Novel insights into Pinus species plastids genome through phylogenetic relationships and repeat sequence analysis.

Pinus is one of the most economical and ecological important conifers, model specie for studying sequence divergence and molecular phylogeney of gymnosperms. The less availability of information for genome resources enable researchers to conduct evolutionary studies of Pinus species. To improve understanding, we firstly reported, previously released chloroplast genome of 72 Pinus species, the sequence variations, phylogenetic relationships and genome divergence among Pinus species. The results displayed 7 divergent hotspot regions (trnD-GUC, trnY-GUA, trnH-GUG, ycf1, trnL-CAA, trnK-UUU and trnV-GAC) in studied Pinus species, which holds potential to utilized as molecular genetic markers for future phylogenetic studies in Pinnus species. In addition, 3 types of repeats (tandem, palindromic and dispersed) were also studied in Pinus species under investigation. The outcome showed P. nelsonii had the highest, 76 numbers of repeats, while P. sabiniana had the lowest, 13 13 numbers of repeats. It was also observed, constructed phylogenetic tree displayed division into two significant diverged clades: single needle (soft pine) and double-needle (hard pine). Theoutcome of present investigation, based on the whole chloroplast genomes provided novel insights into the molecular based phylogeny of the genus Pinus which holds potential for its utilization in future studies focusing genetic diversity in Pinnus species.

Introduction

Pinus L. (Pinaceae) is an important genus of conifers with more than 230 species. It is a broadly distributed in temperate zones of Northern Hemisphere [1]. Pines include important tree species which are commercially used in pharmacology and wood pulp industries around the world. Genus Pinus is divided into two subgenera Strobus, (Haploxylon) and Pinus (Diploxylon) [2]. Moreover, anatomical, molecular, and morphological evidence strongly reinforced divergence of Strobus and Pinus, respectively [3]. Because of ecological importance and diversity, genus Pinus prove a best model for molecular study of conifers. Pinnus genomes are extremely large (c. 20–40 Gb) and shown no evidence of recent polyploidy or chromosomal duplication. Pine chromosomes (2n = 24) are uniform both in number and appearance, owing to lack of major distinguishing physical features [4].

The phylogenetic tree displayed evolutionary relationships among different biological species based on similarities and differences for their maternal characteristics. Moreover, phylogenetic relationships in Pinus species are regularly studied through genome sequencing [5]. The whole chloroplast genome has numerous features e.g., small in size, conserved structure, maternal inheritance, and species identification is broadly applied for evolutionary studies [6]. Recently, an extremely divergent region in plant plastome has been identified called "hotspot region" and served an useful genetic marker for phylogeny and evolutionary studies of genus Pinus [7]. Previous studies showed that the genus Pinus had shared several genomic sequence variations for cp DNAs owing to their recent divergence radiation, regular interspecific and introgression gene flow among species [8]. The low degree of genomic divergence among Pinus species has been attributed to a large number of molecular evolution takeing place in related species [9]. Therefore, it hold integral importance to understand complete phylogenetic relationships of Pinus species to understand the underlying genetic mechanisms controlling its diverse features [10].

Complete chloroplast genome are circular DNA molecules, had a quadripartite shape with large single copy (LSC) region, a small single copy (SSC) region, and two inverted repeats (IRs) regions [6]. Previous studies had revealed that plastid DNA of gymnosperm plants were extremely maintained in genome structure, order and gene contents [11]. The repeat sequence analysis in plastome contributes to various cellular functions including RNA editing, gene mobility and gene evolution [12]. Repetitive sequences are categorized into three modules: local repeats (simple sequence repeats (SSRs) and tandem repeats), families of dispersed repeats (mostly transposable elements and retro-transposed cellular genes), and segmental duplications (duplicated genomic fragments). The large number of repetitive sequences involved during the process of evolution in plant genomes depending on their structure and mode of multiplications [13]. Moroever, long repeat sequences are spread throughout the chloroplast genomes of Pinus species. Recent studies have shown that most repeat sequences were positioned in the intergenic and intron regions whereas, limited repeat sequence were located in the coding regions of gymnosperm plastomes [1]. The diversity of the repeated sequences may provide valuable information for species adaptation to varying environmental condition.

