Comparative Analysis of the Plastid Genomes of Zephyranthes bagnoldii, Zephyranthes sarae, and Paposoa laeta from the Flowering Desert of the Atacama Region, Chile

Comparative Analysis of the Plastid Genomes of Zephyranthes bagnoldii, Zephyranthes sarae, and Paposoa laeta from the Flowering Desert of the Atacama Region, Chile | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative Analysis of the Plastid Genomes of Zephyranthes bagnoldii, Zephyranthes sarae, and Paposoa laeta from the Flowering Desert of the Atacama Region, Chile Mariana Arias-Aburto, Zoë Dennehy-Carr, Liesbeth van den Brink, and 2 more This is a preprint; it has not been peer reviewed by a journal.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-8801993/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background: The Atacama “flowering desert” offers a natural setting to investigate plant genomic diversity and lineage differentiation in endemic geophytes shaped by long-term aridity, fragmented habitats, and ENSO-linked winter rains. We assembled, annotated, and compared complete plastid genomes for three Chilean taxa ( Zephyranthes bagnoldii , Zephyranthes sarae , and Paposoa laeta ) with two objectives: (i) to clarify their relationships within Hippeastreae, and (ii) to identify genome regions and repeat features that can be applied to species identification, population studies, and conservation planning in northern Chile and comparable deserts. Results: All three genomes showed the canonical quadripartite organization with broadly similar sizes (158,144 to 158,678 bp) and gene complements. Whole-genome alignments and synteny comparisons indicated overall collinearity, while boundary visualizations revealed modest shifts at the junctions between the large single-copy, small single-copy, and inverted repeat regions, most often involving rpl22 and ycf1 . Analyses of repetitive elements and simple sequence repeats (microsatellites) identified predominantly adenine/thymine-rich motifs and regions of elevated sequence variability in the single-copy portions, indicating multiple loci as suitable for DNA barcoding and population-level studies. Both subtribes within Hippeastreae are resolved as monophyletic, however, within subtribe Hippeastrinae , Zephyranthes is recovered as polyphyletic. Conclusions: These plastid genomes demonstrate strong architectural stability while revealing informative boundary differences and clear contrasts in repeat and microsatellite profiles. The identified regions of elevated variability provide usable markers for species delimitation, phylogeography, and monitoring of genetic connectivity in Amaryllidaceae Atacama geophytes. The recovered relationships confirm subtribal relationships between Traubiinae and Hippeastrinae and establish a plastid reference framework that supports integrative studies and conservation efforts in the flowering-desert system. Zephyranthes chloroplast genome dryland plastome desert bloom Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction The Atacama Desert, possesses 1,000 native species, of which 54.3% are endemic (Letelier, 2008), with taxa well-adapted to prolonged drought and temperature fluctuations, supporting a unique biodiversity. The region is characterized by extreme aridity, high temperatures, intense solar radiation, salinity, and nutrient-poor soils (Vicencio et al., 2024 ). These extreme climatic and environmental conditions impose severe limitations on plant establishment and growth (Alsharif et al., 2020 ; Kirschner et al., 2021 ). Average annual rainfall ranged between 0.25 to 5 mm a year (Vicencio et al., 2024 ). Rainfall patterns are strongly influenced by the El Niño-Southern Oscillation (ENSO) (Campos & Rondanelli, 2023 ). In years where precipitation exceeds 15 mm can result in mass flowering events also referred to as the “flowering desert” phenomenon and characterized by the emergence of more than 200 species of annual plants and geophytes during spring (Araya et al., 2020 ), including species of the geophytic family Amaryllidaceae J.St.-Hil. (Gutiérrez, 2008 ). Following APG III (2009), Amaryllidaceae includes three subfamilies (Agapanthoideae Endl., Herb., Amaryllidoideae Burnett, and Allioideae Herb), with ~ 70 genera and 1700–1800 species (Meerow, 2023 ; POWO, 2025 ). The subfamily Amaryllioideae has a centres of diversity in the Mediterranean Basin, South Africa, and South America (Meerow et al. 2020 ), including 122 species native to the coastal desert of northern Chile of which 101 are endemics (Rodriguez et al., 2018 ). Current taxonomic treatments recognize 14 tribes within the subfamily, including 6 American tribes (Meerow, 2023 ). Phylogenetic analyses have resolved the American clade of Amaryllioideae sensu Meerow et al. (1999) into two strongly supported monophyletic clades: the Andean tetraploid clade (tribes Clinantheae Meerow, Eucharideae Hutch., Eustephieae Hutch., and Hymenocallideae Meerow) and the Hippeastroid clade (tribes Griffineae Ravenna and Hippeastreae Herb. ex Sweet) (Meerow, 2023 ). Within Hippeastreae, two subtribes are recognized: Traubiinae ( Traubia Moldenke, Paposoa Nic.García, Phycella Lindl., and Rhodolirium Phil.), and Hippeastrinae ( Hippeastrum Herb. and Zephyranthes Herb.). Several species from these subtribes are important parts of the Atacama Desert ecosystem, including the endemics Paposoa laeta (Phil.) Nic. García, Zephyranthes bagnoldii (Herb.) Nic. García, and Zephyranthes sarae J. M. Watson & A. R. Flores. These taxa face environmental and anthropogenic pressures including habitat fragmentation and climate change (Rodriguez et al., 2018 ; Squeo et al., 2008 ). Given the exceptional level of endemism and the current phylogenetic uncertainty within this taxonomically complex group, chloroplast genome analyses can provide complementary molecular data to resolve evolutionary relationships, validating previous phylogenetic hypotheses, and support evidence-based conservation strategies. Plastomes typically exhibit a conserved quadripartite structure comprising two inverted repeat regions (IRA and IRB) separated by large and small single-copy regions (LSC and SSC, respectively) (Ravi et al., 2008 ), and typically range between 120 to 160 kb in size, containing highly conserved genes fundamental to plant life and more variable regions that provide information over broad time scales (Nock et al., 2011 ; Zhang et al., 2023 )(Díaz et al., 2022 ). Comparison of chloroplast DNA sequences from plants inhabiting a range of climatic conditions and habitats, provides valuable information on gene content, genome rearrangement and genetic evolution at the mutational level, making these sequences useful to understand the climatic drivers of plant evolution (Sabater, 2018 ). Recent plastome surveys in Amaryllidaceae have revealed notable structural variations, for example, Strumaria truncata Jacq. exhibits an expansion of the inverted repeat (IR) regions, accompanied by substantial loss of ndh gene family members (Könyves et al., 2021 ). In Hippeastreae, ten complete plastomes have been sequenced to date, including five Hippeastrum species and four Zephyranthes species, with genome sizes ranging from 153,946 bp to 162,215 bp ( http://www.ncbi.nlm.nih.gov/genome/organelle/ ). Comparative plastome analyses within Hippeastrum have revealed phylogenetically informative structural variations, particularly within IR region and junctions. For example, H . reticulatum exhibits an IR expansion of 20 bp longer compared to those observed in H . 'Milady' and H . alberti , suggesting lineage-specific evolutionary processes within the genus (Liu et al., 2022 ). Similarly, the Atacama desert-adapted Zephyranthes phycelloides (Herb.) Nic.García possesses a 158,107 bp plastome, containing 137 genes (87 protein-coding, 8 rRNAs, 38 tRNAs, and 4 pseudogenes), providing initial insights into plastome evolution under extreme arid conditions (Contreras-Díaz et al., 2022 ). Additionally, whole-plastome sequences offer a genome-wide framework for species identification by combining conserved genes with more variable regions, increasing resolving power relative to single-locus plastid markers (Nock et al., 2011 ; Parks et al., 2009 ). In this study, the plastid genomes of Z. bagnoldii , Z. sarae , and P. laeta were sequenced, characterized, and compared with previously published Hippeastreae genomes to provide new plastid genetic resources. Given the scarcity of plastid genomic data in Hippeastreae (Amaryllidoideae), these results improve our understanding of plastome evolution within the tribe and provide resources for marker development that support accurate species identification, phylogeographic analyses, and the traceability of wild-harvested plant material, thereby informing conservation planning and sustainable use. Materials and methods Plant material and DNA isolation Fresh leaves of P. laeta , Z. bagnoldii , and Z. sarae were collected in Caldera, Freirina and Chañaral respectively in the Atacama Region, Chile, October 2024 (supplementary Figure S1 ). Plant material was formally identified by Dr. Roberto Contreras-Díaz (Universidad de Atacama, Chile) following the taxonomic treatment and diagnostic keys provided by García et al. ( 2019 ). Identifications were confirmed by CONAF professionals and by Dr. Nicolás García (Universidad de Chile), curator of the Herbarium EIF. The specimens were deposited in the Departamento de Silvicultura y Conservación de la Naturaleza herbarium EIF of Universidad de Chile (under the names that were correct at the time of deposition: Paposoa laeta , EIF14530; Zephyranthes bagnoldii , EIF14564; and Zephyranthes sarae , EIF14549) (supplementary Table S1 ). Collection and fieldwork were authorized by CONAF (Corporación Nacional Forestal; National Forestry Corporation) under permits Nº 122/2019 (granted on 8 November 2019) and Nº 78/2024 (granted on 14 April 2024); in addition, a research and collection permit was issued by the Municipality of Caldera (Permit No. 161/2024). DNA was isolated from the leaves using the modified cetyl-trimethylammonium bromide (CTAB) protocol (Contreras et al., 2020 ). The DNA concentration was measured using a Qubit™ 3.0 Fluorometer and the Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). DNA integrity was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) prior to sequencing. Genome sequencing, assembling and annotation DNA samples were sent to Novogene Corporation (Sacramento, California, USA) for library construction and subsequent sequencing using the NovaSeq™ X Plus 25B platform (Illumina, San Diego, CA, USA), obtaining paired-end reads of 150 base pairs (bp) in length for each DNA sample (R1 and R2 reads). The plastid genomes were assembled using GetOrganelle v1.7.7.1 (Jin et al., 2020 ). CPGAVAS2 (Shi et al., 2019 ) ( http://47.96.249.172:16019/analyzer/home ) and GeSeq (Tillich et al., 2017 ) ( https://chlorobox.mpimp-golm.mpg.de/geseq.html ) were used to annotate the chloroplast genomes, using Zephyranthes phycelloides (NC_059688.1; Contreras-Díaz et al. ( 2022 )) as the reference genome. The annotation results from both programs were compared, and manual adjustments were made when necessary. The circular genome map and gene structure were visualized using Chloroplot (Zheng et al., 2020 ) ( https://irscope.shinyapps.io/Chloroplot/ ). The resulting genome sequences have been deposited in GenBank under accession numbers PV849990, PV890574, PV890573. Genome comparison and variation analysis To assess overall structural conservation and identify