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Complete chloroplast genomes of five Cuscuta species and their evolutionary significance in the Cuscuta genus

Abstract

Background

Cuscuta, a parasitic plant species in the Convolvulaceae family, grows in many countries and regions. However, the relationship between some species is still unclear. Therefore, more studies are needed to assess the variation of the chloroplast (cp) genome in Cuscuta species and their relationship with subgenera or sections, thus, providing important information on the evolution of Cuscuta species.

Results

In the present study, we identified the whole cp genomes of C. epithymum, C. europaea, C. gronovii, C. chinensis and C. japonica, and then constructed a phylogenetic tree of 23 Cuscuta species based on the complete genome sequences and protein-coding genes. The complete cp genome sequences of C. epithymum and C. europaea were 96,292 and 97,661 bp long, respectively, and lacked an inverted repeat region. Most cp genomes of Cuscuta spp. have tetragonal and circular structures except for C. epithymum, C. europaea, C. pedicellata and C. approximata. Based on the number of genes and the structure of cp genome and the patterns of gene reduction, we found that C. epithymum and C. europaea belonged to subgenus Cuscuta. Most of the cp genomes of the 23 Cuscuta species had single nucleotide repeats of A and T. The inverted repeat region boundaries among species were similar in the same subgenera. Several cp genes were lost. In addition, the numbers and types of the lost genes in the same subgenus were similar. Most of the lost genes were related to photosynthesis (ndh, rpo, psa, psb, pet, and rbcL), which could have gradually caused the plants to lose the ability to photosynthesize.

Conclusion

Our results enrich the data on cp. genomes of genus Cuscuta. This study provides new insights into understanding the phylogenetic relationships and variations in the cp genome of Cuscuta species.

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Background

Chloroplasts are essential in photosynthesis and carbon fixation and thus, promote plant growth and development [1]. Chloroplasts are highly conserved based on gene size, gene content, and sequence order. They comprise a single circular molecule with a quadripartite structure that harbors two copies of inverted repeats (IRs) that separate large and small single-copy (LSC and SSC) [2]. The cp genomes encode 110–130 genes that range from 100 to 180 kb in length [3] that are primarily associated with photosynthesis, transcription, and translation [2]. Since complete cp genome sequences are contained in a single plastid genome, they have recently become popular for plant species identification, taxonomy, and phylogenetic analyses [4].

The genus Cuscuta belongs to the Convolvulaceae family and has approximately 200 species that are widely distributed worldwide [5]. Cuscuta is a holoparasite and obtains nutrients, water, and organic compounds from the host via haustoria [6]. Engelmann (1859) divided 77 Cuscuta species into three groups based on the morphology of their stigma [7]. Yuncker (1932) also divided 158 Cuscuta species into the three subgenera Cuscuta (28 species), Grammica (121 species), and Monogyna (nine species) based on dehiscence of the fruits [8]. Revill (2005) indicated that the molecular phylogeny of 15 species of Cuscuta belonged to three subgenera based on three types of plastid DNA (rbcL, rps2, and matK) [9], consistent with the conclusions of Yuncker [8]. Garcia (2014) also divided 131 Cuscuta species into four subgenera (Monogynella, Grammica, Pachystigma, and Cuscuta) using rbcL, nrLSU, fruit cracking, style number, and stigma shape [5]. Garcia indicated that Pachystigma does not belong to the subgenus Cuscuta but is related to the subgenus Grammica, a conclusion that was inconsistent with those of Yuncker [8], Revill [9], and McNeal [10]. Costea et al. (2015) grouped 194 Cuscuta species into four subgenera (Monogynella, Cuscuta, Pachystigma, and Grammica) based on the morphological and biogeographical predictive value [11], which was consistent with the conclusions of Garcia [5]. Banerjee and Stefanović (2020) classified six Cuscuta species into four subgenera using the whole cp genome sequencing method [12], which was consistent with the previous phylogenetic relationship based on morphological [11] and DNA sequences [5]. However, Banerjee and Stefanović used few Cuscuta species [12]. Therefore, a precise phylogenetic relationship should be assessed by including more species of Cuscuta. Moreover, the phylogenetic analysis of particular Cuscuta species is necessary to clarify the phylogenetic location of each species. For example, the phylogenic location of C. epilinum has been inconsistent in different studies. Revill (2005) showed that C. epilinum belongs in the subgenus Grammica [9], while McNeal (2007) found that C. epilinum belongs in the subgenus Cuscuta based on the nuclear ribosomal internal transcribed spacer (nrITS) rps2, rbcL, and matK [10].

The cp genome encodes numerous structural proteins that are essential for photosynthesis. It also encodes ribosomal proteins and structural RNAs [13]. Therefore, the loss or mutation of genes in chloroplasts could affect photosynthesis. For example, mutants in the single-copy SPPA1 gene in Arabidopsis thaliana maintain a higher level of the quantum efficiency of Photosystem II [14]. The photosynthetic ability of parasitic plants ranges from reduced levels to a complete lack of the ability to photosynthesize [2]. Most Cuscuta species do not have chlorophyll and thus, cannot photosynthesize [15]. However, some Cuscuta species (C. pentagona and C. reflexa) have chloroplasts with photosystems and some chlorophyll [15,16,17,18]. A recent study showed that highly divergent plastid chromosomes exist in non-photosynthetic parasitic plants [19,20,21,22]. The size of plastid genome in Cuscuta species is related to their photosynthetic capacity. Photosynthetic species have more plastomes than non-photosynthetic species [23]. In addition, gene loss is significantly correlated with species in different subgenera or Sects.  [24, 25]. However, Revill et al. identified the loss of photosynthesis and alterations in the structure of the cp genome of 15 Cuscuta species using the DNA dot analysis method but did not find a correlation with the phylogenetic position [9]. Therefore, more studies are needed to assess the variation of the cp genome in Cuscuta species and their relationship with subgenera or sections, thus, providing important information on the evolution of Cuscuta species.

