Reassessing the temporal evolution of orchids with new fossils and a Bayesian relaxed clock, with implications for the diversification of the rare South American genus Hoffmannseggella(Orchidaceae: Epidendroideae)
© Gustafsson et al; licensee BioMed Central Ltd. 2010
Received: 21 August 2009
Accepted: 14 June 2010
Published: 14 June 2010
The temporal origin and diversification of orchids (family Orchidaceae) has been subject to intense debate in the last decade. The description of the first reliable fossil in 2007 enabled a direct calibration of the orchid phylogeny, but little attention has been paid to the potential influence of dating methodology in obtaining reliable age estimates. Moreover, two new orchid fossils described in 2009 have not yet been incorporated in a molecular dating analysis. Here we compare the ages of major orchid clades estimated under two widely used methods, a Bayesian relaxed clock implemented in BEAST and Penalized Likelihood implemented in r8s. We then perform a new family-level analysis by integrating all 3 available fossils and using BEAST. To evaluate how the newly estimated ages may influence the evolutionary interpretation of a species-level phylogeny, we assess divergence times for the South American genus Hoffmannseggella (subfam. Epidendroideae), for which we present an almost complete phylogeny (40 out of 41 species sampled).
Our results provide additional support that all extant orchids shared a most recent common ancestor in the Late Cretaceous (~77 million years ago, Ma). However, we estimate the crown age of the five orchid subfamilies to be generally (~1-8 Ma) younger than previously calculated under the Penalized Likelihood algorithm and using a single internal fossil calibration. The crown age of Hoffmannseggella is estimated here at ~11 Ma, some 3 Ma more recently than estimated under Penalized Likelihood.
Contrary to recent suggestions that orchid diversification began in a period of global warming, our results place the onset of diversification of the largest orchid subfamilies (Orchidoideae and Epidendroideae) in a period of global cooling subsequent to the Early Eocene Climatic Optimum. The diversification of Hoffmannseggella appears even more correlated to late Tertiary climatic fluctuations than previously suggested. With the incorporation of new fossils in the orchid phylogeny and the use of a method that is arguably more adequate given the present data, our results represent the most up-to-date estimate of divergence times in orchids.
Orchidaceae is the largest and one of the ecologically and morphologically most diverse families of flowering plants . Several ages have been proposed for the origin of modern orchid lineages (i.e., their crown age), ranging from ~26 million years (Ma) , ~40 Ma , ~80 Ma  to as much as ~110 Ma . A correct time estimation is essential for our understanding of the mechanisms underlying the diversification of orchids, and could contribute to discern between alternative hypotheses of diversification - such as significant increases in speciation rates temporally correlated to climatic changes, tectonic events, or radiation of pollinators.
Many parameters have been identified to affect divergence time estimates in phylogenies, including taxon sampling, reliability, number and placement of internal calibration points, and dating method [6–16]. Until recently, molecular dating of the Orchidaceae has been challenging due the complete absence of reliable orchid fossils. The finding of a 15-20 Ma fossil of an extinct stingless bee (Proplebeia dominicana), covered with pollinia from an orchid species belonging to the subtribe Goodyerinae, finally allowed for temporal calibration of the family . Using this fossil as an internal calibration point, and departing from a phylogenetic tree obtained from the analysis of plastid DNA sequences (matK and rbcL), Ramirez et al.  estimated the origin of Orchidaceae at 76-84 Ma. These results supported an 'ancient' origin of orchids in the Late Cretaceous.
Although the study by Ramirez et al.  unquestionably constituted a milestone in orchid research, the large discrepancies in age estimates obtained in the last decade - some 80 Ma between the youngest  and oldest  crown ages - suggests that the matter is probably not completely settled. In a recent study in the family Begoniaceae, Goodall-Copestake et al. found that the second largest source of variance in age estimates (after availability and placement of internal calibration points) was derived from the choice of dating method employed, a result consistent with previous evaluations of empirical data [10, 11]. In particular, recent developments in molecular dating techniques have called into question the assumptions and algorithms implemented in Non-parametric Rate Smoothing [NPRS; ] and Penalized Likelihood [PL; ] - the two methods employed by Ramirez et al. . Whereas NPRS has been largely abandoned in favour of its successor PL [see discussion in ], both implemented in the software r8s , PL competes today in popularity with Bayesian dating  implemented in the software BEAST .