potential genomic rearrangements, the three newly sequenced plastomes, along with seven previously published Hippeastreae plastomes (Table 1), were aligned using Mauve v.2.4.0 (Darling et al., 2004 ). A synteny plot was generated using the pyGenomeViz package v.1.5.0 (Shimoyama, 2024 ), employing the MMseqs RBH mode (available at: https://github.com/moshi4/pyGenomeViz ). The boundaries between the large single-copy (LSC), small single-copy (SSC), and inverted repeat (IR) regions were compared and visualized across all 10 species using CPJSdraw v0.0.1 (Li et al., 2023 ). To visualize sequence divergence among the 10 plastomes, a global alignment was performed using the mVISTA ( https://genome.lbl.gov/vista/index.shtml ) program in Shuffle-LAGAN mode, with the annotation of Z. phycelloides used as the reference (Frazer et al., 2004 ). To estimate the differentiation hotspot regions in Amaryllidaceae, the whole plastid genomes of the selected species were aligned using MAFFT v7.525 (Katoh & Standley, 2013 ). Subsequently, nucleotide diversity (π) was calculated across the alignment using DnaSP v.6 (Rozas et al., 2017 ), with a sliding window of 600 bp and a step size of 200 bp. Repeat element and SSR loci analysis Repeated sequence elements were searched using Vmatch v.2.3.1 ( http://www.vmatch.de/ ), which incorporates the REPuter software v2.74 (Kurtz et al., 2001 ). Direct (D) and palindrome (P) sequence repeats were searched for, parameters were set for sequences of n ≥ 30 bp, a sequence identity of ≥ 90% and a Hamming distance of 3. Additionally, simple sequence repeats (SSRs) were identified using the MISA-web v2.1 ( https://webblast.ipk-gatersleben.de/misa/ ) (Beier et al., 2017 ), applying thresholds of 10 repeat units for mononucleotides, 5 for dinucleotides, 4 for trinucleotides, and 3 for tetra-, penta-, and hexanucleotides. Phylogenetic analysis Seventy-six plastome protein-coding genes (PCGs) were extracted from ten Hippeastreae tribe plastomes (supplementary Table S1 ). Individual PCGs were aligned with MAFFT v7.525 (mafft –auto) (Katoh & Standley, 2013 ), and poorly aligned regions were removed using trimAl v1.5 with default parameters(Capella-Gutiérrez et al., 2009 ). Alignments were concatenated prior to phylogenetic inference. The best-fitting nucleotide substitution model for maximum likelihood (ML) analysis was selected with jModelTest v2.1.10 (Darriba et al., 2012 ) based on the Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC), resulting in GTR + I+G. Maximum likelihood (ML) phylogenetic inference was conducted in RAxML-HPC v8.2.13 (Stamatakis, 2014 ) under the GTR + G+I model, with 1,000 bootstrap replicates. Bayesian inference (BI) was conducted in MrBayes v3.2.7 (Ronquist et al., 2012 ) using a single run of four Markov chains for five million generations, sampling every 1,000 generations and discarding the first 25% as burn-in. The resulting ML and BI trees were visualized using FigTree v1.4.4 (Rambaut, 2007 ). Results Plastome genome structure and organization The complete plastid genomes of P. laeta , Z. bagnoldii and Z. sarae all exhibit the typical quadripartite structure composed of a large single-copy (LSC) region, a small single-copy (SSC) region, and two inverted repeats (IRs). Genome lengths ranged from 158,144 bp to 158,678 bp and GC contents between 37.80 – 38.06 % (Table 1; Figure 1). The plastid genomes of Z. bagnoldii , Z. sarae and P. laeta encoded an identical set of 137 genes, of which 87 are protein coding genes (PCGs), 42 tRNAs, 8 rRNAs. Among them, 113 single copy PCGs were identified, 15 genes contain one intron ( rps16 , atpF , rpoC1 , petB , petD , rpl16 , rpl2 , ndhB , ndhA , trnK-UUU , trnG-UCC , trnL-UAA , trnV-UAC , trnI-GAU , trnA-UGC ) and 3 genes contain 2 introns ( rps12 , ycf3 , clpP ) (Table 2). All 11 ndh genes, which encode the NADH dehydrogenase-like complex, were found to be intact and functional in all three species. Genome comparison and variation analyses Mauve analyses revealed that the plastomes of Hippeastrum and Zephyranthes studied do not exhibit significant rearrangements, suggesting high stability in plastid genome organization (Figure 2). Similarly, a high level of sequence similarity and structural conservation was observed with pyGenomeViz (Figure 3). The junctions were determined using CPJSdraw and the genes found at these junctions. These junctions were determined using CPJSdraw (Figure 4). The genes rpl22 , rps19 , ycf1 , ndhF , ycf1 , trnN , psbA , and are consistently found at plastome junctions, reflecting the importance of these genes in IR region expansion/contraction processes. The rpl22 gene was situated in the LSC region for Z. bagnoldii and Z. sarae . In P. laeta this gene extended 52 bp into the IRb boundary. The ycf1 gene extended across the SSC/IRa boundary by 978 bp, 978, bp and 1024 bp for Z. bagnoldii , Z. sarae and P. laeta, respectively. Furthermore, the SSC/IRa junction was observed to be located within the ycf1 gene. The mVISTA alignment revealed high overall sequence conservation among the ten Hippeastreae plastomes, with most regions showing > 90% sequence identity (Figure 5). The complete plastomes ranged from approximately 120-158 kb in length, maintaining typical quadripartite structure across all species. Protein-coding regions displayed the greatest similarity, with most genes showing more than 95% sequence identity across taxa. By contrast, non-coding regions, particularly several intergenic spacers, exhibited reduced identity relative to coding genes. The inverted repeat regions were comparatively less variable than the single-copy regions, consistent with the sliding-window analysis of nucleotide diversity (π), where the mean Pi across the plastome was 0.00408 and values differed among partitions (LSC: 0.00527; SSC: 0.00645; IRb: 0.00132; IRa: 0.00171). Divergence was concentrated in the small single-copy region, including ycf1 and adjacent intergenic segments, in agreement with local Pi peaks detected in this interval (Figure 6). To determine divergent hotspot regions in the ten Amarillydaceae tribe Hippeastreae species, we compared the Pi values of the cp genomes using DNASP software (Figure 6). For the ten Amarillydaceae tribe Hippeastreae species, Pi values ranged from 0.0 to 0.02115. The mean nucleotide diversity value of the whole cp genome was 0.00408, while the corresponding values of the LSC, IRb, SSC, and IRa were 0.00527, 0.00132, 0.00645, and 0.00171, respectively. LSC and SSC regions exhibited greater variation than IR regions, consistent with mVISTA findings. The graph highlights a distinct heterogeneity in evolutionary rates across the plastid genome, where IRs remain conserved while LSCs and SSCs showed greater variability. Variable regions greater than 0.012 were detected. In the LSC region, the psbA gene shows moderate variability (0.015), a notable peak is observed in the trnL-UAA region (~0.018) and the psbJ-psbL-psbF-psbE region shows the highest peak in this region (~0.020). In the SSC region, the ccsA-ndhD-psaC genes show significant peaks of variability (~0.017) and the ycf1 region exhibits multiple peaks of variability (~0.012). Repeat elements analysis Large sequence repeats (LSRs) were identified as repeats with a length of ≥30 bp each. A total of 381 LSRs were detected across the ten Hippeastreae plastomes, including palindromic repeats (P) and forward repeats (D) (Figure 7). Palindromic repeats were the most prevalent type with 224 occurrences, while forward repeats were the least common with 157 occurrences. A total of 38 repeats were recovered in Z. bagnoldii including 22 forward and 16 palindromic. In Z. sarae 41 repeats were detected including 22 forward and 19 palindromic repeats whilst in P. laeta 43 repeats including 25 forward and 18 palindromic repeats were recovered. In the MISA-web analysis, six types of simple sequence repeats (SSRs) were identified, with mononucleotide repeats comprising the majority (399, 64.88%), followed by dinucleotide repeats (111, 18.05%), tetranucleotide repeats (74, 12.03%), pentanucleotide repeats (16, 2.60%), trinucleotide repeats (11, 1.79%), and hexanucleotide repeats (4, 0.65%) (Figure 8a). SSRs have been detected in Z. bagnoldii (58), Z. sarae (62) and P. laeta (69). Z. bagnoldii has 35 (63.79%) mononucleotide (A/T) repeats, whereas Z. sarae and P. laeta have 39 (66.13%) and 44 (63.77%), respectively. Phylogenetic analysis Phylogenetic relationships within the Hippeastreae tribe were inferred using 76 plastome protein-coding genes from ten Hippeastreae plastomes and one outgroup ( Narcissus poeticus ; NC_039825.1), using maximum likelihood (ML) and Bayesian inference (BI) methods. Both analyses produced congruent topologies (Figure 9). Subtribes Hippeastrinae and Traubinnae are resolved as monophyletic. Paposoa laeta (subtribe Traubinnae ) was recovered sister to subtribe Hippeastrinae (100 BS; 1.00 PP). Within subtribe Hippeastrinae , Zephyranthes clade I ( H. reticulatum , Z. candida , Z. mesochloa ) was recovered as sister to the Hippeastrum clade ( H. albertii , H. vittatum , H. rutilum ) and Zephyranthes clade subg. Myostemma ( Z. bagnoldii , Z. phycelloides , Z. sarae ) with maximum support. The sister relationship between the Hippeastrum clade and Z. subgenus Myostemma had recovered support values of 48 BS and 0.59 PP. Both Hippeastrum and Zephyranthes are recovered as polyphyletic. Discussion The three newly assembled plastomes of Z. bagnoldii , Z. sarae and P. laeta exhibit the canonical quadripartite architecture and a narrow range of genome sizes and GC content. These plastomes fall within the size range (153–160 kb) and GC content (around 37%) typical of other Amaryllidaceae species (Cheng et al., 2022 ; Jimenez et al., 2020 ), mirroring the strong structural conservatism long recognized for Amaryllidaceae and other angiosperms (Contreras-Díaz et al., 2022 ; Mower & Vickrey, 2018 ). Mauve alignments and synteny plots revealed no major rearrangements among Hippeastreae plastomes, consistent with the predominantly collinear organization reported for closely related lineages (Huo et al., 2019 ; Namgung et al., 2021 ). Together with the stable gene complement recovered here (137 genes: 87 PCGs, 42 tRNAs, 8 rRNAs), these patterns suggest that most interspecific plastome differences in Hippeastreae arise not from large-scale structural change but from localized boundary shifts and sequence-level variation. All species had intact and functional ndh genes which is in stark contrast to the pattern of ndh gene degradation and loss observed in other Amaryllidaceae taxa like Strumaria truncate (Könyves et al., 2021 ), and Allium paradoxum (Omelchenko et al., 2020 ), suggesting a conserved and photosynthetically competent plastome in the studied species. Small yet informative differences occur at the junctions between single-copy regions and the inverted repeats (IRs). The genes rpl22 , rps19 , ndhF , ycf1 , trnN , psbA , and are consistently found at plastome junctions, reflecting the importance of these genes in IR region expansion/contraction processes. Notably, P. laeta shows a ~ 52 bp extension of rpl22 into IRb, a derived, lineage-specific character state that may serve as a genus-level diagnostic and provides insights into the evolutionary history of Traubiinae. Additionally, this combined with the phylogenetic placement of P. laeta sister to subtribe Hippeastrinae, reinforces subtribal delimitations (García et al., 2017 ; García et al., 2019 ; Meerow, 2023 ). From a plastome-evolution perspective, IR