Dating Suborder Polypodiineae (Eupolypods I) with Its Oldest Fossil ()
1. Introduction
Despite numerous phylogenetic and molecular dating studies that have continuously refined the time tree of ferns, there remain conflicting estimates regarding the diversification times of certain fern groups, particularly the Eupolypods (Table 1) [1]-[10]. Eupolypods, or eupolypod ferns, represent a highly diversified lineage and account for the majority of extant fern diversity, with nearly twice the number of species as all other non-eupolypod ferns combined (Figure 1) [2] [11]. Eupolypods are divided into two major clades: suborder Polypodiineae (eupolypods I) and suborder Aspleniineae (eupolypods II). The Polypodiineae, comprising 4665 species, is more species-rich than the Aspleniineae, which contains 3442 species (Figure 1) [2] [11]. Wang and Li [1] were the first to apply the TED method to estimate the divergence times within Eupolypods. However, their dating of Polypodiineae, the more diverse clade, has two significant limitations: 1) the absence of stem fossils, meaning only relatively recent fossils were used for Eupolypods I, and 2) an uneven fossil representation across lineages, particularly the lack of fossils for the highly diverse Dryopteridaceae.
Moreover, their study presents a controversial placement of Hypodematiaceae. While Wang and Li [1] proposed it as being located at the base of a clade within suborder Polypodiineae, recent studies suggest it forms a basal lineage within suborder Polypodiineae, representing an independent basal clade.
To address these issues, we incorporate four fossils morphologically attributed to Dryopteridaceae: the crown group fossils of Elaphoglossum miocenicum from the Miocene and Polystichum pacltovae from the Oligocene, along with the stem group fossils Cretacifilix fungiformis and Dryopterites beishanensis from the Cretaceous (Table 2) [5] [12]-[26]. We also add sampling extant taxa of Hypodematiaceae, Didymochlaenaceae, and Davalliaceae (Table 3) [27]-[32]. In this study, we continue to apply the integrative tip-dating approach that combines molecular and morphological data to re-estimate divergence times for eupolypods, the most diverse of all major lineages of ferns, in the light of the stratigraphic records. By including newly added taxa fossil and extant taxa, we aim to determine when Eupolypods, especially suborder Polypodiineae, began diversifying, thus enhancing our understanding of their evolutionary history.
Table 1. Summary of fern phylochrological analyses in previous studies and this study.
Study |
Phylegenetic depth |
Eupolypods/ Ferns sampled |
Characters used |
Dating methods |
Eupolypod/Fern Fossils used |
Ages of Total Aspleniineae |
Ages of Crown
Aspleniineae |
Ages of Total Polypodiineae |
Ages of Crown
Polypodiineae |
Wang and Li [1] |
Eupolypods |
214/218 |
Three plastid genes (rbcL, atpA, and atpB), 3841 bp |
Bayesian Inference (MrBayes version 3.2.7a) |
Tip calibrations 9/9 |
146.43 (122.8 - 170.25) Ma* |
64.64 (49.16 - 91.16) Ma* |
109.11 (80.78 - 141.85) Ma* |
109.11 (80.78 - 141.85) Ma* |
Nitta et al. [2] |
Ferns |
3311/5582 |
Plastid genes
12,716 bp |
Penalized likelihood (treePL) |
Node calibrations 16/51 |
~196 Ma** |
163.0 Ma |
~196 Ma** |
161.1 Ma |
Du et al. [3] |
Polypodiales |
162/214 |
