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Organellar genomic studies of the green algae are revealing extensive variability in genome size, architecture and gene order, and phylogenomic analyses are resolving relationships and discovering novel lineages (Fučíková et al., 2014; Lemieux et al., 2015; Turmel, Otis & Lemieux, 2015; Fučíková, Lewis & Lewis, 2016a; Fučíková, Lewis & Lewis, 2016b; Leliaert et al., 2016; Lemieux, Otis & Turmel, 2016; Turmel et al., 2016). Of the five orders comprising the Chlorophyceae, the Sphaeropleales have garnered recent attention, with genomic studies characterizing dynamic evolutionary patterns in both chloroplast and mitochondrial genome architecture and gene content (Farwagi, Fučíková & McManus, 2015; Lemieux et al., 2015; Fučíková, Lewis & Lewis, 2016a; Fučíková, Lewis & Lewis, 2016b). Of these studies, only one has focused on genome evolution at the family level, analyzing the mitochondrial genomes of the Hydrodictyaceae (Farwagi, Fučíková & McManus, 2015).

The freshwater green algal family Hydrodictyaceae, a member of the Sphaeropleales and sister to the Neochloridaceae (Fučíková et al., 2014), includes the well-known genera Hydrodictyon Roth 1797 and Pediastrum Meyen 1829. The Hydrodictyaceae has undergone taxonomic revisions based on molecular phylogenetic studies of individual nuclear and chloroplast genes (Buchheim et al., 2005; McManus & Lewis, 2011); however, several relationships remain unresolved, particularly the paraphyly of Pediastrum duplex Meyen 1829 and its relationship to Hydrodictyon (McManus & Lewis, 2011). Farwagi, Fučíková & McManus (2015) presented the first complete mitochondrial genomes of four representatives from the Hydrodictyaceae. The results revealed size differences and gene rearrangements that carry phylogenetic signal, indicating that whole genome-level studies of the Hydrodictyaceae may be useful in resolving ongoing systematic questions.

To gain further insight into the evolution of the chlorophyte plastome and increase the number of representative plastomes for the Sphaeropleales, we fully sequenced the plastomes of a strain of Hydrodictyon reticulatum (L.) Bory 1824 and Pediastrum duplex. The complete plastomes of these Hydrodictyaceae strains, representing the largest of the Sphaeropleales sequenced thus far, once again highlight the variability in size, architecture, gene order and content across this order.

Materials and Methods

Hydrodictyon reticulatum was collected from the freshwater Geyser Brook, Saratoga Co., NY, USA (43.058117, −73.807914) on 23 July 2014 and DNA was extracted directly from the field collection. A strain of Pediastrum duplex (EL0201CT/HAM0001) was isolated from the freshwater Eagleville Pond, Tolland Co., CT, USA (41.7848239, -72.2805262) in June 2002 and maintained in culture at 20 °C under a 16:8 h light:dark (L:D) cycle on agar slants. The agar slants consisted of a 50:50 mixture of Bold’s basal medium (BBM) (Bold, 1949; Bischoff & Bold, 1963) and soil water prepared following McManus & Lewis (2011) in 3% agar. Voucher material for each strain is deposited in The New York Botanical Garden William and Lynda Steere Herbarium (NY) under barcodes 02334980 and 02334981, respectively. Duplicate specimens of each are deposited in the George Safford Torrey Herbarium at the University of Connecticut (CONN) and in the personal collection of HAM.

Total genomic DNA was extracted from living cells following a CTAB extraction protocol (Doyle & Doyle, 1987). DNA was sent to the Woodbury Genome Center at Cold Spring Harbor Laboratories for TruSeq library preparation followed by sequencing on Illumina HiSeq2500 to produce 2 × 101 bp paired-end reads. Geneious v.9.1.5 (Biomatters, was used to trim, pair, and de novo assemble the reads. Several contigs containing plastome fragments were recovered for each strain after the initial de novo assembly. Geneious was then used to map reads to the plastome fragments in a series of reference assemblies until longer fragments were obtained that could be joined into a single sequence. DOGMA (Wyman, Jansen & Boore, 2004,, BLAST (, tRNAscan-SE 2.0 (Lowe & Chan, 2016), RNAweasel (Lang, Laforest & Burger, 2007,, and Geneious were used to annotate each plastome. OrganellarGenomeDRAW (Lohse et al., 2013, was used to draw plastome maps, and synteny maps were generated using the Mauve plugin (Darling et al., 2004) with default settings in Geneious. Gene order analyses were performed using MLGO: Maximum Likelihood Gene Order Analysis web server ( (Lin, Hu & Moret, 2013). BLASTX homology searches were used to characterize introns and open reading frames (orfs) ≥300 bp with an E-value threshold <1e − 06 (


