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. 2022 Apr 6;11(7):1001. doi: 10.3390/plants11071001

Plastid Genome of Equisetum xylochaetum from the Atacama Desert, Chile and the Relationships of Equisetum Based on Frequently Used Plastid Genes and Network Analysis

Anchittha Satjarak 1,*, Linda E Graham 2, Marie T Trest 2, Patricia Arancibia-Avila 3
Editors: Alex Troitsky, Jaakko Hyvönen
PMCID: PMC9002811  PMID: 35406981

Abstract

The modern pteridophyte genus Equisetum is the only survivor of Sphenopsida, an ancient clade known from the Devonian. This genus, of nearly worldwide distribution, comprises approximately 15 extant species. However, genomic information is limited. In this study, we assembled the complete chloroplast genome of the giant species Equisetum xylochaetum from a metagenomic sequence and compared the plastid genome structure and protein-coding regions with information available for two other Equisetum species using network analysis. Equisetum chloroplast genomes showed conserved traits of quadripartite structure, gene content, and gene order. Phylogenetic analysis based on plastome protein-coding regions corroborated previous reports that Equisetum is monophyletic, and that E. xylochaetum is more closely related to E. hyemale than to E. arvense. Single-gene phylogenetic estimation and haplotype analysis showed that E. xylochaetum belonged to the subgenus Hippochaete. Single-gene haplotype analysis revealed that E. arvense, E. hyemale, E. myriochaetum, and E. variegatum resolved more than one haplotype per species, suggesting the presence of a high diversity or a high mutation rate of the corresponding nucleotide sequence. Sequences from E. bogotense appeared as a distinct group of haplotypes representing the subgenus Paramochaete that diverged from Hippochaete and Equisetum. In addition, the taxa that were frequently located at the joint region of the map were E. scirpoides and E. pratense, suggesting the presence of some plastome characters among the Equiseum subgenera.

Keywords: Equisetum, plastid genome, haplotype map

1. Introduction

Equisetum L. is a genus of vascular plants that represents ancient Sphenopsida, a long-enduring clade known from fossils of the Devonian and later ages and, therefore, is considered useful in understanding the evolution of vascular plants. This genus is comprised of approximately 15 extant species, with a nearly worldwide distribution [1,2]. Previous studies have examined the evolutionary relationships among stem and crown Equisetum species using both morphology and genomic data. However, because morphology can vary as the result of hybridization and climate differences, molecular approaches have become popular. Recent studies have indicated three subgenera, including the primitive subgenus Paramochaete, and the later diverging subgenera Equisetum and Hippochaete. However, such relationships were estimated from relatively few plastid genes, e.g., rbcL, rps4, and trnL-F e.g., [3,4,5,6].

Among reported pteridophyte plastome sequences, only three were from Equisetum species: one from E. hyemale [7] and two from US and Korean E. arvense [8,9], which were placed in subgenera Equisetum and Hippochaete, respectively e.g., [3,4,5,6]. These reports revealed plastome variation among Equisetum species. The two E. arvense genomes differed by 417 bp and the E. hyemale genome was about 1.5 kbp smaller than that of E. arvense. In addition, rpl16 of E. arvense had an intron that is not present in E. hyemale [7,8,9]. These observations indicate that additional chloroplast genomes would be useful in evaluating evolutionary trends in this long-enduring genus.

Previous Equisetum plastome information was obtained using PCR amplification or from organelle-enriched DNA. The advancement of sequencing technologies and computational techniques allowed us to obtain complete organelle genome sequences from the shotgun metagenomic data we have archived for E. xylochaetum, presenting an additional technical option for obtaining Equisetum plastid genomes. Therefore, in this study, we assembled the complete plastid genome of E. xylochaetum, a giant species endemic to the Atacama Desert of Chile, South America, and used the information obtained to explore the evolution of Equisetum plastid genomes and the phylogenetic relationship of Equisetum species, as well as to determine whether the phylogeny estimated by using the popular plastid conserved regions was congruent with the haplotype mapping results of the corresponding sequences. Results showed that we successfully constructed the de novo plastid genome of E. xylochaetum using the shotgun metagenomic data. Phylogenetic estimations and comparison of Equisetum plastid genomes showed that E. xylochaetum was in the subgenus Hippochaete and that the Equisetum plastid genomes from subgenera Hippochaete and Equisetum were conserved in terms of genome structure, gene content, and gene order. Furthermore, results from TCS haplotype mapping showed that some of the taxa had a higher level of nucleotide diversity and some of the taxa shared common nucleotide haplotypes. Therefore, more conserved nucleotide regions and complete plastid genomes are needed for a better understanding of the evolutionary relationships of Equisetum.

2. Results

The chloroplast genome of E. xylochaetum displayed a quadripartite structure. The single-copy regions were 93,902 bp and 9726 bp, with two reverse repeated regions (IRa and IRb) of 14,386 bp in length. The GC contents of the LSC, SSC, and IR regions individually, and of the cp genome as a whole, were 31.5%, 30.9%, 48.4%, and 33.9%, respectively. The E. xylochaetum plastome encoded a total of 119 unique genes, of which nine were duplicated in the IR regions. Seventy-eight were protein-coding genes, 38 were tRNA genes, and eight were rRNA genes. Fourteen genes contain introns (atpF, clpP, ndhA, ndhB, petB, petD, rpl2, rpoC1, rps12, ycf3, trnK(uuu), trnL(uaa), trnV(uac), and trnI(gau)) as shown in Figure 1.

Figure 1.

Figure 1

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Circular map of the Equisetum xylochaetum plastid genome, NCBI accession MW282958, drawn by OGDRAW version 1.3.1 [10]. Genes positioned on the outside of the map are transcribed counterclockwise and those inside the map are transcribed clockwise. The thick lines indicate the extent of the inverted repeat regions.

