Plastomes of the green algae Hydrodictyon reticulatum and Pediastrum duplex (Sphaeropleales, Chlorophyceae).

BACKGROUND: Comparative studies of chloroplast genomes (plastomes) across the Chlorophyceae are revealing dynamic patterns of size variation, gene content, and genome rearrangements. Phylogenomic analyses are improving resolution of relationships, and uncovering novel lineages as new plastomes continue to be characterized. To gain further insight into the evolution of the chlorophyte plastome and increase the number of representative plastomes for the Sphaeropleales, this study presents two fully sequenced plastomes from the green algal family Hydrodictyaceae (Sphaeropleales, Chlorophyceae), one from Hydrodictyon reticulatum and the other from Pediastrum duplex.
METHODS: Genomic DNA from Hydrodictyon reticulatum and Pediastrum duplex was subjected to Illumina paired-end sequencing and the complete plastomes were assembled for each. Plastome size and gene content were characterized and compared with other plastomes from the Sphaeropleales. Homology searches using BLASTX were used to characterize introns and open reading frames (orfs) ≥ 300 bp. A phylogenetic analysis of gene order across the Sphaeropleales was performed.
RESULTS: The plastome of Hydrodictyon reticulatum is 225,641 bp and Pediastrum duplex is 232,554 bp. The plastome structure and gene order of H. reticulatum and P. duplex are more similar to each other than to other members of the Sphaeropleales. Numerous unique open reading frames are found in both plastomes and the plastome of P. duplex contains putative viral protein genes, not found in other Sphaeropleales plastomes. Gene order analyses support the monophyly of the Hydrodictyaceae and their sister relationship to the Neochloridaceae. DISCUSSION: The complete plastomes of Hydrodictyon reticulatum and Pediastrum duplex, representing the largest of the Sphaeropleales sequenced thus far, once again highlight the variability in size, architecture, gene order and content across the Chlorophyceae. Novel intron insertion sites and unique orfs indicate recent, independent invasions into each plastome, a hypothesis testable with an expanded plastome investigation within the Hydrodictyaceae.

Introduction

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, http://www.geneious.com) 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, dogma.ccbb.utexas.edu/), BLAST (http://blast.ncbi.nlm.nih.gov/), tRNAscan-SE 2.0 (Lowe & Chan, 2016), RNAweasel (Lang, Laforest & Burger, 2007, http://megasun.bch.umontreal.ca/cgi-bin/RNAweasel/RNAweaselInterface.pl), and Geneious were used to annotate each plastome. OrganellarGenomeDRAW (Lohse et al., 2013, http://ogdraw.mpimp-golm.mpg.de/) 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 (http://www.geneorder.org/server.php) (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 (https://blast.ncbi.nlm.nih.gov/blast.cgi).

Results

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).

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.

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
	trnY-GUA
ATP synthase	atpA	atpB * in Pd	atpE	atpF
	atpH x2	atpI
Chlorophyll biosynthesis	chlB	chlL	chlN
Cytochrome	petA	petB	petD	petG
	petL
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
	rpoC2
Hypothetical proteins	ftsH	ycf1	ycf3	ycf4
	ycf12
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✜

Notes.

trans-spliced

intron-containing gene in both plastomes

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

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)

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)	193836-189361
Pediastrum duplex	orf300	Replication-associated protein (McMurdo Ice Shelf pond-associated circular DNA virus-8) Sequence ID: YP_009047144.1	142962-143864
Pediastrum duplex	orf432	Replication-associated protein (McMurdo Ice Shelf pond-associated circular DNA virus-8) Sequence ID: YP_009047144.1	141638-142936
Pediastrum duplex	orf1819 (similarity with orf1491 in Hydrodictyon)	Group II intron, maturase-specific domain (pfam08388)Rft protein (pfam04506)	199224-193765

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

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 endonucleaseorf747: 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)

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).

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.

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.

Discussion

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.

Conclusions

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.

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