c/em\u003e (25.935 bp) in comparison to the \u003cem\u003eNetuma\u003c/em\u003e species. Therefore, the SSC regions of the \u003cem\u003eStrombocarpa\u003c/em\u003e species showed contraction, and were the shortest SSC regions compared with those of the \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e genera. Asaf et al \u003csup\u003e57\u003c/sup\u003e did not detect IR expansion in \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e species, however, they detected a slight expansion in the outgroup species of the genus \u003cem\u003eAdenanthera\u003c/em\u003e (with a length of 26,028 bp), similar to what we found the in \u003cem\u003eStrombocarpa\u003c/em\u003e species. The study of Asaf Asaf et al \u003csup\u003e57\u003c/sup\u003e did not, however, include \u003cem\u003eStrombocarpa\u003c/em\u003e species to compare to the \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e species. Similar to Asaf \u003csup\u003e57\u003c/sup\u003e, we found a partially duplicated \u003cem\u003erps19\u003c/em\u003e gene at the beginnings and ends of the IR regions in \u003cem\u003eN. alba\u003c/em\u003e, \u003cem\u003eN. chilensis\u003c/em\u003e, \u003cem\u003eS. strombulifera\u003c/em\u003e and \u003cem\u003eS. tamarugo\u003c/em\u003e (including 91 bp in IR). In of most \u003cem\u003eMimosoideae\u003c/em\u003e species, the \u003cem\u003erps19\u003c/em\u003e is located in the LSC/IRB junction (JLB), with 98\u0026ndash;109 bp of the 5\u0026prime; end of this gene into the IR region \u003csup\u003e67\u003c/sup\u003e. The \u003cem\u003endhF\u003c/em\u003e gene was located closer to the IRB-SSC border (JSB) in \u003cem\u003eStrombocarpa\u003c/em\u003e species (up to 67 bp) than in \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e species (137 to 156 bp). Likewise, the \u003cem\u003endhF\u003c/em\u003e gene in the species of the genera \u003cem\u003eAdenanthera\u003c/em\u003e, \u003cem\u003eParkia\u003c/em\u003e, \u003cem\u003ePiptadenia\u003c/em\u003e, \u003cem\u003eLeucaena\u003c/em\u003e and \u003cem\u003eDichrostachys\u003c/em\u003e (\u003cem\u003eMimosoideae\u003c/em\u003e) was found entirely within the SSC region (ranging 11 to 150 away from the JSB junction), however, in species of the tribe \u003cem\u003eAcacia\u003c/em\u003e and \u003cem\u003eInga\u003c/em\u003e (\u003cem\u003eMimosoideae\u003c/em\u003e) it was found within the JSB junction, resulting in the duplication of this gene \u003csup\u003e67\u003c/sup\u003e. Several models concerning the expansion and contraction of IR regions have been proposed to explain the possible mechanisms that result in shifts in the IR-LSC junctions \u003csup\u003e70\u003c/sup\u003e. In our case, we detected that \u003cem\u003eStrombocarpa\u003c/em\u003e species had a larger contraction of the LSC region then \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e species. The structural differences presented among the plastomes of the \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003e species reinforce the idea and necessity to disintegrate the \u003cem\u003eProsopis\u003c/em\u003e cluster, as proposed by Hughes et al \u003csup\u003e5\u003c/sup\u003e. However, for the new genera it would have been recommendable to have kept the names of the sections (\u003cem\u003eAlgarobia\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003e, as proposed by Burkart \u003csup\u003e4\u003c/sup\u003e for the new genera.\u003c/p\u003e \u003cp\u003eThe nucleotide diversity (Pi) analysis of \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003e plastomes showed more variations in the LSC and SSC regions than the IR regions. In addition, strong differences of nucleotide diversity value were found between \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003e species. The Pi values between \u003cem\u003eNeltuma\u003c/em\u003e species were so low that we found only three variable regions (\u003cem\u003erps16-trnQ\u003c/em\u003e\u003csup\u003eUUG\u003c/sup\u003e, \u003cem\u003eaccD-psaI\u003c/em\u003e and \u003cem\u003eycf2- trnI\u003c/em\u003e\u003csup\u003eCAU\u003c/sup\u003e), whereas in \u003cem\u003eStrombocarpa\u003c/em\u003e species we found ten regions with high Pi values (\u003cem\u003ematK-rps16\u003c/em\u003e, \u003cem\u003etrnK-psbI\u003c/em\u003e, \u003cem\u003etrnS\u003c/em\u003e\u003csup\u003eGCU\u003c/sup\u003e-\u003cem\u003etrnG\u003c/em\u003e\u003csup\u003eGCC\u003c/sup\u003e, \u003cem\u003epetN-psbM\u003c/em\u003e, \u003cem\u003epsaB-psaA\u003c/em\u003e, \u003cem\u003erbcL-accD\u003c/em\u003e, \u003cem\u003epsbE-petG\u003c/em\u003e, \u003cem\u003erpoA-rpl36\u003c/em\u003e, \u003cem\u003erps7-ndhB\u003c/em\u003e and \u003cem\u003eycf2\u003c/em\u003e). We believe that these ten highly variable regions found in \u003cem\u003eStrombocarpa\u003c/em\u003e species, can be of use to resolve uncertainties in phylogenetic analysis of the genus, as well as for DNA barcoding. However, as a very low number of variable regions was found in the species of the \u003cem\u003eNeltuma\u003c/em\u003e genus, it will be necessary for further studies to include a sufficient number of samples in order to identify the best regions for identification within the genus \u003cem\u003eNeltuma\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe phylogenetic results (ML and BI) based on 80 protein-coding genes of the plastid genome of nine \u003cem\u003eMimosoideae\u003c/em\u003e species showed that \u003cem\u003eS. strombulifera\u003c/em\u003e formed a strongly supported group with \u003cem\u003eS. tamarugo\u003c/em\u003e (BP\u0026thinsp;=\u0026thinsp;100; PP\u0026thinsp;=\u0026thinsp;1.00), and the \u003cem\u003eNeltuma\u003c/em\u003e group appeared as paraphyletic because \u003cem\u003eP. cineraria\u003c/em\u003e was part of a well-supported clade (BP\u0026thinsp;=\u0026thinsp;62; PP\u0026thinsp;=\u0026thinsp;1.00) with \u003cem\u003eN. juliflora\u003c/em\u003e, \u003cem\u003eN. alba\u003c/em\u003e and \u003cem\u003eN. chilensis\u003c/em\u003e. \u003cem\u003eP. farcta\u003c/em\u003e, however appeared as sister group of \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003e clade, as expected. Within the \u003cem\u003eNeltuma\u003c/em\u003e clade, \u003cem\u003eN. alba\u003c/em\u003e formed a highly supported clade with \u003cem\u003eN. chilensis\u003c/em\u003e (BP\u0026thinsp;=\u0026thinsp;100; PP\u0026thinsp;=\u0026thinsp;1.00), and so did \u003cem\u003eN. juliflora\u003c/em\u003e with \u003cem\u003eP. cineraria\u003c/em\u003e (BP\u0026thinsp;=\u0026thinsp;100; PP\u0026thinsp;=\u0026thinsp;1.00), whereas \u003cem\u003eN. glandulosa\u003c/em\u003e appeared as a strongly supported sister group to both (BP\u0026thinsp;=\u0026thinsp;100; PP\u0026thinsp;=\u0026thinsp;1.00). With the exception of \u003cem\u003eP. cineraria\u003c/em\u003e (further discussed in the next paragraph), the \u003cem\u003eNeltuma\u003c/em\u003e group was monophyletic with \u003cem\u003eStrombocarpa\u003c/em\u003e group as its sister clade. Although \u003cem\u003eS. strombulifera\u003c/em\u003e and \u003cem\u003eS. tamarugo\u003c/em\u003e formed a well-supported group, these two species showed important differences in genome size, number of genes and nucleotide diversity with high degree of variation. These genetic differences in the chloroplast correspond to the findings of Burkart \u003csup\u003e4\u003c/sup\u003e who separated \u003cem\u003eS. tamarugo\u003c/em\u003e and \u003cem\u003eS. strombulifera\u003c/em\u003e into the \u003cem\u003eCavernicarpae\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003ee series, respectively. The same was observed by Catalano et al \u003csup\u003e13\u003c/sup\u003e through a three-marker analysis (\u003cem\u003etrnS-psbC\u003c/em\u003e, \u003cem\u003eG3pdh\u003c/em\u003e, \u003cem\u003eNIA\u003c/em\u003e), who found two well supported groups, one of them corresponding to the \u003cem\u003eCavenicarpae\u003c/em\u003e series (including \u003cem\u003eProsopis ferox\u003c/em\u003e and \u003cem\u003eP. tamarugo\u003c/em\u003e) and the other formed by North American species of the \u003cem\u003eStrombocarpa\u003c/em\u003ee