boundary shifts represent recurrent expansion–contraction dynamics that change the extent of duplicated sequence and create lineage-specific junction patterns, providing a subtle but informative source of structural variation in otherwise stable plastomes (Goulding et al., 1996 ; Zhu et al., 2016 ). In Hippeastreae, IR boundary shifts are small in absolute size, but the resulting junction configurations (i.e., which loci extend into or out of the inverted repeat) can be consistent within lineages; when compared across Hippeastrinae and Traubiinae, these patterns provide complementary structural characters that help distinguish subtribal groupings and corroborate relationships inferred from sequence-based phylogenies (García et al., 2019 ; Namgung et al., 2021 ). A higher number of palindromic repeats is recovered in the ten Hippeastreae plastomes studied (14–19) compared to the number of forward repeats (21–25). SSRs are strongly A/T-biased, accounting for 81.36–85.07% of SSRs. A higher number of large sequence repeats and SSRs was recovered for P. laeta compared to Z. bagnoldii and Z. sarae , differentiating these plastomes. Specifically, P. laeta had a higher abundance of mononucleotide repeats. The dinucleotide type remains constant at 11 across our studied species. Z. bagnoldii and Z. sarae are most similar to Z. phycelloides when comparing the presence of mono-, di-, tetra- and hexa-nucleotides. Whereas P. laeta is different in having pentanucleotides and no hexanucleotides. Among these SSRs, repeat units of A/T, AT/AT and AAAT/ATTT accounted for 83.57% of the total, indicating a bias towards A/T bases in SSR composition (Fig. 8 b). This suggests that P. laeta has greater genomic dynamism, which could be related to specific ecological adaptations to the environment. To distinguish the species studied in this paper from other Hippeastreae, we suggest two complementary marker panels: (i) a “short-amplicon” set targeting psbA , trnL-UAA , and psbJ-psbL-psbF-psbE for degraded material (herbarium, soil eDNA), and (ii) a “long-amplicon” set that includes the 3′ region of ycf1 and the ccsA-ndhD-psaC interval for high-resolution phylogeography and species delimitation. The convergence of mVISTA conservation profiles and π peaks across our 10-taxon comparison underscores that these loci are robust candidates rather than idiosyncratic outliers. Our phylogenetic analyses support the recovery of subtribes Hippeastrinae and Traubiinae as monophyletic, support the placement of P. laeta within Traubiinae, and align with cytological evidence indicating chromosome numbers of 2n = 18 in Hippeastrinae and 2n = 16 in Traubiinae (Baeza et al., 2017 ; Jara-Seguel et al., 2012 ). The phylogenetic placement and karyology of P. laeta combined with the higher number of SSRs and variation of the IRb junction, provide a reliable genus-specific marker for P. laeta . The polyphyly recovered in subtribe Hippeastrinae concurs with the relationships recovered in García et al. (2014; 2017 ), which has been attributed to reticulation evolution and incomplete lineage sorting. Zephyranthes bagnoldii , Z. sarae , and Z. phycelloides have overlapping distributions and ecological niches, occurring along the Atacama coastal belt and undergo mass flowering events following winter rainfall associated with La Niña conditions (Araya et al., 2020 ; Gutiérrez, 2008 ). Because mass flowering in the Atacama is triggered by infrequent rainfall pulses, above-ground emergence and reproductive output can vary strongly among years. In geophytes, however, this variability may be buffered by below-ground bulbs (a “bud bank”), so fluctuations in visible abundance do not necessarily reflect comparable changes in survival. Instead, rainfall pulses likely drive episodic recruitment and seed production, potentially influencing effective population size and chloroplast haplotype frequencies. Our plastome markers (SSRs and high-variability regions) enable tests of these patterns and connectivity. Although chloroplast variation within species is generally low (Nock et al 2011 ), sampling several individuals would provide a stronger basis for population-level analyses and confirm the stability of boundary positions or repeat counts. Plastome genomes are typically maternally inherited, representing one evolutionary history (Hagemann, 2004; Wicke et al., 2011). The nuclear genome is bi-parentally inherited and can be used to detect reticulate evolution including ancienty hybridization events and introgression (Stull et al., 2023). Given that previous nuclear analyses have documented reticulation within Zephyranthes , comparisons between plastid and nuclear phylogenetic signals provide an important context for interpreting plastome-based relationships within Myostemma (García et al., 2017 ; García et al., 2019 ). That P. laeta is genomically conservative yet phylogenetically divergent offers an additional insight: plastome stability does not preclude lineage-specific boundary characters ( rpl22 at IRb/LSC) or differences in repeat density. Rather, these fine-scale shifts may be especially informative at the subtribal level, where broader structural rearrangements are absent. We therefore advocate explicit use of IR boundary features and repeat profiles as a complementary, low-cost layer of evidence when resolving relationships among Traubiinae and Hippeastrinae genera. Conclusion The plastomes of Z. bagnoldii , Z. sarae , and P. laeta presented in this study deepen our understanding of plastome evolution and structure in Chilean Hippeastreae taxa. Shifts in the inverted repeat boundary and number of repeat and SSRs can form the basis for DNA barcoding and population genetics within this group. Our study shows that small shifts between single-copy regions and the inverted repeats (IRs), can be used to distinguish between species. Repeat and SSR profiles distinguish the three species, with strong A/T bias overall and a motif composition in P. laeta that distinguishes it from Zephyranthes (presence of pentanucleotides and absence of hexanucleotides), suggesting useful loci for downstream barcoding and population-level applications in Atacama geophytes. Phylogenomically, our plastid data support current subtribal descriptions. These results corroborate extensive structural stability, reveal modest yet phylogenetically informative differences among lineages, and identify repeat/SSR features that can be used for species delimitation, phylogeography. The recovered topology, Myostemma cohesion with P. laeta sister to Hippeastrinae, agrees with current subtribal delimitations and provides a plastid reference that can be combined with nuclear data in future phylogenomic analyses to test cytonuclear congruence and refine relationships within Hippeastreae. In a region where above-ground diversity and flowering respond to infrequent rainfall pulses, these resources can help track genetic responses and support conservation strategies, by aided reproduction to keep the genetic pool healthy, for narrowly distributed geophytes of the flowering desert. Declarations 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 PV849990 for Zephyranthes bagnoldii , PV890574 for Zephyranthes sarae and PV890573 for Paposoa laeta . The BioProject, BioSample and SRA accession numbers on NCBI for Zephyranthes bagnoldii are PRJNA1304596, SAMN50563324 and SRR34952303, for Zephyranthes sarae are PRJNA1304964, SAMN50580584 and SRR34962843, and for Paposa laeta are PRJNA1304263, SAMN50554916 and SRR34941586. This study is not a clinical trial. Acknowledgements RC thanks the Atacama Regional Government for funding support through the FIC project BIP 40057824 and ANID through the FONDECYT Initiation grant 11230668. PJ acknowledges support XCEL - Extreme Cryptogram Ecology Lab, University of Applied Sciences Kaiserslautern, Germany. MA thanks the Máster Universitario de Bioinformática y Bioestadística (UOC–UB), Universitat Oberta de Catalunya. RC also thanks Neri Contreras-Ascencio and Ana Díaz-Sánchez for their support during field sampling. The authors are grateful to Mayor Brunilda González Anjel of the Municipality of Caldera for granting permission to conduct field sampling. Ethics approval and consent to participate This article does not contain any studies with human participants or animals performed by any of the authors. This research was conducted in compliance with the relevant research permits, in accordance with national and international standards for the collection of plant material ( Paposoa laeta , Zephyranthes bagnoldii , and Zephyranthes sarae ) and with appropriate safeguards for flora and fauna. Collection and fieldwork were authorized by CONAF (Corporación Nacional Forestal; National Forestry Corporation) under permits Nº 122/2019 (granted on 8 November 2019) and Nº 78/2024 (granted on 14 April 2024). In addition, a research and collection permit was issued by the Municipality of Caldera (Permit No. 161/2024). Consent for publication Not applicable Competing interests The authors declare that they have no potential conflict of interest. Funding This work was funded by the Atacama Regional Government through the FIC Project BIP 40057824, by ANID–FONDECYT (Initiation Project No. 11230668), and by XCEL (Extreme Cryptogram Ecology Lab), University of Applied Sciences Kaiserslautern. Contributions M.A., R.C., Z.D., L.vdB.: data analyses, writing manuscript. M.A., R.C., Z.D., P.J.: data interpretation, writing and editing manuscript. M.A., R.C.: experimental analysis. M.A., R.C., Z.D., L.vdB., P.J.: Analysis and interpretation of result. M.A., R.C., Z.D., L.vdB., P.J.: editing-review original draft. R.C., P.J.: funding acquisition. All the authors have approved the final manuscript. All authors authorize the publication of this manuscript. Authors' information PhD Program in Biotechnology and Sustainable Bioproduction, a consortium between the Universidad de Atacama and the Universidad Arturo Prat, Chile. Mariana Arias-Aburto References Alsharif, W., Saad, M. M., & Hirt, H. (2020). 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Supplementary Files FigureS1.docx TableS1.docx Tables.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 26 Mar, 2026 Reviewers agreed at journal 19 Mar, 2026 Reviewers invited by journal 26 Feb, 2026 Editor assigned by journal 26 Feb, 2026 Editor invited by journal 18 Feb, 2026 Submission checks completed at journal 17 Feb, 2026 First submitted to journal 17 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8801993","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":597758345,"identity":"fbe8a889-62b5-4ec1-8c3c-6762a258d4bd","order_by":0,"name":"Mariana Arias-Aburto","email":"","orcid":"","institution":"Universidad de Atacama","correspondingAuthor":false,"prefix":"","firstName":"Mariana","middleName":"","lastName":"Arias-Aburto","suffix":""},{"id":597758346,"identity":"95d67d8b-43c5-4ea0-860f-8c75ab261f8d","order_by":1,"name":"Zoë Dennehy-Carr","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Zoë","middleName":"","lastName":"Dennehy-Carr","suffix":""},{"id":597758348,"identity":"e4598e53-1d09-44b3-a5fa-9c13ce0356c9","order_by":2,"name":"Liesbeth van den Brink","email":"","orcid":"","institution":"BOKU University","correspondingAuthor":false,"prefix":"","firstName":"Liesbeth","middleName":"van den","lastName":"Brink","suffix":""},{"id":597758350,"identity":"94bd46e6-1f63-4cf8-818b-53a41b772a53","order_by":3,"name":"Roberto Contreras-Diaz","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,"prefix":"","firstName":"Roberto","middleName":"","lastName":"Contreras-Diaz","suffix":""},{"id":597758352,"identity":"67e73aea-421b-4db6-833f-a54ca3309222","order_by":4,"name":"Patrick Jung","email":"","orcid":"","institution":"University of Applied Sciences Kaiserslautern","correspondingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"","lastName":"Jung","suffix":""}],"badges":[],"createdAt":"2026-02-06 02:54:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8801993/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8801993/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103890707,"identity":"77bae97b-05f8-497e-bc0e-a1f7a0be2506","added_by":"auto","created_at":"2026-03-04 08:02:01","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":315269,"visible":true,"origin":"","legend":"\u003cp\u003eCircular gene map of the plastid genomes of \u003cem\u003eP. laeta, Z. bagnoldii and Z. sarae\u003c/em\u003e. Genes were colored according to their functional group. Small single copy (SSC), large single copy (LSC), and inverted repeats (IRA and IRB), genome length, total GC content, were indicated.