Abstract
During the past three decades, molecular taxonomy has made considerable changes in the systematic delimitations of several families in the order Ericales which were formed earlier based on morphology. For instance, the Pentaphylacaceae s.l. has been treated differently by both modern and traditional taxonomists. Modern molecular taxonomists constituted this family by combining the traditionally defined Pentaphylacaceae s.s. (Pentaphylax), Sladeniaceae s.s. (Sladenia), the subfamily Ternstroemioideae with 11 genera of Theaceae s.l. and the genus Ficalhoa. There are also treatments placing the genus Pentaphylax with Ternstroemioideae in Pentaphylacaceae and Ficalhoa with Sladenia in Sladeniaceae. Because most of these genera are poorly studied, investigations on all aspects are important to understand the phylogeny to settle the issues surrounding the treatment of the 14 genera in this family. In the present study, DNA sequences of nrITS and trnL-F genes from species of 11 genera from these 14 genera were generated and analyzed together with sequences from other closely related members of Ericales. The results suggested existence of four distinct lineages viz., Sladenia, Pentaphylax, and tribes Frezierieae (9 genera) and Ternstroemieae (2 genera). Further, it demonstrated that within the biggest lineage, Frezierieae, the Visnea remained sister to the clades Adinandra+Cleyera, Euryodendron+Symplococarpon, Freziera, and finally, Eurya. Based on the evidence, it can be concluded that Sladeniaceae and Pentaphylacaceae are very close to each other and the proposal of merging them into a mega family Pentaphylacaceae s.l. with four tribes, i.e., Sladenieae, Pentaphylaceae, Ternstroemieae, and Freziereae should be considered seriously.
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Introduction
The earth is filled with an overwhelming diversity of life. As biologists cataloged more and more of this life’s diversity, it was felt necessary to have a systematic means to deal with the vast biodiversity available on earth. Consequently, efforts have been made to categorizing them based on their size, forms, habitat, structure, anatomy, and biochemical and molecular features to interpret the relationships among the organisms (Bajpai et al. 2014; Feng et al. 2014). The transition from phenetics to phylogenies has seen sea changes in the concept of classification of organisms and during the past three decades, molecular taxonomy has made considerable changes in the systematic delimitations of several plant species, families, orders, and classes formed earlier based on morphology. The order Ericales, for example, now includes 21–24 families which were originally assigned by pre-molecular classification into Ericales, Ebenales, Primulales, Theales, Lecythidales, Diapensiales, and Sarraceniales (Cronquist 1981; Thorne 1992; Takhtajan 1997). Likewise, Pentaphylacaceae, a monogeneric family in Theales as traditionally defined (Cronquist 1981), are now placed in the Ericales and it included 12 genera according to Weitzman et al. (2004) and APG (2009) or 14 genera according to Schönenberger et al. (2005). Of late, a five-gene sequence analysis of Ericales (Anderberg et al. 2002) grouped Pentaphylax, Cleyera, Eurya, and Ternstroemia into a clade with very high Jackknife (JK) support (97%) and Ficalhoa and Sladenia (71% JK support) in a clade sister to it with weak support (68% JK support). This study has, thus, grouped, for the first time, Pentaphylacaceae s.s. (Pentaphylax), Sladeniaceae s.s. (Sladenia), and subfamily Ternstroemioideae (11 genera) and Ficalhoa from Theaceae into a single family. However, a revision of the angiosperm families based on evidence from molecular studies (Kubitzki 2004), classified these 14 genera into two families, Ficalhoa and Sladenia in Sladeniaceae and the other 12 genera in three tribes under Pentaphylacaceae viz., Pentaphylaceae with Pentaphylax, Ternstroemieae with Anneslea and Ternstroemia, and Frezierieae with Adinandra, Archboldiodendron, Balthsaria, Cleyera, Eurya, Euryodendron, Freziera, Symplococarpon, and Visnea (Stevens and Weitzman 2004; Weitzman et al. 2004). This treatment was further supported