Nannandrous, gynandrosporous; vegetative cells cylindrical; oogonium single, subglobose, poriferous, pore median to inframedian, oospore of the same form as oogonium, nearly or completely filling the oogonium, outer layer reticulate and dentate, teeth spreading form reticulations; suffultory cells slightly inflated or inflated; androsporangia single or up to 2 seriate, scattered; dwarf males on suffultory cells, stipes slightly curved, antheridia exteriors, 1 or 2 continuous; top and base of the filaments often slender; apical and basal cells not observed; vegetative cells generally 5–9 times as long as their width (Figture 1A–D).
Vegetative cells: 14–19 × 35–91 μm
Oogonium: 33–48 × 33–51 μm
Oospore with dentate teeth: 33–49 × 33–51 μm
Oospore without dentate teeth: 27–37 × 29–36 μm
Androsporangia: 13–15 × 12–14 μm
Antheridia: 7 × 11 μm.
Monoecious; oogonium usually single, obovoid-globose, operculate, division superior; oospore globose or subglobose, nearly or completely filling the oogonium, spore wall smooth; antheridium single, subepigynous; sperms 2, division horizontal; terminal cells apically obtuse; basal cells elongate; vegetative cells generally 3–4.5 times as long as their width (Figture 1E–I).
Vegetative cells: 10–14 × 24–36 μm
Oogonium: 33–38 × 30–41 μm
Oospore: 28–34 × 27–32 μm
Antheridium: 5–8 × 10–12 μm.
Dioecious, macrandrous; oogonium single, slightly inflated, rarely 2 continuous, obovoid to subovoid, with superior pore; oospore variable, ovoid-globose, subglobose, or globose, nearly or completely filling the oogonium, spore wall smooth; antheridium 2–7 in a series, often alternating with the vegetative cell; sperms 2, division horizontal; terminal cells apically apiculate; basal cells elongate; vegetative cells generally 1–3 times as long as their width (Figture 2A–E).
Female vegetative cells: 20–29 × 54–95 μm; male vegetative cells: 19–24 × 42–62 μm
Oogonium: 45–53 × 48–69 μm
Oospore: 44–49 × 42–50 μm
Antheridium: 14–17 × 9–13 μm.
The characteristics of all the five Oedogonium species were examined, and strains FACHB-3309, FACHB-3310, and FACHB-3312 were identified as Oe. dentireticulatum, Oe. crispum, and Oe. Capilliforme, respectively. With regard to strains FACHB-3311 (Figture 2F) and FACHB-3313 (Figture 2G), the entire sexual features could not be observed; however, the filaments of both of these strains were unbranched, indicating that they obviously belonged to the genus Oedogonium. In particular, strain FACHB-3313 exhibited unbranched rhizoids that resembled those of Oedocladium.
General characteristics and comparison of Oedogoniales cp genomes
Table 1 summarizes the cp genomes characteristics of the five newly included Oedogonium species, three reported Oedocladium taxa and one Oedogonium species. The complete cp genomes of the nine species of Oedogoniales ranged from 146,367 bp (Oe. crispum) to 204,438 bp (O. carolinianum) in length. All of the five Oedogonium cp genomes displayed typical circular mapping with a large single copy (LSC) region (76,475–98,887 bp), a small single copy (SSC) region (43,305–58,055 bp), and two inverted repeats (IR) regions (12,808–35,492 bp) (Supplementary Figs S1–S5). The overall AT content in each cp genome was comparable and showed a little difference among the species, ranging from 69.98% (strain FACHB-3311) to 72.66% (O. prescottii); besides, difference was noted in coding proportion, which varied from 51.4% (O. carolinianum) to 69.5% (O. prescottii). The cp genomes of six Oedogonium species were moderately compact relative to those of the Oedocladium species. The number of genes located in the plus or minus strand showed some differences. All the cp genomes contained 68 protein-coding genes and three rRNA genes, except for the cp genome of Oe. cardiacum, which had two additional genes (dpoB and int) located in the IR region. With respect to tRNA, the cp genomes showed slight difference as follows: Oe. cardiacum exhibited two additional trnR(ccu) located in the IR regions and Oe. dentireticulatum (strain FACHB-3309) presented an additional trnR(ccu) in the LSC region; Oe. sp. (strain FACHB-3313) contained two additional trnR(ccg) in the IR regions and O. carolinianum had an additional trnR(ccg) in the LSC region; and O. carolinianum had an additional trnS(gga) in the LSC region. Sequence repeats of more than 30 bp were less frequent (3.9%–4.9%) in the cp genomes of the five Oedogonium species when compared with those in the two O. carolinianum cp genomes, but were more frequent, when compared with those in the Oe. cardiacum cp genome.