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8801993/v1/ea0efade38d49e3ff3221a64.jpeg"},{"id":103890699,"identity":"1525f215-c437-4680-8ce8-d6c01dd782ea","added_by":"auto","created_at":"2026-03-04 08:02:00","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":500343,"visible":true,"origin":"","legend":"\u003cp\u003eConservation and rearrangements in plastid genomes of \u003cem\u003eZ. bagnoldii\u003c/em\u003e, \u003cem\u003eZ. sarae\u003c/em\u003e, \u003cem\u003eP. laeta\u003c/em\u003e, and seven Hippeastreae plastomes revealed by Mauve. Homologous regions appear as collinear blocks; protein-coding genes are shown in white, tRNA in black, and rRNA in red.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8801993/v1/5de9e3faa084a4d56173dd92.jpeg"},{"id":103890703,"identity":"f393b5df-4560-4f6d-8ad7-6940c6cdd51a","added_by":"auto","created_at":"2026-03-04 08:02:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":622512,"visible":true,"origin":"","legend":"\u003cp\u003eSynteny diagram of \u003cem\u003eZ. bagnoldii\u003c/em\u003e, \u003cem\u003eZ. sarae\u003c/em\u003e, \u003cem\u003eP. laeta\u003c/em\u003e, and seven other plastomes of the tribe Hippeastreae, generated using pyGenomeViz. The diagram shows normal linkages in orange, inverted linkages in green, coding sequences (CDS) in sky blue, and rRNA in pink.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8801993/v1/11b00cf6b3c9cd65f3468436.png"},{"id":104834986,"identity":"ae020b29-e141-4ddc-9856-17c4596a1eed","added_by":"auto","created_at":"2026-03-17 17:37:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":340387,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the large single-copy (LSC), small single-copy (SSC), and inverted repeat (IR) junctions across ten Hippeastreae tribe plastomes, generated with CPJSdraw.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8801993/v1/38c2571e03080e508127d2fa.png"},{"id":104401418,"identity":"90592b5f-9df5-4313-9992-ca25348de9ca","added_by":"auto","created_at":"2026-03-11 12:12:39","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":585284,"visible":true,"origin":"","legend":"\u003cp\u003eStructural comparisons of ten aligned Amarillydaceae\u003cstrong\u003e \u003c/strong\u003etribe Hippeastreae plastomes with the mVISTA program. The x-axis represents the gene position and sequence length, and the y-axis shows the average percentage identity, ranging from 50% to 100%. Dark blue areas denote exons, light blue signifies untranslated regions (tRNA and rRNA), and pink shows non-coding sequences (CNS). Arrows indicate the annotated genes and their transcription direction.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8801993/v1/458731fbb754b47040cf12d6.jpeg"},{"id":103890706,"identity":"fba598e0-6c7e-474a-9ef7-09089704cfbe","added_by":"auto","created_at":"2026-03-04 08:02:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":103339,"visible":true,"origin":"","legend":"\u003cp\u003eNucleotide diversity (π) across plastomes of ten species belonging to the Hippeastreae tribe, using a sliding window analysis with a sliding window of 600 bp and step size of 200 bp. The x-axis indicates the position of the midpoint of the window, while the y-axis indicates the nucleotide diversity of each window. Gene names are labeled at regions of highest variability. Peaks indicate hypervariable regions, while valleys represent highly conserved sequences.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8801993/v1/e81e269112d6ee5f26d01a5a.png"},{"id":103890701,"identity":"b4414101-7958-4bcb-a72c-668fa97df35a","added_by":"auto","created_at":"2026-03-04 08:02:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":37911,"visible":true,"origin":"","legend":"\u003cp\u003eClassification of long repeats in ten species of the Hippeastreae tribe. D = forward repeats; P = palindromic repeats.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8801993/v1/2fec4ab282ff169fef850acf.png"},{"id":103890705,"identity":"c14b3cc6-cbc8-4421-af3b-81e1d449d79f","added_by":"auto","created_at":"2026-03-04 08:02:00","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":200710,"visible":true,"origin":"","legend":"\u003cp\u003eFrequency of simple sequence repeat (SSR) motifs across repeat classes in plastid genomes of ten Hippeastreae (Amaryllidaceae) species. (a) Distribution by length class. (b) Distribution by specific motifs.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8801993/v1/52fab2f070fb4409a956d609.png"},{"id":104401330,"identity":"efff6fea-4f9e-451c-9078-b00fac701c1b","added_by":"auto","created_at":"2026-03-11 12:12:25","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":90970,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood and Bayesian inference consensus phylogeny inferred using 76 plastid protein-coding genes for ten Hippeastreae species, with\u003cem\u003e Narcissus poeticus\u003c/em\u003e included as the outgroup. Tip labels show updated species names and GenBank accessions (e.g., \u003cem\u003eH. striatum\u003c/em\u003e instead of \u003cem\u003eH. rutilum\u003c/em\u003e; \u003cem\u003eH. reginae\u003c/em\u003e instead of \u003cem\u003eH. alberti\u003c/em\u003e). Node labels show bootstrap support (BS) and posterior probability (PP) values.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8801993/v1/18d525d11d09bd0239355414.png"},{"id":104835754,"identity":"b643b719-a197-4cb2-ba45-59972f33745a","added_by":"auto","created_at":"2026-03-17 17:49:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3588475,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8801993/v1/5cd22dcb-8937-4f66-9b4a-7ea3fd5e45dc.pdf"},{"id":103890710,"identity":"b37a49f1-15fe-4ac6-9cef-9d363fe6472a","added_by":"auto","created_at":"2026-03-04 08:02:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2079213,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8801993/v1/85abb9afc55208b7edd172d9.docx"},{"id":104401667,"identity":"6a9c2f53-bb1c-45c3-b8b1-1d7f48f84268","added_by":"auto","created_at":"2026-03-11 12:13:13","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15681,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8801993/v1/be5368f3c70e22f5eee07e9c.docx"},{"id":103890700,"identity":"2823a4d8-6928-457d-a0c8-fcbf4faf3ef1","added_by":"auto","created_at":"2026-03-04 08:02:00","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":22092,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8801993/v1/d31141c7c80fb5c6a2fac868.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative Analysis of the Plastid Genomes of Zephyranthes bagnoldii, Zephyranthes sarae, and Paposoa laeta from the Flowering Desert of the Atacama Region, Chile","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Atacama Desert, possesses 1,000 native species, of which 54.3% are endemic (Letelier, 2008), with taxa well-adapted to prolonged drought and temperature fluctuations, supporting a unique biodiversity. The region is characterized by extreme aridity, high temperatures, intense solar radiation, salinity, and nutrient-poor soils (Vicencio et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These extreme climatic and environmental conditions impose severe limitations on plant establishment and growth (Alsharif et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kirschner et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Average annual rainfall ranged between 0.25 to 5 mm a year (Vicencio et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Rainfall patterns are strongly influenced by the El Ni\u0026ntilde;o-Southern Oscillation (ENSO) (Campos \u0026amp; Rondanelli, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In years where precipitation exceeds 15 mm can result in mass flowering events also referred to as the \u0026ldquo;flowering desert\u0026rdquo; phenomenon and characterized by the emergence of more than 200 species of annual plants and geophytes during spring (Araya et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), including species of the geophytic family Amaryllidaceae J.St.-Hil. (Guti\u0026eacute;rrez, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFollowing APG III (2009), Amaryllidaceae includes three subfamilies (Agapanthoideae Endl., Herb., Amaryllidoideae Burnett, and Allioideae Herb), with ~\u0026thinsp;70 genera and 1700\u0026ndash;1800 species (Meerow, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; POWO, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The subfamily Amaryllioideae has a centres of diversity in the Mediterranean Basin, South Africa, and South America (Meerow et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), including 122 species native to the coastal desert of northern Chile of which 101 are endemics (Rodriguez et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Current taxonomic treatments recognize 14 tribes within the subfamily, including 6 American tribes (Meerow, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Phylogenetic analyses have resolved the American clade of Amaryllioideae sensu Meerow et al. (1999) into two strongly supported monophyletic clades: the Andean tetraploid clade (tribes Clinantheae Meerow, Eucharideae Hutch., Eustephieae Hutch., and Hymenocallideae Meerow) and the Hippeastroid clade (tribes Griffineae Ravenna and Hippeastreae Herb. ex Sweet) (Meerow, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Within Hippeastreae, two subtribes are recognized: Traubiinae (\u003cem\u003eTraubia\u003c/em\u003e Moldenke, \u003cem\u003ePaposoa\u003c/em\u003e Nic.Garc\u0026iacute;a, \u003cem\u003ePhycella\u003c/em\u003e Lindl., and \u003cem\u003eRhodolirium\u003c/em\u003e Phil.), and Hippeastrinae (\u003cem\u003eHippeastrum\u003c/em\u003e Herb. and \u003cem\u003eZephyranthes\u003c/em\u003e Herb.). Several species from these subtribes are important parts of the Atacama Desert ecosystem, including the endemics \u003cem\u003ePaposoa laeta\u003c/em\u003e (Phil.) Nic. Garc\u0026iacute;a, \u003cem\u003eZephyranthes bagnoldii\u003c/em\u003e (Herb.) Nic. Garc\u0026iacute;a, and \u003cem\u003eZephyranthes sarae\u003c/em\u003e J. M. Watson \u0026amp; A. R. Flores. These taxa face environmental and anthropogenic pressures including habitat fragmentation and climate change (Rodriguez et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Squeo et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Given the exceptional level of endemism and the current phylogenetic uncertainty within this taxonomically complex group, chloroplast genome analyses can provide complementary molecular data to resolve evolutionary relationships, validating previous phylogenetic hypotheses, and support evidence-based conservation strategies.