A phylotranscriptome study using silica gel-dried leaf tissues produces an updated robust phylogeny of Ranunculaceae

The utility of transcriptome data in plant phylogenetics has gained popularity in recent years. However, because RNA degrades much more easily than DNA, the logistics of obtaining fresh tissues has become a major limiting factor for widely applying this method. Here, we used Ranunculaceae to test whether silica-dried plant tissues could be used for RNA extraction and subsequent phylogenomic studies. We sequenced 27 transcriptomes, 21 from silica gel-dried (SD-samples) and six from liquid nitrogen-preserved (LN-samples) leaf tissues, and downloaded 27 additional transcriptomes from GenBank. Our results showed that although the LN-samples produced slightly better reads than the SD-samples, there were no significant differences in RNA quality and quantity, assembled contig lengths and numbers, and BUSCO comparisons between two treatments. Using this data, we conducted phylogenomic analyses, including concatenated- and coalescent-based phylogenetic reconstruction, molecular dating, coalescent simulation, phylogenetic network estimation, and whole genome duplication (WGD) inference. The resulting phylogeny was consistent with previous studies with higher resolution and statistical support. The 11 core Ranunculaceae tribes grouped into two chromosome type clades (T- and R-types), with high support. Discordance among gene trees is likely due to hybridization and introgression, ancient genetic polymorphism and incomplete lineage sorting. Our results strongly support one ancient hybridization event within the R-type clade and three WGD events in Ranunculales. Evolution of the three Ranunculaceae chromosome types is likely not directly related to WGD events. By clearly resolving the Ranunculaceae phylogeny, we demonstrated that SD-samples can be used for RNA-seq and phylotranscriptomic studies of angiosperms.

Traditionally, systematic studies of the Ranunculaceae emphasized morphological 117 characters (especially floral traits and fruit types) (Prantl, 1887). Because of its complex 118 evolutionary history, the infrafamilial classifications of Ranunculaceae have remained 119 highly controversial (Prantl, 1887;Hutchinson, 1923;Takhtajan, 1980;Cronquist, 1981; 120 Tamura, 1995). Since the studies by Langlet (1927Langlet ( , 1932, cytological characters, 121 including chromosome size and base number, have become important for delimiting 122 subfamilies and tribes of the family (Janchen, 1949;Tamura, 1995). Since the 1990s,  Recently, phylogenomic studies using complete plastid genome data have greatly sequences are uniparentally inherited, they may not truly reflect phylogenetic 133 relationships among taxa (Greiner et al., 2015). Therefore, the tribal relationships, 134 especially within the core Ranunculaceae (defined below), still need further 135 clarifications using multiple single-copy nuclear markers.  (Gregory, 1941). The genome sizes between these two chromosome types vary 180- the exploration of genome-wide gene tree discordance is essential for understanding the 161 underlying evolutionary processes of Ranunculaceae. We also need a robust phylogeny 162 within which to investigate how the three chromosome types evolved and whether 163 ancient WGD events were important in that evolution. 164 Here, we extracted RNA from both liquid-nitrogen frozen and silica gel-dried leaf 165 tissues and conducted a phylotranscriptomic study of Ranunculaceae. We compared the 166 quality and quantity of extracted RNA between liquid-nitrogen and silica-preserved 167 samples, assessed whether silica-dried plant tissues can be used for phylogenomic study, 168 used multiple single-copy nuclear datasets to reconstruct the Ranunculaceae 169 phylogenetic framework, analyzed the discordance among the gene trees (nuclear as 170 well as cyto-nuclear discordance), and explored divergence times and WGD events in 171 Ranunculaceae and its closely related taxa.   (Table 1).  SCOGs each with no more than 10% missing taxa (SCOG-10MS published studies (Table S1) and the plastome phylogeny was inferred using the ML 282 method as described above.      Table S2). However, one-way analysis of variance (ANOVA) showed that 399 none of those differences was significant at P < 0.05. 400 We then counted and compared the obtained reads, bases, Q20, Q30, and GC  Table S2). We  Table S3).

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The results showed that, except for the mean value of NC, which is significantly lower 407 in SD-samples than in LN-samples, all the reference indices were not significantly 408 different between the two treatments.  (Table S4). Also, the average number of SCOGs was higher in SD-   Table 1) plus the liquid nitrogen-preserved samples (n = 6). One-way ANOVA, * P < 0.05. OD: optical density;

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Q30: Phred quality score 30 threshold. For detailed sampling information see Table 1 429  Table 1). The purple zone shows The coalescent-based phylogeny (Figure 3b)   Eocene) were characterized by high levels of gene tree discordance (Figure 4). 517 We also compared the Ranunculaceae nuclear and plastid phylogenies ( Figure 5) 518 and found that the plastid phylogeny, having low support values at several nodes, was 519 not better resolved than the nuclear phylogeny. Otherwise, the two phylogenies were 520 largely consistent. The only supported cyto-nuclear discordance is the position of 521 Adonineae (R-type). In the nuclear phylogeny, Adonieae is well-resolved in the R-type 522 clade, but in the plastid phylogeny it is sister to Isopyreae in the T-type clade.  Table   535 S1) 536 537 538 33

Coalescent simulations and phylogenetic network estimating 539
To explain the cyto-nuclear discordance, we compared conflicts among the the SD-samples were higher than six (average RIN = 7.5, Table S2). Although, most of 616 those SD-samples did not yield ≥ 30 μg total RNA, the concentration of the extracted 617 RNA was higher than 131.3 ng/μL (Table S2). Also, we found no significant differences 618 in those RNA indices between the SD and LN treatments (Table S2).

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Our results also showed that RNA does not decay as easily as previously thought, at 620 least not in our Ranunculales samples. All 21 SD-samples yielded usable total RNA and 621 among them, the SD-sample of Clematis leschenaultiana was stored in the -20 ℃ 622 freezer for more than two years (Table 1, Table S2). Although SD-samples generated

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In this study, we tested whether silica gel-dried plant tissues could be used for RNA Ranunculaceae phylogeny that had better statistical support than in previous studies.

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The 11 tribes of the core Ranunculaceae grouped into two clades corresponding to the