Both C. epithymum and C. europaea had been proposed to belong to subgenus Cuscuta [12], however, no complete cp genome was available until now. Both C. chinensis and C. japonica collected in Korea were identified to belong to subgenus Grammica and Monogynella, respectively, based on the complete cp genome sequences [26]. C. gronovii was identified to belong to subgenera Grammica [26] based on the complete cp genome sequences [27]. In this study, five Cuscuta species, including C. epithymum, C. europaea, C. gronovii, C. chinensis and C. japonica, were sequenced, and their cp genomes were assembled. We then compared the whole cp genome of 23 Cuscuta species to determine the following: (1) the novel cp genomes of both C. epithymum and C. europaea; (2) the phylogenetic relationship based on the whole cp genomes of the 23 Cuscuta species and the division of four subgenus; (3) the structural variation of the cp genomes among the 23 Cuscuta species, including C. chinensis, C. japonica, and C. gronovii collected in China; and (4) the loss of genes in the cp genome of the 23 Cuscuta species and its correlation with phylogenetic positions and photosynthetic ability. This study uncovered the phylogenetic relationships and variations in the cp genomes of Cuscuta species.

Results

Cp genome features of five Cuscuta species

The cp genomes of five Cuscuta species were sequenced and the raw data ranged from 11,769,885 (C. gronovii) to 273, 318,504 (C. japonica), while the clean data ranged from 11,646,979 (C. gronovii) to 273,081,185 (C. japonica) (Table 1). Assembled by NOVOPlasty (version 3.7.2), the length of the cp genomes of five Cuscuta species ranged from 86,745 bp (C. gronovii) to 121,031 bp (C. japonica) (Table 2). Among them, the cp genomes of C. chinensis, C. japonica and C. gronovii were 99.87%, 100%, and 99.86% similar with those deposited in the NCBI database (Table 2). The cp genomes of C. epithymum and C. europaea were novel. The cp genome sequences of C. epithymum and C. europaea were similar with that of C. approximata with a similarity of 97.96% and 95.45%, respectively. The complete cp genome sequences of C. epithymum and C. europaea were 96,292 and 97,661 bp long, respectively, and lacked an inverted repeat (IR) region (Fig. 1). The LSC regions were 69,214 bp and 77,514 bp long in C. epithymum and C. europaea, respectively, and those of the SSC region were 2,908 bp and 1,624 bp long, respectively. The IR regions (IRa and IRb) were 24,170 bp and 18,523 bp in C. epithymum and C. europaea, respectively. The GC content of C. epithymum and C. europaea was 37.7% and 37.6%, respectively (Table 2). The gene content and gene order differed substantially between the two Cuscuta Cp genomes. The cp genome of C. epithymum harbored 99 unique genes, including 66 protein-coding genes, four rRNA genes and 27 tRNA genes, whereas that of C. europaea contained 91 unique genes, including 60 protein-coding genes, four rRNA genes and 27 tRNA genes (Table 2). Genetic map of the cp genomes of C. japonica, C. gronovii and C. chinensis were in the supplement (Figure S1, S2 and S3).

Table 1 The data of NGS sequencing of the five Cuscuta species
Table 2 Characteristics of the chloroplast genomes of 23 Cuscuta species and the outgroup
Fig. 1
figure 1

Genetic map of the chloroplast genomes of Cuscuta epithymum and C. europaea. The transcriptional direction of genes in the outside circle is counter-clockwise, while those in the inside circle are clockwise. The outer circle shows the genes at each locus. The inner circle also shows the GC content graph of the genome, where the dark and light gray lines indicate the GC and AT contents, respectively, at each locus

Cuscuta species have genomic sequence lengths that range from 60,905 bp to 125,373 bp. Cuscuta cp genomes have 31–69 protein-coding genes, 23–42 transfer RNAs (tRNAs), and 4–8 ribosomal RNAs (rRNAs) (Table 2). The most diminished cp genome, that of C. erosa (60,959 bp long), has 33 protein-coding genes, 27 tRNAs, and eight rRNAs and was reduced by 62% compared with the chloroplast. The genome of Ipomoea purpurea, a member of the Convolvulaceae family, was used as the reference genome. The cp genome of C. exaltata (125,373 bp long) has 69 protein-coding genes, 42 tRNAs, and eight rRNAs with a reduction in its composition of 22% that demonstrated a significant variation in the genome length and gene composition in the Cuscuta chloroplast. The cp genomes of C. exaltata, C. reflexa, and C. japonica were larger than the genomes of remaining Cuscuta species (24-25% sequence reduction compared with the genome of I. purpurea) (Table 2).

Phylogenetic analysis

The GTR + G + I model was selected as the best-fit substitution model using MEGA 7. Herein, phylogenetic trees based on protein-coding sequences and complete cp genome sequences produced similar topologies (Fig. 2). The 23 Cuscuta species clustered into four subgenera, Monogynella, Cuscuta, Pachystigma, and Grammica. The subgenus Monogynella contains C. exaltata, C. reflexa, and C. japonica. The subgenus Cuscuta includes C. epithymum, C. europaea, C. approximata and C. pedicellata. The subgenus Pachystigma includes C. nitida and C. africana. The remaining species form the subgenus Grammica.