series (including \u003cem\u003eProsopis pubescens\u003c/em\u003e and \u003cem\u003eProsopis palmeri\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eUndoubtedly, the biggest inconsistency observed in our phylogenetic analysis was the nesting of \u003cem\u003eP. cineraria\u003c/em\u003e within the \u003cem\u003eNeltuma\u003c/em\u003e clade. According to the results of Asaf et al \u003csup\u003e57\u003c/sup\u003e, \u003cem\u003eP. cineraria\u003c/em\u003e forms a group with high support with \u003cem\u003eN. juliflora\u003c/em\u003e. It is interesting and unexpected that \u003cem\u003eP. cineraria\u003c/em\u003e did not form a group with \u003cem\u003eP. farcta\u003c/em\u003e, both of them being Old World species, but nested with \u003cem\u003eN. juliflora\u003c/em\u003e, \u003cem\u003eN. glandulosa\u003c/em\u003e, \u003cem\u003eN. alba\u003c/em\u003e and \u003cem\u003eN. chilensis\u003c/em\u003e, which are New World species. However, according to the phylogenetic analysis performed by Catalano et al\u003csup\u003e13\u003c/sup\u003e, there are more distant relationships among species from the Old World sections and closer relationships among species of the American sections (\u003cem\u003eStrombocarpa\u003c/em\u003e, \u003cem\u003eAlgarobia\u003c/em\u003e, and \u003cem\u003eMonilicarpa\u003c/em\u003e sections). \u003cem\u003eProsopis cineraria\u003c/em\u003e is one of the most common trees of the Indian desert, Arabian Peninsula and, in general, is abundant throughout the middle east \u003csup\u003e57,71\u003c/sup\u003e, whereas \u003cem\u003eN. juliflora\u003c/em\u003e is native to the Caribbean, Central and northern South America \u003csup\u003e72\u003c/sup\u003e. However, \u003cem\u003eNeltuma juliflora\u003c/em\u003e was introduced to Ethiopia and the Middle East around 1970 and over the years this species has spread outside the plantation areas, adversely affecting natural habitats and rangelands \u003csup\u003e73\u003c/sup\u003e. This invasive plant is characterized by vigorous growth which helps it to outcompete indigenous plant species \u003csup\u003e74\u003c/sup\u003e. \u003cem\u003eNeltuma juliflora\u003c/em\u003e seeds survive in livestock and warthogs\u0026rsquo; droppings, which serve as a vehicle for the plant to reach distant areas and to expand their distribution throughout the region \u003csup\u003e74,75\u003c/sup\u003e. We hypothesize that \u003cem\u003eN. juliflora\u003c/em\u003e might have crossed with some individuals of \u003cem\u003eP. cineraria\u003c/em\u003e in a natural way, giving offspring to a hybrid with a phenotype resembling \u003cem\u003eP. cineraria\u003c/em\u003e but, when \u003cem\u003eN. juliflora\u003c/em\u003e acted as the maternal part, with the cp genome of \u003cem\u003eN. juliflora\u003c/em\u003e. This could be a logic explanation for the nesting of \u003cem\u003eP. cineraria\u003c/em\u003e within the \u003cem\u003eNeltuma\u003c/em\u003e clade, if the samples used by Asaf et al. \u003csup\u003e57\u003c/sup\u003e were obtained from a \u003cem\u003eP. cineraria\u003c/em\u003e resembling hybrid.\u003c/p\u003e \u003cp\u003eEstimate of divergence time in plant groups have been important in order to understand their phylogeographic history and evolutionary biology \u003csup\u003e76\u003c/sup\u003e. Our molecular dating analysis suggests that \u003cem\u003eLeucaena trichandra\u003c/em\u003e as root species diverged in the Middle Eocene (mean\u0026thinsp;=\u0026thinsp;43.11 Mya; 95% HPD\u0026thinsp;=\u0026thinsp;37.72\u0026ndash;48.07 Mya). Later, \u003cem\u003eP. farcta\u003c/em\u003e diverged in the Early Oligocene (mean\u0026thinsp;=\u0026thinsp;33.52 Mya; 95% HPD\u0026thinsp;=\u0026thinsp;29.48\u0026ndash;37.70 Mya), while \u003cem\u003eP. cineraria\u003c/em\u003e diverged together with the \u003cem\u003eNeltuma\u003c/em\u003e species in the Pleistocene. Sudalaimuthuasari et al \u003csup\u003e71\u003c/sup\u003e, using whole genome sequencing with 76,554 genes, estimated that \u003cem\u003eP. cineraria\u003c/em\u003e