Phylotranscriptomic analysis of Dillenia indica L. (Dilleniales, Dilleniaceae) and its systematics implication.

The recent massive development in the next-generation sequencing platforms and bioinformatics tools including cloud based computing have proven extremely useful in understanding the deeper-level phylogenetic relationships of angiosperms. The present phylotranscriptomic analyses address the poorly known evolutionary relationships of the order Dilleniales to order of the other angiosperms using the minimum evolution method. The analyses revealed the nesting of the representative taxon of Dilleniales in the MPT but distinct from the representative of the order Santalales, Caryophyllales, Asterales, Cornales, Ericales, Lamiales, Saxifragales, Fabales, Malvales, Vitales and Berberidopsidales.

Introduction

The family Dilleniaceae (Parad. Lond. sub t. 73, 1806) previously placed in subclass Dilleniidae (Phytologia 74(3): 171, 1993) (Cronquist and Takhtadzhi︠, 1981), were unplaced in Angiosperm Phylogeny Group, APG (APG, 1998; APG II, 2003; APG III, 2009) to as an order; later APG IV (APG IV, 2016) recognized as the new monofamilial order Dilleniales (Prir. Rostlin 223, 1820) (characterized as mostly woody; lvs if veins strong, proceed to apex of teeth flw mostly K5, persisting, mostly Androceium ∞, Gynoceium mostly slightly connate seeds often with aril; fruits usually follicles) under clade core eudicots containing family Dilleniaceae only (-the family of trees, shrubs, and lianas) based on molecular phylogenetic studies (Soltis et al., 2011, Ruhfel et al., 2014). The APG system recognizes 12 genera [viz. 1. Acrotrema Jack (Malayan Misc. 1(5): 36, 1820), 2. Curatella Loefl. (Iter Hispan. 229, 260, 1758), 3. Davilla Vand. (Fl. Lusit. Bras. Spec. 35, f. 14, 1788), 4. Didesmandra Stapf (Hooker's Icon. Pl. 27:, ad t. 2646, 1900), 5. Dillenia L. (Sp. Pl. 1: 535, 1753), 6. Doliocarpus Rol. (Kongl. Svenska Vetensk. Acad. Handl. 17: 260–261, 1756), 7. Hibbertia Andrews (Bot. Repos. 2:, t. 126, 1800), 8. Neodillenia Aymard (Harvard Pap. Bot. 10: 121, 1997), 9. Pachynema R. Br. ex DC. (Syst. Nat. 1: 397, 411, 1818), 10. Pinzona Mart. & Zucc. (Abh. Math.-Phys. Cl. Königl. Bayer. Akad. Wiss. 1: 371, 1832), 11. Schumacheria Vahl (Skr. Naturhist.-Selsk. 6: 122, 1810) and 12. Tetracera L. (Sp. Pl. 1: 533, 1753)] and c. 430 species of the family Dilleniaceae (http://www.mobot.org/MOBOT/research/APweb/welcome.html). Since, the evolutionary relationship of the order Dilleniales among the angiosperms phylogeny remains poorly known, the present study explores the relationship of the order Dilleniales in the angiosperm phylogeny based on phylotrasncriptomic analyses.

Materials and methods

Taxon selection

The RNA transcriptome SRA data of Dillenia indica L. (order Dilleniales, family Dilleniaceae) was available from the study of ‘One Thousand Plant Transcriptomes Initiative’ (Leebens-Mack, 2019) were retrieved, and analyzed together with representatives from the order Asterales, Berberidopsidales, Caryophyllales, Cornales, Ericales, Fabales, Gunnerales, Lamiales Malvales, Santalales, Saxifragales and Vitales. The RNA transcriptome data of Gunnera manicata (order Gunnerales, family Gunneraceae) was used as the outgroup in the phylotranscriptomic analysis (Table 1).

Table 1

Taxon used in the phylotranscriptomic analyses to infer relationships of Dilleniales.