A comparison of Equisetum plastid genomes showed a collinear relationship, forming only one syntenic block in whole genome alignment. The genomes had similar genome size, % GC, gene content, gene length, and had identical gene order (Table 1 and Table 2). The protein-coding regions of E. xylochaetum plastid genes were subjected to purifying selection when compared against the corresponding protein-coding genes of E. hyemale and E. arvense.

Table 1.

Comparison of general characters of Equisetum plastid genomes.

E. xylochaetum E. hyemale E. arvense (US) E. arvense (Korea)
Accession MW282958 KC117177 GU191334 JN968380
genome size 132,400 131,760 133,309 132,726
LSC 93,902 92,580 93,542 92,961
SSC 9,726 18,994 19,469 19,477
IRs 14,386 10,093 10,149 10,144
%GC 33.9 33.7 33.4 33.4
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Table 2.

Protein-coding gene content and introns of Equisetum plastid genomes. Comparison showed percent identity and size of the gene and its derived proteins.

DNA Protein
No. Gene (# of Intron) Identical Site (%) Mean (bp) SD (bp) min (bp) max (bp) Identical Site (%) Mean (aa) SD (aa) min (aa) max (aa)
1. accD 92.8 1021.5 81 948 1143 89.9 340 26.5 316 380
2. atpA 95.5 1539 20.8 1527 1575 98 512 6.9 508 524
3. atpB 94.4 1470 0 1470 1470 96.3 489 0 489 489
4. atpE 93.4 396 0 396 396 93.1 131 0 131 131
5. atpF (1) 97.5 555 0 555 555 73.9 184 0 184 184
6. atpH 96.3 246 0 246 246 100 81 0 81 81
7. atpI 95.4 747 0 747 747 99.1 248 0 284 248
8. ccsA 93.3 943.5 1.5 942 945 91.1 313.5 0.5 313 314
9. cemA 89.5 1438.5 21.7 1425 1476 82.7 478.5 7.2 474 491
10. chlB 94.3 1549.5 4.5 1545 1554 93.2 515.5 1.5 514 517
11. chlL 91.6 879 6 873 885 93.9 292 2 290 294
12. chlN 92.8 1300.5 10.5 1290 1311 90.4 432.5 3.5 429 436
13. clpP (1) 95.1 615 0 615 615 98.5 204 0 204 204
14. infA 94.7 243 0 243 243 96.3 80 0 80 80
15. matK 88.7 1470 3 1467 1473 81.4 489 1 488 490
16. ndhA (1) 92.7 1101.8 1.3 1101 1104 91.8 366.3 0.4 366 367
17. ndhB (1) 94.8 1473 0 1473 1473 94.3 490 0 490 490
18. ndhC 95.9 363 0 363 363 98.3 120 0 120 120
19. ndhD 95.1 1497 0 1497 1497 95.2 498 0 498 498
20. ndhE 98.3 303 0 303 303 100 100 0 100 100
21. ndhF 92.8 2221.5 1.5 2220 2223 92.2 739.5 0.5 739 740
22. ndhG 91.4 606 17.2 585 633 85.7 201 5.7 194 210
23. ndhH 95.3 1182 0 1182 1182 97.2 393 0 393 393
24. ndhI 97.3 549 0 549 549 98.4 182 0 182 182
25. ndhJ 93.9 520.5 4.5 516 525 94.8 172.5 1.5 171 174
26. ndhK 90.6 747.8 9.1 732 753 86.8 248.3 3 243 250
27. petA 92.4 955.5 7.5 948 963 93.8 317.5 2.5 315 320
28. petB (1) 96 648 0 648 648 100 215 0 215 215
29. petD (1) 96.9 483 0 483 483 100 160 0 160 160
30. petG 97.4 114 0 114 114 100 37 0 37 37
31. petL 93.8 96 0 96 96 93.5 31 0 31 31
32. petN 99 96 0 96 96 100 31 0 31 31
33. psaA 96.2 2253 0 2253 2253 99.6 750 0 750 750
34. psaB 95.7 2205 0 2205 2205 99.2 734 0 734 734
35. psaC 95.1 246 0 246 246 98.8 81 0 81 81
36. psaI 91.9 111 0 111 111 94.4 36 0 36 36
37. psaJ 97.7 129 0 129 129 100 42 0 42 42
38. psaM 96 99 0 99 99 96.9 32 0 32 32
39. psbA 98.1 1062 0 1062 1062 100 353 0 353 353
40. psbB 96.1 1527 0 1527 1527 99 508 0 508 508
41. psbC 95 1422 0 1422 1422 99.4 473 0 473 473
42. psbD 95.6 1062 0 1062 1062 87.3 353 0 353 353
43. psbE 97.2 246 0 246 246 100 81 0 81 81
44. psbF 98.3 120 0 120 120 100 39 0 39 39
45. psbH 94.7 225 0 225 225 89.2 74 0 74 74
46. psbI 97.3 111 0 111 111 100 36 0 36 36
47. psbJ 99.2 123 0 123 123 100 40 0 40 40
48. psbK 97 168 0 168 168 96.4 55 0 55 55
49. psbL 98.3 117 0 117 117 100 38 0 38 38
50. psbM 98.2 111 0 111 111 94.4 36 0 36 36
51. psbN 95.5 132 0 132 132 93 43 0 43 43
52. psbT 97.4 112.5 1.5 111 114 97.3 36.5 0.5 36 37
53. psbZ 94.2 189 0 189 189 100 62 0 62 62
54. rbcL 96.1 1428 0 1428 1428 99.2 475 0 475 475
55. rpl14 97.6 369 0 369 369 99.2 122 0 122 122
56. rpl16 (1 in E. arvense) 93.1 423 0 423 423 95 140 0 140 140
57. rpl2 (1) 94.3 834.8 1.3 834 837 95.3 277.3 0.4 277 278
58. rpl20 89.7 347.3 1.3 345 348 85.2 114.8 0.4 114 115
59. rpl21 91.3 364.5 1.5 363 366 86 120.5 0.5 120 121
60. rpl22 94.1 372 0 372 372 96.6 123 0 123 123
61. rpl23 94.9 273 0 273 273 93.3 90 0 90 90
62. rpl32 95.3 171 0 171 171 98.2 56 0 56 56
63. rpl33 95.5 201 0 201 201 90.9 66 0 66 66
64. rpl36 93.9 114 0 114 114 100 37 0 37 37
65. rpoA 93.5 1.18.5 4.5 1014 1023 93.5 338.5 1.5 337 340
66. rpoB 93.9 3235.5 33.8 3216 3294 92.9 1.00.5 11.3 1071 1097
67. rpoC1 (1) 93.3 2060.3 3.9 2058 2067 91.3 685.8 1.3 685 688
68. rpoC2 92.3 4143 21 4122 4164 87.2 1380 7 1373 1387
69. rps11 94.7 396 0 396 396 95.4 131 0 131 131
70. rps12 98.1 372 0 372 372 100 123 0 123 123
71. rps14 92.2 306 0 306 306 93.1 101 0 101 101
72. rps15 95.2 270 0 270 270 92.1 89 0 89 89
73. rps18 96.5 228 0 228 228 98.7 75 0 75 75
74. rps19 95.7 279 0 279 279 98.9 92 0 92 92
75. rps2 95.2 708 0 708 708 97 235 0 235 235
76. rps3 95.4 657 0 657 657 96.3 218 0 218 218
77. rps4 94.1 624 0 624 624 92.3 207 0 207 207
78. rps7 94.9 468 0 468 468 94.8 155 0 155 155
79. rps8 95.7 399 0 399 399 95.5 132 0 132 132
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Protein-coding regions of Equisetum species were similar in size, ranging between having the same length in atpB, E, F, H, I, clpP, infA, ndhB, C, D, E, H, I, petB, D, G, L, N, psaA, B, C, I, J, M, psbA, B, C, D, E, F, H, I, J, K, L, M, N, Z, rbcL, rpl14, 16, 22, 23, 32, 33, 36, rps2, 3, 4, 7, 8, 11, 12, 14, 15, 18, and 19 to having a 195 bp or 64 amino acids difference in accD. These genes have a similar number and position of introns except for the presence of 753 bp of intron in rpl16 in E. arvense. The percentage of the identical nucleotide of the aligned sites ranged from 88.7 percent in matK to 99.2 percent in psbJ, while the percentage of the identical derived amino acid of the aligned sites ranged from 73.9 percent in atpF to 100 percent in atpH, ndhE, petB, D, G, N, psaJ, psbA, E, F, I, J, L, Z, rpl36, and rps2 (Table 1). Phylogenetic estimation of Equisetum using plastome protein-coding sequences suggested that the known complete plastid genomes of Equisetum species formed a monophyletic clade of the two subgenera, Hippochaete and Equisetum. The newly assembled E. xylochaetum plastome indicates placement within Hippochaete with E. hyemale (Figure 2).