In the present study, we will analyzed complete chloroplast genomes of or seventy-two Pinus species to identify structural variations and theircomparative genome analysis. We aimed toinvestugae comprehensive structural variations in Pinus genomes, examination of large repeat sequence variation in the plastid genome of Pinus, and reconstruction of phylogeny of major lineages of Pinus species based on complete chloroplast genome.

Materials and methods

Materials

The whole plastid genome dataset of seventy-two Pinus species and their three outgroups (Picea glauca, Abies koreana and Abies nephrolepis) were identified and downloaded from the NCBI (https://www.ncbi.nlm.nih.gov/). The Pinus complete cp genomes sequencing was annotated and further utilized for analysis.

Chloroplast genome sequencing, annotation and divergence analysis

Data were used to generate a consensus sequence inside the software Geneious R v 8.0.2 (Biomatters Ltd., Auckland, New Zealand). Preliminary, the plastome annotation was turned using the program DOGMA (https://domainworld-services.uni-muenster.de/dogma/.). The stop and start codons are manually adjusted in Geneious R v 8.0.2. The round plastid genome map was drawn with the Organellar Genome DRAW v1.1 (OGDRAW) [14]. The sequence rearrangement of seventy-two plastomes was undertaken on Mauve Alignment [15]. To display interspecific variation, the alignments of the plastid DNA of the seventy-two genus Pinus were envisioned by mVISTA online software (https://genome.lbl.gov/vista/mvista/about.shtml) in the Shuffle-LAGAN mode and P. squamata specie was used as reference. The percentages of variable characters for non-coding and coding regions were counted via procedure given by Zhang et al. [16].

Repeat sequence analysis

We found three types of repeats in complete chloroplast genome of seventy-two Pinus species: dispersed, tandem, and palindromicwhereas, web-based REPuter (https://bibiserv.cebitec.uni-bielefeld.de/reputer/) programmed was used to investigate these repeat sequences. The dispersed and palindromic repeats were used on following condition; (1) sequence identity 90%; (2) Hamming distance = 1 (3) repeat size minimum = 30 bp [17]. Tandem motifs (>10 bp in length) was identified using online software Tandem Repeats Finder (https://tandem.bu.edu/trf/trf.html) [18].

Phylogenetic analysis

The complete dataset of Pinus genome sequence was aligned using MAFFT V 7.0.0 programmed [19]. Phylogenetic analysis was carried out using the cpDNA of all seventy two Pinus species (Table 1). These species were aligned with the Clustal W method of MEGA v7.0.18 software with manual inspection [20]. In addition, we included sequences from Abies koreana, Abies nephrolepis and Picea gluca as an outgroups. Maximum likelihood (ML) and maximum parsimony (MP) analysis were performed with the Akaike Information Criterion and an appropriate sequence evolution model selected by Model Test version 3.7. (AIC) [21]. Subsequently, one thousand (1000) bootstrap replicate was used to evaulate the support value of both ML and MP branches. PAUP* was used to calculate the phylogenetic reconstruction. Furthermore, the Bayesian phylogenetic analysis was operated using MrBayes v3.1.2 [22]. Markov Chain Monte Carlo (MCMC) was run over 3,000,000 generations, starting with an arbitrary tree and sampling topologies for every 100 generations. The first 2,500 trees (containing 25% of our samples) were burned (as recommended by MrBayes), and the remaining trees were used to build the 50% majorityrule consensus tree and estimate Bayesian posteriors of nodal support probabilities.

Table 1

The features of complete chloroplast genomes of seventy-two Pinus species.