Plastid 84
protein-coding genes and four rRNA genes,76 448 bp |
Penalized likelihood (treePL), Bayesian inference (BEAST), three root age
constraints |
Node calibrations 6/14 |
Between 144.22 and 200.3 Ma from six different dating schemes |
Between 138.86 and 155.9 Ma from six different dating schemes |
Between 144.22 and 200.3 Ma from six different dating schemes |
Between 134.76 and 151.3 Ma from six different dating schemes |
Qi et al. [4] |
Vascular plants: ferns, lycophytes, seed plants |
70/129 |
935, 501, 348, 267 and 146 nuclear gene sets from transcriptomes |
Penalized
likelihood (treePL) |
Node calibrations 4/17 |
Cretaceous*** |
Cretaceous*** |
Cretaceous*** |
Cretaceous*** |
Regalado
et al. [5] |
Eupolypods |
199/203 |
Three plastid genes (rbcL, atpA, and atpB), 3826 bp |
Rating dating, i.e., using the standard substitution rate for plastid DNA |
No calibrations 0/0 |
165.02 (108.87 247.74) Ma |
128.44 (85.16 192.02) Ma |
165.02 (108.87 247.74) Ma |
140.91 (93.96 211.16) Ma |
Testo and Sundue [6] |
Ferns, lycophytes |
2468/3973 |
Six chloroplast
markers (atpB, rbcL, rps4+rps4-trnS IGS, trnL+ trnL-trnF IGS), 8059 bp |
Penalized likelihood (treePL) |
Node calibrations 7/26 |
196.55 (194.82, 201.87) Ma |
185.79 (183.78, 196.08) Ma |
196.55 (194.82, 201.87) Ma |
160.94 (158.62, 172.49) Ma |
Rothfels
et al. [7] |
Ferns, seed plants |
31/73 |
25 nuclear loci
35 877 bp from
transcriptomes |
Bayesian methods (MrBayes
version 3.2.2) |
Second node
calibrations 2/12 |
112.42 (92.08, 133.13) Ma |
94.96 (82.01, 109.91) Ma |
112.42 (92.08, 133.13) Ma |
96.09 (80.83, 109.82) Ma |
Schuettpelz and Pryer [8] |
Leptosporangiate ferns |
242/400 |
Three plastid genes (rbcL, atpA, and atpB), >4000 bp |
Penalized likelihood in r8s version 1.71 |
Node calibrations 5/24 |
116.7 (105.6, 144.9) Ma |
103.1 (96.8, 126.5) Ma |
116.7 (105.6, 144.9) Ma |
98.9 (88.2, 127.9) Ma |
Pryer
et al. [9] |
Vascular plants: ferns, lycophytes, seed plants |
6/51 |
Four genes (plastid rbcL, atpB, rps4, and nuclear 18S rDNA), 4747 bp |
Penalized likelihood in r8s version 1.60 |
Node calibrations 1/21 |
75.49 ± 7.66 Ma |
/ |
75.49 ± 7.66 Ma |
/ |
Schneider
et al. [10] |
Ferns, seed plants |
19/42 |
Two plastid genes (rbcL, rps4),
~2500 bp |
Penalized likelihood in r8s version 1.60 |
Node calibrations 2/14 |
104.69 Ma |
94.52 Ma |
104.69 Ma |
93.61 Ma |
This study |
Eupolypods |
228/232 |
Three plastid genes (rbcL, atpA, and atpB), 3841 bp |
Bayesian Inference (MrBayes version 3.2.7a) |
Tip calibrations 13/13 |
171.62 (149.28, 190.34)**** |
134.52 (108.80, 171.37)**** |
157.03 (142.63, 176.95)**** |
157.03 (142.63, 176.95)**** |
*Ages are got from the dating scheme 3/72F (Table 4). **Ages are estimated based on Figure 3 of Nitta et al. [10]. ***Ages are estimated based on suppl. Fig. S16 of Qi et al. [6]. “/” No ages were provided. ****Ages are got from the dating scheme 7/82F (Table 4).
Figure 1. Diversity of Eupolypod Ferns (Order/Suborder). The 16 green bars represent species counts for 16 fern clades, while the blue bars indicate the relative proportions of each clade in relation to the total number of fern species. Numerical species counts and proportions are presented for two suborders of Eupolypods. Data and taxonomy are based on Nitta et al. [2] and the Pteridophyte Phylogeny Group I [11].
