Complete Plastid Genome Sequencing of Trochodendraceae Reveals a Significant Expansion of the Inverted Repeat and Suggests a Paleogene Divergence between the Two Extant Species

Abstract

The early-diverging eudicot order Trochodendrales contains only two monospecific genera, Tetracentron and Trochodendron. Although an extensive fossil record indicates that the clade is perhaps 100 million years old and was widespread throughout the Northern Hemisphere during the Paleogene and Neogene, the two extant genera are both narrowly distributed in eastern Asia. Recent phylogenetic analyses strongly support a clade of Trochodendrales, Buxales, and Gunneridae (core eudicots), but complete plastome analyses do not resolve the relationships among these groups with strong support. However, plastid phylogenomic analyses have not included data for Tetracentron. To better resolve basal eudicot relationships and to clarify when the two extant genera of Trochodendrales diverged, we sequenced the complete plastid genome of Tetracentron sinense using Illumina technology. The Tetracentron and Trochodendron plastomes possess the typical gene content and arrangement that characterize most angiosperm plastid genomes, but both genomes have the same unusual ∼4 kb expansion of the inverted repeat region to include five genes (rpl22, rps3, rpl16, rpl14, and rps8) that are normally found in the large single-copy region. Maximum likelihood analyses of an 83-gene, 88 taxon angiosperm data set yield an identical tree topology as previous plastid-based trees, and moderately support the sister relationship between Buxaceae and Gunneridae. Molecular dating analyses suggest that Tetracentron and Trochodendron diverged between 44-30 million years ago, which is congruent with the fossil record of Trochodendrales and with previous estimates of the divergence time of these two taxa. We also characterize 154 simple sequence repeat loci from the Tetracentron sinense and Trochodendron aralioides plastomes that will be useful in future studies of population genetic structure for these relict species, both of which are of conservation concern.

Introduction

The eudicot order Trochodendrales [1] contains only two extant genera, both of which are monotypic: Trochodendron Sieb. & Zucc. and Tetracentron Oliver. Historically, these two genera have been treated either as the separate families Trochodendraceae and Tetracentraceae, or as the combined family Trochodendraceae [1]–[7]. The Trochodendraceae sensu APG III [1] appear to have been widespread in the Northern Hemisphere during the Paleogene and Neogene [7]–[15]. However, the two extant species of the family have small geographic ranges and are restricted to eastern Asia [16]. Trochodendron aralioides Sieb. & Zucc. is a large, evergreen shrub or small tree native to the mountains of Japan to South Korea and Taiwan, and the Ryukyu Islands [2], [17], whereas Tetracentron sinense Oliver is a deciduous tree occurring in southwestern and central China and the eastern Himalayan regions. Both species are characterized by apetalous flowers arranged in cymose inflorescences and by loculicidal capsules that dehisce to release winged seeds [2], [5], [7], [18]. Although earlier researchers reported that wood of Trochodendrales wood lacked vessels and thus suggested that Trochodendrales were among the earliest-diverging angiosperms, recent research has documented the presence of vessels in the wood of both genera [2], [7], [19].

Molecular phylogenetic studies, including analyses of complete plastid genome sequences, have routinely recovered Trochodendrales as an early-diverging member of the clade Eudicotyledoneae (sensu [20]; all italicized clade names follow this system), specifically as part of a strongly supported clade with Buxales and Gunneridae, or core eudicots [21]–[27]. However, the relationships among Trochodendrales, Buxales, and Gunneridae have often been only weakly supported. In the 17-gene analysis of Soltis et al. [28], which included data from all three plant genomes, Trochodendrales and Buxales were subsequent sisters to Gunneridae, with 100% and 98% BS support, respectively. However, other studies have found Buxales to be sister to Gunneridae with only weak support [24], [26], [29]–[30], whereas in other analyses Trochodendrales have appeared as sister to Gunneridae [27], [31]–[32].