Fig. 2
figure 2

The phylogenetic trees of 23 Cuscuta species and Ipomoea purpurea as determined from different data based on protein-coding sequences (left) and the complete chloroplast genome sequences (right). The support values are shown for nodes as maximum likelihood bootstrap (approach branches). The species with the same color belong to the same subgenus. Brown, purple, orange and blue represent the subgenera Monogynella, Cuscuta, Pachystigma, and Grammica, respectively. * indicates that the chloroplast genomes of these Cuscuta species were sequenced in this study

Simple sequence repeats (SSRs) analysis

A total of 471 SSRs were detected in the 23 Cuscuta species (Table 3). Among them, more than 80% were mononucleotide SSRs and belonged to the A or T types. Only one SSR in C. approximata and one SSR in C. europaea were polynucleotide repeats belonging to the c type.

Table 3 Chloroplast SSRs in 23 Cuscuta species

Sequence inversions

Compared with the genome of I. purpurea, the structural changes in the sequences among C. epithymum, C. europaea, C. approximata, and C. pedicellata belonging to subgenus Cuscuta were shown in Fig. 3. Two sequence inversions were detected in the subgenera of Cuscuta. One inversion included trnL-UAA, trnT-UGU and trnF-GAA (inversion A), while the other included ccsA, psaC and rps15 (inversion B). There were four inversions (black region) that did not contain any genes (Fig. 3).

Fig. 3
figure 3

Structural variation of chloroplast genomes among C. epithymum, C. europaea, C. approximata, and C. pedicellata belonging to subgenus Cuscuta compared with genome of Ipomoea purpurea. The yellow region represents inversion A, and the brown region represents inversion B. The four black regions represent the four inversions. The colored regions (red, green, blue and pink) do not contain gene inversions

IR expansion and contraction

Expansion and contraction at the IR region boundaries are common and influence the variation in the sizes of cp genomes. A detailed comparison between the IR-SSC and IR-LSC borders of genomes among the 23 intact four-part structures (IR-SSC-IR-LSC) of the Cuscuta chloroplasts is shown in Fig. 4. Similar to the sequence inversions, the IR borders were highly conserved within the Cuscuta subgenus. The ycf2 gene crossed the LSC/IRb region of species in the subgenus Monogynella, including C. reflexa, C. japonica, and C. exaltata. The length of extension of the ycf2 gene into the LSC region was based on the genome (C. reflexa, 3,519 bp; C. japonica, 4,228 bp; and C. exaltata, 4,227 bp). The IRb/SSC junction of the subgenus Pachystigma was located in the ycf1 gene and extended into the IRb regions (1,534 bp in C. africana and 1,062 bp in C. nitida). The IR boundaries varied significantly more in subgenus Grammica (four ycf1 distribution patterns that crossed the IR boundaries) compared with the subgenera Monogynella and Pachystigma. The first one was located inside the SSC regions (C. erosa, C. boldinghii, C. strobilacea, C. carnosa, C. bonafortunae, C. mexicana, and C. chapalana). The second extended by ~ 1,000 bp from the SSC region to the IRa region (C. gronovii, C. campestris, and C. costaricensis); the third one extended by less than 1,000 bp from the SSC region to IRb region (C. obtusiflora and C. australis), and the last one crossed both the SSC/IRa (extended by ~ 1,000 bp to IRa) and the SSC/IRb regions (extended by 258 and 956 bp to the IRb, respectively) (C. chinensis and C. pentagona). In addition, in contrast to the other Cuscuta species, the cp genomes of C. epithymum, C. europaea, C. approximata and C. pedicellata lacked an IR.

Fig. 4
figure 4

The IR borders in chloroplast genomes of 23 Cuscuta species. The gray, purple, and light blue blocks represent the LSC region, IR region and SSC region, respectively. Blocks with different colors represent different genes. IR, inverted repeat; LSC, large single-copy; SSC, small single-copy

Patterns of reduction of the Cuscuta cp. genome

The 23 Cuscuta species had a significantly reduced genome size and gene content (Tables 2 and 4). Notably, the entire NAD(P)H dehydrogenase complex gene (ndh) family related to land adaptation and photosynthesis was lost in all 23 Cuscuta species. The photosystem genes (ycf15 and ycf1) and ribosomal protein-coding genes (rpl23, rps15, and rps16) were lost in the 23 Cuscuta cp genomes. In addition, matK that encodes maturase and the photosynthesis-related gene psaI were lost in all the species in subgenus Grammica. The photosystem gene ycf2 was lost in the three subgenera Cuscuta, Pachystigma, and Monogynella. The lost genes were relatively conserved inside subgenus Cuscuta (Fig. 3). However, all the ATP synthase genes (atp) were present in all 23 Cuscuta species, even in the smallest cp genome C. erosa (except for atpF in C. boldinghii). TrnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA, trnV-UAC were lost in all 23 species. Two copies of trnR-ACG were only lost in all the species of subgenus Grammica.

Table 4 Loss of chloroplast protein coding genes and transfer RNA genes across the Cuscuta spp

Discussion

Molecular phylogeny of 23 Cuscuta species

Phylogenetic analysis of specific Cuscuta species is necessary to clarify the phylogenetic location of each species. To date, 23 plastomes have been identified [4, 10, 12, 25, 27, 28], including C. europaea and C. epithymum obtained in this study. Here, we found that C. europaea was closely related to C. epithymum based on protein coding genes and whole cp genome. This result was consistent with the findings of Neumann [29]. We also found that C. australis is closely related to C. pentagona, which is consistent with the findings of previous research [4], and not closely related to C. epithymum, which was mentioned by Revill et al. [9]. These comparative genomic analyses provide new insights into understanding the phylogeny of Cuscuta species. However, it is necessary to increase the sample size of Cuscuta species and use data based on nuclear genome sequencing to strengthen the understanding of their phylogenetic relationships.