2. Analysis Methods
2.1. Sampling Taxa Set and Assembling Dataset
The data matrix of this study is assembled on the dataset from our previous work [1]. Four fossil taxa (Table 2) [5] [12]-[26] and ten extant taxa (Table 3) [27]-[32] are added to that combined dataset of Wang and Li [1]. The four newly added fossils, morphologically attributed to Dryopteridaceae, include crown group fossils Elaphoglossum miocenicum from the Miocene and Polystichum pacltovae from the Oligocene, as well as stem group fossils Cretacifilix fungiformis and Dryopterites beishanensis from the Cretaceous. All fossils included in this study, along with their respective information, are presented in Table 2 [5] [12] [26]. The ten additional extant species, which were not included in the dataset of Wang and Li [1], belong to Davalliaceae (two taxa), Didymochlaenaceae (three taxa), and Hypodematiaceae (five taxa). Their DNA sequences were obtained from GenBank, with accession numbers and references provided in Table 3 [27]-[32]. The data matrix consists of 15 morphological characters, and the DNA sequence data, totaling 3826 bp from three plastid genes (rbcL, atpA, and atpB), remains identical to that used by Wang and Li [1].
Table 2. Fossils included in this study and their information.
Fossil taxa* |
Selected references |
Geological age and locality |
Fossil ages (Ma), prior assignments, and affinities** |
Athyrium cretaceum
Chen and Meng |
Chen et al. [12], Deng and Chen [13], Li et al. [14] |
Neocomian (Hauterivian–Barremian), Lower Cretaceous, Liaoning, northeastern China |
Uniform (100.00, 145.00),
Aspleniineae |
Cretacifilix fungiformis
G. O. Poinar and R. Buckley |
Regalado et al. [15], Poinar and Buckley [16] |
Late Albian to earliest Cenomanian, Lower Cretaceous, Kachin State, northern Myanmar |
Fixed (100.00), Polypodiineae |
Davallia walkeri
Conran, U. Kaulfuss,
Bannister, Mildenhall and D. E. Lee |
Conran et al. [17] |
Early Miocene, Foulden Maar diatomite deposit, Otago, New Zealand |
Uniform (20.44, 23.03),
Polypodiineae |
Drynaria dimorpha
J. Y. Wu and B. N. Sun |
Wu et al. [18] |
Mangbang Formation, upper Pliocene, Yunnan Province, China |
Uniform (2.58, 3.60),
Polypodiineae |
Dryopterites beishanensis Ren and Sun |
Ren et al. [19] |
Early Cretaceous (Hauterivian-Barremian), Zhongkouzi Basin, Beishan area, Northwest China |
Uniform (129.40, 132.90), Polypodiineae |
Elaphoglossum miocenicum
Lóriga, A. R. Schmidt, R. C. Moran, K. Feldberg, H. Schneid and Heinrichs |
Lóriga et al. [20] |
Early Miocene (Burdigalian-Aquitanian), Dominincan Republic, Santiago area |
Uniform (15.97, 23.03),
Polypodiineae |
Holttumopteris burmensis L. Regalado, H. Schneid., M. Krings and Heinrichs |
Regalado et al. [5] |
Late Albian to earliest Cenomanian, Lower Cretaceous, Kachin State, northern Myanmar |
Fixed (100.00), Aspleniineae |
Onoclea sensibilis L. |
Pigg and Rothwell [21], Rothwell and Stockey [22] |
Paleocene, Paskapoo Formation, central Alberta, Canada |
Fixed (55.80), Aspleniineae |
Polystichum pacltovae Kvacek |
Kvacek and
Teodoridis [23] |
Oligocene, Děčín Formation of the České středohoří Mts, Czech Republic |
Uniform (23.03, 33.9),
Polypodiineae |
Protodrynaria takhtajanii Vikulin and Bobrov |
Vikulin and Bobrov [24] |
Paleogene flora of Tim in Russia |
Fixed (33.90), Polypodiineae |
Thelypteris sp.