Figure 2.

Figure 2

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Maximum-likelihood tree inferred from all Equisetum plastome protein-coding regions using a GTR+I+F model. The scale bar represents the estimated number of nucleotide substitutions per site. The bootstrap and posterior probability values are reported at the respective nodes. The values include the ML bootstrap values of nucleotide and protein data and the BI posterior probability of the nucleotide and protein data, respectively.

Single-gene ML phylogenetic analysis of atpB, matK, rpoB, rps4, and trnL-F resolved the known subgenera of Equisetum, including Paramochaete, Hippochaete, and Equisetum (Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7). The majority of the Equisetum species were resolved with ML bootstrap values of at least 50. However, the monophyly of some Equisetum species could not be resolved. The monophyly of E. arvense and E. variegatum was not resolved in the matK tree, the monophyly of E. bogotense, E. laevigatum, E. myriochaetum, E. hyemale, and E. giganteum was not resolved in the rps4 tree, and the monophyly of E. hyemale, E. praealtum, E. ramosissimum, E. trachyodon, and E. xylochaetum was not resolved in the trnL-F tree. All hybrid taxa were phylogenetically placed within the clade consisting of the majority of their maternal parent, if the monophyly of the taxa was absent. In the case of the rps4 tree, these hybrids included Equisetum x fontqueri isolate 26093 located within the clade of E. telmateia, Equisetum x litorale isolates 41084 and 41085 with E. arvense, Equisetum x schaffneri isolates 40813 and 40824 with E. giganteum, and Equisetum x schaffneri isolate 40814 with E. myriochaetum. For trnL-F, the hybrid taxa Equisetum x ferrissii (AY226113) located in the clade with E. laevigatum, Equisetum x litorale isolates 41084 and 41085 with E. arvense, and Equisetum x schaffneri isolate 40814 with E. myriochaetum.

Figure 3.

Figure 3

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Phylogenetic estimation and TCS network of Equisetum atpB sequences. The scale bar of the tree represents the estimated number of nucleotide substitutions per site. The maximum-likelihood bootstrap values are reported at the respective nodes. The colours of taxa present in the tree correspond with the colours in the TCS haplotype map. The size of the circle represents the number of the taxa that share the same haplotype.

Figure 4.