Division of four subgenera

The division of four subgenera (Monogynella, Cuscuta, Pachystigma, and Grammica) was always controversial. Banerjee and Stefanović classified six Cuscuta species into four subgenera using the whole cp genome sequencing method and found it was consistent with the morphological and DNA sequences-based phylogenetic method, however, the number of studied species was limited [12]. In this study, we classified 23 Cuscuta species into four subgenera based on the complete cp genome sequences and found it was not consistent with the phylogenetic relationship.

Neumann (2020) divided C. approximate and C. pedicellata into the subgenus Cuscuta [29] and both C. epithymum and C. europaea were identified to belong to subgenus Cuscuta by using sequences of the nuclear ribosomal internal transcribed spacer and plastid rps2, rbcL and matK [10]. The cp genomes of C. europaea, C. epithymun, C. approximate and C. pedicellata had the same pattern of gene inversion and both lacked one IR region (Figs. 3 and 4). Therefore, we agreed that C. europaea and C. epithymum belonged to subgenus Cuscuta. However, in this study, C. europaea and C. epithymum grouped together, while C. approximate and C. pedicellata formed another group, however, the two clades were not sister to each other. McNeal et al. found that subgenus Cuscuta is unequivocally paraphyletic with subgenus Grammica and subgenus Pachystigma nested within it [10]. Thus, we hypothesized that C. europaea and C. epithymum might be classified into another subgenus and played special role in the morphological and plastid genome evolution. More studies including more numbers of Cuscuta species are needed in the future.

The subgenus Monogynella, containing of C. exaltata, C. reflexa, and C. japonica, was monophyletic and was the most basal clade with fewer speciation events. The ycf2 gene crossed the LSC/IRb region of species in C. reflexa, C. japonica, and C. exaltata. The results were consistent with the findings of McNeal et al. [10] and Park et al. [26].

In this study, the molecular phylogeny showed C. nitida and C. africana were closed to C. approximate and C. pedicellate (Fig. 2), however, the cp genome of C. nitida and C. africana didn’t lose one IR region (Fig. 4). Thus, we suggested that C. nitida and C. africana belonged to subgenus Pachystigma, which was consistent with the findings of Banerjee and Stefanovic [12].

Wang et al. [4] and Banerjee and Stefanovic [25] revealed that C. bonafortuna was closed to C. strobilacea and were belonged to subgenus Grammica. In our research, the cp genome of C. bonafortuna didn’t loss one IR region which was different from C. approximate and C. pedicellata (Fig. 4). The four subgenus of Cuscuta occurred different gene loss event (Table 4). C. bonafortuna lost ndh, psaI, psbL, rpl23, rpl32, rps15, rps16, ycf1, matK, ycf15, but C. nitida and C. africana both lost rpl23, rps15, rps16, ycf1 and ycf15. The IR borders of C. bonafortuna is similar to C. strobilacea (Fig. 4). These results supported that C. bonafortuna belonged to subgenus Grammica. As mentioned by McNeal et al. [10], subgenera Cuscuta, Grammica and Pachystigma were not monophyletic, indicating that more evolution events related with the gene loss might happened among them, which might drive the changes of morphological and physiological traits of Cuscuta species. More studies are needed to elucidate the relationships among Cuscuta species based on taxonomic, morphological, physiological and molecular evidences.

Variation in chloroplast gene structure

The uniparentally inherited SSRs in cp genomes are valuable molecular markers owing to their high degree of variations even within an individual species [30, 31]. Herein, most of the cp genomes of the 23 Cuscuta species had single nucleotide repeats of A and T, which are similar to those of other species [32, 33]. The validation of SSRs of cp genomes should be done before it can be used to identify species and in population genetics and evolutionary research of Cuscuta and its relatives.

Most cp genomes had a quadripartite structure, which consisted of two IR regions separated by one LSC and one SSC region (Fig. 2). The genomic structure, gene content, gene order, and base composition are highly conserved in the IR regions in most plant chloroplasts [34]. Herein, four Cuscuta species (C. epithymum, C. europaea, C. approximata and C. pedicellata) lacked an IR region (Table 2). The loss of IR could be found in the cp genomes of higher plants [35]. These results further confirmed the structural plasticity of the chloroplast. Owing to unequal recombination and replication slippage, the expansion and contraction of IR regions caused the structural variations in the IR boundaries (IRb) (Fig. 4).

Inversion events could be owing to the activity of tRNA [36] and high GC content [37]. The regions that flank two inversions (inversion A and inversion B) contained tRNA gene sequences (Fig. 3), indicating that tRNA recombination promotes inversions in the plastid genomes [36]. However, the GC content in inversion flanking sequences was not consistently higher than the average GC content in the cp genome. Therefore, the patterns of sequence variations at inversion boundaries are more consistent with tRNA activity and not intragenomic recombination between regions with a high content of GC [36, 37].

Cp genome reduction and gene selection in Cuscuta species

Cuscuta is a parasitic angiosperm that exists as a hemiparasite or holoparasite [38]. Herein, the 23 Cuscuta species had a significantly reduced genome size and gene content (Tables 2 and 4), which is consistent with the findings of previous studies [10, 25, 39]. The lost genes were conserved in the Cuscuta subgenus and correlated with the species classification and phylogenetic relationships. Previous studies showed that Cuscuta species is a phenomenon of irreversibly reducing their genes, i.e., genes cannot be regained once they are lost [25, 40]. According to our results, ndh, ycf1, ycf15, rpl23, rps15, and rps16 were lost in the species in all four subgenera, while the matK and pasI genes were lost in the subgenus Grammica. Therefore, it can be inferred that the ndh, ycf15, ycf1, rpl23, rps15, and rps16 genes were lost before the pasI, rpo, and matK genes, considering that the genes could not be regained once they are lost.