and \u003cem\u003eP. alba\u003c/em\u003e diverged\u0026thinsp;~\u0026thinsp;23Mya. Undoubtedly, this divergence time is closer to what would be expected for species of the genus \u003cem\u003eProsopis\u003c/em\u003e that belongs to the Old World, but not for \u003cem\u003eP. cineraria\u003c/em\u003e, whose complete chloroplast genome data show a divergence time of 1.85 Mya (95% HPD: 1.56\u0026ndash;2.12 Mya), strengthening our suspicion of this sample being a natural hybrid. Although a previous study indicates that the divergence between \u003cem\u003eStrombocarpa\u003c/em\u003e and \u003cem\u003eNeltuma\u003c/em\u003e genera occurred in the Oligocene \u003csup\u003e13\u003c/sup\u003e, our results show that these genera diverged in the early Miocene (mean\u0026thinsp;=\u0026thinsp;22.32 Mya; 95% HPD\u0026thinsp;=\u0026thinsp;19.55\u0026ndash;25.08 Mya). The molecular divergence time found in \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003e genera is close to the diversification of the major clades in the subfamily \u003cem\u003eMimosoideae\u003c/em\u003e, which occurred in the Late Miocene \u003csup\u003e13,50\u003c/sup\u003e. Our results showed that \u003cem\u003eStrombocarpa\u003c/em\u003e diverged in the Late Miocene (mean\u0026thinsp;=\u0026thinsp;8.70 Mya; 95% HPD\u0026thinsp;=\u0026thinsp;7.52\u0026ndash;9.89 Mya), which is supported by the fossil \u003cem\u003eProsopisinoxylon anciborae\u003c/em\u003e, a \u003cem\u003eMimosoideae\u003c/em\u003e species with a high similarity to genus \u003cem\u003eProsopis\u003c/em\u003e L. (currently re-delimitated), reported to have occurred during the Late Miocene in the Catamarca Province, Argentina \u003csup\u003e77\u003c/sup\u003e. Additionally, a similar divergence time, around 9.21 Mya (8.35\u0026ndash;10.07), for the genus \u003cem\u003eStrombocarpa\u003c/em\u003e was found Catalano et al \u003csup\u003e13\u003c/sup\u003e. Our results also showed that the \u003cem\u003eNeltuma\u003c/em\u003e genus started diverging in the Pliocene (mean\u0026thinsp;=\u0026thinsp;2.96 Mya; 95% HPD\u0026thinsp;=\u0026thinsp;2.62\u0026ndash;3.33 Mya) and continued in the Pleistocene. This corresponds to the Mesquite species (e.g. \u003cem\u003eN. alba\u003c/em\u003e, \u003cem\u003eN. juliflora\u003c/em\u003e, \u003cem\u003eN. glandulosa\u003c/em\u003e, \u003cem\u003eN. chilensis\u003c/em\u003e, \u003cem\u003eN. alpataco\u003c/em\u003e and \u003cem\u003eN. nigra\u003c/em\u003e) whose divergence time started in the Pliocene and continued in the Pleistocene, (mean\u0026thinsp;=\u0026thinsp;3.65 Mya; 95% HPD\u0026thinsp;=\u0026thinsp;3.31\u0026ndash;3.99 Mya) \u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTree species such as \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003e are subject to a number of ecological selective pressures due to the hostile conditions of the Atacama Desert. Chloroplast genes are involved in regulatory responses to various abiotic stresses, including heat, chilling, salinity, drought and radiation \u003csup\u003e78,79\u003c/sup\u003e. Therefore, the here presented chloroplast genomes of the \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003e species can play an important role in understanding the plants adaptations to these hostile environments.\u003c/p\u003e \u003cp\u003eThe chloroplast genome structure of legumes is particularly interesting, because it contains multiple rearrangements, expansions, contractions, and loss of genetic content, which are all very useful for phylogenetic studies \u003csup\u003e79\u003c/sup\u003e. Phylogenetic analysis can aid conservation of species through the confirmation of taxonomic status, clarification of evolutionary relationships and consequently the determination fo conservation priorities \u003csup\u003e80\u003c/sup\u003e. Additionally, phylogeographic studies offer valuable information for conservation purposes as they describe the geographical distribution of genetic variability among species populations \u003csup\u003e81\u003c/sup\u003e. With this study, we discovered differences in chloroplast genomes of \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003e, species improving our understanding of its phylogeny and evolution, in hope to aid the conservation of these valuable species before it is too late and they disappear.