Aline M. Homes et al. |
Homes et al. [25] |
Late Eocene Pikopiko Fossil Forest, southern New Zealand |
Fixed (34.40), Aspleniineae |
Woodwardia
changchangensis
Naugolnykh and Song |
Song et al. [26] |
Middle Eocene of the Changchang Basin, Hainan Island, South China |
Uniform (33.90, 56.00),
Aspleniineae |
Woodwardia virginica (L.) J. E. Smith |
Pigg and Rothwell [21] |
Middle Miocene Yakima Canyon flora of central Washington State, USA |
Fixed (15.60), Aspleniineae |
*Fossils in pink are newly added this study. **The fossils are ascribed to Aspleniineae or Polypodiineae based on originally described.
2.2. Set 12 Analytical Schemes for Bayesian Tip-Dating Analyses
Since the focus of this study is on the earliest divergence times of suborder Polypodiineae, or the total ages of Polypodiineae, we based our analysis on the dataset from Wang and Li [1]. The suborder Aspleniineae (eupolypods II) section of the dataset was kept unchanged, while we restructured the dataset by adding newly incorporated fossils belonging to suborder Polypodiineae. Each fossil or fossil group was combined with the constraint sets for family Dryopteridaceae or suborder Polypodiineae, resulting in 12 different analysis schemes (Table 4). Meanwhile, we used stepping-stone analysis [34] [35] to estimate marginal likelihoods for each model (Table 4). Our 12 tip dating analyses were performed in Mrbayes 3.2.7a [35]-[37] following the manuals downloaded from http://mrbayes.net. The FBD model was used as the tree prior, the ages of fossil terminals were provided as uniform or fixed priors with bounds equal to the limits of the estimated ages of their deposits (Table 2). We used a “diversity” setting in the sampling strategy since we strived to include as many eupolypods terminals as possible, and set the sample probability prior to 0.0178; this was done because we included 214 terminals, while the diversity of the eupolypods lineage is currently of about 6000 species and we expected it contains about that same number of undescribed species. Our analyses were run for 20 million generations, sampling every 5000 generations. Bayesian posterior probabilities (PP) were calculated for the majority rule (>50%) consensus tree of all sampled trees after discarding the first 25% as burn-in. Visualized trees and all the nodes were checked in FigTree v.1.4 [38].
Table 3. GenBank accession numbers and references for extant taxa newly added in tip-dating analyses for this study.
Family |
Representative species |
GenBank accession numbers |
Reference* |
rbcL |
atpA |
atpB |
Davalliaceae |
Davallia repens (L.f.) Kuhn |
MH392498 |
JF304018 |
MH392498 |
Ma et al. [27]; Kuo et al. [28] Ma et al. [27] |
D. multidentata Wall.
ex Hook |
MH392507 |
/ |
MH392507 |
Ma et al. [27] |
Didymochlaenaceae |
Didymochlaena alpina Li Bing Zhang & H. Shang |
OP595169 |
OP595261 |
OP595231 |
Shang et al. [29] |
D. amazonica Li Bing Zhang & H. Shang |
OP595168 |
OP595260 |
OP595230 |
Shang et al. [29] |
D. solomonensis Li Bing Zhang & H. Shang |
MW323310 |
MW323335 |
MW323322 |
Shang et al. [30] |
Hypodematiaceae |
Hypodematium glandulosum Ching ex K. H. Shing |
MZ957158 |
/ |
MZ957050 |
Fan et al. [31] |
H. hirsutum (Don) Ching |
MZ957200 |
/ |
MZ957085 |
Fan et al. [31] |
H. shingii Li Bing Zhang, X. P. Fan & X. F. Gao |
MZ957236 |
/ |
MZ957121 |
Fan et al. [31] |
Hypodematiaceae |
Leucostegia amplissima (Christ) C. W. Chen |
MZ957135 |
/ |
MZ957032 |
Fan et al. [31] |
L. immersa Wall. ex C. Presl |
AB232388 |
JF304009 |
MZ957036 |
Tsutsumi and Kato [32]; Kuo et al. [28] Fan et al. [31] |
*Three references are for three DNA accessions respectively. “/” No accessions were provided.