The absence of ndh genes in angiosperms is primarily related to the loss of photosynthetic function in parasitic plants [41]. The ndh genes encode NDH dehydrogenase complexes, which are closely related to light and action [42]. The loss of NDH complex reduces the dependence of Cuscuta on photosynthesis, which is lower than that of green plants [43]. All the Cuscuta species lost ndh genes, indicating the loss of photosynthetic capacity during their evolution from autotrophy to heterotrophy, which is similar to the results of a previous study [25]. However, the functions of ycf1 and ycf15 genes are unclear. The loss of rpl23, rps15, and rps16 genes related to ribosomal protein formation could enable the adaptation of Cuscuta to parasitic life. Herein, the types of gene reduction that were tracked included psaI, rpo and matK in the subgenus Grammica. psaI is related to the function of photosystem I (PS I). The rpo gene (rpoA, rpoB, rpoC1, and rpoC2) is crucial for the production of chloroplasts [44]. The loss of rpo gene can affect protein synthesis in the chloroplast, thus, affecting the development of chloroplasts and photosynthesis [45]. MatK is related to RNA processing [46]. The loss of psaI, rpo, and matK can decrease the ability of chloroplasts to photosynthesize and produce materials, which renders Cuscuta more parasitic. Herein, all the Cuscuta species that lost ndh, psaI, rpo, and matK (except for C. erosa, C. boldinghii, C. strobilacea) could be hemiparasitic. However, C. erosa, C. boldinghii, C. strobilacea lost several genes associated with photosynthesis, such as psa, psb, pet, rbcL, which indicates the transition from hemiparasitic to holoparasitic. These results indicate that the loss of genes related to photosynthesis is a continuous process [25]. The ndh gene was lost first (in all species of Cuscuta), followed by the psaI, rpo and matK genes (C. africana and across the subgenera Grammica and Cuscuta), and the substantial loss of psa, psb, pet, rbcL, and other genes (C. erosa, C. boldinghii, C. strobilacea). This study supports the model of plastid evolution proposed by Banerjee and Stefanović [25].

Materials and methods

Plant materials and cp. genome sequences

C. chinensis seeds were purchased in Aohanqisidaowanzi town, Chifeng City, China. C. japonica seeds were collected from a field in Sanmen County, China. C. gronovii seeds were collected from the field of Taizhou University, Taizhou City, China. After germination, the plants were identified by Professor Beifen Yang from Taizhou University based on their morphological traits according to a standard reference. Voucher herbarium specimens from the three species were deposited at Zhejiang Provincial Key Laboratory of Plant Evolutionary Ecology and Conservation in Taizhou University. Cuscuta epithymum from a wild population was collected in the region of Eleshnitsa, Pirin Mt., Bulgaria, while Cuscuta europaea was collected in Zlatni Mostove locality, Vitosha Mt., Bulgaria. Voucher herbarium specimens from the two species were deposited at Department of Biochemistry, Faculty of Biology, Sofia University. All the five Cuscuts species are parasitic weeds without regulations of ecological protection.

The whole cp genome sequences of 21 Cuscuta genes, including C. exaltata (EU189132), C. reflexa (AM711640), C. japonica (MH780080), C. nitida (NC052869), C. africana (NC052870), C. approximata (NC052871), C. pedicellata (MN464181), C. chinensis (MH780079), C. gronovii (AM711639), C. campestris (NC052920), C. costaricensis (MK881072), C. pentagona (MH121054), C. obtusiflora (EU189133), C. australis (NC045885), C. chapalana (MK887214), C. mexicana (MK887213), C. bonafortunae (MK887215), C. carnosa (MK887212), C. strobilacea (MK867795), C. boldinghii (MK881074), C. erosa (MK881073), and I. purpurea (EU118126) were downloaded from the NCBI database.

Cp genome sequencing, assembly, and annotation

The total DNA was extracted from the stem samples collected from the three species using a modified CTAB method [47]. Pair-end sequencing (insert size: 350 bp) was then performed using an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA). Raw paired-end reads of 150 bp were processed using SOAPnuke (version 1.5.2) to remove adapters and low‐quality sequences (the unknown base ratio was higher than 5% and the low‐quality base ratio [Q < = 5] was more than 20%) [48]. The raw reads were filtered to obtain high-quality clean data. The cp genome was assembled using NOVOPlasty (version 3.7.2) [49]. C. exaltata was used as the reference sequence. The other setting was default (K-mer = 39). The genes in the cp genomes of C. epithymun, C. europaea, C. chinensis, C. japonica and C. gronovii were annotated using GeSeq software. The start and stop codons of the gene were identified using automated tools. Circular maps of the cp genomes were obtained using OGDRAW (https://chlorobox.mpimp-golm.mpg.de/OGDraw. html) [50].

Phylogenetic analysis

Molecular phylogenetic trees were constructed using the whole cp genomes and all the protein-coding sequences of the 23 Cuscuta species. Ipomoea purpurea was used as an outgroup [29]. A total of 27 cp genomes were aligned using MAFFT v.7.450 [51] and manually adjusted using Geneious Prime 2021.1.1 (Biomatters, Ltd., Auckland, New Zealand). The maximum likelihood (ML) analysis was performed using 1,000 bootstrap replicates after selecting the best-fit substitution model via MEGA 7 [52].

Cp genome comparison and SSR searching

GENEIOUS software was used to determine the GC content. MAVUE was used to align the cp genome and identify inversions [53]. MISA software was used to detect the SSRs in the cp genome using the following parameters: minimum SSR motif length of 10 bp and repeat times of mono-10, di-6, tri-5, tetra-5, penta-5, and hexa-5 [54].