Figure 4

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Phylogenetic estimation and TCS network of Equisetum matK sequences. The scale bar of the tree represents the estimated number of nucleotide substitutions per site. The maximum-likelihood bootstrap values are reported at the respective nodes. The colours of taxa present in the tree correspond with the colours in the TCS haplotype map. The size of the circle represents the number of the taxa that share the same haplotype.

Figure 5.

Figure 5

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Phylogenetic estimation and TCS network of Equisetum rpoB sequences. The scale bar of the tree represents the estimated number of nucleotide substitutions per site. The maximum-likelihood bootstrap values are reported at the respective nodes. The colours of taxa present in the tree correspond with the colours in the TCS haplotype map. The size of the circle represents the number of the taxa that share the same haplotype.

Figure 6.

Figure 6

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Phylogenetic estimation and TCS network of Equisetum rps4 sequences. The scale bar of the tree represents the estimated number of nucleotide substitutions per site. The maximum-likelihood bootstrap values are reported at the respective nodes. The colours of taxa present in the tree correspond with the colours in the TCS haplotype map. The size of the circle represents the number of the taxa that share the same haplotype.

Figure 7.

Figure 7

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Phylogenetic estimation and TCS network of Equisetum trnL-trnF. The scale bar of the tree represents the estimated number of nucleotide substitutions per site. The maximum-likelihood bootstrap values are reported at the respective nodes. The colours of taxa present in the tree correspond with the colours in the TCS haplotype map. The size of the circle represents the number of the taxa that share the same haplotype.

TCS haplotype network analyses using atpB, matK, and rpoB resolved distinct clades representing each of the Equisetum subgenera. At the species level, haplotype networks constructed using atpB and rpoB showed one haplotype for each Equisetum species. In contrast, maps of matK, rps4, and trnL-F resolved more than one haplotype for some species and resolved some haplotypes that consisted of more than one species. For matK, there was more than one haplotype for E. arvense and E. hyemale and there was one haplotype that consisted of sequences from E. arvense and E. variegatum (Figure 4).

Haplotype maps of rps4 and trnL-F seemed to be more complex compared to those of atpB, matK, and rpoB. In the map of rps4 (Figure 6), we observed 10 haplotypes, of which, haplotypes 1–3 of E. bogotense appeared as a distinct group representing subgenus Paramochaete. A few haplotypes consisted of only one Equisetum species, which were haplotype 4 for E. palustre, haplotype 5 for E. diffusum, and haplotype 8 for E. scirpoides. The hybrid taxa were embedded within the same haplotypes as their maternal taxa. These included Equisetum x fontqueri isolate 26093 that was in haplotype 6 with E. telmateia, Equisetum x litorale isolates 41084 and 41085 in haplotype 7 with E. arvense, Equisetum x schaffneri isolates 40813 and 40824 in haplotype 9 with E. giganteum, and Equisetum x schaffneri isolate 40814 in haplotype 9 with E. myriochaetum. Some haplotypes consisted of many plant species, i.e., haplotype 7 and 9, where the majority of Equisetum and Hippochaete were placed together, respectively. Interestingly, a rps4 sequence from E. hyemale grouped with other sequences of that species but also was present as a unique haplotype, as haplotype 10 with E. praealtum isolate 41501.

The map of trnL-F (Figure 7) resolved two distinct groups of haplotypes representing subgenus Paramochaete (haplotype 1) and subgenus Equisetum (haplotypes 2–8). Many of the Equisetum species were present as unique haplotypes, including E. bogotense (haplotype 1), E. palustre (haplotype 2), E. pratense (haplotype 3), E. telmateia (haplotype 4), E. sylvaticum (haplotype 5), E. fluviatile (haplotype 6), Equisetum x dycei (haplotype 7), and E. scirpoides (haplotype 9).

Some Equisetum species were resolved as more than a single haplotype. E. hyemale isolate 20201 was resolved as a unique haplotype 14 while E. hyemale isolate 1273o was located in haplotype 10 with E. variegatum. For E. variegatum, in addition to its member in haplotype 10, E. variegatum isolates 40820 and 40823 were resolved as additional unique haplotypes 11 and 12. In addition, E. myriochaetum isolate 40826 was present as haplotype 15, while most members were located in haplotype 13.

Most of the hybrid taxa in the trnL-F map were placed in the same haplotypes as their maternal taxa. Equisetum x ferrissii (AY226113) was located in haplotype 16 with its maternal taxon, E. laevigatum, Equisetum x ferrissii in haplotype 13 with the majority of E. hyemale, Equisetum x litorale isolates 41084 and 41085 in haplotype 8 with E. hyemale, Equisetum x schaffneri isolates 40813 and 40824 in haplotype 13 with E. giganteum, and Equisetum x schaffneri isolate 40814 in haplotype 13 with the majority of E. myriochaetum.

3. Discussion

In this study, we assembled the complete plastid genome of E. xylochaetum from shotgun metagenomes of E. xylochaetum sampled from two Atacama Desert locales exhibiting different degrees of disturbance. Results showed that the plastid genomes constructed from these two E. xylochaetum metagenome accessions were identical, suggesting that the Equisetum samples were from the same Equisetum population. Comparison of nucleotide, and their derived protein, sequences of this newly assembled E. xylochaetum plastid genome to those of E. hyemale and E. arvense showed that the Equisetum plastid genomes were highly conserved in terms of structure and function, even though the two subgenera (Hippochaete and Equisetum) might have diverged as early as 135 Mya during the early Cretaceous [4,6]. All the plastid protein-coding sequences were subjected to purifying selection, with genes of the same type having identical nucleotide percentages and having nucleotide identity ranging from 88.7–99.2 percent. The only major difference in gene structure was presence of the intron in E. arvense rpl16. To determine where and when the intron of rp116 originated in the Equisetum lineage, more Equisetum rpl16 sequences or complete plastid genomes are required.