Table 4. Ages (in Ma) of total Eupolypods and its two subclades (median and 95% HPD) from tip dating under fossilized birth-death (FBD) priors*.
Number of Fossil/extant taxa of Polypodiineae, fossil newly added** |
Fossil
Constraints$ |
Marginal likelihood$ $ |
Ages of Total Eupolypods |
Ages of Total
Aspleniineae |
Ages of Crown Aspleniineae |
Ages of Total
Polypodiineae |
Ages of Crown Polypodiineae |
3/72, no newly added fossil |
Suborder |
−75046.58 |
152.15 (128.76, 173.80) |
145.29 (121.05, 165.88) |
65.04 (52.48, 86.28) |
102.44 (71.96, 136.22) |
102.44 (71.96, 136.22) |
3/72, no newly added fossil |
Family |
−73065.95 |
152.95 (131.24, 174.44) |
146.15 (135.31, 169.37) |
78.74 (50.14, 113.44) |
108.33
(81.63, 141.65) |
108.33 (81.63, 141.65) |
4/72, Cretacifilix |
Suborder |
−75046.58 |
156.57 (137.62, 179.14) |
149.02 (127.03, 174.71) |
47.01 (35.04, 91.13) |
123.90 (105.05, 147.08) |
99.01 (68.94, 116.70) |
4/72, Cretacifilix |
Family |
−73754.00 |
157.61 (137.49, 178.41) |
150.16 (126.87, 172.67) |
63.51 (42.41, 111.12) |
121.60 (101.88, 148.44) |
86.19 (69.47, 106.80) |
4/72, Dryopterites |
Suborder |
−75805.55 |
162.50 (147.15, 179.51) |
154.26 (133.72, 173.91) |
80.40 (57.86, 111.85) |
156.04 (139.78, 171.47) |
118.12 (88.58, 148.50) |
4/72, Dryopterites |
Family |
−73856.49 |
183.46 (171.22, 196.84) |
174.77 (154.40, 192.85)) |
79.23 (55.51, 122.62) |
168.02 (149.23, 188.79) |
168.02 (149.23, 188.79) |
5/72, Cretacifilix, Elaphoglossum |
Suborder |
−75106.40 |
156.40 (134.84, 177.11) |
147.55 (123.12, 170.25) |
74.87 (55.37, 100.49) |
114.81 (86.36, 145.38) |
114.81 (86.36, 145.38) |
5/72, Cretacifilix, Elaphoglossum |
Family |
−73525.47 |
158.01 (143.23, 175.36) |
150.96 (130.57, 170.04) |
81.38 (62.48, 109.26) |
129.33 (115. 28, 145,19) |
129.33 (115. 28, 145,19) |
5/80, Cretacifilix, Elaphoglossum |
Suborder |
−77760.32 |
153.20 (133.22, 171.62) |
146.74 (127.71, 166.00) |
62.92 (46.05, 90.17) |
118.34 (100.06, 147.65) |
97.95 (75.71, 124.61) |
5/80, Cretacifilix, Elaphoglossum |
Family |
−75491.86 |
158.73 (142.94, 176.80) |
150.80 (131.14, 169.85) |
75.39 (56.58, 108.10) |
126.24 (112.26, 142.02) |
126.24 (112.26, 142.02) |
7/82, Cretacifilix, Dryopterites,
Elaphoglossum, Polystichum |
Suborder |
−79006.37 |
162.42 (143.92, 180.91) |
154.15 (132.85, 176.14) |
154.15 (132.85, 176.14) |
152.88 (135.78, 173.45) |
107.1 (75.58, 134.99) |
7/82, Cretacifilix, Dryopterites,
Elaphoglossum, Polystichum |
Family |
−76503.86 |
181.78 (167.04, 195.63) |
171.62 (149.28, 190.34) |
134.52 (108.80, 171.37) |
157.03 (142.63, 176.95) |
157.03 (142.63, 176.95) |
*All Bayesian analyses were conducted using relaxed clock model TK02 (autocorrelated lognormal; Thorne and Kishino [33]) with an offset exponential tree age prior, as implemented in MrBayes3.2.7a [35], ages of Total Suborder Polypodiineae in Jurassic were in bold. **The four newly added fossils—Cretacifilix fungiformis, Dryopterites beishanensis, Elaphoglossum miocenicum, and Polystichum pacltovae—are listed by genus name only in the table. $Fossils were set constraint to family Dryopteridaceae or suborder Polypodiineae. $$Marginal likelihood (in natural log units, ln) were estimated using stepping-stone sampling (Xie et al. [34]).