Conclusions

In this study, the cp genomes of C. epithymum, C. europaea C. gronovii, C. chinensis and C. japonica were sequenced and assembled. We analyzed the cp genomes of five Cuscuta species and compared them with the previously released cp genomes of 21 Cuscuta species. The complete cp genome sequences of C. epithymum and C. europaea were 96,292 and 97,661 bp long, respectively, and lacked an inverted repeat (IR) region. Based on the cp genome structure and genes loss events, we divided the 23 Cuscuta species into four subgenera (Monogynella, Pachystigma, Cuscuta, and Grammica). We found that C. epithymum and C. europaea belonged to subgenus Cuscuta for the lack of one IR region and the presence of two inversions. Furthermore, the 23 Cuscuta species had substantial variations in the length of their cp genome and its gene composition. Most of the reduced cp genomes lost several photosynthetic genes (ndh, rpo, psa, psb, pet, and rbcL), thus, gradually decreasing their photosynthetic capacity. This study will guide future comparative genomic investigation into the evolution of Cuscuta species.

Data Availability

Cp genome data of five Cuscuta species were deposited in the NCBI database (OL752638-OL752640, OP620588, OP620589).

References

  1. Neuhaus HE, Emes MJ. Nonphotosynthetic metabolism in plastids. Annu. Rev. Plant Physiol. Plant Mol Biol. 2000;51(1):111–40. https://doi.org/10.1146/annurev.arplant.51.1.111

    Article  CAS  Google Scholar 

  2. Daniell H, Lin CS, Yu M, Chang WJ. Chloroplast genomes: diversity, evolution, and applications in genetic engineering. Genome Biol. 2016;17(1):134. https://doi.org/10.1186/s13059-016-1004-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Wicke S, Schneeweiss GM, dePamphilis CW, Muller KF, Quandt D. The evolution of the plastid chromosome in land plants: gene content, gene order, gene function. Plant Mol Biol. 2011;76(3–5):273–97. https://doi.org/10.1007/s11103-011-9762-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wang FF, Huang DJ, Qin BL, Wang XW, Qian J, Zhang ZH, Wang XH. The complete chloroplast genome of Cuscuta australis R. Br. (Convolvulaceae) and its phylogenetic implication. Mitochondrial DNA B. 2020;5(1):766–7. https://doi.org/10.1080/23802359.2020.1715865

    Article  Google Scholar 

  5. Garcia MA, Costea M, Kuzmina M, Stefanovic S. Phylogeny, character evolution, and biogeography of Cuscuta (dodders; Convolvulaceae) inferred from coding plastid and nuclear sequences. Am J Bot. 2014;101(4):670–90. https://doi.org/10.3732/ajb.1300449

    Article  PubMed  Google Scholar 

  6. Clarke CR, Timko MP, Yoder JI, Axtell MJ, Westwood JH. Molecular Dialog between parasitic plants and their hosts. Annu Rev Phytopathol. 2019;57(1):279–99. https://doi.org/10.1146/annurev-phyto-082718-100043

    Article  CAS  PubMed  Google Scholar 

  7. Engelmann G. Systematic arrangement of the species of the genus Cuscuta with critical remarks on old species and descriptions of new ones. Trans Acad Sci St Louis. 1859;1:453–523.

    Google Scholar 

  8. Yuncker TG. The genus Cuscuta. Mem Torrey Bot Club. 1932;18:113–331.

    Google Scholar 

  9. Revill MJW, Stanley S, Hibberd JM. Plastid genome structure and loss of photosynthetic ability in the parasitic genus Cuscuta. J Exp Bot. 2005;56(419):2477–86. https://doi.org/10.1093/jxb/eri240

    Article  CAS  PubMed  Google Scholar 

  10. McNeal JR, Arumugunathan K, Kuehl JV, Boore JL, Depamphilis CW. Systematics and plastid genome evolution of the cryptically photosynthetic parasitic plant genus Cuscuta (Convolvulaceae). BMC Biol. 2007;5(1):55. https://doi.org/10.1186/1741-7007-5-55

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Costea M, Garcia MA, Stefanovic S. A phylogenetically based infrageneric classification of the parasitic plant genus Cuscuta (dodders, Convolvulaceae). Syst Bot. 2015;40(1):269–85. https://doi.org/10.1600/036364415X686567

    Article  Google Scholar 

  12. Banerjee A, Stefanovic S. Reconstructing plastome evolution across the phylogenetic backbone of the parasitic plant genus Cuscuta. (Convolvulaceae) Bot J Linn Soc. 2020;194:423–38. https://doi.org/10.1093/botlinnean/boaa056

    Article  Google Scholar 

  13. Wicke S, Schneeweiss GM, DePamphilis CW, Muller KF, Quandt D. The evolution of the plastid chromosome in land plants: gene content, gene order, gene function. Plant Mol Biol. 2011;76(3–5):273–97. https://doi.org/10.1007/s11103-011-9762-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wetzel CM, Harmacek LD, Yuan LH, Wopereis JLM, Chubb R, Turini P. Loss of chloroplast protease SPPA function alters high light acclimation processes in Arabidopsis thaliana L. (Heynh). J Exp Bot. 2009;60(6):1715–27. https://doi.org/10.1093/jxb/erp051

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Van der Kooij TAW, Krause K, Dorr I, Krupinska K. Molecular, functional and ultrastructural characterisation of plastids from six species of the parasitic flowering plant genus Cuscuta. Planta. 2000;210(5):701–7. https://doi.org/10.1007/s004250050670

    Article  PubMed  Google Scholar 

  16. Panda MM, Choudhury NK. Effect of irradiance and nutrients on chlorophyll and carotenoid content and Hill reaction activity in Cuscuta reflexa. Int J Dent. 1992;2014:385687.