PL and BEAST operate in very different ways: i) PL requires a fixed phylogram as input, whereas BEAST samples topologies simultaneously as it calculates divergence times under a MCMC analysis, and allows the choice of several different priors and models; ii) PL assumes autocorrelation of rates within the phylogeny (i.e. that mutational rates are inherited, resulting in closely related taxa exhibiting similar evolutionary rates), whereas BEAST allows branches to vary in evolutionary rate; iii) in PL, nodes can be calibrated to be either fixed to a certain age, or constrained by a maximum or a minimal bound; whereas in BEAST, several additional alternatives are available for calibrating a node, because such calibrations represent age priors drawn from distributions of various shapes (e.g., normal, lognormal, exponential, or uniform). The methodological and conceptual differences between r8s, BEAST, and some other methods available today for molecular dating have been reviewed by several authors [12, 14, 22–24].
Although the methodology and assumptions implemented in each molecular dating method can be readily compared, our knowledge of how time estimates are influenced by the choice of method is still poor. For instance, Goodall-Copestake et al. obtained younger ages in the Begoniaceae using PL than using NPRS, but as these authors noted the inverse situation was found by Clement et al. [25, 26] on the same taxonomic group. According to Goodall-Copestake , this surprising discrepancy was probably caused by differences in density of sampled taxa and calibration points. Similarly, it may be very difficult to predict differences in age estimates using PL and BEAST: in the study by Goodall-Copestake , PL produced considerably younger ages than BEAST, whilst the opposite situation was found within family Caryophyllaceae . These results exemplify the potential influence of methodology on age estimations.
In this study we aim at reassessing the temporal origin and diversification of Orchidaceae, using the Bayesian uncorrelated relaxed molecular clock approach implemented in BEAST. In addition to choosing a different dating method, we conduct a new analysis on an expanded taxon sampling by adding two internal calibration points in the orchid phylogeny. We base these calibrations on fossil leaves described subsequent to the study by Ramirez et al.  from Early Miocene deposits of New Zealand, which were confidently assigned to genera Dendrobium and Earina . Then, to explore how the high-level age estimates obtained here may affect the evolutionary interpretation of a species-level orchid clade, we date the origin and diversification of the rare South American orchid genus Hoffmannseggella.
Hoffmannseggella belongs to the Epidendroideae, the largest subfamily within Orchidaceae, which comprises over half of all orchid species . The subfamily has been divided into 'lower' and 'higher' Epidendroids  and this latter clade includes the monophyletic subtribe Laeliinae, where Hoffmannseggella is nested . The genus is endemic to Brazil, where it is confined to the High Altitude Rocky Complexes (Brazilian Campos Rupestres and Campos de Altitude) of Minas Gerais, Rio de Janeiro, Espírito Santos and Bahia states. It comprises exclusively rupicolous species, i.e. growing among rocks . Adding to the 32 different species recognized by Chiron and Castro Neto , several new species have recently been described and today Hoffmannseggella comprises 41 species [31, 33–39]. Half of these are "micro-endemic" - known from a single natural population, and some only from the type collection. We have been able to obtain or generate sequences for all but a single species, thus reaching a 98% complete species sampling.
Crown group ages (in million of years) estimated for the family Orchidaceae, the five orchid subfamilies, the 'Higher Epidendroids' and the subtribe Goodyerinae, as compared to previous estimates using Penalized Likelihood .
Ramirez et al.: Penalized Likelihood
This study: BEAST, same data set as Ramirez et al.
This study: BEAST, with additional taxa and calibration points
Oldest & youngest mean ages (± standard deviations)
Median (95% HPD)
Median (95% HPD)
84 ± 6; 76 ± 5
49 ± 5; 45 ± 4
71 ± 5; 65 ± 4
37 ± 4; 34 ± 4
58 ± 5; 52 ± 4
59 ± 8; 51 ± 7
50 ± 7; 42 ± 6
38 ± 4; 34 ± 3
Evolution of major orchid clades
According to our new estimates based on the uncorrelated relaxed molecular clock approach, and incorporating three internal calibration points, extant Orchidaceae shared a MRCA in the Late Cretaceous, about 77 Ma (95% CI: 63 - 92 Ma). Except for Orchidoideae, median age estimates for the remaining orchid subfamilies are consistently younger in our study as compared to the youngest mean ages obtained by Ramirez et al.  (Table 1). Also in the first analysis where we used the same matrix as Ramirez et al.  and obtained precisely the same topology for the phylogenetic tree, median age estimates for the five orchid subfamilies are younger (Table 1).
