Tao Sang

Phylogeny and Biogeography of Paeonia (PAEONIACEAE)

DISSERTATION

chapter 2


COMPLEX RETICULATE EVOLUTION IN PEONIES REVEALED BY NUCLEAR AND CHLOROPLAST DNA SEQUENCES

ABSTRACT. Although speciation via hybridization is an important evolutionary mechanism in plants, accurate reconstruction of reticulate evolution has been remarkably challenging. Here complex reticulate evolution in peonies is reconstructed based on sequences of the internal transcribed-spacers (ITS) of nuclear ribosomal DNA and the chloroplast matK gene. In addition to the hybrid species detected by ITS sequence additivity, those ones that have fixed the paternal type of ITS sequences were identified by comparing their different positions on the ITS and matK phylogenies. The study reveals surprising complexity of reticulation in plant evolution that requires careful interpretation with independent gene phylogenies.

INTRODUCTION

Phylogenetic reconstructions are accumulating rapidly for all types of organisms due to developments in evolutionary theory, new sources of molecular data, sophisticated algorithms for data analysis (l; Reconstructing reticulate evolution, however, remains remarkably-challenging (2,3 Although application of molecular data has provided significant insights into this problem, our ability to reconstruct accurately reticulate evolution is still limited by a lack of explicit methods and an understanding of the complex dynamic of molecular evolution (3) . Because speciation via hybridization, particularly when associated with polyploidy, is an important evolutionary mechanism in plants (4) , effective approaches for reconstructing reticulate evolution need further exploration.

The common garden peonies (Paeonia,, Paeoniaceae), provide a favorable system for studying reticulate evolution. This genus comprises approximately 35 species of shrubs perennial herbs in three taxonomic sections occurring widely in several disjunct areas of the Northern Hemisphere (5). A number of hybridization events have been documented recently in the largest section, Paeonia, using ITS sequences (Fig. 3) (6). Full or partial nucleotide additivity from different parental sequences is found to have been maintained in the hybrid species. Partial additivity was considered to be a result of gradients of gene conversion following hybridization (6, 7) . To understand further these complex reticulate evolutionary patterns and their molecular consequences in section Paeonia, the coding region of the rapidly evolving chloroplast gene, matK. has been sequenced for 32 peony species (8).


MATERIALS AND METHODS

For most species, fresh leaves used as sources of DNA were collected from natural populations in California, Bulgaria, China, Greece, and Spain. The voucher specimens are deposited in OS. The remaining species were collected from The Royal Botanic Gardens, Kew (Table 1) . Methods of DNA extraction, amplification, and sequencing have been described (6) . PCR and sequencing primers are: three forward primers, (matKlF 5'-ACTGTATCGCACTATGTATCA-3') , (matK2F S'-GTTCACTAATTGTGAAACGT, (matK3F 5'-AAGATGCCTCTTCTTTGCAT-3'); three reverse primers, (matK2R 5'-GATCCGCTGTGATAATGAGA-3') , (matKIR 5'-TTCATGATTGGCCAGATCA-3'), (trnK3R S-GAACTAGTCGGATGGAGTAG-) . The matK sequences of peonies were aligned with those of tobacco and mustard (9) .

Variable nucleotide sites were analyzed by unweighted Wagner parsimony using PAUP version 3.1.1 (10). The shortest trees were searched with the Heuristic method, and character changes were interpreted with the ACCTRAN optimization. The section Oneapia is used as the outgroup for cladistic analysis (6, 11). Bootstrap analyses were carried out with 1000 replications using TBR Branch Swapping of the Heuristic search (12).

RESULTS AND DISCUSSION

The matK coding region of all peony species is 1491 bp long with 53 variable nucleotide sites found among them. Five equally most parsimonious trees, with length of 59, CI of 0.914, and RI of 0.972, were obtained. The strict consensus tree of these five trees has a length of 62, CI of 0.903, and RI of 0.945 (Fig. 5).

Phylogenies of section Paeonia obtained from ITS and matK sequences are concordant in certain respects and discrepant in others. A synthesis of both gene phylogenies leads to a more accurate species phylogeny that reflects both divergent and reticulate evolution (Fig. 6). Hybridization events in addition to those detected from ITS sequence additivity are suggested to account for discrepancy between the nuclear and organelle gene phylogenies (13).