Complete plastid genome sequences have been used increasingly over the past decade to resolve deep-level phylogenetic relationships that have been unclear based on only a few genes. For example, recent plastid phylogenomic studies have helped to resolve key relationships among the earliest-diverging Mesangiospermae [33] as well as early-diverging Eudicotyledoneae and Pentapetalae [26], [34]. Indeed, the plastid genome represents an excellent source of characters for plant phylogenetics due to the generally strong conservation of plastid genome structure and its mix of sequence regions that vary tremendously in evolutionary rate [35]–[37], which enable plastid genome sequence data to be applied to phylogenetic problems at almost any taxonomic level in plants [26], [38]–[43]. It is now relatively inexpensive to generate complete plastid genome sequence due to rapid improvements in next-generation sequencing (NGS) technologies [25], [44]–[45] and due to the relatively small size of the plastid genome (∼150 kb) and its structural conservation, which enable dozens of plastomes to be multiplexed per sequencing lane and facilitate relatively straightforward genome assembly [45]–[48].

Despite the promise of NGS technology for plastid genomics, the complete plastomes of only eight genera of early-diverging eudicots have been reported: Ranunculus (Ranunculaceae, Ranunculales), Megaleranthis (Ranunculaceae, Ranunculales), Nandina (Berberidaceae, Ranunculales), Nelumbo (Nelumbonaceae, Proteales), Platanus (Platanaceae, Proteales), Meliosma (Sabiaceae, Sabiales), Trochodendron (Trochodendraceae, Trochodendrales) and Buxus (Buxaceae, Buxales). Previous phylogenetic analyses based on some of these complete genomes have not fully resolved the relationships among early-diverging eudicots, however; in addition to the uncertainty surrounding relationships of Buxales, Trochodendrales, and Gunneridae, the positions of Sabiales and Proteales remain poorly supported [26]–[27]. Plastome taxon sampling is still sparse in these clades, however, and additional sampling may help elucidate these recalcitrant relationships.

In addition to their important role in phylogenetics, plastid genomes may be rich sources of population-level data. The non-recombination and uniparental inheritance of most plastid genomes can make plastid genomes extremely useful for population genetics, particularly for tracing maternal lineages [49]–[50]. For example, chloroplast simple sequence repeats (cpSSR) have been widely used in plant population genetics [51], including within early-diverging eudicots, where numerous cpSSR loci have been reported from the plastid genome of the endangered species Megaleranthis saniculifolia (Ranunculaceae) [52].

Here we report the complete plastid genome sequences of Tetracentron sinense and Trochodendron aralioides (the protein-coding and rRNA genes of Trochodendron cp genome were used for phylogenetic analyses in Moore et al. [26], but the cp genome structure of this genus has never been reported), as well as the results of new phylogenetic analyses based on adding Tetracentron and Megaleranthis genomes [52] to the 83-gene data set of Moore et al. [26]. We also compare the plastid genome structure of Trochodendron and Tetracentron, including the characterization of a significant expansion of the inverted repeat in both taxa, and we estimate the divergence time between the two genera. Finally, we characterize the distribution and location of cpSSRs in both Tetracentron sinense and Trochodendron aralioides, which provided further opportunity to study the population genetic structures of these two ancient relict species.

Results

Sequencing and Genome Assembly

Illumina paired-end sequencing produced 892.11 Mb of data for Tetracentron sinense. We obtained 9912310 raw reads of 90 bp in length. The N50 of contigs was 13,981 bp and the summed length of contigs was 143,709 bp. The mean coverage of this genome was 5424.2×. After de novo and reference-guided assembly, we obtained a cp genome containing nine gaps. PCR and Sanger sequencing were used for filling the gaps. Four junction regions between IRs and SSC/LSC were first determined based on de novo contigs, and subsequently confirmed by PCR amplifications and Sanger sequencing, sequenced results were compared with the assembled genome directly and no mismatch or indel was observed, which validated the accuracy of our assembly. The genome sequences of Tetracentron sinense and Trochodendron aralioides have been submitted to GenBank (GenBank IDs: KC608752 and KC608753).