S. No.	Taxon	Family	Order	Clade	GenBank
	Ingroup
1.	Dillenia indica L.	Dilleniaceae	Dilleniales	Eudicots,Core eudicots	ERS1829252
2.	Phoradendron serotinum (Raf.) M.C. Johnst.	Santalaceae	Santalales	Eudicots,Superasterids	ERS1829342
3.	Silene latifolia Poir.	Caryophyllaceae	Caryophyllales	Superasterids	ERS1829296
4.	Inula helenium L.	Asteraceae	Asterales	Asterids,Campanulids	ERS368261
5.	Cornus florida L.	Cornaceae	Cornales	Eudicots,Asterids	ERS1829540
6.	Cavendishia cuatrecasasii A.C. Sm.	Ericaceae	Ericales	Eudicots,Asterids	ERS1829554
7.	Lavandula angustifolia Mill.	Lamiaceae	Lamiales	Asterids,Lamiids	ERS1829627
8.	Astilbe chinensis (Maxim.) Franch. & Sav.	Saxifragaceae	Saxifragales	Eudicots,Core eudicots	ERS1829362
9.	Lupinus polyphyllus C.E. Anderson	Fabaceae	Fabales	Rosids,Fabids	ERS631113
10.	Hibiscus cannabinus L.	Malvaceae	Malvales	Rosids,Malvids	ERS368256
11.	Cissus quadrangularis L.	Vitaceae	Vitales	Superrosids	ERS1829366
12.	Berberidopsis beckleri (F. Muell.) Veldkamp	Berberidopsidaceae	Berberidopsidales	Eudicots,Superasterids	ERS1829251
	Outgroup
13.	Gunnera manicata Linden ex André	Gunneraceae	Gunnerales	Eudicots,Core eudicots	ERS1829249

Phylotranscriptomic analyses

The retrieved aligned data were then imported to MEGA X (Kumar et al., 2018) for pairwise sequence alignment. The aligned sequence data set saved into mega format and the evolutionary analyses (outgroup at Gunnera manicata, order Gunnerales, family Gunneraceae) were performed using Maximum Parsimony (MP) bootstrap methods (Felsenstein, 1985) in MEGA X (Kumar et al., 2018) in order to detect proximity of Dillenia indica (the representative of the order Dilleniales with the representatives of the order Asterales Link, Berberidopsidales Doweld, Caryophyllales Benth. & Hook. f., Cornales Link, Ericales Bercht. & J. Presl, Fabales Bromhead, Gunnerales Takht. ex Reveal, Lamiales Bromhead, Malvales Juss., Santalales R. Br. ex Bercht. & J. Presl, Saxifragales Bercht. & J. Presl and Vitales Juss. ex Bercht. & J. Presl.

Results and discussion

The present phylotranscriptomic analyses of 28,176 parsimony informative sites out of a total of 1,42,796 positions in the aligned (the aligned data set contains 64,202 conserved, 71,580 variable and 41,870 singleton sites, Fig. 1) transcriptome dataset of D. indica, P. serotinum, S. latifolia, I. helenium, C. florida, C. cuatrecasasii, L. angustifolia, A. chinensis, L. polyphyllus, H. cannabinus, C. quadrangularis, B. beckleri and G. manicata inferred using the MP method recovered the MPT with length of 1,72,735 [consistency index 0.795270 (0.647002), retention index 0.253898, composite index 0.201917]. The MPT revealed the nesting of the representative taxon of Dilleniales (D. indica) but distinct from the representative of Santalales (P. serotinum), Caryophyllales (S. latifolia), Asterales (I. helenium), Cornales (C. florida), Ericales (C. cuatrecasasii), Lamiales (L. angustifolia), Saxifragales (A. chinensis), Fabales (L. polyphyllus), Malvales (H. cannabinus), Vitales (C. quadrangularis) and Berberidopsidales (B. beckleri) (Fig. 2) which is also evident from the estimates of evolutionary divergence between the sequences (Zuckerkandl and Pauling, 1965) used to infer the phylogeny using MEGA X (Kumar et al., 2018) (Table 2).

Fig. 1

The final transcriptome data set showing number of sites.

Fig. 2

The evolutionary history of the order Dilleniales inferred from phylotranscriptomic analysis.

Table 2

The estimates of evolutionary divergence between the sequences (Zuckerkandl and Pauling, 1965) used to infer the phylogeny sung MEGA X (Kumar et al., 2018).