In a broad sense, the phylogenetic positions of Equisetum species inferred by using all protein-coding sequences along with their derived proteins and the single-gene analysis present in this study were congruent with results from previous studies that used a single-gene approach [11] or a combination of multi-genes and morphological characters e.g., [4,5,6], where Equisetum formed monophyletic clades of each subgenus and placed E. xylochaetum in Hippochaete. Despite the presence of the high conservation level of Equisetum plastid genes, it was surprising to us that the single-gene phylogenetic approach was not sufficient to resolve relationships among Equisetum taxa, especially those closely related taxa placed in subgenus Hippochaete, e.g., E. giganteum, E. variegatum, and E. hyemale. Therefore, it is evident that more Equisetum plastid genomes, plus additional molecular information from other genetic compartments, are needed.

The addition of haplotype mapping provided in this study enhanced the understanding of how plastid genes from each taxon are related. In general, the haplotype maps reflected the relationship resolved from phylogenetic estimation using the corresponding nucleotide regions. Even so, these new maps aid the visualisation of how these plastome nucleotide data were interrelated to each other at the level of isolate, species, and subgenus. The presence of only one shared distinct haplotype of an Equisetum species, though its samples were collected from different locales, suggested a high conservation level of the corresponding genes within its plastid genomes. On the other hand, the presence of more than one haplotype at the specific level suggested the presence of nucleotide diversity, indicating the need to further examine the populations of E. arvense, E. bogotense, E. hyemale, E. variegatum, and E. myriochaetum. In addition, the presence of a haplotype consisting of more than one Equisetum species, e.g., haplotypes 8 and 9 of the rps4 map and haplotypes 8 and 13 of the trnL-F map, suggested that these conserved regions alone were not sufficient for studying the relationship and diversity of Equisetum taxa. These findings emphasize the need for more Equisetum plastid genomes.

The presence of distinct haplotype(s) in the early-diverging species E. bogotense in rps4 and trnL-F suggested that these plastid sequences might not represent the ancestral characters of Equisetum. Instead, these E. bogotense samples may only represent the survival representatives of the extinct members that also evolved during the course of time. In contrast, according to the rps4 and trnL-F maps, the taxa that frequently occurred at the junction region between each subgenus were E. scirpoides and E. pratense, suggesting that these taxa might be particularly helpful for understanding how the Equisetum subgenera diverged.

4. Materials and Methods

Nucleotide data for Equisetum xylochaetum Mett. were obtained from GenBank BioProject PRJNA555713 [12], generated by metagenomic shotgun sequencing of the microbiome of giant Equisetum xylochaetum sampled from two streambed locales in the Atacama Desert of northern Chile that differed in the degree of human disturbance. The two raw data sets, separately archived in accessions SRX6486516 and SRX6486517, each represented pooled replicate DNA extractions from both above-ground green and below-ground non-green tissues. To obtain the complete chloroplast genome of E. xylochaetum, metagenomic sequences were trimmed using Trimmomatic v. 0.39 [13] using the parameter sliding window:4:30. Next, the trimmed sequences from the two raw data sets were independently assembled using MEGAHIT ver. 1.2.9 [14] with the parameter “bubble-level equal to 0” in order to prevent the merging of sequences that were highly similar, e.g., sequences from closely related species or sequences that display single nucleotide polymorphisms. Each assembly yielded a contig of the complete plastid genome of E. xylochaetum, and these two contigs were identical in sequence. To validate the assembly, we calculated the coverage of the plastid genomes using the methods described in Satjarak and Graham [15]. One of the two contigs, which had the mean coverage of 706 fold, was then selected for annotation of protein-coding genes using proteins inferred from E. arvense [8,9] and E. hyemale [7] as references. The tRNAs and rRNA genes were annotated using tRNAscan-SE On-line [16] and the RNAmmer 1.2 Server [17], respectively. The complete plastid genome of E. xylochaetum was deposited in GenBank under accession number MW282958. A representative plant specimen has been deposited at the University of Concepción herbarium under accession number CONC-CH 6005.

We compared the plastid genome of E. xylochaetum obtained from this study to other complete Equisetum plastid genomes, including E. hyemale (KC117177), E. arvense from the US (GU191334), and E. arvense from Korea (JN968380). We examined general characteristics of the genomes, including the genome size, %GC, the gene content, gene length, gene order, and polymorphism of nucleotides within coding regions and their derived proteins. To consider nucleotide polymorphisms, we aligned the protein-coding sequences (Table 1) using Geneious translation alignment: global alignment with free end gap, standard genetic code, and Identity (1.0/0.0) cost matrix (Geneious ver. 9.1.3; https://www.geneious.com; accessed in 31 January 2022).

The mode of evolution of protein-coding regions was performed using the method described in Mekvipad and Satjarak [18]. For the polymorphism of protein sequences, we aligned the derived protein sequences using MAFFT alignment: auto algorithm and Blosum62 scoring matrix [19]. To investigate the relationship of E. xylochaetum and other Equisetum complete chloroplast genomes, Psilotum nudum (NC_003386.1) was used as an outgroup. The protein-coding sequences and protein sequences (Table 1) were similarly aligned, trimmed using Trimal ver. 1.2 [20], and concatenated. The nucleotide data matrix was 60,987 bp and the protein data matrix consisted of 19,435 amino acids. Phylogenetic relationships were estimated using maximum likelihood and Bayesian frameworks as described in Satjarak and Graham [15].