General Features of the Tetracentron and Trochodendron Plastomes

The plastid genome size of Tetracentron sinense is 164,467 base pairs (bp) (Figure 1), and that of Trochodendron aralioides is 165,945 bp (Figure 2). Both genomes show typical quadripartite structure, consisting of two copies of an inverted repeat (IR) separated by the large single-copy (LSC) and small single-copy (SSC) regions (Table 1). The IR exhibits a significant expansion relative to most other angiosperms at the LSC/IR junction; specifically, the IR in both Tetracentron and Trochodendron has expanded to include the entirety of the rps19, rpl22, rps3, rpl16, rpl14, and rps8 genes (Figures 1, 2). The SSC/IR boundary occurs within the ycf1 gene, as is typical in angiosperms, but is slightly expanded in the Trochodendron genome to include 1461 bp of the 5′ end of ycf1 (versus 1083 bp in Tetracentron; Figure 3). This expansion of the IR at the SSC junction contributes to the difference in length between the two Trochodendrales plastomes; the remainder of the difference is largely the result of length differences among various noncoding regions (Table 2).

Figure 1. Map of the Tetracentron sinense plastid genome.

Figure 2. Map of the Trochodendron aralioides plastid genome.

Table 1. Basic characteristic of the Tetracentron sinense and Trochodendron aralioides plastid genomes.

	Tetracentron	Trochodendron
total genome length	164467	165945
IR length	30231	30744
SSC length	19539	18974
LSC length	84466	85483
total length of coding sequence	94699	95168
total length of noncoding sequence	69768	70777
overall G/C content	38.1%	38.0%

All values given are in base pairs (bp), unless otherwise noted.

Figure 3. Comparison of the IR junctions in Tetracentron and Trochodendron.

Table 2. The principal noncoding regions contributing to the size difference between the Tetracentron and Trochodendron plastid genomes.

Spacer region or intron names	Tetracentron	Trochodendron	length difference
trnK-UUU/rps16 spacer	870	1308	438
rps16/trnQ-UUG spacer	1529	1797	268
trnS-GCU/trnG-UCC spacer	505	658	153
trnE-UUC/trnT-GGU spacer	957	1316	359
trnT-UGU/trnL-UAA spacer	1199	1309	110
petA/psbJ spacer	1146	754	−392
ycf1/ndhF spacer	440	325	−115
*rpl16 intron	865	972	107

All sizes are in base pairs. The only locus residing in the IR is marked with an asterisk (*).

Both genomes contain 119 genes (79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes) arranged in the same order, of which 24 are duplicated in the IR regions (Table 3). Sequence divergence between Tetracentron and Trochodendron in coding regions is low (Table 4, Figures 4, 5). Only 7 genes (rps11, rpoA, rpl32, rps16, ndhF, ycf1, and rpl36) exhibit divergences of more than 2%, and 12 genes have an identical sequence (Table 4, Figure 4). The genes ndhF, ycf1, and rpl36 have the highest sequence divergences (2.7%, 3.5% and 4.4%, respectively). The coding regions account for 57.5% and 57.3% of the Tetracentron and Trochodendron plastid genomes, respectively. For both cp genomes, single introns are present in 18 genes, whereas three genes (rps12, clpP, and ycf3) have two introns (Table 5). The overall genomic G/C nucleotide composition is 38.1% and 38.0% for Tetracentron and Trochodendron, respectively; detailed A/T contents of different regions of the plastome for both genomes are listed in Table 6. Due to the lower A/T content of the four rRNA genes, the IR regions possess lower A/T content than the single-copy regions.

Table 3. List of genes present in the plastid genomes of Tetracentron sinense and Trochodendron aralioides.