DNA sequence data collection resulted in 12.5 million paired-end reads for Hydrodictyon reticulatum and 9.9 million paired-end reads for Pediastrum duplex. The plastome for each strain was assembled with no gaps, and the average coverage was 195X (225,641 bp) for H. reticulatum (Fig. 1; GenBank accession KY114065) and 134X (232,554 bp) for P. duplex (Fig. 2; GenBank accession KY114064). Each plastome comprised two copies of an inverted repeat (IR) separated by two single-copy (SC) regions. Hydrodictyon reticulatum contained 102,823 bp and 86,226 bp SC regions and P. duplex 98,587 bp and 94,307 bp SC regions. Inferred protein translations indicated the universal genetic code was used in both plastomes, and RNA editing did not appear to be necessary. All protein-coding regions used the AUG start codon, with the exception of psbC that used GUG. The coding regions for each plastome included genes for 3 rRNAs, 25 unique tRNAs and 68 functionally identifiable protein genes, including ycf1, ycf3, ycf4 and ycf12 (Table 1). Fifty-nine putative open reading frames (orfs) ≥300 bp of unknown function were identified in the plastome of H. reticulatum and 32 were identified in the plastome of P. duplex (Table 1).

Gene map of Hydrodictyon reticulatum plastome (KY114065).

Figure 1: Gene map of Hydrodictyon reticulatum plastome (KY114065).

The inverted repeats (IRA and IRB) which separate the genome into two single copy regions are indicated on the inner circle along with the nucleotide content (G/C dark grey, A/T light grey). Genes shown on the outside of the outer circle are transcribed clockwise and those on the inside counter clockwise. Gene boxes are color coded by functional group as shown in the key.
Gene map of Pediastrum duplex plastome (KY114064).

Figure 2: Gene map of Pediastrum duplex plastome (KY114064).

The inverted repeats (IRA and IRB) which separate the genome into two single copy regions are indicated on the inner circle along with the nucleotide content (G/C dark grey, A/T light grey). Genes shown on the outside of the outer circle are transcribed clockwise and those on the inside counter clockwise. Gene boxes are color coded by functional group as shown in the key.
Table 1:
List of plastid-encoded genes annotated for Hydrodictyon reticulatum and Pediastrum duplex.
Open reading frames (orfs) ≥300 bp are indicated separately for each plastome.
Gene class Genes
Ribosomal RNAs rrf x2IR rrl x2IR * in Pd rrs x2IR
Transfer RNAs trnA-UGC x2IR trnC-GCA trnD-GUC trnE-UUC x2
trnF-GAA trnG-UCC trnH-GUG trnI-CAU
trnI-GAU x2IR trnK-UUU trnL-UAA * trnL-UAG
trnMe-CAU trnMf-CAU trnN-GUU trnP-UGG
trnQ-UUG trnR-ACG trnR-UCU trnS-GCU x2IR
trnS-UGA trnT-UGU trnV-UAC trnW-CCA
ATP synthase atpA atpB * in Pd atpE atpF
atpH x2 atpI
Chlorophyll biosynthesis chlB chlL chlN
Cytochrome petA petB petD petG
Photosystem I psaA ts psaB * in Pd psaC psaJ
Photosystem II psbA * in Hr psbB * in Hr psbC psbD
psbE psbF psbH psbI
psbJ psbK psbL psbM
psbN psbT psbZ
Ribosomal proteins rpl2 rpl5 rpl14 rpl16
rpl20 rpl23 rpl32 rpl36
rps2 rps3 rps4 rps7
rps8 rps9 rps11 rps12
rps14 rps18 rps19
RNA polymerase rpoA rpoBa rpoBb rpoC1
Hypothetical proteins ftsH ycf1 ycf3 ycf4
Miscellaneous proteins ccsA cemA clpP infA
rbcL tufA
orfs (H. reticulatum) orf102a orf102b orf104 orf106
orf106 orf110 orf112 orf116
orf117 orf119a orf119b orf120a
orf120b orf121 orf122 orf123
orf126 orf127 orf128 orf133
orf139a orf139b x2IR orf140 orf149a
orf149b orf150 x2IR orf151a orf151b
orf153 orf154 orf161 orf162
orf163 orf166 orf168 orf170
orf174 orf176 orf182 orf187
orf192 orf194 orf197 orf200
orf202 orf208 orf219 orf228
orf200 orf234 x2IR orf244 orf311 x2IR
orf327 x2IR orf374 orf383 x2IR orf483
orf598 orf602 orf1491
orfs (P. duplex) orf99 orf103 orf106 orf107a
orf107b x2IR orf113a orf113b orf118
orf119 orf120 orf121 x2IR orf124
orf127 orf129 x2IR orf132 orf143
orf145 orf151 orf153 x2IR orf164
orf171 x2IR orf184 x2IR orf186 x2IR orf201 x2IR
orf270 orf275 x2IR orf300 orf327
orf432 orf747 orf854 orf1819
DOI: 10.7717/peerj.3325/table-1