\u003c/p\u003e \u003cp\u003ePlastomes typically exhibit a conserved quadripartite structure comprising two inverted repeat regions (IRA and IRB) separated by large and small single-copy regions (LSC and SSC, respectively) (Ravi et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and typically range between 120 to 160 kb in size, containing highly conserved genes fundamental to plant life and more variable regions that provide information over broad time scales (Nock et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)(D\u0026iacute;az et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Comparison of chloroplast DNA sequences from plants inhabiting a range of climatic conditions and habitats, provides valuable information on gene content, genome rearrangement and genetic evolution at the mutational level, making these sequences useful to understand the climatic drivers of plant evolution (Sabater, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Recent plastome surveys in Amaryllidaceae have revealed notable structural variations, for example, \u003cem\u003eStrumaria truncata\u003c/em\u003e Jacq. exhibits an expansion of the inverted repeat (IR) regions, accompanied by substantial loss of \u003cem\u003endh\u003c/em\u003e gene family members (K\u0026ouml;nyves et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In Hippeastreae, ten complete plastomes have been sequenced to date, including five \u003cem\u003eHippeastrum\u003c/em\u003e species and four \u003cem\u003eZephyranthes\u003c/em\u003e species, with genome sizes ranging from 153,946 bp to 162,215 bp (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/genome/organelle/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/genome/organelle/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Comparative plastome analyses within \u003cem\u003eHippeastrum\u003c/em\u003e have revealed phylogenetically informative structural variations, particularly within IR region and junctions. For example, \u003cem\u003eH\u003c/em\u003e. \u003cem\u003ereticulatum\u003c/em\u003e exhibits an IR expansion of 20 bp longer compared to those observed in \u003cem\u003eH\u003c/em\u003e. 'Milady' and \u003cem\u003eH\u003c/em\u003e. \u003cem\u003ealberti\u003c/em\u003e, suggesting lineage-specific evolutionary processes within the genus (Liu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similarly, the Atacama desert-adapted \u003cem\u003eZephyranthes phycelloides\u003c/em\u003e (Herb.) Nic.Garc\u0026iacute;a possesses a 158,107 bp plastome, containing 137 genes (87 protein-coding, 8 rRNAs, 38 tRNAs, and 4 pseudogenes), providing initial insights into plastome evolution under extreme arid conditions (Contreras-D\u0026iacute;az et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, whole-plastome sequences offer a genome-wide framework for species identification by combining conserved genes with more variable regions, increasing resolving power relative to single-locus plastid markers (Nock et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Parks et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, the plastid genomes of \u003cem\u003eZ. bagnoldii\u003c/em\u003e, \u003cem\u003eZ. sarae\u003c/em\u003e, and \u003cem\u003eP. laeta\u003c/em\u003e were sequenced, characterized, and compared with previously published Hippeastreae genomes to provide new plastid genetic resources. Given the scarcity of plastid genomic data in Hippeastreae (Amaryllidoideae), these results improve our understanding of plastome evolution within the tribe and provide resources for marker development that support accurate species identification, phylogeographic analyses, and the traceability of wild-harvested plant material, thereby informing conservation planning and sustainable use.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material and DNA isolation\u003c/h2\u003e \u003cp\u003eFresh leaves of \u003cem\u003eP. laeta\u003c/em\u003e, Z. \u003cem\u003ebagnoldii\u003c/em\u003e, and \u003cem\u003eZ. sarae\u003c/em\u003e were collected in Caldera, Freirina and Cha\u0026ntilde;aral respectively in the Atacama Region, Chile, October 2024 (supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Plant material was formally identified by Dr. Roberto Contreras-D\u0026iacute;az (Universidad de Atacama, Chile) following the taxonomic treatment and diagnostic keys provided by Garc\u0026iacute;a et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Identifications were confirmed by CONAF professionals and by Dr. Nicol\u0026aacute;s Garc\u0026iacute;a (Universidad de Chile), curator of the Herbarium EIF. The specimens were deposited in the Departamento de Silvicultura y Conservaci\u0026oacute;n de la Naturaleza herbarium EIF of Universidad de Chile (under the names that were correct at the time of deposition: \u003cem\u003ePaposoa laeta\u003c/em\u003e, EIF14530; \u003cem\u003eZephyranthes bagnoldii\u003c/em\u003e, EIF14564; and \u003cem\u003eZephyranthes sarae\u003c/em\u003e, EIF14549) (supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Collection and fieldwork were authorized by CONAF (Corporaci\u0026oacute;n Nacional Forestal; National Forestry Corporation) under permits N\u0026ordm; 122/2019 (granted on 8 November 2019) and N\u0026ordm; 78/2024 (granted on 14 April 2024); in addition, a research and collection permit was issued by the Municipality of Caldera (Permit No. 161/2024). DNA was isolated from the leaves using the modified cetyl-trimethylammonium bromide (CTAB) protocol (Contreras et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The DNA concentration was measured using a Qubit\u0026trade; 3.0 Fluorometer and the Qubit\u0026trade; dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). DNA integrity was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) prior to sequencing.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenome sequencing, assembling and annotation\u003c/h3\u003e\n\u003cp\u003eDNA samples were sent to Novogene Corporation (Sacramento, California, USA) for library construction and subsequent sequencing using the NovaSeq\u0026trade; X Plus 25B platform (Illumina, San Diego, CA, USA), obtaining paired-end reads of 150 base pairs (bp) in length for each DNA sample (R1 and R2 reads). The plastid genomes were assembled using GetOrganelle v1.7.7.1 (Jin et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). CPGAVAS2 (Shi et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://47.96.249.172:16019/analyzer/home\u003c/span\u003e\u003cspan address=\"http://47.96.249.172:16019/analyzer/home\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and GeSeq (Tillich et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://chlorobox.mpimp-golm.mpg.de/geseq.html\u003c/span\u003e\u003cspan address=\"https://chlorobox.mpimp-golm.mpg.de/geseq.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used to annotate the chloroplast genomes, using \u003cem\u003eZephyranthes phycelloides\u003c/em\u003e (NC_059688.1; Contreras-D\u0026iacute;az et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)) as the reference genome. The annotation results from both programs were compared, and manual adjustments were made when necessary. The circular genome map and gene structure were visualized using Chloroplot (Zheng et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://irscope.shinyapps.io/Chloroplot/\u003c/span\u003e\u003cspan address=\"https://irscope.shinyapps.io/Chloroplot/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The resulting genome sequences have been deposited in GenBank under accession numbers PV849990, PV890574, PV890573.\u003c/p\u003e\n\u003ch3\u003eGenome comparison and variation analysis\u003c/h3\u003e\n\u003cp\u003eTo assess overall structural conservation and identify potential genomic rearrangements, the three newly sequenced plastomes, along with seven previously published Hippeastreae plastomes (Table\u0026nbsp;1), were aligned using Mauve v.2.4.0 (Darling et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). A synteny plot was generated using the pyGenomeViz package v.1.5.0 (Shimoyama, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), employing the MMseqs RBH mode (available at: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/moshi4/pyGenomeViz\u003c/span\u003e\u003cspan address=\"https://github.com/moshi4/pyGenomeViz\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The boundaries between the large single-copy (LSC), small single-copy (SSC), and inverted repeat (IR) regions were compared and visualized across all 10 species using CPJSdraw v0.0.1 (Li et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To visualize sequence divergence among the 10 plastomes, a global alignment was performed using the mVISTA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://genome.lbl.gov/vista/index.shtml\u003c/span\u003e\u003cspan address=\"https://genome.lbl.gov/vista/index.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) program in Shuffle-LAGAN mode, with the annotation of \u003cem\u003eZ. phycelloides\u003c/em\u003e used as the reference (Frazer et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). To estimate the differentiation hotspot regions in Amaryllidaceae, the whole plastid genomes of the selected species were aligned using MAFFT v7.525 (Katoh \u0026amp; Standley, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Subsequently, nucleotide diversity (π) was calculated across the alignment using DnaSP v.6 (Rozas et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), with a sliding window of 600 bp and a step size of 200 bp.\u003c/p\u003e\n\u003ch3\u003eRepeat element and SSR loci analysis\u003c/h3\u003e\n\u003cp\u003eRepeated sequence elements were searched using Vmatch v.2.3.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.vmatch.de/\u003c/span\u003e\u003cspan address=\"http://www.vmatch.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which incorporates the REPuter software v2.74 (Kurtz et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Direct (D) and palindrome (P) sequence repeats were searched for, parameters were set for sequences of n\u0026thinsp;\u0026ge;\u0026thinsp;30 bp, a sequence identity of \u0026ge;\u0026thinsp;90% and a Hamming distance of 3. Additionally, simple sequence repeats (SSRs) were identified using the MISA-web v2.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://webblast.ipk-gatersleben.de/misa/\u003c/span\u003e\u003cspan address=\"https://webblast.ipk-gatersleben.de/misa/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Beier et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), applying thresholds of 10 repeat units for mononucleotides, 5 for dinucleotides, 4 for trinucleotides, and 3 for tetra-, penta-, and hexanucleotides.