To investigate whether the Equisetum relationship resolved from frequently-used nucleotide sequences reported in previous studies exhibited grades or evolutionary intermediates, we performed haplotype network analysis of selected, frequently-used Equisetum conserved regions. These included atpB, matK, rpoB, rps4, and trnL-F (Table 3). To prepare the data matrices, the conserved nucleotide regions were extracted from the complete plastid genomes and from DNA sequences from other published studies (Table 3). Next, the data were aligned, and the phylogenetic trees were estimated using the methods described above. The haplotype network analysis was calculated using (Templeton, Crandall, and Sing; TCS) [21] and visualized in PopArt v1.7 [22].

Table 3.

Nucleotide sequences used in the single gene phylogenetic analysis and TCS haplotype mapping.

No. Name GenBank Accession Locality References
atpB
1. E. arvense GU191334 USA [8]
2. E. arvense JN968380 Korea [9]
3. E. hyemale KC117177 unknown [7]
4. E. ramosissimum subsp. debile EU439074 unknown [23]
5. E. telmateia AF313542 unknown [24]
6. E. xylochaetum MW282958 Chile This study
7. Equisetum x ferrissii AF313541 unknown [24]
matK
1. E. arvense JX392862 China [25]
2. E. arvense JX392863 Europe [25]
3. E. arvense AY348551 unknown [26]
4. E. arvense GU191334 USA [8]
5. E. arvense JN968380 Korea [9]
6. E. bogotense KP757846 unknown [27]
7. E. hyemale EU749486 unknown [28]
8. E. hyemale EU749485 unknown [28]
9. E. hyemale EU749484 unknown [28]
10. E. hyemale EU749487 unknown [28]
11. E. hyemale HF585136 unknown [29]
12. E. hyemale KC117177 unknown [7]
13. E. palustre MZ400482 Sweden [30]
14. E. ramosissimum JF303895 unknown [31]
15. E. scirpoides MZ400480 Sweden [30]
16. E. variegatum MZ400481 Sweden [30]
17. E. xylochaetum MW282958 Chile This study
rpoB
1. E. arvense HQ658110 China [32]
2. E. arvense GU191334 USA [8]
3. E. arvense JN968380 Korea [9]
4. E. hyemale KC117177 Unknown [7]
5. E. ramossissimum HQ658109 China [32]
6. E. xylochaetum MW282958 Chile This study
rps4
1. E. arvense subsp. arvense isolate 41072 MH750111 Finland [5]
2. E. arvense AJ583677 unknown [3]
3. E. arvense JN968380 Korea [9]
4. E. arvense GU191334 USA [8]
5. E. arvense subsp. arvense isolate 26084 MH750108 India (Himachal Pradesh) [5]
6. E. arvense subsp. arvense isolate 40833 MH750109 USA (California) [5]
7. E. arvense subsp. arvense isolate 41071 MH750110 Finland [5]
8. E. arvense subsp. boreale isolate 41073 MH750112 Finland/Norway (border) [5]
9. E. arvense subsp. boreale isolate 41074 MH750113 Finland/Norway (border) [5]
10. E. arvense x E. telmateia subsp. braunii isolate 40834 MH750114 USA (California) [5]
11. E. bogotense AF231898 unknown [33]
12. E. bogotense AF313603 unknown [24]
13. E. bogotense AJ583678 unknown [3]
14. E. bogotense isolate 40800 MH750115 Argentina [5]
15. E. bogotense isolate 40802 MH750116 Ecuador [5]
16. E. bogotense isolate 40827 MH750117 Colombia [5]
17. E. diffusum AJ583679 unknown [3]
18. E. diffusum isolate 40804 MH750118 India [5]
19. E. fluviatile AJ583680 unknown [3]
20. E. fluviatile isolate 41075 MH750119 Finland [5]
21. E. fluviatile isolate 41076 MH750120 Finland [5]
22. E. giganteum AJ583681 unknown [3]
23. E. giganteum isolate 40806 MH750121 Chile [5]
24. E. hyemale AJ583682 unknown [3]
25. E. hyemale KC117177 unknown [7]
26. E. hyemale isolate 23252 MH750123 Norway [5]
27. E. laevigatum AJ583683 unknown [3]
28. E. laevigatum isolate 40812 MH750125 USA (California) [5]
29. E. myriochaetum AJ583684 unknown [3]
30. E. myriochaetum isolate 40825 MH750126 Mexico [5]
31. E. myriochaetum isolate 40936 MH750127 El Salvador [5]
32. E. palustre AJ583685 unknown [3]
33. E. palustre isolate 17671 MH750128 UK (England, Norfolk) [5]
34. E. palustre isolate 39349 MH750129 UK (England, Surrey) [5]
35. E. praealtum isolate 41501 MH750122 USA (Ohio) [5]
36. E. pratense AJ583686 unknown [3]
37. E. pratense isolate 39348 MH750130 Finland [5]
38. E. ramosissimum subsp. debile AJ583687 unknown [3]
39. E. ramosissimum subsp. debile EU439173 unknown [23]
40. E. ramosissimum subsp. debile isolate 24579 MH750131 Sri Lanka [5]
41. E. ramosissimum subsp. debile isolate 40837 MH750132 New Caledonia [5]
42. E. ramosissimum subsp. ramosissimum isolate 36802 MH750133 Spain (Andalucia) [5]
43. E. scirpoides AJ583688 unknown [3]
44. E. scirpoides isolate 26090 MH750134 Greenland [5]
45. E. scirpoides isolate 40830 MH750124 Russia (Kamtschatka) [5]
46. E. sylvaticum AJ583689 unknown [3]
47. E. telmateia subsp. braunii AJ583690 unknown [3]
48. E. telmateia subsp. braunii isolate 40817 MH750136 USA (California) [5]
49. E. telmateia subsp. braunii isolate 40828 MH750137 Canada (British Columbia) [5]
50. E. telmateia subsp. braunii isolate 40832 MH750138 USA (California) [5]
51. E. telmateia subsp. braunii isolate 40836 MH750139 USA (California) [5]
52. E. telmateia subsp. telmateia isolate 41082 MH750140 Ireland [5]
53. E. variegatum AJ583691 unknown [3]
54. E. variegatum isolate 11639 MH750141 UK (Wales) [5]
55. E. variegatum isolate 40819 MH750148 Ireland [5]
56. E. variegatum isolate 40820 MH750142 France (Pyrenees) [5]
57. E. variegatum isolate 40823 MH750143 USA (Keweenaw, Michigan) [5]
58. E. variegatum isolate 41083 MH750144 Ireland [5]
59. E. variegatum subsp. alaskanum isolate 40818 MH750145 USA (Alaska) [5]
60. E. variegatum subsp. alaskanum isolate 40821 MH750146 Canada (British Columbia) [5]
61. E. variegatum subsp. alaskanum isolate 40822 MH750147 Canada (Banff) [5]
62. E. xylochaetum MW282958 Chile This study
63. Equisetum scirpoides isolate 41089 MH750135 Finland [5]
64. Equisetum x ferrissii AF313590 unknown [24]
65. Equisetum x fontqueri isolate 26093
(E. telmateia x E. palustre) 		MH750149 UK (Scotland) [5]
66. Equisetum x litorale isolate 41084
(E. arvense x E. fluviatile) 		MH750150 Ireland [5]
67. Equisetum x litorale isolate 41085