by evidences from morphological distinctions in stem anatomy (deep seated vs. superficial phellogen), type of inflorescence (cymose vs. racemose or solitary) and type of embryos (straight vs. curved). Later on, a more intensive analysis of Ericales with 11-gene sequences was carried out (Schönenberger et al. 2005), which also showed that the six genera i.e., Cleyera, Eurya, Ficalhoa, Pentaphylax, Sladenia, and Ternstroemia formed a single clade with a Bootstrapping (BS; Felsenstein 1985) value of 65% in parsimony analysis and a posterior probability (PP) of 1.0 in Bayesian analysis. Based on this study, Ficalhoa and Sladenia were placed in Pentaphylacaceae s.l. (herein Pentaphylacaceae s.l. referred to the family with Ficalhoa and Sladenia and Pentaphylaceae without Ficalhoa and Sladenia to avoid confusion). However, some other authors such as Angiosperm Phylogenetic Group (APG) (www.mobot.org/MOBOT/research/APweb) still placed Ficalhoa and Sladenia under Sladeniaceae sister to Pentaphylacaceae (with 12 genera) in the Ericales. The discrete treatments are probably because of the supports from the molecular taxonomy and also based on the differences on the anther and ovule development between Pentaphylacaceae and Sladeniaceae that Sladenia possessed the Monocot-type anther-wall formation and tetrasporic Adoxa-type embryo-sac formation (Li et al. 2003), which differed from the Basic- or Dicot-type anther-wall formation and monosporic, Polygonum-type embryo-sac formation in Adinandra, Cleyera, and Eurya of Pentaphylacaceae (Tsou 1995).
Thus, the expanded Pentaphylacaceae s.l. now comprise 14 genera distributed in Asia, Africa, the Pacifics, and Central and South America. Several of these genera are poorly known because they have only a single or few species, that occur in remote and limited area, and were not subjected to detailed investigation due to less economic importance. In order to resolve the confusion on the position of the member genera of this newly described family of Pentaphylacaceae s.l., present study was undertaken with both plastid and nuclear gene sequences to provide a better understanding on the intra-familial relationships among them as molecular data offer a large and essentially limitless set of characters to elucidate complex relationships among organisms.
Materials and Methods
DNA Sequencing Samples
Forty samples representing 36 species from 11 of the 14 genera of Pentaphylacaceae s.l. were included in this study (Table 1). The nuclear ribosomal ITS and chloroplast trnL-F sequences were analyzed. The DNA sequences newly generated in this study have HM series GenBank accession numbers (Table 1). DNA sequences of five other families in the Ericales, Fouquieriaceae, Lecythidaceae, Marcgraviaceae, Sapotaceae, and Theaceae, obtained from GenBank were also incorporated in the analysis. Sequences of Marcgraviaceae were used as the outgroup because this family is an early branch among these Ericalean families (Schönenberger et al. 2005). These sequences acquired from the GenBank are shown with their accession numbers in Figs. 1 and 2.
DNA Isolation and PCR Amplification
The protocols for genomic DNA extraction and PCR amplification with nrITS primers were previously described (Vijayan and Tsou 2008), as well as those for PCR amplification with trnL-trnF primers (Wu et al. 2007).
Sequence Alignment and Analysis
The complete sequences for both nrITS and trnL-trnF genes for each species were generated by sequencing both forward and reverse sequences. For each species, the sequencing was repeated twice or thrice to eliminate PCR artifacts. If any variation existed between forward and reverse sequences or between first and second results, the sequence was corrected manually by examining corresponding peaks in the chromatogram; if the differences still existed, the process was repeated starting from the DNA extraction. Multiple alignments were constructed with use of the PILEUP program of GCG version 8.1 (Genetic Computer Group 1994). Final alignment of the sequences was performed manually following Simmons (2004) in BioEdit version 5.0.9 (Hall 1999). The data matrixes are provided as online supplemental materials S1 and S2.