    Google Scholar 

  17. Hibberd JM, Bungard RA, Press MC, Jeschke WD, Scholes JD, Quick WP. Localization of photosynthetic metabolism in the parasitic angiosperm Cuscuta reflexa. Planta. 1998;205:506–13.

    Article  CAS  Google Scholar 

  18. Sherman TD, Pettigrew WT, Vaughn KC. Structural and immunological characterization of the Cuscuta pentagona L. chloroplast. Plant Cell Physiol. 1999;40:592–603.

    Article  CAS  Google Scholar 

  19. Depamphilis CW, Palmer JD. Loss of photosynthetic and chlororespiratory genes from the plastid genome of a parasitic flowering plant. Nature. 1990;348(6299):337–9.

    Article  CAS  PubMed  Google Scholar 

  20. dePamphilis CW, Press MC, Graves JD. ; Parasitic Plants: Chapman and Hall, London, 1995; 176–205.

  21. Nickrent DL, Ouyang Y, Duff RJ, DePamphilis CW. Do nonasterid holoparasitic flowering plants have plastid genomes? Plant Mol Biol. 1997;34(5):717–29. https://doi.org/10.1023/A:1005860632601

    Article  CAS  PubMed  Google Scholar 

  22. Krause K. Piecing together the puzzle of parasitic plant plastome evolution. Planta. 2011;234(4):647–56. https://doi.org/10.1007/s00425-011-1494-9

    Article  CAS  PubMed  Google Scholar 

  23. Berg S, Krupinska K, Krause K. Plastids of three Cuscuta species differing in plastid coding capacity have a common parasite-specific RNA composition. Planta. 2003;218(1):135–42. https://doi.org/10.1007/s00425-003-1082-8

    Article  CAS  PubMed  Google Scholar 

  24. Braukmann T, Kuzmina M, Stefanovic S. Plastid genome evolution across the genus Cuscuta (Convolvulaceae): two clades within subgenus Grammica exhibit extensive gene loss. J Exp Bot. 2013;64(4):977–89. https://doi.org/10.1093/jxb/ers391

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Banerjee A, Stefanovic S. Caught in action: fine–scale plastome evolution in the parasitic plants of Cuscuta section Ceratophorae (Convolvulaceae). Plant Mol Biol. 2019;100:621–34. https://doi.org/10.1007/s11103-019-00884-0

    Article  CAS  PubMed  Google Scholar 

  26. Park I, Song JH, Yang SY, Kim WJ, Choi G, Moon BC. Cuscuta Species Identification based on the morphology of Reproductive Organs and complete chloroplast genome sequences. Int J Mol Sci. 2019;20(11):2726. https://doi.org/10.3390/ijms20112726

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Funk HT, Berg S, Krupinska K, Maier UG, Krause K. Complete DNA sequences of the plastid genomes of two parasitic flowering plant species, Cuscuta reflexa and Cuscuta gronovii. BMC Plant Biol. 2007;7(1):45. https://doi.org/10.1186/1471-2229-7-45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Park I, Yang SY, Kim WJ, Noh P, Lee HO, Moon BC. The complete chloroplast genome of Cuscuta pentagona Engelm. Mitochondrial DNA B. 2018;3(2):523–4. https://doi.org/10.1080/23802359.2018.1467229

    Article  Google Scholar 

  29. Neumann P, Oliveira L, Cizkova J, Jang TS, Klemme S, Novak P, Stelmach K, Koblizkova A, Dolezel J, Macas J. Impact of parasitic lifestyle and different types of centromere organization on chromosome and genome evolution in the plant genus Cuscuta. New Phytol. 2020;4:2365–77. https://doi.org/10.1111/nph.17003

    Article  CAS  Google Scholar 

  30. Powell W, Morgante M, McDevitt R, Vendramin GG, Rafalski JA. Polymorphic simple sequence repeats regions in chloroplast genomes: applications to the population genetics of pines. Proc Natl Acad Sci. 1995;92(17):7759–63. https://doi.org/10.1073/pnas.92.17.7759

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Xue JH, Wang S, Zhou SL. Polymorphic chloroplast microsatellite loci in Nelumbo (Nelumbonaceae). Am J Bot. 2012;99(6):e240–4. https://doi.org/10.3732/ajb.1100547

    Article  PubMed  Google Scholar 

  32. Kuang DY, Wu H, Wang YL, Gao LM, Zhang SZ, Lu L. Complete chloroplast genome sequence of Magnolia kwangsiensis (Magnoliaceae): implication for DNA barcoding and population genetics. Genome. 2011;54(8):663–73. https://doi.org/10.1139/G11-026

    Article  PubMed  Google Scholar 

  33. Qian J, Song J, Gao H, Zhu Y, Xu J, Pang X, et al. The complete chloroplast genome sequence of the medicinal plant Salvia miltiorrhiza. PLoS ONE. 2013;8(2):e57607. https://doi.org/10.1371/journal.pone.0057607

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Jeon JH, Kim SC. Comparative analysis of the complete chloroplast genome sequences of three closely related East-Asian Wild Roses (Rosa sect. Synstylae; Rosaceae). Genes. 2019;10(1):23. https://doi.org/10.3390/genes10010023

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Lin ZC, Zhou P, Ma XY, et al. Comparative analysis of chloroplast genomes in Vasconcellea pubescens A.DC. And Carica papaya L. Sci Rep. 2020;0(1):15799. https://doi.org/10.1038/s41598-020-72769-y

    Article  CAS  Google Scholar 

  36. Hiratsuka J, Shimada H, Whittier R, et al. The complete sequence of the rice (Oryza satira) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals. Mol Gen Genet. 1989;217:185–94.