Radiation of the genusHoffmannseggella
Our estimate for the crown age of Hoffmannseggella indicates a Late Miocene radiation for the genus (~11 Ma; Figure 3). This is some 3 Ma younger than the dates obtained using a Penalized Likelihood analysis over a sample of Bayesian phylograms [43, 44] (mean 14.2 Ma, 95% CI: 9.69 - 18.6).
Antonelli et al.  postulated a strong correlation between climate cooling following the Mid-Miocene Climatic Optimum and range expansion and diversification in Hoffmannseggella. This younger age estimate may imply an even stronger link than originally conceived, since the intensity of climatic oscillations augmented towards the end of the Tertiary [40, 41].
Reliability of results
The age estimation of genus Hoffmannseggella is based on a single, secondary calibration point (the crown group age of 'Higher Epidendroids' obtained in the high-level dating analysis), which should have a direct effect on all internal divergence times. However, the node age estimations between Cattleya and Masdevallia are strikingly similar in both analyses (25 Ma in the high-level Orchidaceae data set, CI:14 - 35; and 26.5 Ma in the Hoffmannseggella data set, CI: 14 - 41). This agreement provides some cross-validation for the use of the 'Higher Epidendroids' as a calibration point for the Hoffmannseggella data set.
As outlined in the Introduction, PL and BEAST make different evolutionary assumptions and have very different algorithms. This precludes categorical assertions on which of these methods yields the most correct divergence time estimates. One way to assess the autocorrelation assumption made by PL is to examine the covariance between parent and child branch in each phylogeny. This value is calculated by the software Tracer v1.4  from the log files of the MCMC analyses, and should be significantly positive when rates are autocorrelated, and near zero when there is no evidence of autocorrelation (see BEAST manual). For the Orchidaceae data set, this covariance had a mean of 0.10 and 95% confidence intervals ranging from -0.05 to 0.26. Although the covariance has been criticized as a weak measure of autocorrelation and more critical discussion on this subject is needed , the low covariance found in this study does not provide positive evidence for autocorrelation, thus favouring the BEAST results reported here.
Influence of internal fossil calibrations
Molecular dating techniques have greatly improved in the last years, offering novel opportunities to study the temporal evolution of taxa. However, it is essential to critically evaluate the impact of methodology and other parameters (taxon sampling, fossil calibrations, sequence regions) on the reliability of results. This study has shown that age estimations for orchid clades vary by several million years when using BEAST or Penalized Likelihood. While the addition of two new internal calibration points makes our study the most up-to-date estimate of the temporal evolution of orchids, additional studies may be required before a stable chronogram of this charismatic plant family is achieved.
Taxon sampling and genetic markers
Species list for the additional species included in the new Orchidaceae dataset, with GenBank accession numbers.
1. Agrostophyllum majus Hook. f.
2. Anigozanthos flavidus DC.
3. Dendrobium crystallinum Rchb. f.
4. Dendrobium kingianum Bidwill ex Lindl.
5. Dendrobium officinale Kimura & Migo
6. Earina autumnalis Hook. f.
7. Earina valida Rchb. f.
8. Elaeis oleifera (Kunth) Cortés
9. Musella lasiocarpa (Franch.) H.W.Li.
10. Nypa fruticans Wurmb
Species list for the Hoffmannseggella data set with voucher and GenBank accession numbers, indicating ITS sequences obtained from van den Berg  and the species sequenced for this study.