![]()
Figure 2. Divergence times (median ages in Ma) of Eupolypods and its two subclades, estimated using a Bayesian tip-dating approach across 12 analytical schemes. The horizontal axis represents the 12 analytical schemes (see Table 3), where “S” and “F” respectively indicate whether fossils were constrained to the family Dryopteridaceae (indicated by F) or the suborder Polypodiineae (indicated by S).
3. Results
3.1. Comparison among Different Tip-Dating Schemes
The divergence times (median ages in Ma) for Eupolypods and its two subclades obtained from the 12 analytical schemes (Table 4, Figure 2) show significant variation. However, all results indicate that the diversification of total Eupolypods occurred in the Jurassic, ranging from 152.15 - 183.46 Ma. The diversification of total eupolypod II (suborder Aspleniineae) also took place in the Jurassic, between 145.29 - 174.77 Ma, while eupolypod I diversified later. Among the 12 analytical schemes (Table 4), only four show that the earliest divergence time of suborder Polypodiineae occurred in the Jurassic, ranging from 158.88 - 168.02 Ma, and all four of these schemes include the earliest fossil of suborder Polypodiineae, Dryopterites beishanensis Ren et Sun from Early Cretaceous (Hauterivian-Barremian) of Northwest China. Our analysis suggests that the earliest divergence time of suborder Polypodiineae is determined by its earliest fossil record and the combinations that include this fossil, rather than being strongly influenced by whether the fossil or fossil combination is assigned to the constraint sets of family Dryopteridaceae or suborder Polypodiineae. However, the estimated divergence times are slightly higher when the fossil or fossil combination is assigned to the family Dryopteridaceae compared to suborder Polypodiineae.
Based on our 12 analytical schemes, the marginal likelihoods (in natural log units, ln) estimated using stepping-stone sampling [34] consistently show higher values for the six analytical schemes where the constraint set is family, compared to the six schemes where the constraint set is suborder (Table 4). On the other hand, the values of marginal likelihoods decrease as the dataset size increases (Table 4).
(a)
(b)
Figure 3. Chronogram of eupolypod ferns constructed using a tip-dating approach and the Fossilized Birth-Death model within a Bayesian framework. Clades of the Suborder Polypodiineae (Eupolypods I) are shown in green, while those of the Suborder Aspleniineae (Eupolypods II) are shown in blue. The different systematic positions of Hypodematiaceae on chronogram (a) and (b) are highlighted with red dashed frames. (a) The chronogram of Eupolypods, modified from Wang and Li [1], includes only extant fern families and nine fossil taxa (in red), along with their relative extant genera. (b) The chronogram of Eupolypods I (Polypodiineae). Node bars represent 95% highest posterior density (HPD) intervals. Four newly added fossil taxa (in pink) are incorporated in this study. The three main lineages of eupolypods are indicated with their mean estimated ages, and the focal group of this study topic of this study, Suborder Polypodiineae, is highlighted with a red star.