	Group of genes	Name of genes
Protein synthesis and DNA replication	Ribosomal RNAs	rrn4.5 (×2) rrn5 (×2) rrn16 (×2) rrn23 (×2)
	Transfer RNAs	trnH-GUG trnK-UUU* trnQ-UUG trnS-GCU trnG-UCC* trnR-UCU trnC-GCA trnD-GUC trnY-GUA trnE-UUC trnT-GGU trnS-UGA trnG-GCC trnfM-CAU trnS-GGA trnT-UGU trnL-UAA*trnF-GAA trnV-UAC* trnM-CAU trnW-CCA trnP-UGG trnI-GAU* (×2) trnL-CAA (×2) trnV-GAC (×2) trnI-GAU (×2) trnA-UGC* (×2) trnR-ACG (×2) trnN-GUU (×2) trnL-UAG
	small subunit	rps2 rps3 rps4 rps7 (×2) rps8 rps11 rps12* (×2) rps14 rps15 rps16* rps18 rps19
	Ribosomal proteins large subunit	rpl2* (×2) rpl14 rpl16* rpl20 rpl22 rpl23 (×2) rpl32 rpl33 rpl36
	RNA polymerase	rpoA rpoB rpoC1* rpoC2
Photosynthesis	Photosystem I	psaA psaB psaC psaI psaJ
	Photosystem II	psbA psbB psbC psbD psbE psbF psbH psbI psbJ psbK psbL psbM psbN psbT psbZ
	Cytochrome b6/f	petA petB* petD* petG petL petN
	ATP synthase	atpA atpB atpE atpF* atpH atpI
	NADH dehydrogenase	ndhA* ndhB*(×2) ndhC ndhD ndhE ndhF ndhG ndhH ndhI ndhJ ndhK
	Large subunit of Rubisco	rbcL
Miscellaneous proteins	Subunit of Acetyl-CoA-carboxylase	accD
	c-type cytochrome synthesis gene	ccsA
	Envelope membrane protein	cemA
	Protease	clpP*
	Translational initiation factor	infA
	Maturase	matK
Genes of unknown function	Hypothetical conserved coding frame	ycf1 ycf2(×2) ycf3* ycf4

Genes with introns are marked with asterisks (*).

Table 4. Comparisons of the protein-coding genes of Tetracentron and Trochodendron.

Gene	Length in Tetracentron	Length in Trochodendron	Number of nucleotide differences	Proportion of nucleotide differences	Number of indel differences
petL	102	102	0	0	0
psaI	111	111	0	0	0
psaJ	129	129	0	0	0
psbE	252	252	0	0	0
psbF	120	120	0	0	0
psbJ	123	123	0	0	0
psbL	117	117	0	0	0
psbT	108	108	0	0	0
rpl23	288	288	0	0	0
rps19	279	279	0	0	0
rps7	468	468	0	0	0
rps8	399	399	0	0	0
rpl2	825	825	1	0.00121	0
rps3	657	657	1	0.00152	0
petD	504	504	1	0.00198	0
rpl16	501	501	1	0.00249	0
rpl14	369	369	1	0.00271	0
ycf2	6879	6897	19	0.00276	1
ndhB	1533	1533	5	0.00326	0
ycf3	507	507	2	0.00394	0
rpl33	201	201	1	0.00498	0
psbZ	189	189	1	0.00529	0
psaA	2253	2253	12	0.00533	0
psbK	186	186	1	0.00538	0
rps12	372	372	2	0.00538	0
psbA	1062	1062	6	0.00565	0
rpl20	354	354	2	0.00565	0
rpoC1	2049	2070	12	0.00586	1
atpA	1524	1524	9	0.00591	0
rpl22	486	480	3	0.00625	1
ndhJ	477	477	3	0.00629	0
psbD	1062	1062	7	0.00659	0
petA	963	963	7	0.00727	0
rpoB	3213	3213	24	0.00747	0
psbN	132	132	1	0.00758	0
psaB	2205	2205	17	0.00771	0
psbC	1422	1422	11	0.00774	0
atpH	246	246	2	0.00813	0
psaC	246	246	2	0.00813	0
ndhA	1095	1095	9	0.00822	0
rps4	606	606	5	0.00825	0
infA	234	234	2	0.00855	0
atpB	1497	1497	13	0.00868	0
cemA	690	690	6	0.0087	0
petG	114	114	1	0.00877	0
psbI	111	111	1	0.00901	0
rbcL	1428	1428	13	0.0091	0
petB	648	648	6	0.00926	0
atpI	744	744	7	0.00941	0
clpP	609	609	6	0.00985	0
rps14	303	303	3	0.0099	0
atpE	402	402	4	0.00995	0
ccsA	966	966	10	0.01035	0
psbB	1527	1527	16	0.01048	0
accD	1491	1491	16	0.01073	0
ndhK	822	858	9	0.01095	1
ndhC	363	363	4	0.01102	0
petN	90	90	1	0.01111	0
ndhG	531	531	6	0.0113	0
rpoC2	4137	4146	50	0.01209	1
ndhD	1503	1503	18	0.01264	0
rps2	711	711	9	0.01266	0
psbH	222	222	3	0.01351	0
ndhI	543	543	8	0.01473	0
atpF	555	555	9	0.01622	0
matK	1536	1536	25	0.01628	0
ndhE	306	303	5	0.0165	1
rps18	303	303	5	0.0165	0
ndhH	1182	1182	20	0.01692	0
ycf4	555	555	10	0.01805	0
rps15	273	273	5	0.01832	0
psbM	105	105	2	0.01905	0
rps11	417	417	9	0.02158	0
rpoA	1014	1014	24	0.02367	0
rpl32	162	162	4	0.02469	0
rps16	227	227	6	0.02622	0
ndhF	2223	2223	61	0.02744	0
ycf1	5688	5691	195	0.0345	6
rpl36	114	114	5	0.04386	0