Phylogenetic Analysis
Phylogenetic analyses of maximum parsimony involved use of PAUP* b10 (Swofford 2003) and the Bayesian MCMC analysis (Yang and Rannala 1997) with MrBayes version 3.0b4 (Huelsenbeck and Ronquist 2001). In the parsimony analysis, all characters were considered unordered and weighted equally (Fitch and Ye 1991). Heuristic searches were performed with stepwise addition of random sequences with 1000 replicates. Tree bisection reconnection (TBR) branch swapping, MULTREE, collapse and steepest descent options, and ACCTRAN character optimizations were in effect. JK support (Farris et al. 1996) was estimated with 1000 replicates of heuristic search with 10 random additions of sequences per replicate. For Bayesian analysis, four Markov Chain Monte Carlo (MCMC) runs were performed for 1.5 million generations with four incrementally heated chains, starting from random trees and sampling 1 in every 100 generations. The evolutionary model that best fit the dataset for ITS1-5.8S-ITS2 was GTR+I+G and for trnL-trnF sequences GTR+G, determined by Akaike information criterion (AIC) (Akaike 1974) in Modeltest 3.6 (Posada and Crandall 1998). The first 200,000 generations were discarded as “burn in”, on the basis of log-likelihood plots. To estimate the posterior probability of the recovered branches, 50% majority-rule consensus trees were created from the remaining 13,000 trees. Analyses were repeated three times to confirm the results. To test for potential incongruity between the two datasets, we performed the incongruence length difference test (Farris et al. 1996; Cunningham 1997).
Results
nrITS Sequence Variation and Molecular Phylogeny
The length of the nrITS sequences varied from 621 bp in Adinandra formosa to 644 bp in Adinandra bockeana. The GC content of the sequences was the lowest (55%) for Freziera undulata and highest (66%) for Pentaphylax euryoides (Table 2). The length of the most parsimonious tree was 1187 steps (Fig. 1). The ensemble consistency index (Kluge and Farris 1969) based on informative characters was 0.50 and the ensemble retention index (Farris 1989) was 0.75. In the nrITS phylogenetic tree, among the ingroup families, Fouquieriaceae were sister to the remaining taxa, followed in order by clades of Lecythidaceae, Sapotaceae, Theaceae, and a large but weakly supported clade of Sladenia+Pentaphylacaceae (JK <50%, PP = 0.53). Within this large clade, the clade of Pentaphylax with two samples from a single species (JK = 100%, PP = 1.00) was sister to two other clades. One including Sladenia and tribe Ternstroemieae (Ternstroemia+Anneslea) is weakly supported (JK <50%, PP <0.5), but both of its subclades, Sladenia and Ternstroemieae, are highly supported (JK = 100%, PP = 1.00). The other clade including all samples of tribe Frezierieae is highly supported (JK = 93%, PP = 0.94). Within Frezierieae, Visnea mocanera is sister to the remaining taxa followed in order by four well-supported subclades, Cleyera+Adinandra (JK = 100%, PP = 1.00), Euryodendron+Symplococarpon (JK = 72%, PP = 0.99), then Freziera (JK = 100%, PP = 1.00) and Eurya (JK = 71%, PP = 0.98). Among the 12 genera of Pentaphylacaceae and Sladeniaceae, Euryodendron, Pentaphylax, and Visnea, each with a single species sampled, appear as singletons, whereas the remaining nine genera, each with 2–11 species sampled, form their respective subclades with strong support (JK = 98–100% for all but 71% for Eurya, PP = 0.98–1.00 for all) and without any mix-up with other genera.