1. H. alvaroana F. E. L. Miranda
van den Berg C227 (ESA)
2. H. angereri Pabst
C223-Machado s. n. (ESA)
3. H. bahiensis Schultr.
C221-Machado s. n. (ESA)
4. H. blumenscheinii Pabst
C209-Machado s. n. (ESA)
5. H. bradei Pabst
C215-Machado s. n. (ESA)
6. H. brevicaulis (H. G. Jones) Withner
C208-Machado s. n. (ESA)
7. H. briegeri Blumensch. ex Pabst
Brieger Coll. 4612 (ESA)
8. H. cardimii Pabst & A. F. Mello
C205-Machado s. n. (ESA)
9. H. caulescens Lindl.
Brieger Coll. 1916 (ESA)
10. H. cinnabarina Batem. Ex Lindl
Brieger Coll. 1395 (ESA)
11. H. colnagoi Chiron & astro
A. L. S. Gustafsson 09 (GB)
12. H. conceicionensis Castro & ampacci
A. L. S. Gustafsson 14 (GB)
13. H. crispilabia (A. Rich. ex Rchb. f.) Warner
Brieger Coll. 4837 (ESA)
14. H. diamantinensis Castro & Marçal
A. L. S. Gustafsson 08 (GB)
15. H. duveenii Fowlie
C213-Machado s. n. (ESA)
16. H. endsfeldzii (Pabst) Castro & hiron
A. L. S. Gustafsson 06 (GB)
17. H. esalqueana Blumensch. Ex Pabst
Brieger Coll. 4980 (ESA)
18. H. flavasulina Miranda & acerda
A. L. S. Gustafsson 05 (GB)
19. H. fournieri (Cogniaux) Castro & hiron
A. L. S. Gustafsson 01 (GB)
20. H. ghillanyi Pabst
C214-Machado s. n. (ESA)
21. H. gloedeniana Hoehne
van den Berg C35(ESA)
22. H. gracilis (Pabst) Castro & hiron
A. L. S. Gustafsson 04 (GB)
23. H. itambana Pabst
C-Machado s. n. (ESA)
24. H. kautskyana Castro & hiron
A. L. S. Gustafsson 10 (GB)
25. H. kettieana Pabst
C210-Machado s. n. (ESA)
26. H. kleberi Miranda
A. L. S. Gustafsson 11 (GB)
27. H. liliputana Pabst
A. L. S. Gustafsson 03 (GB)
28. H. longipes Rchb. f.
Brieger Coll. 5183 (ESA)
29. H. mantiqueirae Pabst ex D. C. Zappi
van den Berg C224 (ESA)
30. H. milleri Blumensch. ex Pabst
Brieger Coll. 5070 (ESA)
31. H. mirandae Lacerda & astro
A. L. S. Gustafsson 07 (GB)
32. H. mixta Hoehne ex Ruschi
C220-Machado s. n. (ESA)
33. H. pabstii Miranda & acerdo
A. L. S. Gustafsson 12 (GB)
34. H. pfisteri Pabst & Senghas
van den Berg C226 (ESA)
35. H. presidentensis Camacci
not available for sequencing
36. H. reginae Pabst
C218-Machado s. n. (ESA)
37. H. rupestris Lindl.
van den Berg C33 (ESA)
38. H. sanguiloba Withner
C216-Machado s. n. (ESA)
39. H. tereticaulis Hoehne
van den Berg C222 (ESA)
40. H. verboonenii (Miranda) Castro & hiron
A. L. S. Gustafsson 13 (GB)
41. H. viridiflora Verola & Semir
A. L. S. Gustafsson 02 (GB)
42. Cattleya dowiana Batem
Chase O-282 (K)
(X) only rbcL
43. Cattleya bowringiana Veitch
Brieger Coll. 96 (ESA)
44. Dungsia kautskyi Pabst
van den Berg C286 (K spirit)
45. Masdevallia floribunda Lindl.
Chase O-296 (K)
46. Cymbidium kanran Makino
47. Oncidium ornithorhynchum Kunth
48. Gongora gratulabunda Rchb.f.
49. Stanhopea ecornuta Lem.
50. Zygopetalum maculatum (Kunth) Garay
Chase 160 (K)
51. Bifrenaria tyrianthina Rchb. f.
52. Maxillaria porrecta Lindl.
53. Lycaste cruenta Lindl.
DNA extraction, amplification and sequencing
Total genomic DNA was extracted exclusively from fresh plant material using a 2% CTAB protocol (adapted from ). Amplification was performed using PuReTaq™Ready-To-Go™PCR beads (Amersham Biosciences) for 25 μL reactions using 20 pmol of each primer. The two primers used were 'P17' (5'-CTACCGATTGAATGGTCCGGTGAA-3') and '26S-82R' (5'-TCCCGGTTCGCTCGCCGTTACTA3') of . PCR-products were analysed by electrophoresis using a 1% agarose gel and purified using QIAquick® PCR Purification Spin Columns (QIAGEN®). Quantification of the PCR-products was then done using GeneQuant II (Pharmacia Biotech).