Introns content and insertion sites
The introns content and insertion sites of the nine Oedogoniales cp genomes are listed in Table 1 and Supplementary Tables S1 and S2. The nine cp genomes significantly differed with respect to the introns content. Oe. sp. (strain FACHB-3311) had the maximum introns content with 26 group I introns and 11 group II introns. When compared with the other six Oedogonium cp genomes, multiple intron losses were observed in the cp genome of Oe. crispum (strain FACHB-3310), with four group I introns in trnL(uaa), psbC, atpA, and psbD, respectively, and four group II introns in psbI, petD, psaC, and psaB, respectively. Besides, similar to O. prescottii, Oe. crispum also exhibited introns losses in psbA. Oe. sp. (strain FACHB-3311) presented two additional group II introns in chlB and chlL, introns were first observed in them. All the nine cp genomes included group I introns in trnL(uaa), which is common across all algal lineages and is considered to originate from the common ancestor of cp . The nine cp genomes showed a certain variation in insertion sites. The common group I introns in trnL(uaa) and group II introns in petB, psaC, and psbI (only strain FACHB-3311 lost the intron in psbI) showed the same insertion sites. With regard to the other genes with introns, the insertion sites in different species showed similarities and variations. For instance, in psbA, the number of introns (introns in psbA are all group I) differed among the species, whereas the insertion sites of the first intron in Oe. dentireticulatum (strain FACHB-3309), Oe. sp. (strain FACHB-3311), and Oe. sp. (strain FACHB-3313) were identical. The two O. carolinianum were the same; however, the insertion site of the first intron in Oe. capilliforme was similar to that of the fourth intron in Oe. dentireticulatum and sp. (strain FACHB-3311).
Synteny analysis and average nucleotide identity analysis
ProgressiveMauve was used to analyze the Oedogoniales cp genomes synteny, with Oe. cardiacum used as a reference to compare gene order among the cp genomes (Fig. 3). More than 19 locally collinear blocks (LCBs) were identified in the cp genomes of the nine species of Oedogoniales, including six taxa from Oedogonium and three taxa from Oedocladium. The nine cp genomes showed high degree of syntenic conservation on the whole, with Oe. capilliforme exhibiting high similarity to Oe. cardiacum, and Oe. dentireticulatum resembling Oe. sp. (strain FACHB-3311). However, some rearrangements and inversions were still observed among certain short LCBs mainly owing to the inversion or loss of introns. The genes order and number were almost identical except for that an inversion between trnE(uuc) and petL with a length of less than 3 kb and including the genes petD and trnR(ucg) was detected in O. carolinianum (MT364369) and O. carolinianum (NC_031510).
The average nucleotide identity (ANI) of the nine species of Oedogoniales was calculated using FastANI (Supplementary Fig. S6). Oe. crispum showed high ANI with Oe. dentireticulatum and Oe. sp. (strain FACHB-3311) (90.64% and 90.56%, respectively), Oe. dentireticulatum was similar to Oe. sp. (strain FACHB-3311) with 92.57% ANI, and Oe. capilliforme was similar to Oe. cardiacum with 97.03% ANI.