trnL-trnF Sequence Variation and Molecular Phylogeny
The length of trnL-trnF sequences varied from 888 bp in Sladenia integrifolia to 918 bp in Pentaphylax euryoides. The GC content ranges from 34% in P. euryoides to 36% in Eurya subintegra (Table 2). The length of the most parsimonious tree (Fig. 2) is 392 steps. The ensemble consistency index calculated from the informative bases is 0.80 and the ensemble retention index 0.92. Among the five ingroup families in the most parsimonious tree, Lecythidaceae are sister to the other four families, followed in order by the clades of Fouquieriaceae, Theaceae, Sapotaceae, and finally a well-supported clade of Sladenia+Pentaphylacaceae (JK = 96, PP = 0.71) (Fig. 2). Within the clade of Sladenia+Pentaphylacaceae, four major groups can be recognized. The clade of Sladenia with three samples (JK = 100%, PP = 1.00) is sister to all taxa of Pentaphylacaceae, in which the clade of Pentaphylax (JK = 100%, PP = 1.00) is sister to the clades of Ternstroemieae (JK = 100%, PP = 1.00) and Frezierieae (JK = 100%, PP = 0.99). In the clade of Frezierieae, the seven genera are grouped into three parallel subclades. One including two singletons (Euryodendron, Visnea) and a big branch with 11 Eurya species receives high support (JK = 98%, PP = 0.93). The second subclade including three species of Freziera and two species of Symplococarpon shows poor support (JK <50%, PP <0.5). The third subclade with three species of Cleyera and six species of Adinandra is highly supported (JK = 100%, PP = 1.0). Among the 12 genera of Pentaphylacaceae, the genera Euryodendron, Pentaphylax, and Visnea, with a single species sampled, appear as singletons, and the remaining nine genera with multiple species sampled form their own well-supported clade (JK = 97–100%, PP = 0.67–1.00), except for Ternstroemia, with T. kwangtungensis sister to the remaining species of Anneslea and Ternstroemia.
Discussion
Pentaphylacaceae and Sladenia form a Monophyletic Clade
The 11 genera of Pentaphylacaceae s.l. formed a monophyletic group in both nrITS and trnL-trnF phylogenetic trees, and the supports were high in the latter (JK = 96%, PP = 0.71), but weak in the former (JK <50%, PP = 0.53). However, these results are in conformity with the earlier molecular phylogenetic studies of Anderberg et al. (2002) and Schönenberger et al. (2005) in suggesting that Sladenia and Pentaphylacaceae are closer enough to produce a monophyletic clade. As for the inter-familial relationships, both trees suggest that Theaceae and Sapotaceae are closer to Pentaphylacaceae s.l. than Lecythidaceae and Fouquieriaceae.
In the present study, because of the low incongruence between nrITS and trnL-trnF datasets was significant (p = 0.001), the data sets were not concatenated as it may have given high support values to wrong nodes if the internal branches are short as compared with the rate of gene tree coalescence (Degnan and Rosenberg 2006). Comparing the nrITS tree and the trnL-trnF tree, the former provides deeper interspecies differentiation, with Pentaphylax sister to all the remaining taxa of Pentaphylacaceae s.l., and Euryodendron joining Symplococarpon with good support (JK = 72%, PP = 0.99); on the other hand, the latter is weaker in interspecific differentiation, with Sladenia sister to all the remaining taxa and followed by Pentaphylax, and Euryodendron as an isolate. In general, the intra-tribal resolutions are clearer in the nrITS than trnL-trnF tree.
Molecular Phylogeny of Clades of Sladenia, Pentaphylax, Ternstroemieae and Frezierieae
In the study, the trnL-trnF sequences seem more conservative than the nrITS sequences. With the nrITS sequences, 327 parsimony-informative sites are present, whereas with trnL-trnF sequences, only 177 such sites are observed. Nonetheless, phylogenetic analyses with both sequences show four major lineages and the monophyly of each of the four lineages is highly supported by both analyses (JK = 93–100%, PP = 0.94–1). These four lineages are Sladenia, Pentaphylax, and tribes Frezierieae (Adinandra, Cleyera, Eurya, Euryodendron, Freziera, Symplococarpon, and Visnea) and Ternstroemieae (Anneslea and Ternstroemia). The circumscription of the tribes Frezierieae and Ternstroemieae as proposed by Melchior (1925) and later adopted by Keng (1962), Luna and Ochoterena (2004), and Weitzman et al. 2004 is well supported by our molecular phylogenetic trees.