Section	Species	Size (bp)	LSC (bp)	SSC (bp)	IR (bp)	Number of Protein Coding Genes	Number of rRNA Genes	Number of tRNA Genes	GC Contents (%)	Gene bank number
Double needle Section (Subgenera Pinus)
	P. jaliscana	119,697	64,805	54,092	403	75	4	37	38.5	NC_035948
	P. pringlei	119,580	65,084	53,718	389	75	4	36	38.5	JN854189
	P. lawsonii	119,411	65,135	53,498	389	75	4	36	38.5	JN854188
	P.oocarpa	120,596	-	-	-	-	-	-	-	NC_035949
	P. palustris	119,149	65,190	53,181	389	75	4	36	38.5	JN854176
	P. greggii	119,480	64,849	53,853	389	74	4	36	38.5	NC_035947
	P. patula	119,356	65,130	53,448	389	75	4	36	38.5	JN854175
	P. occidentalis	119,826	65,204	53,844	389	75	4	36	38.5	JN854177
	P. taeda	120,534	65,610	54,146	389	75	4	36	38.5	NC_021440
	P. pungens	119,456	65,224	53,454	389	75	4	36	38.5	JN854167
	P. caribaea	119,528	64,924	53,634	399	75	4	36	38.5	JN854222
	P. elliottii	119,523	65,155	53,590	389	75	4	36	38.5	JN854202
	P. glabra	121,976	64,936	56,070	485	75	4	36	38.8	JN854199
	P. muricata	118,328	65,039	52,745	244	75	4	35	38.5	JN854180
	P. radiata	119,678	65,164	53,736	389	75	4	36	38.5	JN854165
	P. coulteri	119,785	65,141	53,866	389	75	4	36	38.5	JN854215
	P. sabiniana	118,929	64,830	53,129	485	75	4	36	38.5	JN854161
	P. jeffreyi	119,767	65,140	53,849	389	75	4	36	38.5	JN854193
	P. engelmanii	119,742	65,140	53,824	389	75	4	36	38.5	JN854201
	P.douglasiana	119,624	65,076	53,658	444	76	4	36	38.5	JN854205
	P. arizonica	119,965	64,899	54,084	492	75	4	37	36.4	JN854216
	P. devoniana	119,688	65,116	53,794	389	75	4	36	36.0	JN854208
	P. montezeumae	119,181	65,103	53,255	523	75	4	35	38.5	JN854183
	P. hartweggii	119,460	64,869	53,623	485	75	4	36	38.5	JN854206
	P.pseudostrobus	117,391	64,712	51,901	389	74	4	35	38.5	JN854178
	P. clausa	118,899	65,027	52,918	484	75	4	35	38.5	JN854217
	P. roxburgii	119,409	64,886	53,776	384	75	4	36	38.6	JN854162
	P. pinea	119,195	64,843	53,564	394	75	4	36	38.5	JN854173
	P. heldrichii	117,823	65,065	51,952	406	75	4	35	38.6	JN854195
	P. halepensis	118,947	64,750	53,237	394	75	4	36	38.5	JN854197
	P. brutia	120,570	64,990	54,610	485	75	4	36	38.5	JN854224
	P. pinaster	119,212	64,932	53,492	399	73	4	36	38.5	FJ899583
	P. latteri	119,279	65,069	53,432	389	75	4	36	38.6	JN854190
	P. resinosa	119,527	65,057	53,681	402	75	4	36	38.5	FJ899556
	P. tropicalis	118,924	65,002	53,133	389	75	4	36	38.5	JN854156
	P. massoniana	119,025	65,139	53,108	389	75	4	36	38.6	NC_021439
	P. sylvestris	115,909	64,415	50,661	420	75	4	37	38.6	KR476379
	P. densiflora	119,124	65,179	53,147	399	75	4	19	38.5	JN854210
	P. fragilissima	119,038	65,143	53,097	399	75	4	36	38.5	JN854200
	P. kesiya	118,986	65,179	53,009	399	75	4	36	38.6	JN854191
	P.hwangshanensis	118,993	65,175	53,020	399	75	4	36	38.5	JN854194
	P. yunnanensis	118,614	65,061	52,763	395	74	4	36	38.5	JN854151
Single needle Section (Subgenera Strobus)
	P. culminicola	115,155	64,364	50,035	362	75	4	36	38.7	JN854213
	P. discolor	115,154	64,297	50,111	357	75	4	36	38.7	JN854207
	P. cembroides	115,919	64,394	50,581	460	75	4	36	38.6	JN854220
	P. remota	115,422	64,493	50,178	357	75	4	36	38.6	JN854164
	P. quadrifolia	115,508	64,385	50,367	362	75	4	36	38.7	JN854166
	P.maximartinezii	115,620	64,575	50,269	382	75	4	35	38.7	JN854184
	P. rzedowskii	115,934	64,652	50,508	380	75	4	36	38.6	FJ899557
	P. nelsonii	116,210	64604	50,845	367	74	4	35	38.7	EU998746
	P. aristata	116,918	64,251	51,707	480	75	4	35	38.7	FJ899567
	P. bungeana	116,751	64,311	51,490	475	75	4	36	38.8	NC_028421.
	P. gerardiana	116,668	64,296	51,339	516	75	4	36	38.7	EU998741
	P. strobiformis	116,200	64,230	51,108	474	75	4	36	38.7	JN854159
	P. chiapensis	116,197	64,524	50,895	392	75	4	36	38.8	JN854219
	P. parviflora	120,724	66,364	53,409	475	74	4	36	38.6	MG897304
	P. wallichiana	116814	-	-	-	-	-	-	-	JN854154
	P. squamata	117,327	64,706	51,825	398	74	4	36	38.7	MG897303
	P. lambertiana	116,958	64,604	51,592	379	75	4	35	38.8	EU998743
	P. pumila	114,087	64,553	48,762	384	75	4	36	38.7	JN854168
	P. dalatensis	116,657	64,533	51,321	393	75	4	36	38.8	JN854211
	P. armandii	116,998	64,337	51,711	389	75	4	36	37	NC_029847
	P. morrisonicola	116,636	64,104	51,770	381	74	4	36	38.7	MG897305
	P. wangii	118,073	65,598	51,521	476	74	4	36	38.7	MG897302
	P. fenzeliana	117,805	64,490	52,565	375	75	4	35	36.8	KX255674