\u003c/p\u003e\n\u003ch3\u003ePhylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eSeventy-six plastome protein-coding genes (PCGs) were extracted from ten Hippeastreae tribe plastomes (supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Individual PCGs were aligned with MAFFT v7.525 (mafft \u0026ndash;auto) (Katoh \u0026amp; Standley, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and poorly aligned regions were removed using trimAl v1.5 with default parameters(Capella-Guti\u0026eacute;rrez et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Alignments were concatenated prior to phylogenetic inference. The best-fitting nucleotide substitution model for maximum likelihood (ML) analysis was selected with jModelTest v2.1.10 (Darriba et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) based on the Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC), resulting in GTR\u0026thinsp;+\u0026thinsp;I+G. Maximum likelihood (ML) phylogenetic inference was conducted in RAxML-HPC v8.2.13 (Stamatakis, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) under the GTR\u0026thinsp;+\u0026thinsp;G+I model, with 1,000 bootstrap replicates. Bayesian inference (BI) was conducted in MrBayes v3.2.7 (Ronquist et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) using a single run of four Markov chains for five million generations, sampling every 1,000 generations and discarding the first 25% as burn-in. The resulting ML and BI trees were visualized using FigTree v1.4.4 (Rambaut, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003ch3\u003ePlastome genome structure and organization\u003c/h3\u003e\n\u003cp\u003eThe complete plastid genomes of \u003cem\u003eP. laeta\u003c/em\u003e, \u003cem\u003eZ. bagnoldii\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Z. sarae\u003c/em\u003e all exhibit the typical quadripartite structure composed of a large single-copy (LSC) region, a small single-copy (SSC) region, and two inverted repeats (IRs). Genome lengths ranged from 158,144 bp to 158,678 bp and GC contents between 37.80 \u0026ndash; 38.06 % (Table 1; Figure 1). The plastid genomes of \u003cem\u003eZ. bagnoldii\u003c/em\u003e, \u003cem\u003eZ. sarae\u003c/em\u003e and \u003cem\u003eP. laeta\u003c/em\u003e encoded an identical set of 137 genes, of which 87 are protein coding genes (PCGs), 42 tRNAs, 8 rRNAs. Among them, 113 single copy PCGs were identified, 15 genes contain one intron (\u003cem\u003erps16\u003c/em\u003e, \u003cem\u003eatpF\u003c/em\u003e, \u003cem\u003erpoC1\u003c/em\u003e, \u003cem\u003epetB\u003c/em\u003e, \u003cem\u003epetD\u003c/em\u003e, \u003cem\u003erpl16\u003c/em\u003e, \u003cem\u003erpl2\u003c/em\u003e, \u003cem\u003endhB\u003c/em\u003e, \u003cem\u003endhA\u003c/em\u003e, \u003cem\u003etrnK-UUU\u003c/em\u003e, \u003cem\u003etrnG-UCC\u003c/em\u003e, \u003cem\u003etrnL-UAA\u003c/em\u003e, \u003cem\u003etrnV-UAC\u003c/em\u003e, \u003cem\u003etrnI-GAU\u003c/em\u003e, \u003cem\u003etrnA-UGC\u003c/em\u003e) and 3 genes contain 2 introns (\u003cem\u003erps12\u003c/em\u003e, \u003cem\u003eycf3\u003c/em\u003e, \u003cem\u003eclpP\u003c/em\u003e) (Table 2). All 11 \u003cem\u003endh\u003c/em\u003e genes, which encode the NADH dehydrogenase-like complex, were found to be intact and functional in all three species.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eGenome comparison and variation analyses\u003c/h3\u003e\n\u003cp\u003eMauve analyses revealed that the plastomes of \u003cem\u003eHippeastrum\u003c/em\u003e and \u003cem\u003eZephyranthes\u003c/em\u003e studied do not exhibit significant rearrangements, suggesting high stability in plastid genome organization (Figure 2). Similarly, a high level of sequence similarity and structural conservation was observed with pyGenomeViz (Figure 3). The junctions were determined using CPJSdraw and the genes found at these junctions. These junctions were determined using CPJSdraw (Figure 4). The genes \u003cem\u003erpl22\u003c/em\u003e, \u003cem\u003erps19\u003c/em\u003e, \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003endhF\u003c/em\u003e, \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003etrnN\u003c/em\u003e, \u003cem\u003epsbA\u003c/em\u003e, and are consistently found at plastome junctions, reflecting the importance of these genes in IR region expansion/contraction processes. The rpl22 gene was situated in the LSC region for \u003cem\u003eZ. bagnoldii\u003c/em\u003e and \u003cem\u003eZ. sarae\u003c/em\u003e. In P. laeta this gene extended 52 bp into the IRb boundary. The \u003cem\u003eycf1\u0026nbsp;\u003c/em\u003egene extended across the SSC/IRa boundary by 978 bp, 978, bp and 1024 bp for \u003cem\u003eZ. bagnoldii\u003c/em\u003e, \u003cem\u003eZ. sarae\u003c/em\u003e and \u003cem\u003eP. laeta,\u003c/em\u003e respectively. Furthermore, the SSC/IRa junction was observed to be located within the \u003cem\u003eycf1\u003c/em\u003e gene. The mVISTA alignment revealed high overall sequence conservation among the ten Hippeastreae plastomes, with most regions showing \u0026gt; 90% sequence identity (Figure 5). The complete plastomes ranged from approximately 120-158 kb in length, maintaining typical quadripartite structure across all species. Protein-coding regions displayed the greatest similarity, with most genes showing more than 95% sequence identity across taxa. By contrast, non-coding regions, particularly several intergenic spacers, exhibited reduced identity relative to coding genes. The inverted repeat regions were comparatively less variable than the single-copy regions, consistent with the sliding-window analysis of nucleotide diversity (\u0026pi;), where the mean Pi across the plastome was 0.00408 and values differed among partitions (LSC: 0.00527; SSC: 0.00645; IRb: 0.00132; IRa: 0.00171). Divergence was concentrated in the small single-copy region, including \u003cem\u003eycf1\u003c/em\u003e and adjacent intergenic segments, in agreement with local Pi peaks detected in this interval (Figure 6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine divergent hotspot regions in the ten Amarillydaceae\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003etribe Hippeastreae species, we compared the Pi values of the cp genomes using DNASP software (Figure 6). For the ten Amarillydaceae\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003etribe Hippeastreae species, Pi values ranged from 0.0 to 0.02115. The mean nucleotide diversity value of the whole cp genome was 0.00408, while the corresponding values of the LSC, IRb, SSC, and IRa were 0.00527, 0.00132, 0.00645, and 0.00171, respectively. LSC and SSC regions exhibited greater variation than IR regions, consistent with mVISTA findings. The graph highlights a distinct heterogeneity in evolutionary rates across the plastid genome, where IRs remain conserved while LSCs and SSCs showed greater variability. Variable regions greater than 0.012 were detected. In the LSC region, the \u003cem\u003epsbA\u003c/em\u003e gene shows moderate variability (0.015), a notable peak is observed in the \u003cem\u003etrnL-UAA\u003c/em\u003e region (~0.018) and the \u003cem\u003epsbJ-psbL-psbF-psbE\u003c/em\u003e region shows the highest peak in this region (~0.020). In the SSC region, the \u003cem\u003eccsA-ndhD-psaC\u003c/em\u003e genes show significant peaks of variability (~0.017) and the \u003cem\u003eycf1\u003c/em\u003e region exhibits multiple peaks of variability (~0.012).\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eRepeat elements analysis\u003c/h3\u003e\n\u003cp\u003eLarge sequence repeats (LSRs) were identified as repeats with a length of \u0026ge;30 bp each. A total of 381 LSRs were detected across the ten Hippeastreae plastomes, including palindromic repeats (P) and forward repeats (D) (Figure 7). Palindromic repeats were the most prevalent type with 224 occurrences, while forward repeats were the least common with 157 occurrences. A total of 38 repeats were recovered in \u003cem\u003eZ. bagnoldii\u003c/em\u003e including 22 forward and 16 palindromic. In \u003cem\u003eZ. sarae\u0026nbsp;\u003c/em\u003e41 repeats were detected including 22 forward and 19 palindromic repeats whilst in \u003cem\u003eP. laeta\u0026nbsp;\u003c/em\u003e43 repeats including 25 forward and 18 palindromic repeats were recovered.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the MISA-web analysis, six types of simple sequence repeats (SSRs) were identified, with mononucleotide repeats comprising the majority (399, 64.88%), followed by dinucleotide repeats (111, 18.05%), tetranucleotide repeats (74, 12.03%), pentanucleotide repeats (16, 2.60%), trinucleotide repeats (11, 1.79%), and hexanucleotide repeats (4, 0.65%) (Figure 8a). \u0026nbsp;SSRs have been detected in \u003cem\u003eZ. bagnoldii\u003c/em\u003e (58), \u003cem\u003eZ. sarae\u003c/em\u003e (62) and \u003cem\u003eP. laeta\u003c/em\u003e (69). \u003cem\u003eZ. bagnoldii\u003c/em\u003e has 35 (63.79%) mononucleotide (A/T) repeats, whereas \u003cem\u003eZ. sarae\u003c/em\u003e and \u003cem\u003eP. laeta\u003c/em\u003e have 39 (66.13%) and 44 (63.77%), respectively.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003ePhylogenetic analysis\u003c/h3\u003e\n\u003cp\u003ePhylogenetic relationships within the Hippeastreae tribe were inferred using 76 plastome protein-coding genes from ten Hippeastreae plastomes and one outgroup (\u003cem\u003eNarcissus poeticus\u003c/em\u003e; NC_039825.1), using maximum likelihood (ML) and Bayesian inference (BI) methods. Both analyses produced congruent topologies (Figure 9). Subtribes \u003cem\u003eHippeastrinae\u0026nbsp;\u003c/em\u003eand \u003cem\u003eTraubinnae\u003c/em\u003e are resolved as monophyletic. \u003cem\u003ePaposoa laeta\u0026nbsp;\u003c/em\u003e(subtribe \u003cem\u003eTraubinnae\u003c/em\u003e) was recovered sister to subtribe \u003cem\u003eHippeastrinae\u0026nbsp;\u003c/em\u003e(100 BS; 1.00 PP). Within subtribe \u003cem\u003eHippeastrinae\u003c/em\u003e, \u003cem\u003eZephyranthes\u0026nbsp;\u003c/em\u003eclade I (\u003cem\u003eH.\u003c/em\u003e\u003cem\u003e\u0026nbsp;reticulatum\u003c/em\u003e, \u003cem\u003eZ. candida\u003c/em\u003e, \u003cem\u003eZ. mesochloa\u003c/em\u003e) was recovered as sister to the \u003cem\u003eHippeastrum\u0026nbsp;\u003c/em\u003eclade (\u003cem\u003eH. albertii\u003c/em\u003e, \u003cem\u003eH. vittatum\u003c/em\u003e, \u003cem\u003eH. rutilum\u003c/em\u003e) and \u003cem\u003eZephyranthes\u003c/em\u003e clade subg. \u003cem\u003eMyostemma\u003c/em\u003e (\u003cem\u003eZ. bagnoldii\u003c/em\u003e, \u003cem\u003eZ. phycelloides\u003c/em\u003e, \u003cem\u003eZ. sarae\u003c/em\u003e) with maximum support. The sister relationship between the \u003cem\u003eHippeastrum\u0026nbsp;\u003c/em\u003eclade and \u003cem\u003eZ.\u0026nbsp;\u003c/em\u003esubgenus \u003cem\u003eMyostemma\u0026nbsp;\u003c/em\u003ehad recovered\u003cem\u003e\u0026nbsp;\u003c/em\u003esupport values of 48 BS and 0.59 PP. Both \u003cem\u003eHippeastrum\u0026nbsp;\u003c/em\u003eand \u003cem\u003eZephyranthes\u0026nbsp;\u003c/em\u003eare recovered as polyphyletic.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe three newly assembled plastomes of \u003cem\u003eZ. bagnoldii\u003c/em\u003e, \u003cem\u003eZ. sarae\u003c/em\u003e and \u003cem\u003eP. laeta\u003c/em\u003e exhibit the canonical quadripartite architecture and a narrow range of genome sizes and GC content. These plastomes fall within the size range (153\u0026ndash;160 kb) and GC content (around 37%) typical of other Amaryllidaceae species (Cheng et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Jimenez et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), mirroring the strong structural conservatism long recognized for Amaryllidaceae and other angiosperms (Contreras-D\u0026iacute;az et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mower \u0026amp; Vickrey, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Mauve alignments and synteny plots revealed no major rearrangements among Hippeastreae plastomes, consistent with the predominantly collinear organization reported for closely related lineages (Huo et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Namgung et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Together with the stable gene complement recovered here (137 genes: 87 PCGs, 42 tRNAs, 8 rRNAs), these patterns suggest that most interspecific plastome differences in Hippeastreae arise not from large-scale structural change but from localized boundary shifts and sequence-level variation. All species had intact and functional \u003cem\u003endh\u003c/em\u003e genes which is in stark contrast to the pattern of \u003cem\u003endh\u003c/em\u003e gene degradation and loss observed in other Amaryllidaceae taxa like \u003cem\u003eStrumaria truncate\u003c/em\u003e (K\u0026ouml;nyves et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and \u003cem\u003eAllium paradoxum\u003c/em\u003e (Omelchenko et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), suggesting a conserved and photosynthetically competent plastome in the studied species.