Genes are ranked from lowest to highest proportion of nucleotide differences.

Figure 4. Amount of sequence divergence between the protein-coding genes of Tetracentron and Trochodendron.

Figure 5. Sequence identity plot between Trochodendron and Tetracentron.

Table 5. Exon and intron lengths (bp) in plastid genes containing introns in Tetracentron sinense and Trochodendron aralioides, respectively.

Gene	Exon 1 (Te/Tr)	Intron 1 (Te/Tr)	Exon 2 (Te/Tr)	Intron 2 (Te/Tr)	Exon 3 (Te/Tr)
trnK-UUU	37/37		35/35
trnG-UCC	24/24	698/698	48/48
trnL-UAA	35/35	444/442	50/50
trnV-UAC	39/39	583/585	37/37
trnI-GAU	42/42	954/954	35/35
trnA-UGC	38/38	794/794	35/35
petB	6/6	793/797	642/642
petD	8/8	704/709	496/496
atpF	145/145	727/724	410/410
ndhA	553/553	1106/1084	542/542
ndhB	777/777	700/700	756/756
rpl2	391/391	671/674	434/434
rpl16	9/9	865/972	402/402
rps12	114/114		232/232	538/536	26/26
rpoC1	432/432	728/714	1617/1638
clpP	71/71	682/710	292/292	659/650	246/246
ycf3	124/124	734/725	230/230	731/758	153/153
rps16	40/40	831/844	227/227

The rps12 gene is trans-spliced, and hence the length of intron 1 is unknown.

Table 6. A/T content (%) of different regions in Tetracentron and Trochodendron.

Region	Tetracentron	Trochodendron
overall	61.86	61.98
LSC	63.50	63.74
IR	57.63	57.83
SSC	67.84	67.48
Protein-coding regions	61.58	61.53

Characterization of SSR Loci

In all, 154 SSR loci (77 each from Tetracentron sinense and Trochodendron aralioides) were detected in the two plastid genomes, of which 123 are mononucleotide repeats, 28 are dinucleotide repeats, two are trinucleotide repeats, and one is a tetranucleotide repeat (Table 7). Nearly all of the SSR loci are composed of A/T repeats (Table 7), and these SSR loci are mostly present in noncoding regions. The tetranucleotide locus identified in Tetracentron is in the first intron of ycf3. The two trinucleotide loci in Trochodendron are both located in the spacer region between trnK-UUU and rps16. The unique C mononucleotide repeat from Trochodendron is present in the trnV-ndhC intergenic spacer region.

Table 7. Distribution of SSR loci in the plastid genomes of Tetracentron and Trochodendron.