Regarding the phylogenetic relationship among the four major lineages, in the trnL-trnF tree (Fig. 2) Sladenia are sister to the remaining taxa and Pentaphylax are sister to Frezierieae and Ternstroemieae. However, in the nrITS tree (Fig. 1), Pentaphylax are the earliest branch, followed by Sladenia, and the other two tribes. With respect to the reverse position of Pentaphylax and Sladenia in trnL-trnF and nrITS trees, the relationship revealed by the trnL-trnF tree could be more acceptable because it not only agrees with earlier reports with five genes and 11 genes, respectively (Anderberg et al. 2002; Schönenberger et al. 2005) but also of the support values.
For the intra-tribal relationships, only the tribe Frezierieae is relevant because the other three lineages contain only one or two genera. Earlier reports of Anderberg et al. (2002) and Schönenberger et al. (2005) did not provide details on the phylogeny of Frezierieae because of the limited sapling as only Cleyera and Eurya among the nine genera were included. In this study, Euryodendron from Southeast China, Visnea from the Canary Islands and other five genera out of the nine genera of Frezierieae were sampled. The intra-tribal relationships of Frezierieae are more resolved in the nrITS than trnL-trnF tree. In the former tree (Fig. 1), the clade of Visnea is sister to the remaining taxa, then followed by clades Adinandra+Cleyera, Euryodendron+Symplococarpon, Freziera, and finally, Eurya. The intimate relationship between Adinandra and Cleyera, first suggested by Merrill (1918) but questioned later by Keng (1962), is supported by an earlier embryological study (Tsou 1995) as well as the present analysis. The close relationship between Euryodendron and Symplococarpon is reported for the first time in this study. Our results also indicate a close relationship between Freziera and Eurya, which has been mentioned by Weitzman (1987) and Luna and Ochoterena (2004).
Conclusions
In this study, we sequenced nrITS and trnL-trnF segments of 36 species from 11 of the 14 genera of Pentaphylacaceae s.l. The monophyly of this family is supported, which is compatible with earlier molecular phylogenetic analyses of Anderberg et al. (2002) and Schönenberger et al. (2005). Our molecular phylogenetic trees recognize four lineages, Sladenia, Pentaphylax, and tribes Frezierieae and Ternstroemieae. Among these four major groups, Pentaphylax appear to be an intermediate taxa between Sladenia and Ternstroemieae-Freziereae. Among the biggest clade of Freziereae, the Visnea is placed sister to the remaining, which is followed by Adinandra+Cleyera, and Eurodendron+Symplococarpon, clades. Thus, it is concluded that Sladeniaceae and Pentaphylacaceae are very close to each other and the proposal of merging them into a single family can be given a serious thought based on other evidence from embryological and morphological traits as these two groups share several important common embryological characters such as bitegmic–tenuinucellate ovule, micropyle formed by the inner integument alone, Polygonum-type of embryo-sac formation, ovule vascular rays ended at chalaza. The mega family Pentaphylacaceae s.l. can be divided into four tribes, i.e., Sladenieae, Pentaphylaceae, Ternstroemieae, and Freziereae. The tribe Sladenieae should include Sladenia and Ficalhoa and the other three tribes can be retained as such as defined by Weitzman et al. (2004).
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Acknowledgments
The authors thank Arne Anderberg, Bruce Bartholomew, Jens Bittner, Dawn Frame, James Maxwell, Hua Peng, Hong Wang, Hua-Gu Ye, and Kwok L. Yip for help with field collections and/or providing samples; the curators of the herbaria of CAS, GH, NY, and S for providing specimens; the anonymous reviewers for giving useful comments; the National Science Council, Republic of China (Grant NSC 96-2312-B-001-012) and Academia Sinica, Republic of China, for financial support.
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Tsou, Ch., Li, L. & Vijayan, K. The Intra-familial Relationships of Pentaphylacaceae s.l. as Revealed by DNA Sequence Analysis. Biochem Genet 54, 270–282 (2016). https://doi.org/10.1007/s10528-016-9717-1
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DOI: https://doi.org/10.1007/s10528-016-9717-1