Sequencing was performed using a CEQ™8000 (Genetic Analysis System, software 8.0, Beckham Coulter®) automated sequencer. Reactions were made with GenomeLab™DTCS-Quick Start Kit (Beckham Coulter®) according to manufacturer's instructions, except that 10 μL reactions were used, with 50 ng template and 1.6 pmol per reaction. The two primers used for sequencing ITS were 'P16b' (5'-CCAYTGAACCTTATCATTKAGAGGA-3') of  and 'ITS4R' (5'- TCCTCCGCTTATTGATATGC-3') of . Editing and compilation of the sequences was done using Sequencher™version 4.1 (Gene Codes Corporation).
Sequence alignment and dating analyses
The matrix for Orchidaceae  was analysed using a relaxed molecular clock approach with the software BEAST v1.5.3 . The input data were compiled in BEAUti v1.5.3 with the tree priors set as follows: i) age for the monophyletic subtribe Goodyerinae (corresponding to the age of the fossil orchid pollinia 15 - 20 Ma old; ): uniform prior distribution with a lower bound of 15 Ma and an upper bound of 120 Ma; ii) age for the root of the tree (corresponding to the oldest known fossil record for Asparagales; see discussion in ): normal prior distribution with mean 106.5 Ma and standard deviation of 8.21 (giving a 95% CI ranging from 93 - 120 Ma). The second family-level matrix (with the additional taxa) was analysed with the following tree priors: i) age for the monophyletic subtribe Goodyerinae set as above; ii) the two additional calibration points for Dendrobium and Earina set as uniform prior distributions with a lower bound of 20 Ma and an upper bound of 120 Ma (phylogenetic placement following ); iii) age for the root of the tree set to an uniform prior distribution with a lower bound of 93 Ma and an upper bound of 120 Ma. The upper (maximum) age constraint of 120 Ma for the calibrations above corresponds to the oldest known monocot fossils . We acknowledge that this constraint may be questionable since fossils generally provide minimal ages, but in absence of further evidence such upper bounds are technically advantageous for preventing the root of the tree to assume unreasonably old ages.
The ITS sequences generated here for 14 Hoffmannseggella species were completely re-aligned with those of van den Berg et al. , along with outgroup sequences from the 'Higher Epidendroids' downloaded from GenBank. The alignment was performed using MAFFT version 5.64 . The age for the root of the tree was set to a normal prior distribution with mean 39 Ma and standard deviation of 5.5 (giving a 95% CI ranging from c. 31 - 49 Ma) corresponding to the resulting age estimate for the 'Higher Epidendroids' in the second analysis of the Orchidaceae matrix (see under Results).
The Yule process was chosen as speciation process for all three data sets. The Akaike Information Criterion in MrModelTest v2.3  was used to choose the best-fitting evolutionary model for each sequence region (GTR+Γ+I for both partitions in the Orchidaceae data set and GTR+Γ for ITS in the Hoffmannseggella data set). Five separate runs were performed in BEAST with 20 million generations each. Log files were analysed with Tracer v1.5 , to assess convergence and confirm that the combined effective sample sizes for all parameters were larger than 200, in order to ensure that the MCMC chain had run long enough to get a valid estimate of the parameters . All resulting trees were then combined with LogCombiner v1.5.3, with a burn-in of 25%. A maximum credibility tree was then produced using TreeAnnotator v1.5.3 .
Bayesian evolutionary analysis by sampling trees
highest posterior density
million years (from mega-annum)
most recent common ancestor
We thank Santiago Ramirez for providing us with the molecular data set for Orchidaceae; Bengt Oxelman, Christian Parisod, Bente Eriksen, Anne-Cathrine Scheen, Vivian Aldén, Mats Töpel, Magnus Popp and Kaiser Schwarcz for their various help during this project; Andrew Rambaut for advice using BEAST; Maria do Rosário de Almeida Braga, Tim Moulton and Glauco Batalha Altman for their kind help in Petrópolis, Brazil; Hervé Sauquet, two anonymous reviewers and the associate editor for constructive criticism; and the Swedish Research Council (grant to Claes Persson) and the Carl Skottbergs foundation (travel grant to ALSG) for financial support.
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