Base	Length	Position in plastid genome
SSR loci in Tetracentron
A	10	2085–2094 7164–7173 9478–9487 17266–17275 39220–39229 47812–47821 58880–58889 69930–69939 124816–124825 136417–136426 141648–141657
	11	9611–9621 46892–46902 47147–47157 50813–50823 75797–75807 80873–80883 82302–82312 133069–133079 160432–160442
	12	217–228 49977–49988 50332–50343 118899–118910 162450–162461 163452–163463 163940–163951
	14	65157–65170
	15	38842–38856
	17	39891–39907
	18	74838–74855
	22	72886–72907
T	10	5266–5275 6724–6733 9153–9162 19332–19341 54468–54477 63461–63470 67706–67715 107277–107286 112508–112517 117373–117382 118300–118309 121204–121213 126456–126465 130614–130623
	11	7004–7014 7679–7689 13144–13154 31361–31371 37925–37935 47779–47789 67810–67820 76013–76023 88492–88502
	12	55307–55318 71723–71734 84983–84994 85471–85482 86473–86484 118884–118895 119027–119038
	13	13902–13914
	14	72926–72939
AT	10	1734–1743 20833–20842 50404–50413–63181–63190
	12	4862–4873 12996–13007 114822–114833
	14	60686–60699
TA	10	34083–34092 34111–34120 114741–114750
	14	49132–49145
TAAA	20	46875–46894
SSR loci in Trochodendron
A	10	118854–118863 126258–126267 142993–143002 163821–163830 18142–18151 40389–40398 41060– 41069 51091–51100 6136–6145 68969–68978 76681–76690 86529–86538
	11	134406–134416 16427–16437 30306–30316 39963–39973 51490–51500 70911–70921 81823–81833 9789–9799
	12	10420–10431 48058–48069 48322–48333
	13	164932–164944
	16	161805–161820 73777–73792 75726–75741
	15	46189–46203
	17	214–230 83299–83315 9304–9320
T	10	108427–108436 120424–120433 121028–121037 122665–122674 131951–131960 164891–164900 20189–20198 40375–40387 48933–4894253154–53163 53339–53348 5700–5709 6030–6039 68604–68613 72934–72943 83282–83291 87599–87608
	11	127885–127895 14709–14719 55604–55614 57547–57557
	12	50271–50282
	13	73814–73826 86485–86497
	14	76896–76909
	15	48889–48903
	16	89609–89624
AT	10	1724–1733 51556–51565 64459–64468
	12	4921–4932 4943–4954 4984–4995 4998–5009 5044–5055 5085–5096 5099–5110 5145–5156 5186–5197 5200–5211
	18	73275–73292
TA	10	1738–1747 21689–21698
TAA	18	5016–5033 5218–5235
C	10	55999–56008

Phylogenetic and Molecular Dating Analyses

ML analyses of the 83-gene, 88-taxon data set yielded a tree with a similar topology and bootstrap support (BS) values (Figure 6) as that of the plastid phylogenomic study of Moore et al. [26]. The clades of Trochodendron+Tetracentron and Ranunculus+Megaleranthis were supported with 100% ML BS support. Trochodendrales are sister to the remaining angiosperms with high support (BS = 100%), but Buxaceae are sister to Gunneridae with only 67% BS support.

Figure 6. A maximum likelihood tree determined by GARLI (−ln L = −1095466.026) for the 83-gene, 88-taxon data set.

Numbers associated with branches are ML bootstrap support values. Error bars around nodes correspond to 95% highest posterior distributions of divergence times based on 6 fossils using the program BEAST. Eo = Eocene, Mi = Miocene, Ol. = Oligocene, Pa = Paleocene, Pl = Pliocene.

Molecular dating analyses suggest that Trochodendron and Tetracentron diverged between 44-30 million ago. The crown group 95% highest posterior density (HPD) age estimates for other major lineages of Pentapetalae were as follows: Superasteridae (115-109 mya), Dilleniaceae+Superrosidae (116-112 mya), Superrosidae (114-111 mya), Santalales (98-75 mya), Caryophyllales (76-60 mya), Asteridae (104-99 mya), Rosidae (111-108 mya), Vitaceae+Saxifragales (114-110 mya), and Saxifragales (109-107 mya).