Results

Genome features of seventy-two Pinus species

The complete chloroplast genomes of seventy-two Pinus species ranged in size from 114,087 (P. pumila) to 121,976 bp (P. glabra) (Table 1 and Fig 1). Plastid genomes had a quadripartite structure which present in most of the gymnosperm species. The complete genomes of Pinus species comprised of a large single copy (LSC) region ranged from 64,415 (P. sylvestris) to 65,610 bp (P. taeda), and a small single copy (SSC) region ranged from 50,661 (P. sylvestris) to 56,070 bp (P. glabra), and inverted repeats (IRs) ranged from 244 (P. muricata) to 492 bp (P. arizonica) in size (Table 1). The whole plastome of GC content was comparable to the Pinus species.

Fig 1

Gene map of 72 Pinus species.

Genes drawn outside of the external circle are transcribed clockwise direction, and inside genes are transcribed clockwise directions. The Genes belong to varies functional group are colored coded. The darker gray region inside circle indicates GC content while the lighter gray color to AT content of the cp genome. Large single copy (LSC), small single copy (SSC), inverted repeat (IRs).

Pinus species complete cp genome consisted of 114 functional genes, with 36 tRNA, 4 rRNA and 74 protein-coding. Among 114 genes, 11 genes for small ribosome subunits, 9 genes for large ribosome subunits, 4 genes for DNA-dependent RNA polymerase subunits and 50 genes fragments were related to self-replication. The translational initiation factor (infA) gene, 38 genes for photosynthesis, 6 genes for ATP synthesis, and 11 genes encoding subunits of photosystem I (Table 2).

Table 2

Genes present in the seventy-two Pinus complete chloroplast genomes.

Gene group	Gene name
Ribosomal RNA genes	rrn16	rrn23	rrn5	rrn4.5
Transfer RNA genes	trnI-CAU	trnL-UAA	trnI-GAU	trnL-UAG	trnL-CAA
	trnR-UCU	trnR-ACG	trnA-UGC	trnW-CCA	trnE–UUC
	trnV-GAC	trnV-UAC	trnT-UGU	trnF-GAA	trnT-GGU
	trnfM-CAU	trnP-UGG	trnG-GCC	trnP-GGG	trnS-GGA
	trnS-UGA	trnS-GCU	trnD-GUC	trnC-GCA	trnN-GUU
	trnE-UUC	trnY-GUA	trnQ-UUG	trnK-UUU	trnH-GUG
	trnG-GCC	trnM-CAU
Small Subunit of ribosome	rps2	rps3	rps4	rps7	rps8
	rps11	rps12	rps14	rps15	rps18
	rps19
Large Subunit of ribosome	rp12	rp114	rp116	rp120	rp122
	rp123	rp132	rp133	rp136
DNA-dependent RNA polymerase	rpoA	rpoB	rpoC1	rpoC2
Translational initiation factor	infA
Subunits of photosystem I	psaA	psaB	psaC	psaI	psaJ
	psaM	ycf1	ycf2	ycf3	ycf4
	ycf10
Subunits of photosystem II	psbA	psbB	psbC	psbD	psbE
	psbF	psbH	psbI	psbJ	psbL
	psbM	psbN	psbT
Subunits of cytochrome	petA	petB	petD	petG	petL
	petN
Subunits of ATP synthase	atpA	atpB	atpE	atpF	atpH
	atpI
Large subunit of Rubisco	rbcL
Maturase	matk
Protease	clpP
Subunit of acetyl-CoA	accD
C-type cytochrome synthesis gene	ccsA