(E. arvense x E. fluviatile) 		MH750151 Ireland [5]
68. Equisetum x schaffneri isolate 40813
(E. giganteum x E. myriochaetum) 		MH750152 Mexico [5]
69. Equisetum x schaffneri isolate 40814
(E. myriochaetum x E. giganteum) 		MH750153 Peru (cult RBG Edinburgh) [5]
70. Equisetum x schaffneri isolate 40824
(E. giganteum x E. myriochaetum) 		MH750154 Mexico [5]
trnL-trnF
1. E. arvense JN968380 Korea [9]
2. E. arvense GU191334 USA [8]
3. E. arvense AY226125 Franc [34]
4. E. arvense GQ428069 unknown [35]
5. E. arvense HM590277 Estonia [36]
6. E. arvense GQ244921 unknown [37]
7. E. arvense subsp boreale isolate 41074 MH750043 Finland/Norway [5]
8. E. arvense subsp. arvense isolate 26084 MH750038 India [5]
9. E. arvense subsp. arvense isolate 26085 MH750039 UK [5]
10. E. arvense subsp. arvense isolate 40833 MH750040 USA [5]
11. E. arvense subsp. arvense isolate 41071 MH750041 Finland [5]
12. E. arvense subsp. boreale isolate 41073 MH750042 Finland/Norway [5]
13. E. arvense x E. telmateia subsp. braunii isolate 40834 MH750044 USA [5]
14. E. bogotense AY226124 Colombia [34]
15. E. bogotense isolate 40800 MH750045 Argentina [5]
16. E. bogotense isolate 40801 MH750046 Chile [5]
17. E. bogotense isolate 40802 MH750047 Ecuador [5]
18. E. bogotense isolate 40803 MH750048 Chile [5]
19. E. bogotense isolate 40827 MH750049 Colombia [5]
20. E. diffusum AY226126 India [34]
21. E. diffusum isolate 40804 MH750050 India [5]
22. E. fluviatile AY226121 Canada [34]
23. E. fluviatile GQ244922 unknown [37]
24. E. fluviatile isolate 41075 MH750051 Finland [5]
25. E. fluviatile isolate 41076 MH750052 Finland [5]
26. E. giganteum AY226118 Ecuador [34]
27. E. giganteum isolate 40805 MH750053 Jamaica [5]
28. E. giganteum isolate 40806 MH750054 Chile [5]
29. E. giganteum isolate 40807 MH750055 Peru [5]
30. E. giganteum isolate 40810 MH750057 Argentina [5]
31. E. giganteum isolate 40811 MH750058 Argentina [5]
32. E. hyemale KC117177 unknown [7]
33. E. hyemale AY327837 unknown [34]
34. E. hyemale isolate 0796g GQ244923 unknown [37]
35. E. hyemale isolate 1273o GQ244924 unknown [37]
36. E. hyemale isolate 20201 MH750061 France [5]
37. E. hyemale isolate 23252 MH750062 Norway [5]
38. E. hyemale isolate 41088 MH750063 Finland [5]
39. E. hyemale subsp. affine AY226110 USA [34]
40. E. iganteum isolate 40809 MH750056 Argentina [5]
41. E. laevigatum AY226112 USA [34]
42. E. laevigatum isolate 40812 MH750065 USA [5]
43. E. myriochaetum AY226114 USA [34]
44. E. myriochaetum isolate 40815 MH750066 USA [5]
45. E. myriochaetum isolate 40816 MH750067 USA [5]
46. E. myriochaetum isolate 40825 MH750068 Mexico [5]
47. E. myriochaetum isolate 40826 MH750069 Ecuador [5]
48. E. myriochaetum isolate 40936 MH750070 El Savador [5]
49. E. myriochaetum isolate 41080 MH750071 Guatemala [5]
50. E. palustre AY226123 Canada [34]
51. E. palustre GQ244925 unknown [37]
52. E. palustre isolate 39349 MH750072 UK [5]
53. E. praealtum isolate 40831 MH750059 USA [5]
54. E. praealtum isolate 41501 MH750060 USA [5]
55. E. pratense AY226122 Canada [34]
56. E. pratense GQ244926 unknown [37]
57. E. pratense HM590278 Estonia [36]
58. E. pratense isolate 39348 MH750073 Finland [5]
59. E. pratense isolate 41086 MH750074 Finland [5]
60. E. pratense isolate 41087 MH750075 Finland [5]
61. E. ramosissimum subsp. debile AY226115 Taiwan [34]
62. E. ramosissimum subsp. debile isolate 23679 MH750076 Reunion [5]
63. E. ramosissimum subsp. debile isolate 24579 MH750077 Sri Lanka [5]
64. E. ramosissimum subsp. debile isolate 40837 MH750078 New Caledonia [5]
65. E. ramosissimum subsp. ramosissimum isolate 36802 MH750079 Spain [5]
66. E. ramosissimum subsp. ramosissimum isolate 40829 MH750080 Turkey [5]
67. E. scirpoides AY226116 Canada [34]
68. E. scirpoides GQ244927 unknown [37]
69. E. scirpoides isolate 26090 MH750082 Greenland [5]
70. E. scirpoides isolate 40830 MH750064 Russia [5]
71. E. scirpoides isolate10933 MH750081 UK [5]
72. E. sylvaticum MH750083 UK [5]
73. E. sylvaticum AY226120 France [34]
74. E. sylvaticum GQ244928 unknown [37]
75. E. sylvaticum isolate 41081 MH750084 Finland [5]
76. E. telmateia isolate 11642 MH750089 China [5]
77. E. telmateia isolate 41082 MH750090 Ireland [5]
78. E. telmateia subsp. braunii AY226119 USA [34]
79. E. telmateia subsp. braunii isolate 40817 MH750085 USA [5]
80. E. telmateia subsp. braunii isolate 40828 MH750086 Canada [5]
81. E. telmateia subsp. braunii isolate 40832 MH750087 USA [5]
82. E. telmateia subsp. braunii isolate 40836 MH750088 USA [5]
83. E. trachyodon isolate 41092 MH750106 Finland [5]
84. E. variegatum AY226117 USA [34]
85. E. variegatum isolate 0584g GQ244929 unknown [37]
86. E. variegatum isolate 0977o GQ244930 unknown [37]
87. E. variegatum isolate 11639 MH750091 UK [5]
88. E. variegatum isolate 40819 MH750098 Ireland [5]
89. E. variegatum isolate 40820 MH750092 France [5]
90. E. variegatum isolate 40823 MH750093 USA [5]
91. E. variegatum isolate 41083 MH750094 Ireland [5]
92. E. variegatum subsp. alaskanum isolate 40818 MH750095 USA [5]
93. E. variegatum subsp. alaskanum isolate 40821 MH750096 Canada [5]
94. E. variegatum subsp. alaskanum isolate 40822 MH750097 Canada [5]
95. E. xylochaetum MW282958 Chile This study
96. E. xylochaetum isolate 40614 MH750107 Chile [5]
97. Equisetum sp. AY327838 unknown [34]
98. Equisetum x dycei isolate 26083 MH750099 UK [5]
99. Equisetum x ferrissii (E. laevigatum x E. hyemale) AY226113 USA [34]
100. Equisetum x ferrissii (Equisetum hyemale x laevigatum) AY226111 Canada [34]
101. Equisetum x litorale isolate 41084 MH750101 Ireland [5]
102. Equisetum x litorale isolate 41085 MH750102 Ireland [5]
103. Equisetum x schaffneri isolate 40813 MH750103 Mexico [5]
104. Equisetum x schaffneri isolate 40814 MH750104 Peru [5]
105. Equisetum x schaffneri isolate 40824 MH750105 Mexico [5]
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5. Conclusions