D. indica	0.256
P. serotinum	0.309	0.326
S. latifolia	0.309	0.322	0.359
I. helenium	0.282	0.300	0.345	0.332
C. florida	0.233	0.259	0.308	0.312	0.266
C. cuatrecasasii	0.244	0.269	0.311	0.310	0.260	0.211
L. angustifolia	0.283	0.292	0.344	0.327	0.292	0.270	0.262
A. chinensis	0.232	0.252	0.302	0.302	0.277	0.226	0.235	0.281
L. polyphyllus	0.283	0.289	0.337	0.315	0.303	0.273	0.274	0.290	0.270
H. cannabinus	0.259	0.272	0.318	0.304	0.289	0.251	0.254	0.281	0.244	0.268
C. quadrangularis	0.243	0.261	0.309	0.312	0.289	0.239	0.246	0.289	0.229	0.276	0.249
B. beckleri	0.202	0.228	0.278	0.294	0.264	0.186	0.208	0.269	0.192	0.255	0.227

The recent massive development in the next-generation sequencing platforms (Eid et al., 2009, Rothberg et al., 2011, Shendure and Aiden, 2012, Pattnaik et al., 2014, Jain et al., 2016), and bioinformatics tools (Mavromatis et al., 2007, Knudsen et al., 2010, Hu et al., 2012, Huang et al., 2012, McElroy et al., 2012, Shendure and Aiden, 2012, Yang and Rannala, 2012, Caboche et al., 2014, Shcherbina, 2014, Kwon et al., 2015, Escalona et al., 2016, Langmead and Nellore, 2018) and Cloud computing (Langmead and Nellore, 2018) have proven extremely useful in evaluating assembly, mapping, phasing or genotyping (Nielsen et al., 2011, Angly et al., 2012, Caboche et al., 2014, Shcherbina, 2014, Li et al., 2014), and have led to public archives (Kodama et al., 2012), collaborations (Collins and Varmus, 2015, Melé et al., 2015, Gaziano et al., 2016, Lek et al., 2016; Trans-Omics for Precision Medicine (TOPMed) Program, 2017), and thus have entirely changed the face of plant systematics (Stuessy, 2020), and enhanced understanding of tree of life and plant genomics (Ali et al., 2020) and the transcriptomics based deeper-level phylogenetic relationships (Cannon et al., 2015, Smith et al., 2015, Yang et al., 2018).

The present phylotranscriptomic analyses revealed the nesting of the representative taxon of Dilleniales but distinct from the representative of Santalales, Caryophyllales, Asterales, Cornales, Ericales, Lamiales, Saxifragales, Fabales, Malvales, Vitales and Berberidopsidales. The monofamilial order Dilleniales (characterized as mostly woody; lvs if veins strong, proceed to apex of teeth flw mostly K5, persisting, mostly A∞, G mostly slightly connate seeds often with aril; fr usually follicles) under clade core eudicots containing family Dilleniaceae. The diagnostic taxonomic features of monophyletic Dilleniaceae (Hoot et al., 1999, Savolainen et al., 2000a, Savolainen et al., 2000b, Ingrouille et al., 2002, Hilu et al., 2003) are: combination of scabrous leaves and primary stems, leaf venation with more or less straight, parallel secondaries and scalariform tertiaries, petioles with a broad insertion at the stem, orangish inner or outer bark that exfoliates thin plates or strips, uniformly persistent and typically accrescent calyces, apopetalous, caduceus corollas, polymerous, marcescent, or tardily deciduous androecia, free stylodia, arillate seeds (Horn, 2009). Moreover, The majority of molecular phylogenetic studies resolve the family Dilleniaceae as sister to Vitaceae (Chase et al., 1993, Savolainen et al., 2000b, Hilu et al., 2003), or Caryophyllales (Hoot et al., 1999, Soltis et al., 2000, Soltis et al., 2003, Soltis et al., 2007) or under within the core group of eudicots (APG II, 2003, Moore et al., 2008), the present analyses supports the placement of Dilleniales as a new order in the angiosperm phylogeny.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.