\u003c/p\u003e \u003cp\u003eSmall yet informative differences occur at the junctions between single-copy regions and the inverted repeats (IRs). The genes \u003cem\u003erpl22\u003c/em\u003e, \u003cem\u003erps19\u003c/em\u003e, \u003cem\u003endhF\u003c/em\u003e, \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003etrnN\u003c/em\u003e, \u003cem\u003epsbA\u003c/em\u003e, and are consistently found at plastome junctions, reflecting the importance of these genes in IR region expansion/contraction processes. Notably, \u003cem\u003eP. laeta\u003c/em\u003e shows a\u0026thinsp;~\u0026thinsp;52 bp extension of \u003cem\u003erpl22\u003c/em\u003e into IRb, a derived, lineage-specific character state that may serve as a genus-level diagnostic and provides insights into the evolutionary history of Traubiinae. Additionally, this combined with the phylogenetic placement of \u003cem\u003eP. laeta\u003c/em\u003e sister to subtribe Hippeastrinae, reinforces subtribal delimitations (Garc\u0026iacute;a et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Garc\u0026iacute;a et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Meerow, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). From a plastome-evolution perspective, IR boundary shifts represent recurrent expansion\u0026ndash;contraction dynamics that change the extent of duplicated sequence and create lineage-specific junction patterns, providing a subtle but informative source of structural variation in otherwise stable plastomes (Goulding et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In Hippeastreae, IR boundary shifts are small in absolute size, but the resulting junction configurations (i.e., which loci extend into or out of the inverted repeat) can be consistent within lineages; when compared across Hippeastrinae and Traubiinae, these patterns provide complementary structural characters that help distinguish subtribal groupings and corroborate relationships inferred from sequence-based phylogenies (Garc\u0026iacute;a et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Namgung et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA higher number of palindromic repeats is recovered in the ten Hippeastreae plastomes studied (14\u0026ndash;19) compared to the number of forward repeats (21\u0026ndash;25). SSRs are strongly A/T-biased, accounting for 81.36\u0026ndash;85.07% of SSRs. A higher number of large sequence repeats and SSRs was recovered for \u003cem\u003eP. laeta\u003c/em\u003e compared to \u003cem\u003eZ. bagnoldii\u003c/em\u003e and \u003cem\u003eZ. sarae\u003c/em\u003e, differentiating these plastomes. Specifically, \u003cem\u003eP. laeta\u003c/em\u003e had a higher abundance of mononucleotide repeats. The dinucleotide type remains constant at 11 across our studied species. \u003cem\u003eZ. bagnoldii\u003c/em\u003e and \u003cem\u003eZ. sarae\u003c/em\u003e are most similar to \u003cem\u003eZ. phycelloides\u003c/em\u003e when comparing the presence of mono-, di-, tetra- and hexa-nucleotides. Whereas \u003cem\u003eP. laeta\u003c/em\u003e is different in having pentanucleotides and no hexanucleotides. Among these SSRs, repeat units of A/T, AT/AT and AAAT/ATTT accounted for 83.57% of the total, indicating a bias towards A/T bases in SSR composition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). This suggests that \u003cem\u003eP. laeta\u003c/em\u003e has greater genomic dynamism, which could be related to specific ecological adaptations to the environment. To distinguish the species studied in this paper from other Hippeastreae, we suggest two complementary marker panels: (i) a \u0026ldquo;short-amplicon\u0026rdquo; set targeting \u003cem\u003epsbA\u003c/em\u003e, \u003cem\u003etrnL-UAA\u003c/em\u003e, and \u003cem\u003epsbJ-psbL-psbF-psbE\u003c/em\u003e for degraded material (herbarium, soil eDNA), and (ii) a \u0026ldquo;long-amplicon\u0026rdquo; set that includes the 3\u0026prime; region of \u003cem\u003eycf1\u003c/em\u003e and the \u003cem\u003eccsA-ndhD-psaC\u003c/em\u003e interval for high-resolution phylogeography and species delimitation. The convergence of mVISTA conservation profiles and π peaks across our 10-taxon comparison underscores that these loci are robust candidates rather than idiosyncratic outliers.\u003c/p\u003e \u003cp\u003eOur phylogenetic analyses support the recovery of subtribes Hippeastrinae and Traubiinae as monophyletic, support the placement of \u003cem\u003eP. laeta\u003c/em\u003e within Traubiinae, and align with cytological evidence indicating chromosome numbers of 2n\u0026thinsp;=\u0026thinsp;18 in Hippeastrinae and 2n\u0026thinsp;=\u0026thinsp;16 in Traubiinae (Baeza et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Jara-Seguel et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The phylogenetic placement and karyology of \u003cem\u003eP. laeta\u003c/em\u003e combined with the higher number of SSRs and variation of the IRb junction, provide a reliable genus-specific marker for \u003cem\u003eP. laeta\u003c/em\u003e. The polyphyly recovered in subtribe \u003cem\u003eHippeastrinae\u003c/em\u003e concurs with the relationships recovered in Garc\u0026iacute;a et al. (2014; \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), which has been attributed to reticulation evolution and incomplete lineage sorting.\u003c/p\u003e \u003cp\u003e \u003cem\u003eZephyranthes bagnoldii\u003c/em\u003e, \u003cem\u003eZ. sarae\u003c/em\u003e, and \u003cem\u003eZ. phycelloides\u003c/em\u003e have overlapping distributions and ecological niches, occurring along the Atacama coastal belt and undergo mass flowering events following winter rainfall associated with La Ni\u0026ntilde;a conditions (Araya et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Guti\u0026eacute;rrez, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Because mass flowering in the Atacama is triggered by infrequent rainfall pulses, above-ground emergence and reproductive output can vary strongly among years. In geophytes, however, this variability may be buffered by below-ground bulbs (a \u0026ldquo;bud bank\u0026rdquo;), so fluctuations in visible abundance do not necessarily reflect comparable changes in survival. Instead, rainfall pulses likely drive episodic recruitment and seed production, potentially influencing effective population size and chloroplast haplotype frequencies. Our plastome markers (SSRs and high-variability regions) enable tests of these patterns and connectivity.\u003c/p\u003e \u003cp\u003eAlthough chloroplast variation within species is generally low (Nock et al \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), sampling several individuals would provide a stronger basis for population-level analyses and confirm the stability of boundary positions or repeat counts. Plastome genomes are typically maternally inherited, representing one evolutionary history (Hagemann, 2004; Wicke et al., 2011). The nuclear genome is bi-parentally inherited and can be used to detect reticulate evolution including ancienty hybridization events and introgression (Stull et al., 2023). Given that previous nuclear analyses have documented reticulation within \u003cem\u003eZephyranthes\u003c/em\u003e, comparisons between plastid and nuclear phylogenetic signals provide an important context for interpreting plastome-based relationships within \u003cem\u003eMyostemma\u003c/em\u003e (Garc\u0026iacute;a et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Garc\u0026iacute;a et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThat \u003cem\u003eP. laeta\u003c/em\u003e is genomically conservative yet phylogenetically divergent offers an additional insight: plastome stability does not preclude lineage-specific boundary characters (\u003cem\u003erpl22\u003c/em\u003e at IRb/LSC) or differences in repeat density. Rather, these fine-scale shifts may be especially informative at the subtribal level, where broader structural rearrangements are absent. We therefore advocate explicit use of IR boundary features and repeat profiles as a complementary, low-cost layer of evidence when resolving relationships among Traubiinae and Hippeastrinae genera.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe plastomes of \u003cem\u003eZ. bagnoldii\u003c/em\u003e, \u003cem\u003eZ. sarae\u003c/em\u003e, and \u003cem\u003eP. laeta\u003c/em\u003e presented in this study deepen our understanding of plastome evolution and structure in Chilean Hippeastreae taxa. Shifts in the inverted repeat boundary and number of repeat and SSRs can form the basis for DNA barcoding and population genetics within this group. Our study shows that small shifts between single-copy regions and the inverted repeats (IRs), can be used to distinguish between species. Repeat and SSR profiles distinguish the three species, with strong A/T bias overall and a motif composition in \u003cem\u003eP. laeta\u003c/em\u003e that distinguishes it from \u003cem\u003eZephyranthes\u003c/em\u003e (presence of pentanucleotides and absence of hexanucleotides), suggesting useful loci for downstream barcoding and population-level applications in Atacama geophytes. Phylogenomically, our plastid data support current subtribal descriptions. These results corroborate extensive structural stability, reveal modest yet phylogenetically informative differences among lineages, and identify repeat/SSR features that can be used for species delimitation, phylogeography. The recovered topology, \u003cem\u003eMyostemma\u003c/em\u003e cohesion with \u003cem\u003eP. laeta\u003c/em\u003e sister to Hippeastrinae, agrees with current subtribal delimitations and provides a plastid reference that can be combined with nuclear data in future phylogenomic analyses to test cytonuclear congruence and refine relationships within Hippeastreae. In a region where above-ground diversity and flowering respond to infrequent rainfall pulses, these resources can help track genetic responses and support conservation strategies, by aided reproduction to keep the genetic pool healthy, for narrowly distributed geophytes of the flowering desert.\u003c/p\u003e"},{"header":"Declarations","content":"\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 PV849990 for \u003cem\u003eZephyranthes bagnoldii\u003c/em\u003e, PV890574 for \u003cem\u003eZephyranthes sarae\u003c/em\u003e and PV890573 for \u003cem\u003ePaposoa laeta\u003c/em\u003e. The BioProject, BioSample and SRA accession numbers on NCBI for \u003cem\u003eZephyranthes bagnoldii\u003c/em\u003e are PRJNA1304596, SAMN50563324 and SRR34952303, for \u003cem\u003eZephyranthes sarae\u003c/em\u003e are PRJNA1304964, SAMN50580584 and SRR34962843, and for \u003cem\u003ePaposa laeta\u003c/em\u003e are PRJNA1304263, SAMN50554916 and SRR34941586. This study is not a clinical trial.