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

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.

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

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

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

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

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

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

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

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

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

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]

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.