Repeat sequence variations and genome structure comparison

In this study, we calculated three types of repetitions, i.e. dispersed, palindromic and tandem repeats. Among these repeat variations, a number of divisions and repeats were analyzed (S1 Table and Fig 2). We identified 5,943 repeats, among these repeats dispersed were most common with 2,612 (43.95%), followed by palindromic repeats with 1,921 (32.32%), and tandem repeats with 1,410 (23.72%) (Fig 1). Majority of repeats found circulated in intergenic regions and few were situated within generic regions. P. nelsonii were the most dispersed repeated sequences (76) followed by P. pseudostrobus (63) palindromic repeats whereas, P. sabiniana showed lowest number tandem repeats with only (13) tandem repeats (S1 Table).

Fig 2

A histogram of the number of repeats found in the seventy-two Pinus chloroplast genomes.

(a) The number of repeats in subgenus Pinus (b) Number of repeats in subgenus Strobus.

For sequence identity analysis mVISTA was used with P.squamata sequence as reference (S1 Fig). It was observed that 72 Pinus species hold large number of sequence similarity however, lesser degree of variation was also observed. It is worthy to mention that non-coding regions displayed high levels of divergence compared to coding regions. The outcome helped to identified hotspot divergent regions on Pinus cp genome (S1 Fig). The non-coding regions displayed sequence divergence, and percentage of variation ranged from 0 to 13.78% with an average of 4.96%, whereas, the percentage variation in coding region ranged from 0 to 9.98% with an average of 2.54% (Fig 3). Furthermore, we discovered that IR region has a lower number of mutations and is highly conserved in Pinus species. It noteworthy, we identified seven genes(trnD-GUC, trnY-GUA, trnH-GUG, ycf1, trnL-CAA, and trnV-GAC) at LSC and SSC region located within the non-coding regions showing greater levels of variation, with ability to act as divergence hotspot regions.

Fig 3

Variable characters percentage in homologous regions of Seventy-two Pinus species of chloroplast genome (a) Coding region (b) Non coding region. The homologous regions are oriented according to their locations in the chloroplast genome.

Phylogenetic relationships of Pinus species

The 72 Pinus chloroplast genome sequences were used for phylogenetic analysis. Under the GTR+G+I model, we re-constructed three independent phylogenetic trees through different analytical methods: maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) (Fig 4). Among investigated species, the phylogenetic analysis displayed congruent topologies, although the bootstrap value was kept slighlty different for all phylogenetic trees. The phylogenetic tree further divided into two clades, single-needle section (subgenus Strobus) and double-needle section (subgenus Pinus species) (Fig 4). We found that P. wangii, P. fenzeliana, P. morrisonicola and P. armandii posses close relationships and catageorized in the single needle section. In addition, the P. parviflora, P. chiapensis and P. wallichiana were closely related to subgenus Pinus.

Fig 4

Phylogenetic tree obtained from whole cp genome (A) The numbers above the branches represent bootstrap values greater than 50% for ML (left) and MP (middle) analyses, and Bayesian posterior probabilities (right). A dash shows a bootstrap value less than 50%.