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this work, we present for the first time the assembly and characterization of the chloroplast genomes of \u003cem\u003eNeltuma alba\u003c/em\u003e, \u003cem\u003eNeltuma chilensis\u003c/em\u003e and \u003cem\u003eStrombocarpa strombulifera\u003c/em\u003e. We found enough variation in genome size, GC content, repetitive elements and nucleotide diversity to support the disintegration of the former genus \u003cem\u003eProsopis\u003c/em\u003e L. The chloroplasts presented in this study provide a better understanding of the diversification of \u003cem\u003eNeltuma\u003c/em\u003e, \u003cem\u003eStrombocarpa\u003c/em\u003e and \u003cem\u003eProsopis\u003c/em\u003e as well as important information for evolutionary, phylogenomic and biogeographic studies for other species of the \u003cem\u003eFabaceae\u003c/em\u003e family.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRC thanks projects ANID-FONDECYT initiation 11230668. RC and FC thanks AFOREST, a Millenium Nucleus supported by ANID \u0026ndash; MILENIO \u0026ndash; NCS2022_024. WH thanks \u0026quot;Convenio ESR UTA2295\u0026quot;. RC thanks Neri Contreras-Ascencio and Ana D\u0026iacute;az-Sanchez for their sampling support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the \u003cstrong\u003eANID-FONDECYT Initiation\u003c/strong\u003e into Research grant 11230668 and by the Universidad de Atacama (DIUDA 22423 project). PJ was funded by the German Research Foundation (DFG) with the grant number JU 3228/1-1. LvdB was funded by ANID PIA/ACT 210038.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eR.C., L.vdB., F.C.: data analyses, writing manuscript. R.C., L.vdB., F.C., W.H., P.J.: data interpretation, writing and editing manuscript. R.C., F.C., L.vdB: experimental analysis. R.C., L.vdB., W.H.: Analysis and interpretation of result. R.C., L.vdB., P.J.: editing-review original draft. All the authors have approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest/Competing interests\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThere is no conflict of interest between the authors of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch Permit\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research complies with the corresponding research permits according to national and international standards, for the collection of material from Neltuma alba, Neltuma chilensis and Strombocarpa strombulifera, and the care of flora and fauna. The research permit was granted by CONAF (National Forestry Corporation) N\u0026deg; N00024/08-11-2019 (JBH/FAP/JVO) and N\u0026deg; N00003-2023/27-01-2023 (NOO/FAP/JVO).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll the authors of this manuscript declare that we participated in the design and preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors authorize the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available in the Genome Database on National Center for Biotechnology Information (NCBI) repository under the accession number OP672364 for Neltuma alba, OP672365 for Neltuma chilensis and OP672366 for Strombocarpa strombulifera. The BioProject and BioSample accession numbers on NCBI for Neltuma alba are PRJNA1026123 and SAMN37734720, for Neltuma chilensis are PRJNA1026131 and SAMN37735133, and for Strombocarpa strombulifera are PRJNA1026137 and SAMN37735326. The identification of the plant material was carried out by Roberto Contreras-D\u0026iacute;az, according to the keys described by Burkart (1976). It was also confirmed by CONAF professionals and recognized by the researcher Nicol\u0026aacute;s Garc\u0026iacute;a of the Universidad de Chile.