Discussion

Expansion of the IR Region in Trochodendrales Plastomes

The plastid genomes of Tetracentron and Trochodendron exhibit the typical gene content and genome structure of angiosperms [37], [53]–[54], with the notable exception of a significantly expanded IR region (Figures 1, 2, 3). This ∼4 kb expansion is responsible for the relatively large size of both Trochodendrales plastomes, which are ∼4–5 kb larger than the typical upper size range of angiosperm plastid genomes, including those of nearly all other early-diverging eudicots (Table 8). Significant expansion, contraction, and even loss of the IR appears to be an evolutionarily uncommon phenomena but are nonetheless associated with much of the more significant variation in plastome size in angiosperms. For example, the largest known angiosperm plastome, that of Pelargonium x hortorum, also possesses the largest known IR, at ∼76 kb in length [55]. Other significant IR expansions and contractions have been found in Campanulaceae [56]–[57], Apiaceae [58], and Lemna (Araceae) [59].

Table 8. Numbers of genes (including genes that span IR/SC junctions) in the IR regions of early-diverging eudicots.

Basal eudicot lineages	Species	Genes in IR region	cp genome size (bp)
Ranunculales	Ranunculus macranthus	20	155129
	Megaleranthis saniculifolia	19	159924
	Nandina domestica	19	156599
Proteales	Nelumbo lutea	18	163206
	Platanus occidentalis	19	161791
Sabiales	Meliosma aff. cuneifolia	18	160357
Buxales	Buxus microphylla	18	159010
Trochodendrales	Tetracentron sinense	24	164467
	Trochodendron aralioides	24	165945

Impact of Additional Taxon Sampling on Basal Eudicot Phylogeny

The inclusion of Megaleranthis and Tetracentron in our analyses had no effect on the relationships among the major early-diverging eudicot lineages, and very little effect on support values. Of the basal splits among the eudicots with BS values less than 100% in both the current tree and that of Moore et al. [26], all were within 3% BS value. For example, the sister relationship of Buxales and Gunneridae is 70% in Moore et al. [26] vs. 67% with the inclusion of Megaleranthis and Tetracentron, and the sister relationship of Sabiales and Proteales has BS support of 80% in Moore et al. [26] vs. 83% in the current analyses. These similar values are unsurprising given that Tetracentron and Trochodendron are found to be relatively closely related in our analyses. Indeed, the relatively low sequence divergence between the Tetracentron and Trochodendron plastid genomes supports the taxonomic placement of Tetracentraceae within Trochodenraceae, as advocated by APG III [1]. Although it is possible that the addition of the noncoding regions of the plastid genome (or at least those noncoding regions that can be aligned) to our data set may improve support for these relationships, we may have to look to the other plant genomes for a confident resolution of relationships among the early-diverging eudicots. In fact, the sister relationship of Buxales and Gunneridae received high support (BS = 98%) in the 17-gene analyses of Soltis et al. [28], which employed a combination of 11 plastid genes, 18S and 26S nuclear rDNA, and 4 mitochondrial genes. However, the sister relationship of Sabiales and Proteales were more poorly supported (BS = 59%) in Soltis et al. [28].

Divergence Time Between Tetracentron and Trochodendron

Cenozoic Trochodendrales fossils are known throughout the Northern Hemisphere, with the Paleocene Nordenskioldia the earliest certain fossil of the order [7]–[15]. Both Tetracentron and Trochodendron had wide distributions in the Northern Hemisphere during the Paleogene and Neogene. Fossil remains of Tetracentron have been found in Japan [60]–[61], Idaho [62], Princeton, British Columbia and Republic, Washington [63], and Iceland [15]; Trochodendron fossil remains have been reported from Kamchatka [64], Japan [11], Idaho and Oregon [11]–[12], Washington [7], and British Columbia [63]. Our estimate of the divergence time between the two genera of Trochodendraceae (44-30 mya) encompasses the recent estimate of 37-31 mya from Bell et al. [65], which was based on analysis of 567 taxa and three genes, as well as the mid-Eocene estimate of ∼45 mya derived from the rbcL analysis of Anderson et al. [66], which employed numerous fossil constraints from the early-diverging eudicots. The congruence among these studies and with the fossil record suggests that a mid- to late Eocene divergence for the two extant Trochodendraceae lineages may be a reasonable estimate.