Discussion

Features of cp genomes of Pinus species

The chloroplast genome of higher plants is circular molecule with a length of 120–160 kb with approximately 130 genes [23]. The structure and organization of thes genes found similar among the 72 Pinus species under investigation. Moreover, similar GC level for 72 Pinus species was observed which is less common for most of the terrestrial plants [23]. IRs contraction and expansion are extensively exhibited in many lands plant species. The, large IRs played a significant role in maintaining the constancy of whole plastome [24]. Small IR region may cause variations in genome structure and content of plastome [25]. Interestingly, in present study, we detected small IR regions in all investigated Pinus species (244 to 492 bp). Following results displayed that in certain genes have variations for structure and contents compared to whole cp genome of Pinus species [26].

previous investigations has exhibited that repeat sequences have performed significant roles in genome re-organization and recombination [27]. Among 72 Pinus species P. nelsonii genome had large numbers of repeats (76), whereas, P. pseudostrobus genome have (63) repeats. In contrary, P. sabiniana displayed lowest number of (13) repeats (S1 Table and Fig 2). However, the tandem, dispersed, and palindromic repeats distributions were comparable for all Pinus species. A large number of repeats could maintain cp genomes constants, similar results reported by Zhang et al [16]. The repeat sequence displayed similar genes function rearrangement for further study in population genetics and Pinus species evolution [2].

Comparative analysis of the genomic structure

The complete chloroplast genome of Pinus species displayed a very low genetic divergence. Sequence alignment of 72 plastids genomes were compared, and used for sequence identity analysis via mVISTA programe, keeping P. squamata as a reference specie (S1 Fig). The similarity analysis exhibited a high sequence comparison across the plastid genomes having sequence identities below 90%. However, a low divergence region identified in LSC and less mutation rate in IRs region. In addition, the divergent hotspot regions (trnD-GUC, trnY-GUA, trnH-GUG, ycf1, trnL-CAA, trnK-UUU and trnV-GAC) were found in non-coding regions of some tRNA sequences. Several repetitive sequences were equally distributed in the divergence hotspot regions. These hotspot regions can be utilized for phylogenetic study and provide DNA barcoding for future evolutionary studies of gymnosperm species [28].

The whole plastome phylogenetic analysis has been commonly undertaken in land plants [29]. During recent decade, a study has revealed phylogenetic relationship and comparisons of numerous protein-coding genes present in the chloroplast genomes [30]. That improved our understanding for phylogenetic relationship and molecular studies among Pinus species [31].

The current study used phylogenetic analysis based on entire cp genome sequence of 72 Pinus specieshaving P. glauca, A. nephrolepis, and A. koreana serving as outgroups. Using ML, MP, and BI methods, we created a concurrent phylogenetic tree with a wide range of supported values (Fig 4). The phylogenetic tree of Pinus species was divided into two groups that corresponded to single needle sections and double needle sections. Among these sequenced species, single-needle section species i.e., P. morrisonicola and P. wangii catagorized in the same clade, showing a close relationship with each other. Moreover, these two species showed a high similarity in their chloroplast genome sequences [31]. In addition, the phylogenetic tree revealed P. bungeana and P. gerardiana has a close relationship with each other [32]. The phylogenetic results exhibited P. clausa showed a sister clade to the Pinus species [33].

Conclusion

The present study determined the whole chloroplast genome a rich source to understanf the evolutionary history. The cp genomes of Pinus species, genome structure and order were similar in nature. Moreover, the location and distribution of repeat sequences were determined, and common pairwise sequence divergences among cp genomes of interrelated species were identified. The whole genome sequencing proved to be a significant knowledge for plant taxonomic positioning. The main findings based on complete chloroplast genome of Pinus species divided into two sections, single needle sections and double-needle sections of Pinus species. The phylogenetic relationships dependent on the cp genome greatly developed our understanding on phylogeny of Pinus species. Comparative analyses of plastid genome sequences provide DNA markers for easy identification and classification. These results will provide supportable confirmations and prove a solid basis for the improvement of chloroplast genome in Pinus species.

Sequence alignment of chloroplast genomes of Pinus species.

mVISTA-based identity plots viewing identity between seventy-two Pinus species cp genomes. The vertical scale indicates the percentage identity, ranging from 50% to 100%. Divergent hotspot refers to the places with more variable sites compared to another region.

(DOCX)

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Repeat sequences analysis in seventy-two Pinus species based on complete chloroplast.

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