intron-containing gene in both plastomes

* in Hr

intron(s) in H. reticulatum but not P. duplex

* in Pd

intron(s) in P. duplex but not H. reticulatum


duplicated gene not in inverted repeat (IR)


duplicated gene in IR

shares 58.6% similarity between H. reticulatum and P. duplex (see Table 4)

The coding region made up 59.6% of the Hydrodictyon reticulatum plastome and 53.3% of the Pediastrum duplex plastome (Table 2). Gene content of known genes was similar to that of other Sphaeroplealean plastomes, but the trnG (gcc) gene was not detected in either plastome, similar to Neochloris aquatica (Fučíková, Lewis & Lewis, 2016a). The IR in H. reticulatum was 18,296 bp and contained atpH, rrf, rrl, rrs, trnA (ugc), trnI (gau), and trnS (gcu). The IR in P. duplex was 19,830 bp and included the same genes as H. reticulatum, plus an additional four introns in rrl not found in H. reticulatum. Like other members of the Sphaeropleales, psaA was trans-spliced in both plastomes with exon 1 in the smaller SC and exons 2 and 3 in the larger SC.

Table 2:
Summary of Hydrodictyon reticulatum, Pediastrum duplex and other Sphaeropleales plastomes.
%Coding includes all CDS (including orfs), tRNAs and rRNAs (both IRs); %GC content includes both IRs; Genes includes CDS (including orfs), tRNAs and rRNAs (both IRs).
Species Strain GenBank Size (bp) %GC %Coding Non-Coding (bp) Genes Introns IR (bp)
Acutodesmus obliquus UTEX 393 DQ396875 161,452 26.9 56.0 55,454 106 10 12,022
Ankyra judai SAG 17.84 KT199255 157,224 28.3 57.0 64,708 109 2 8,247
Bracteacoccus aerius UTEX 1250 KT199254 165,732 31.7 54.9 74,226 103 2 7,271
Bracteacoccus minor UTEX B66 KT199253 192,761 31.9 48.4 96,439 104 3 9,577
Chlorotetraedron incus SAG 43.81 KT199252 193,197 27.1 46.7 94,081 106 10 13,490
Chromochloris zofingiensis UTEX 56 KT199251 188,937 30.9 47.2 97,693 106 2 6,375
Hydrodictyon reticulatum HAM0289 KY114065 225,641 32.1 59.6 91,268 111 5 18,296
Kirchneriella aperta SAG 2004 KT199250 207,516 34.1 42.3 76,270 106 27 35,503
Mychonastes homosphaera CAUP H 6502 KT199249 102,718 39.8 80.1 20,264 105 1 6,472
Neochloris aquatica UTEX 138 KT199248 166,767 30.3 50.9 54,026 102 32 18,217
Pediastrum duplex EL0201CT KY114064 232,554 32.6 53.3 108,632 125 8 19,830
Pseudomuriella schumacherensis SAG 2137 KT199256 220,357 31.2 41.6 117,502 109 8 22,004
DOI: 10.7717/peerj.3325/table-2