Analysis of Plastid SSR Loci in the Trochodendrales

Because microsatellite loci, including cpSSRs, often exhibit high variation within species, they are considered valuable molecular markers for population genetics [67]–[69]. A limited number of SSR loci were recently characterized for Tetracentron [70], but no cpSSR loci are available for Trochodendraceae. The 77 cpSSR loci that were identified in both Tetracentron and Trochodendron represent ∼42% more loci than the 54 loci reported in the plastid genome of Megaleranthis (Ranunculaceae), the only other early-diverging eudicot for which a comprehensive analysis of cpSSR loci is available. The abundant and varied cpSSR loci identified in Trochodendrales will be useful in characterizing the population genetics of both extant species, which are of conservation interest in the wild because of their relatively narrow, presumably relictual distributions, and decreasing numbers [71]. Tetracentron is officially afforded second-class protection in China.

Materials and Methods

Sample Preparation, Sequencing, and Assembly

Fresh leaves of Tetracentron sinense were collected from the Kunming Institute of Botany at the Chinese Academy of Sciences, and a voucher was deposited at the Herbarium of Wuhan Botanical Garden, Chinese Academy of Science (HIB). Chloroplast DNA was isolated following the protocol of Zhang et al. [45], and an Illumina library was constructed following the manufacturer’s protocol (Illumina). The DNA was indexed by tag and sequenced together with eight other species in one lane of an Illumina Genome Analyzer IIx at Beijing Genomics Institute (BGI) in Shenzhen, China. Illumina Pipeline 1.3.2 was used conducting image analysis and base calling. Raw sequence reads produced by Illumina paired-end sequencing were filtered for high quality reads which were subsequently assembled into contigs with a minimum length of 100 bp using SOAPdenovo [72] with the Kmer = 57. Contigs were aligned to the Trochodendron aralioides plastid genome using BLAST (http://blast.ncbi.nlm.nih.gov/), and aligned contigs were ordered according to the reference genome.

Genome Annotation and Analysis

The Tetracentron and Trochodendron plastid genomes were annotated with DOGMA [73] and BLAST tools from NCBI (the National Center for Biotechnology Information). Physical maps were generated using GenomeVx [74] with subsequent manual editing. Sequence divergence between the Tetracentron and Trochodendron plastid genomes was evaluated using DnaSP version 5.10 [75], and genome sequence identity plots were generated using mVISTA [76] (http://genome.lbl.gov/vista/mvista/submit.shtml). Msatfinder ver. 1.6.8 [77] was used to identify SSR loci by manually setting repeat units.

Phylogenetic and Divergence Time Analyses

All protein-coding sequences, as well as all rRNA sequences, were extracted from the Tetracentron and Megaleranthis plastome [52] and added manually to the 83-gene, 86-taxon alignment of Moore et al. [26]. ML analyses were performed on the concatenated 83-gene data set using the following partitioning strategy: (1) codon positions 1 and 2 together; (2) codon position 3; and (3) rRNA genes. The optimal nucleotide sequence model was selected for each partition using jModelTest 2.1.1 using the Decision Theory (DT) criterion [78]. The following models were selected: TVM+I+Γ for codon positions 1+2 and for codon position 3, and TIM1+ I+Γ for rRNA.

Partitioned ML analyses were conducted using GARLI 2.0 [79]. A total of ten search replicates were conducted to find the optimal tree, and nonparametric bootstrap support was assessed with 100 replicates [80]. All ML searches used random taxon addition to build starting trees.

Divergence times were estimated using BEAST version 1.7.4 [81], using the same dating strategies employed in Moore et al. [26]. In addition to the three calibration points (used in Moore et al. [26]) of minimum ages of 131.8 mya for angiosperms [82]–[85], 125 mya for eudicots [83], [86], and 85 mya for the most recent common ancestor of Quercus and Cucumis [26], we additionally constrained the stem lineage of Malpighiales using a minimum of 89.3 my [87] and the node uniting Calycanthus and Liriodendron using 98 my [88], and set the age of Proteales to a minimum of 98 my [89].

Funding Statement

This research was supported by Knowledge Innovation Project of Chinese Academy of Sciences (KSCX2-EW-J-20), National Natural Science Foundation of China grant (31070191) and U.S. National Science Foundation grant (ER-0431266). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.