One difference between the two phylogenies is the absence in the matK phylogeny of the two major basal clades of the ITS phylogeny (Figs. 3, 5). We suggest that hybridization occurred between the ancestor of the smaller clade (including MAI, JAP, and OBO) and an early evolutionary lineage of the larger clade on the ITS phylogeny, with the latter serving as the maternal parent. The hybrid obtained the maternally heredited chloroplast genuine (from the larger clade), and fixed the paternal type of ITS sequences (of the smaller clade) through gene conversion (6, 14, 15). Determination of the maternal parent in this hybridization event relies on comparison of the number of nucleotide substitutions supporting the major clades on the ITS and matK phylogenies. A comparable number of substitutions in ITS and matK are found to support section Oneapia and section Moutan. respectively, and thus would be expected to also support the same major clade of section Paeonia. Seven substitutions supporting the larger ITS clade of this section is the same as the number of substitutions supporting the entire section on the matK phylogeny, suggesting that the larger ITS clade served as the maternal parent in this hybridization.

The same explanation for fixing paternal ITS sequences following hybridization can account for the origin of hybrid species, P. xinjiangensis, P. japonica. P. obovata. P. wittmanniana,, and P. tenuifolia. which also have different positions between the two gene phylogenies (Figs. 3, 5, 6). Paeonia xinjiangensis forms a strongly supported sister group with P. veitchii on the ITS phylogeny, but its sister group relationship switches to P. lactiflora on the matK phylogeny, suggesting that P. xinjiangensis is a hybrid with P. veitchii as the paternal parent and P. lactiflora as the maternal parent. Likewise, P. japonica and P. obovata that are separated from four species (ARI, HUM, OFF, PAR) on the ITS phylogeny become the sister groups to these species on the matK phylogeny, indicating that P. japonica and P. obovata were derived from hybridization between the lineage containing these four species as the maternal parent and a paternal lineage that maintained the type of ITS sequences of the smaller clade in the ITS phylogeny (Fig. 6). By the same reasoning, the hybrid origin and parentage of P. wittmanniana is also postulated.

Paeonia tenuifolia is placed with other four species (ARI, HUM, OFF, and PAR) in a strongly supported clade (100% bootstrap value) on the ITS phylogeny, but forms its own lineage on the matK phylogeny. Since the species does not form a sister group with any other species on the matK phylogeny, its maternal parent may be an extinct basal lineage on the matK phylogeny.

While contrasting the discrepancies of the nuclear and organelle gene phylogenies identified a number of hybrid species which no longer maintain additivity in their ITS sequences, a comparison of the concordant portions of the gene phylogenies also provides additional insights into the species phylogeny. The matK phylogeny helps identify the maternal parents of P. emodi. P. sterniana, and P. banatica, species detected as hybrids by ITS sequence additivity (Fig. 5) . The remaining hybrid species identified by ITS sequence additivity, however, do not share nucleotide substitutions with either putative parents in the matK phylogeny. This may be due to the occurrence of hybridization prior to the accumulation of. .novel substitutions in maternal parents.

This phylogenetic study demonstrates that reticulate evolution has played an essential role in enhancing species diversity in peonies. Extensive hybridization is likely to have been triggered by drastic climatic changes in Europe during Pleistocene glaciation (6, 16). The analysis of both nuclear and chloroplast DNA sequences helped reconstruct the complex reticulate patterns within section Paeonia. However, this reconstruction (Fig. 6) may still be an underestimate of reticulate evolution in this group. If a hybrid species fixes the maternal parental ITS sequences, it will form the sister group to its maternal parent in both ITS and matK phylogenies and the hybridization will not be detected by this comparison. The ultimate solution of this problem may come from sequencing multiple nuclear genes and generating their phylogenies independently. If either maternal or paternal sequences of the genes are fixed in a hybrid species, comparisons of the multiple nuclear gene phylogenies as well as the organelle phylogeny should identify the hybrid species and its parentage.


intro

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