Two introns were present in atpB of Pediastrum duplex. Intron 1 contained two open reading frames (orf), one with a putative reverse transcriptase, intron maturase and HNH endonuclease (orf145) and the other with a reverse transcriptase with Group II origin (orf747) (Table 3). Intron 2 contained a reverse transcriptase of Group II intron origin (orf854). No introns were found in atpB of Hydrodictyon reticulatum (Table 3). Pediastrum duplex contained one intron in psaB that contained a putative GIY-YIG homing endonuclease, and both plastomes harbored an intron that lacked an orf in trnL (uaa). The psbB gene in H. reticulatum contained an intron housing a Group II intron reverse transcriptase (orf598). Three introns were present in psbA of H. reticulatum. The first contained a putative HNH homing endonuclease (orf228). The second and third intron each harbored a reverse transcriptase with Group II intron origin (orf483 and orf602, respectively) (Table 3). Four introns were identified in rrl of P. duplex and not found in H. reticulatum. Intron 1 contained two putative site-specific DNA endonucleases (orf184, orf186), introns 2 and 4 each contained a LAGLIDADG superfamily homing endonuclease (orf171 and orf275, respectively); intron 3 did not contain a detectable orf (Table 3).

Table 3:
List of introns and contained conserved domains determined with BLASTX searches (E-value threshold < 1e − 06).
Hydrodictyon reticulatum Pediastrum duplex
atpB intron 1 orf145: putative reverse transcriptase, intron maturase and HNH endonuclease
orf747: Reverse transcriptases (RTs) with group II intron origin (cd01651)
atpB intron 2 orf854: Reverse transcriptases (RTs) with group II intron origin (cd01651); Type II intron maturase (pfam01348)
psaB no orf; putative GIY-YIG homing endonuclease
psbB orf598: Reverse transcriptases (RTs) with group II intron origin (cd01651)
psbA intron 1 orf228: putative HNH homing endonuclease
psbA intron 2 orf483: Reverse transcriptases (RTs) with group II intron origin (cd01651)
psbA intron 3 orf602: Reverse transcriptases (RTs) with group II intron origin (cd01651)
trnL (uaa) no orf no orf
rrl intron 1 orf184, orf186: putative site-specific DNA endonuclease
rrl intron 2 orf171: LAGLIDADG DNA endonuclease (pfam00961)
rrl intron 3 no orf
rrl intron 4 orf275: LAGLIDADG DNA endonuclease family (pfam03161)
DOI: 10.7717/peerj.3325/table-3
Synteny map of Hydrodictyaceae with Neochloridaceae and Acutodesmus obliquus.

Figure 3: Synteny map of Hydrodictyaceae with Neochloridaceae and Acutodesmus obliquus.

Blocks represent regions that align to a corresponding region in another genome and colored bars within each block indicate level of sequence similarity. Lines connecting blocks indicate putative homology.
Table 4:
List of freestanding open reading frames ≥300 bp that harbor a conserved domain determined by BLASTX searches (E-value threshold < 1e − 06).
Taxon orf Conserved domain Position
Hydrodictyon reticulatum orf374 TolA protein (TIGR02794) 97334-96210
Hydrodictyon reticulatum orf244 Putative reverse transcriptase, intron maturase and HNH endonuclease 152114-151380
Hydrodictyon reticulatum orf1491 (similarity with orf1819 in Pediastrum) Group II intron maturase-specific domain (pfam08388)
putative reverse transcriptase and intron maturase (cl02808)
Pediastrum duplex orf300 Replication-associated protein (McMurdo Ice Shelf pond-associated circular DNA virus-8)
Sequence ID: YP_009047144.1
Pediastrum duplex orf432 Replication-associated protein (McMurdo Ice Shelf pond-associated circular DNA virus-8)
Sequence ID: YP_009047144.1
Pediastrum duplex orf1819 (similarity with orf1491 in Hydrodictyon) Group II intron, maturase-specific domain (pfam08388)
Rft protein (pfam04506)
DOI: 10.7717/peerj.3325/table-4