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRC thanks the Atacama Regional Government for funding support through the FIC project BIP 40057824 and ANID through the FONDECYT Initiation grant 11230668. PJ acknowledges support XCEL - Extreme Cryptogram Ecology Lab, University of Applied Sciences Kaiserslautern, Germany. MA thanks the M\u0026aacute;ster Universitario de Bioinform\u0026aacute;tica y Bioestad\u0026iacute;stica (UOC\u0026ndash;UB), Universitat Oberta de Catalunya. RC also thanks Neri Contreras-Ascencio and Ana D\u0026iacute;az-S\u0026aacute;nchez for their support during field sampling. The authors are grateful to Mayor Brunilda Gonz\u0026aacute;lez Anjel of the Municipality of Caldera for granting permission to conduct field sampling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors. This research was conducted in compliance with the relevant research permits, in accordance with national and international standards for the collection of plant material (\u003cem\u003ePaposoa laeta\u003c/em\u003e, \u003cem\u003eZephyranthes bagnoldii\u003c/em\u003e, and \u003cem\u003eZephyranthes sarae\u003c/em\u003e) and with appropriate safeguards for flora and fauna. Collection and fieldwork were authorized by CONAF (Corporaci\u0026oacute;n Nacional Forestal; National Forestry Corporation) under permits N\u0026ordm; 122/2019 (granted on 8 November 2019) and N\u0026ordm; 78/2024 (granted on 14 April 2024). In addition, a research and collection permit was issued by the Municipality of Caldera (Permit No. 161/2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Atacama Regional Government through the FIC Project BIP 40057824, by ANID\u0026ndash;FONDECYT (Initiation Project No. 11230668), and by XCEL (Extreme Cryptogram Ecology Lab), University of Applied Sciences Kaiserslautern.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.A., R.C., Z.D., L.vdB.: data analyses, writing manuscript. M.A., R.C., Z.D., P.J.: data interpretation, writing and editing manuscript. M.A., R.C.: experimental analysis. M.A., R.C., Z.D., L.vdB., P.J.: Analysis and interpretation of result. M.A., R.C., Z.D., L.vdB., P.J.: editing-review original draft. R.C., P.J.: funding acquisition. All the authors have approved the final manuscript. All authors authorize the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhD Program in Biotechnology and Sustainable Bioproduction, a consortium between the Universidad de Atacama and the Universidad Arturo Prat, Chile.\u003c/p\u003e\n\u003cp\u003eMariana Arias-Aburto\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlsharif, W., Saad, M. M., \u0026amp; Hirt, H. (2020). 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Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes. \u003cem\u003eBMC Biology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;7\u003c/em\u003e(1), 84. https://doi.org/10.1186/1741-7007-7-84\u003c/li\u003e\n \u003cli\u003ePOWO. (2025). \u003cem\u003ePlants of the World Online. Facilitated by the Royal Botanic Gardens, Kew\u003c/em\u003e. Retrieved Retrieved 12 August 2025 from https://powo.science.kew.org\u003c/li\u003e\n \u003cli\u003eRambaut, A. (2007). FigTree (v. 1.4.4). Institute of Evolutionary Biology, University of Edinburgh, Edinburgh. \u003cem\u003ehttp://tree.bio.ed.ac.uk/software/figtree/\u003c/em\u003e.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRavi, V., Khurana, J. P., Tyagi, A. K., \u0026amp; Khurana, P. 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(2017).\u0026nbsp;DnaSP 6: DNA Sequence Polymorphism Analysis of Large Data Sets. \u003cem\u003eMol Biol Evol\u003c/em\u003e,\u003cem\u003e\u0026nbsp;34\u003c/em\u003e(12), 3299-3302. https://doi.org/10.1093/molbev/msx248\u003c/li\u003e\n \u003cli\u003eSabater, B. (2018). Evolution and Function of the Chloroplast. Current Investigations and Perspectives. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e,\u003cem\u003e\u0026nbsp;19\u003c/em\u003e(10), 3095. https://doi.org/10.3390/ijms19103095\u003c/li\u003e\n \u003cli\u003eShi, L., Chen, H., Jiang, M., Wang, L., Wu, X., Huang, L., \u0026amp; Liu, C. (2019).\u0026nbsp;CPGAVAS2, an integrated plastome sequence annotator and analyzer. \u003cem\u003eNucleic Acids Research\u003c/em\u003e,\u003cem\u003e\u0026nbsp;47\u003c/em\u003e(W1), W65-W73. https://doi.org/10.1093/nar/gkz345\u003c/li\u003e\n \u003cli\u003eShimoyama, Y. (2024). \u003cem\u003epyGenomeViz: A genome visualization python package for comparative genomics\u003c/em\u003e.\u003cem\u003e\u0026nbsp;\u003c/em\u003eIn GitHub. https://github.com/moshi4/pyGenomeViz\u003c/li\u003e\n \u003cli\u003eSqueo, F. A., Arroyo, M. T., Marticorena, A., Arancio, G., Mu\u0026ntilde;oz-Schick, M., Negritto, M.,\u0026hellip;Huma\u0026ntilde;a, A. (2008). Cat\u0026aacute;logo de la Flora Vascular de la Regi\u0026oacute;n de Atacama. \u003cem\u003eLibro rojo de la flora nativa y de los sitios prioritarios para su conservaci\u0026oacute;n: Regi\u0026oacute;n de Atacama\u003c/em\u003e, 97-120.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eStamatakis, A. (2014). RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. \u003cem\u003eBioinformatics\u003c/em\u003e,\u003cem\u003e\u0026nbsp;30\u003c/em\u003e(9), 1312-1313. https://doi.org/10.1093/bioinformatics/btu033\u003c/li\u003e\n \u003cli\u003eStevens, P. F. (2023). \u003cem\u003eAngiosperm Phylogeny Website. Version 14, July 2017 [and more or less continuously updated since]\u003c/em\u003e. Retrieved Retrieved 12 August 2025 from https://www.mobot.org/MOBOT/research/APweb\u003c/li\u003e\n \u003cli\u003eThe Angiosperm Phylogeny, G., Chase, M. W., Christenhusz, M. J. M., Fay, M. F., Byng, J. W., Judd, W. S.,\u0026hellip;Stevens, P. F. (2016). An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV. \u003cem\u003eBotanical Journal of the Linnean Society\u003c/em\u003e,\u003cem\u003e\u0026nbsp;181\u003c/em\u003e(1), 1-20. https://doi.org/10.1111/boj.12385\u003c/li\u003e\n \u003cli\u003eTillich, M., Lehwark, P., Pellizzer, T., Ulbricht-Jones, E. S., Fischer, A., Bock, R., \u0026amp; Greiner, S. (2017).\u0026nbsp;GeSeq \u0026ndash; versatile and accurate annotation of organelle genomes. \u003cem\u003eNucleic Acids Research\u003c/em\u003e,\u003cem\u003e\u0026nbsp;45\u003c/em\u003e(W1), W6-W11. https://doi.org/10.1093/nar/gkx391\u003c/li\u003e\n \u003cli\u003eVicencio, J., B\u0026ouml;hm, C., Schween, J. H., L\u0026ouml;hnert, U., \u0026amp; Crewell, S. (2024). A comparative study of the atmospheric water vapor in the Atacama and Namib Desert. \u003cem\u003eGlobal and Planetary Change\u003c/em\u003e,\u003cem\u003e\u0026nbsp;232\u003c/em\u003e, 104320. https://doi.org/https://doi.org/10.1016/j.gloplacha.2023.104320\u003c/li\u003e\n \u003cli\u003eZhang, Y., Tian, L., \u0026amp; Lu, C. (2023). Chloroplast gene expression: Recent advances and perspectives. \u003cem\u003ePlant Communications\u003c/em\u003e,\u003cem\u003e\u0026nbsp;4\u003c/em\u003e(5). https://doi.org/10.1016/j.xplc.2023.100611\u003c/li\u003e\n \u003cli\u003eZheng, S., Poczai, P., Hyv\u0026ouml;nen, J., Tang, J., \u0026amp; Amiryousefi, A. (2020). Chloroplot: An Online Program for the Versatile Plotting of Organelle Genomes [Technology and Code]. \u003cem\u003eFrontiers in Genetics\u003c/em\u003e,\u003cem\u003e\u0026nbsp;11\u003c/em\u003e. https://doi.org/10.3389/fgene.2020.576124\u003c/li\u003e\n \u003cli\u003eZhu, A., Guo, W., Gupta, S., Fan, W., \u0026amp; Mower, J. P. (2016). Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates. \u003cem\u003eNew Phytologist\u003c/em\u003e,\u003cem\u003e\u0026nbsp;209\u003c/em\u003e(4), 1747-1756. https://doi.org/https://doi.org/10.1111/nph.13743\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the supplementary files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Zephyranthes, chloroplast genome, dryland, plastome, desert bloom","lastPublishedDoi":"10.21203/rs.3.rs-8801993/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8801993/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: The Atacama “flowering desert” offers a natural setting to investigate plant genomic diversity and lineage differentiation in endemic geophytes shaped by long-term aridity, fragmented habitats, and ENSO-linked winter rains. We assembled, annotated, and compared complete plastid genomes for three Chilean taxa (\u003cem\u003eZephyranthes bagnoldii\u003c/em\u003e, \u003cem\u003eZephyranthes sarae\u003c/em\u003e, and \u003cem\u003ePaposoa laeta\u003c/em\u003e) with two objectives: (i) to clarify their relationships within Hippeastreae, and (ii) to identify genome regions and repeat features that can be applied to species identification, population studies, and conservation planning in northern Chile and comparable deserts.\u003c/p\u003e\n\u003cp\u003eResults: All three genomes showed the canonical quadripartite organization with broadly similar sizes (158,144 to 158,678 bp) and gene complements. Whole-genome alignments and synteny comparisons indicated overall collinearity, while boundary visualizations revealed modest shifts at the junctions between the large single-copy, small single-copy, and inverted repeat regions, most often involving \u003cem\u003erpl22\u003c/em\u003e and \u003cem\u003eycf1\u003c/em\u003e. Analyses of repetitive elements and simple sequence repeats (microsatellites) identified predominantly adenine/thymine-rich motifs and regions of elevated sequence variability in the single-copy portions, indicating multiple loci as suitable for DNA barcoding and population-level studies. Both subtribes within Hippeastreae are resolved as monophyletic, however, within subtribe Hippeastrinae\u003cem\u003e, \u003c/em\u003eZephyranthes is recovered as polyphyletic.\u003c/p\u003e\n\u003cp\u003eConclusions: These plastid genomes demonstrate strong architectural stability while revealing informative boundary differences and clear contrasts in repeat and microsatellite profiles. The identified regions of elevated variability provide usable markers for species delimitation, phylogeography, and monitoring of genetic connectivity in Amaryllidaceae Atacama geophytes. The recovered relationships confirm subtribal relationships between Traubiinae and Hippeastrinae and establish a plastid reference framework that supports integrative studies and conservation efforts in the flowering-desert system.\u003c/p\u003e","manuscriptTitle":"Comparative Analysis of the Plastid Genomes of Zephyranthes bagnoldii, Zephyranthes sarae, and Paposoa laeta from the Flowering Desert of the Atacama Region, Chile","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-04 08:01:50","doi":"10.21203/rs.3.rs-8801993/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-03-27T03:41:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"327810889167115611742009680660953464401","date":"2026-03-19T17:28:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-26T19:53:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-26T19:49:28+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-18T12:28:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-18T01:08:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-02-18T01:05:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f6659a9c-722f-4b84-8655-db5e8f920042","owner":[],"postedDate":"March 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-04T08:01:50+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-04 08:01:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8801993","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8801993","identity":"rs-8801993","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}