    Article  CAS  PubMed  Google Scholar 

  37. Fullerton SM, Antonio BC, Clark AG. Local rates of recombination are positively correlated with GC content in the Human Genome. Mol Biol Evol. 2001;18(6):1139–42. https://doi.org/10.1093/oxfordjournals.molbev.a003886

    Article  CAS  PubMed  Google Scholar 

  38. Westwood JH, Yoder JI, Timko MP, DePamphilis CW. The evolution of parasitism in plants. Trends Plant Sci. 2010;15(4):227–35. https://doi.org/10.1016/j.tplants.2010.01.004

    Article  CAS  PubMed  Google Scholar 

  39. Costea M, Spence I, Stefanović S. Systematics of Cuscuta chinensis species complex (subgenus Grammica, Convolvulaceae): evidence for long-distance dispersal and one new species. Organisms Divers Evol volume. 2011;11(5):373–86. https://doi.org/10.1007/s13127-011-0061-3

    Article  Google Scholar 

  40. Wicke S, Naumann J. Molecular Evolution of Plastid Genomes in parasitic flowering plants. Adv Bot Res. 2017;85:315–47. https://doi.org/10.1007/s11103-011-9762-4

    Article  CAS  Google Scholar 

  41. Peredo EL, King UM, Les DH. The Plastid Genome of Najas flexilis: adaptation to submersed environments is accompanied by the complete loss of the NDH complex in an aquatic angiosperm. PLoS ONE. 2013;8(7):e68591. https://doi.org/10.1371/journal.pone.0068591

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Peltier G, Aro EM, Shikanai T. NDH-1 and NDH-2 plastoquinone reductases in oxygenic photosynthesis. Annu Rev Plant Biol. 2016;67(1):55–80. https://doi.org/10.1146/annurev-arplant-043014-114752

    Article  CAS  PubMed  Google Scholar 

  43. Yu JJ, Wang CB, Gong X. Degeneration of photosynthetic capacity in mixotrophic plants, Chimaphila japonica and Pyrola decorata (Ericaceae). Plant Divers. 2017;39(2):80–8. https://doi.org/10.1016/j.pld.2016.11.005

    Article  PubMed  PubMed Central  Google Scholar 

  44. Tadini L, Jeran N, Peracchio C, Masiero S, Colombo M, Pesaresi P. The plastid transcription machinery and its coordination with the expression of nuclear genome: plastid-encoded polymerase, nuclear-encoded polymerase and the genomes uncoupled 1-mediated retrograde communication. Phil Trans R Soc B. 2020;75(1801):20190399. https://doi.org/10.1098/rstb.2019.0399

    Article  CAS  Google Scholar 

  45. Pfannschmidt T, Blanvillain R, Merendino L, Courtois F, et al. Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle. J Exp Bot. 2015;66(2):6957–73. https://doi.org/10.1093/jxb/erv415

    Article  CAS  PubMed  Google Scholar 

  46. Li DM, Zhao CY, Liu XF. Complete chloroplast genome sequences of Kaempferia Galanga and Kaempferia Elegans: molecular structures and comparative analysis. Moleculars. 2019;24(3):474. https://doi.org/10.3390/molecules24030474

    Article  CAS  Google Scholar 

  47. Doyle JJ, Doyle JL. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bull. 1987;19:11–5.

    Google Scholar 

  48. Chen YX, Chen YS, Shi CM, Huang ZB, et al. SOAPnuke: a MapReduce acceleration-supported software for integrated quality control and preprocessing of high-throughput sequencing data. Oxf Open. 2018;7(1). https://doi.org/10.1093/gigascience/gix120

  49. Dierckxsens N, Mardulyn P, Amits G. Nucleic Acids Res. 2017;45(4). https://doi.org/10.1093/nar/gkw955. NOVOPlasty: de novo assembly of organelle genomes from whole genome data.

  50. Greiner S, Lehwark P, Bock R. Organellar Genome DRAW (OGDRAW) version 1.3.1: expanded toolkit for the graphical visualization of organellar genomes. Nucleic Acids Res. 2019;47:W59–W64. https://doi.org/10.1093/nar/gkz238

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30(4):772–80. https://doi.org/10.1093/molbev/mst010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Kumar S, Stecher G, Tamura K. Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870–4. https://doi.org/10.1093/molbev/msw054

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Darling A, Mau B, Blattner FR, Perna NT. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 2004;14(7):1394–403. https://doi.org/10.1101/gr.2289704

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. MISA—Microsatellite searching tool. Available online: http://pgrc.ipk-gatersleben.de/misa/ (accessed on 12 May 2016).

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This work was financially supported by the National Natural Science Foundation of China (32271590), and the Ten Thousand Talent Program of Zhejiang Province (2019R52043).

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Conceptualization, L.Z. and J.L.; methodology, Y.T., C.C., M.J., Z.S. and H.P.; software, L.C., Y.T. and C.C.; validation, C.C., L.Z. and J.L.; formal analysis, H.P.; investigation, H.P.; data curation, H.P.; writing—original draft preparation, H.P; writing—review and editing, J.L. All authors have read and agreed to the published version of the manuscript.

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Pan, H., Zagorchev, L., Chen, L. et al. Complete chloroplast genomes of five Cuscuta species and their evolutionary significance in the Cuscuta genus. BMC Genomics 24, 310 (2023). https://doi.org/10.1186/s12864-023-09427-w

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