Multiple orfs greater than 300 bp were identified outside of intron regions, some of which contained HNH homing endonucleases or intron maturase proteins similar to those found in other green algae (Table 4). A reciprocal 50% protein similarity comparison of all orfs showed that none were shared between H. reticulatum and P. duplex, with one exception. The largest in both plastomes (orf1491 in H. reticulatum and orf1819 in P. duplex) shared 58.6% pairwise identity. Additionally, a 50% protein similarity search for each set of orfs with other complete sphaeroplealean plastomes resulted in no matches. In Pediastrum duplex, orf300 and orf432 each showed similarity with a virus replication-associated protein isolated from a freshwater pond on McMurdo ice shelf in Antarctica (circular DNA virus-8, YP_009047144, sequence similarity 1 × 10−22; Zawar-Reza et al., 2014).

Both plastomes shared identical gene order, while there were extensive rearrangements when compared with the closely related Acutodesmus obliquus, Chlorotetraedron incus and Neochloris aquatica (Fig. 3). The phylogenetic analysis of gene order recovered Hydrodictyon reticulatum and Pediastrum duplex as sister lineages with bootstrap support of 100, these in turn were found sister to a clade including C. incus plus N. aquatica, also with bootstrap support of 100. Acutodesmus obliquus was recovered sister to the above-mentioned taxa with bootstrap support of 57 (Fig. 4).

Maximum likelihood tree using plastome gene order within Sphaeropleales.

Figure 4: Maximum likelihood tree using plastome gene order within Sphaeropleales.

ML bootstrap support values >50, based on 1,000 replicates, are indicated above each node.


The addition of the two new Hydrodictyaceae plastomes permits a more rigorous analysis of plastomes across the Sphaeropleales, and highlights the importance of increased taxon sampling to aid in understanding plastome evolutionary trends. The plastomes of Hydrodictyon reticulatum and Pediastrum duplex were considerably larger in size compared with sister sphaeroplealean lineages Neochloris aquatica, Chlorotetraedron incus and Acutodesmus obliquus, and represent the largest plastomes thus far reported from the Sphaeropleales (Table 2). The size differences can be attributed to several factors, including relatively large intergenic regions (Table 2) and the infiltration of each plastome by numerous novel orfs (Table 3).

The relatively larger IR in the Hydrodictyaceae is consistent with the dynamic evolution of IRs discussed in Fučíková, Lewis & Lewis (2016b), and are larger than the ∼14 kb IR regions found in most fully-sequenced Sphaeropleales plastomes (with the exception of Kirchneriella aperta and Pseudomuriella schumacherensis), while similar to the IR found in Neochloris aquatica (∼18 kb). The IRs in Hydrodictyon reticulatum and Pediastrum duplex differ by 1,534 bp, and this difference is mainly due to the presence of four rrl introns in P. duplex. Presence and number of rrl introns across the Sphaeropleales does not appear to follow a clear phylogenetic pattern (see Fig. 1 of Fučíková, Lewis & Lewis, 2016a). This holds true for Hydrodictyaceae as well, but dense sampling within the family may uncover local phylogenetic patterns.

Intron number and distribution vary across the Sphaeropleales as well as within the Hydrodictyaceae. Of the five introns identified in Hydrodictyon reticulatum and eight introns in Pediastrum duplex, only the trnL (uaa) intron is shared by both. Six of the remaining 11 introns, one each in atpB and psaB, and two each in psbA and rrl, share identical insertion sites with other members of the order, suggesting possible ancestral origin of these introns. The last five introns have unique insertion sites in either H. reticulatum with two in psbA and one in psbB, or P. duplex with one in atpB and two in rrl. These introns with unique insertion sites could represent recent independent invasions into each plastome, a hypothesis testable with an expanded plastome investigation within the Hydrodictyaceae. The presence of a trnL (uaa) intron at base position 34 in both H. reticulatum and P. duplex is similar to other Sphaeroplealeaen plastomes, with the exception of Ankyra judayi, Mychonastes homosphaera, Mychonastes jurisii, and Ourococcus multisporus (Fučíková, Lewis & Lewis, 2016a). Based on available data, the phylogenetic distribution of this intron indicates that it is of ancestral origin and independently lost at least three times across the Sphaeropleales.