In summary, our study demonstrated that metagenomic data can be a useful way to obtain plastid genomes. The comparison of the de novo plastid genome of E. xylochaetum with other reported Equisetum plastomes showed a high degree of conservation in terms of structure, gene content, gene order, and nucleotide polymorphisms. Even so, this new plastid genome provided additional information about the evolution and diversity of Equisetum, e.g., the presence of an intron in rpl16. Haplotype analyses of the selected conserved nucleotides showed that some Equisetum species were distantly related to other taxa, inferred from the presence of distinct haplotypes. Many of the taxa appeared as shared haplotypes, suggesting that the molecular data we currently have might not be sufficient for a full understanding of the evolutionary relationship of Equisetum and that more Equisetum plastid genomes are needed.

Acknowledgments

We thank Karnjana Ruen-pham for the illustration.

Author Contributions

Conceptualization, A.S.; methodology, A.S.; validation, A.S.; formal analysis, A.S.; resources, L.E.G., M.T.T. and P.A.-A.; data curation, A.S.; writing—original draft preparation, A.S.; writing—review and editing, A.S., L.E.G., M.T.T. and P.A.-A.; visualization, A.S.; funding acquisition, L.E.G. and P.A.-A. All authors have read and agreed to the published version of the manuscript.

Funding

This work was partially supported by US NSF grant DEB1119944 (to L.E.G.) and Chilean CONICYT-FONDECYT grant 1120619 (to P.A.-A.).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The complete plastid genome of Equisetum xylochaetum is publicly available in NCBI GenBank (https://www.ncbi.nlm.nih.gov accessed on 12 March 2022) accession number MW282958. Nucleotide data for analysis are available at GenBank BioProject PRJNA555713 accessions SRX6486516 and SRX6486517.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The complete plastid genome of Equisetum xylochaetum is publicly available in NCBI GenBank (https://www.ncbi.nlm.nih.gov accessed on 12 March 2022) accession number MW282958. Nucleotide data for analysis are available at GenBank BioProject PRJNA555713 accessions SRX6486516 and SRX6486517.

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