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe sequence data is available in GenBank with the codes OP672364, OP672365, OP672366\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLPWG (The Legume Phylogeny Working Group). A new subfamily classification of the leguminosae based on a taxonomically comprehensive phylogeny. 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Conservation and Phylogeography of Plants: From the Mediterranean to the Rest of the World. \u003cem\u003eDiversity\u003c/em\u003e 14, (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3422232/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3422232/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eNeltuma alba\u003c/em\u003e (Algarrobo blanco), \u003cem\u003eNeltuma chilensis\u003c/em\u003e (Algarrobo Chileno) and \u003cem\u003eStrombocarpa strombulifera\u003c/em\u003e (Fortuna) are some of the few trees found in small highly fragmented populations, throughout the Atacama Desert, indicating their drought resistance. We found that the complete chloroplast genomes of \u003cem\u003eN. alba\u003c/em\u003e and \u003cem\u003eN. chilensis\u003c/em\u003e are larger in size compared to species of the \u003cem\u003eStrombocarpa\u003c/em\u003e genus. However, the \u003cem\u003eStrombocarpa\u003c/em\u003e species presented slightly more GC content than the \u003cem\u003eNeltuma\u003c/em\u003e species. Therefore, we assume that \u003cem\u003eStrombocarpa\u003c/em\u003e species have been exposed to stronger evolution than \u003cem\u003eNeltuma\u003c/em\u003e species. We observed high variation values in the number of cpSSRs (chloroplast simple sequence repeats) and repeated elements among \u003cem\u003eNeltuma\u003c/em\u003e and \u003cem\u003eStrombocarpa\u003c/em\u003e species. Very low nucleotide diversity values were found in \u003cem\u003eNeltuma\u003c/em\u003e, while ten highly variable regions found in \u003cem\u003eStrombocarpa\u003c/em\u003e, can likely be used to resolve uncertainties in phylogeny, and for DNA barcoding. Although in general our study supports the phylogeny of other studies, the biggest inconsistency was the nesting of \u003cem\u003eProsopis cineraria\u003c/em\u003e within the \u003cem\u003eNeltuma\u003c/em\u003e clade and showed a divergence time of 1.85 Mya. With this study we provide valuable information about isolated populations of tree species that provide important ecosystem services in hostile environments before they disappear, due to an ongoing fragmentation of their populations.\u003c/p\u003e","manuscriptTitle":"Comparative analysis of the complete plastid genomes of desert trees Neltuma and Strombocarpa genera","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-27 20:21:07","doi":"10.21203/rs.3.rs-3422232/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-01-25T07:41:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-01-19T10:14:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"515d410b-c659-4ad5-9b0c-eb3d346981d0","date":"2024-01-09T08:58:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"b3de438c-a395-49b7-9608-d43cca62650d","date":"2023-12-22T14:05:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-12-21T20:44:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-10-31T08:22:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-10-23T04:17:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-10-23T04:05:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2023-10-08T22:26:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f6659a9c-722f-4b84-8655-db5e8f920042","owner":[],"postedDate":"October 27th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":25568284,"name":"Biological sciences/Genetics/Plant genetics"},{"id":25568285,"name":"Biological sciences/Ecology"},{"id":25568286,"name":"Biological sciences/Genetics"}],"tags":[],"updatedAt":"2024-06-06T14:59:17+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-27 20:21:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3422232","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3422232","identity":"rs-3422232","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}