Many of the orfs 300 bp in size or larger and not located within an intron were identified as putative homing endonucleases and reverse transcriptases similar to those found in Group I and Group II introns (Table 4). The presence of these freestanding intron-like domains may indicate the translocation of in situ genic introns, or the invasion of intergenic spacer regions by novel elements (Turmel, Otis & Lemieux, 2015). Only two of the orfs (orf1819 in Hydrodictyon reticulatum and orf1491 in Pediastrum duplex) were similar to each other and not found in other sphaeroplealean plastomes, suggesting a common origin in the Hydrodictyaceae. The conserved maturase domain found in both suggests a functional importance in each plastome. The remaining orfs, 58 in H. reticulatum and 31 in P. duplex, were unique to each plastome. Because of their sister relationship in our study, we would expect to find homologous orfs if they were present in the common ancestor. Given the lack of shared orfs, it seems more likely that each lineage was independently invaded and that Hydrodictyaceae may be particularly susceptible to plastid viral infiltration. There is evidence that suggests chloroplasts are common targets of viruses (Li et al., 2016) and viral proteins have been reported in green algal plastomes of the Oedogoniales (Brouard et al., 2008), Trebouxiophyceae (Turmel, Otis & Lemieux, 2015), prasinophytes (Lemieux, Otis & Turmel, 2014; Turmel et al., 2009), and Zygnematophyceae (Lemieux, Otis & Turmel, 2016). orf300 and orf432 in P. duplex are the first report of genes putatively coding viral proteins in a plastome of the Sphaeropleales. Further analyses of sphaeroplealean plastomes are necessary to determine additional occurrences, functionality and origin of these novel orfs.

Four mitochondrial genomes of Hydrodictyaceae showed structural variability similar to that seen across the order (Farwagi, Fučíková & McManus, 2015). Thus far the structure of the plastomes is conserved between Hydrodictyon reticulatum and Pediastrum duplex, though additional plastomes within the family are anticipated to shed light on intrafamilial plastome evolution. The gene-order phylogenetic analysis presented here resulted in several well-supported relationships (Fig. 4) also recovered in individual gene and phylogenomic studies (Fučíková, Lewis & Lewis, 2016a; Fučíková, Lewis & Lewis, 2016b), indicating evolutionary relationships can be recovered using genome structure for this group. Incorporating additional Hydrodictyaceae (i.e., Pseudopediastrum and Stauridium) will determine if phylogenetic signal is reflected in plastome structure within the family.


The plastome data reported here for two representatives from the Hydrodictyaceae, Hydrodictyon reticulatum and Pediastrum duplex, provide further insights into the evolution of plastomes in the Sphaeropleales and highlight plastome variability across the order. These plastomes represent the largest thus far sequenced from the Sphaeropleales, with the increased size being attributable to not only expansion of the IR and non-coding regions but also to infiltration of numerous novel open reading frames, many identified as putative homing endonucleases and reverse transcriptases, in both plastomes. Though both plastomes have acquired many orfs, the lack of similarity between these suggests independent acquisition in each lineage and further suggests a potential susceptibility of the hydrodictyaceaen plastome to invasion by novel elements. Phylogenetic analysis using plastome gene order in the Sphaeropleales is consistent with currently accepted phylogenetic schemes and provides an additional source of data for tree reconstruction across the order. More plastomes will need to be sequenced for the Hydrodictyaceae in order to test whether orf infiltration is common across the family or restricted to the Hydrodictyon/Pediastrum assemblage.

Supplemental Information

Sequence data for Hydrodictyon reticulatum plastome (KY114065)

DOI: 10.7717/peerj.3325/supp-1

Sequence data for Pediastrum duplex plastome (KY114064)

DOI: 10.7717/peerj.3325/supp-2

Sphaeropleales plastome gene order dataset used for Fig. 4

DOI: 10.7717/peerj.3325/supp-3