IR expansion and contraction
The IR boundary regions of the nine species of Oedogoniales were compared as illustrated in Fig. 4. Oe. cardiacum and Oe. capilliforme (strain FACHB-3312) showed larger IRs reaching 35,000 bp, whereas Oe. crispum and O. prescottii exhibited smaller IRs reaching 13,284 and 12,808 bp, respectively. The IRs of all the nine cp genomes contained the same four protein-coding genes, three tRNAs, and three rRNAs. However, in Oe. capilliforme and Oe. sp. (strain FACHB-3313), an additional trnR(ccg) was observed between psbA and rbcL; Oe. cardiacum included two additional protein-coding genes (int and dpoB) and one tRNA (trnR(ccu)); and the IRa of four cp genomes included parts of the 5’-end of ccsA (390 bp in Oe. cardiacum, Oe. capilliforme, and O. prescottii and 383 bp in Oe. crispum).
The nine Oedogoniales cp genomes showed high conservation at four regional boundaries, with little variation. The LSC/IRb junctions (JLBs) in the cp genomes of Oe. cardiacum, Oe. capilliforme, O. prescottii, and Oe. sp. (strain FACHB-3311) were located in trnR(ucu); as a result, 2 bp of the 3’-end of this gene were a part of the IR region. In Oe. sp. (strain FACHB-3311), the IR region contained 6 bp of the 3’-end of trnR(ucu), and in the other five cp genomes, the LSC/IRb boundaries occurred between trnR(ccu) and psbA. The IRb/SSC boundaries in all the nine cp genomes occurred between trnL(caa) and psaA, and the SSC/IRa junctions were located in rpoA. The IRa/LSC junctions (JSAs) of the two O. carolinianum cp genomes occurred between psbA and ccsA, while those of the other seven genomes were located in ccsA, with 390 bp of the 5’-end of this gene being a part of the IR region in Oe. cardiacum, Oe. capilliforme, and O. prescottii, and 383, 388, 608, and 389 bp of the 5’-end of this gene being a part of the IR region in Oe. crispum, strain FACHB-3313, strain FACHB-3311, and Oe. dentireticulatum, respectively.
Phylogenetic analysis and adaptive evolution analysis
Phylogenetic assays based on 54 cp protein-coding genes were conducted using maximum likelihood (ML) and Bayesian analyses with amino acid and nucleotide datasets, which generated two kinds of phylogenetic trees showing the same results (Figs 5-6). Phylogenetic trees based on amino acid and nucleotide datasets both indicated that the nine species of Oedogoniales clustered into three clades Oe. sp. (MW250873) formed a separate clade with absolute high support value, the two O. carolinianum clustered together and formed another clade, and the other six Oedogoniales formed the third clade. With regard to the third clade, the two datasets showed a little difference in the location of O. prescottii. Based on nucleotide dataset, O. prescottii clustered with Oe. cardiacum and Oe. capilliforme, whereas according to the amino acid dataset, O. prescottii clustered with Oe. dentireticulatum, Oedogonium sp. (MW250875), and Oe. crispum. These results indicated that both Oedocladium and Oedogonium are polyphyletic, which is in accordance with that reported in a previous study .
Positive selection analysis was performed based on branch-site model, and the null and alternative models were compared. The null model considered that the foreground branch only has dN/dS = 1, and the alternative model assumed that sites on the foreground branch have dN/dS > 1 (positive selection). When the two Oedocladium species and MW250875 were labelled as the foreground branch, the FDR-adjusted P value of psbA was less than 0.05. When the foreground branch only included the three Oedocladium spp., the FDR-adjusted P value of psbA was less than 0.05. Based on Bayes empirical Bayes (BEB) assay, the two methods were noted to indicate that psbA may possibly contain sites under positive selection, with 291SER showing posterior probability higher than 99%. However, owing to the lack of related functional sites information on closely related species such as Chlamydomonas reinhardtii and Stigeoclonium helveticum in UniProt, the positively selected sites of psbA require further investigation.