3.2. Phylogenetic Positions of Dryopteridaceae Fossils
Our tip-dating tree (Figure 3(b)) shows that the phylogenetic placements of fossil taxa are mostly in accordance with their previous taxonomic attributions. Among the four newly added Dryopteridaceae fossils, the crown group fossils Elaphoglossum miocenicum [20] and Polystichum pacltovae [23], which are expected to belong to Dryopteridaceae, appear in the corresponding Dryopteridaceae lineage regardless of whether constraint sets are applied. Moreover, these two crown fossils have more concrete and accurate phylogenetic placements. For example, Elaphoglossum miocenicum [20] clusters with its extant Elaphoglossum relatives, and Polystichum pacltovae [23] clusters with its extant Cyrtomium-Polystichum relatives (Figure 3(b)). However, the situation is different for the stem group fossils Cretacifilix fungiformis [15] [16] and Dryopterites beishanensis [19] from the Cretaceous. The systematic positions of these stem group fossils in the tree depend on whether they are assigned to the constraint sets of family Dryopteridaceae or suborder Polypodiineae. If the two fossils are constrained to family Dryopteridaceae, they both occupy stem positions within Dryopteridaceae. If they are constrained to suborder Polypodiineae, they occupy stem positions within suborder Polypodiineae in our tip-dating tree (Figure 3(b)).
3.3. Phylogenetic Position of Hypodematiaceae
By incorporating ten additional extant taxa related to Hypodematiaceae (Table 2), our tip-dating analyses identify Didymochlaenaceae as the sister group to all other lineages within Polypodiineae, with Hypodematiaceae as the next earliest diverging lineage (Figure 3(b)). This suggests that Didymochlaenaceae represents the earliest divergence within Polypodiineae, followed by Hypodematiaceae. These findings contrast with those of Wang and Li [1], which placed Hypodematiaceae as the earliest diverging lineage within a clade of Polypodiineae, excluding Dryopteridaceae (Figure 3(a)).
4. Discussion
4.1. Diversification of Suborder Polypodiineae (Eupolypods I)
Polypodiineae (Eupolypods I) is the most species-rich lineage of ferns at the subordinal level (Figure 1), and analyses of its phylogenetic relationships and diversification times have been ongoing, resulting in considerable debate (Table 1). We will refrain from commenting on previous studies; instead, we focus on our findings in conjunction with our earlier analysis in Wang and Li [1] to explore the discrepancies in the diversification times of Polypodiineae. Among the current 12 analytical schemes (Table 4), only four indicate that the earliest divergence time of suborder Polypodiineae occurred in the Jurassic, ranging from 158.88 to 168.02 Ma (Table 4), and all four include the earliest fossil of suborder Polypodiineae, Dryopterites beishanensis [19]. Our study highlights the significant role of fossil abundance and taxonomic composition in molecular dating analyses. The results of our tip-dating analysis align with a few divergence time estimates derived from different molecular dating methods (node-dating [2] [3] [6] and rate-dating [5]). For the ages of crown Polypodiineae, all analytical schemes, except for 7/82S (Table 4, Figure 2), indicate that they are earlier than those of crown Aspleniineae, suggesting that crown Polypodiineae has a longer evolutionary history than crown Aspleniineae. This may explain its greater species richness compared to Aspleniineae and its more ecological opportunistic response to the establishment of complex, angiosperm-dominated ecosystems.
4.2. Ongoing Controversy on Phylogenetic Position of
Hypodematiaceae
While our results in this study clarify the systematic position of Hypodematiaceae, indicating that it, along with Didymochlaenaceae, represents the basal lineages of Polypodiineae, this finding is only one of four analytical outcomes regarding its phylogenetic placement. Earlier studies have placed Hypodematiaceae nested with Didymochlaenaceae [8] [39]. Later, analyses of multiple chloroplast genes resolved Didymochlaenaceae as sister to the rest of Eupolypods I, Hypodematiaceae following [6] [28] [40], a result consistent with our findings (Figure 2(b)). However, recent plastid phylogenomic [2] [3] and nuclear phylotranscriptomic [4] analyses, based on more extensive sampling, have identified Hypodematiaceae as the most basal family within Polypodiineae. It is quite uncommon for analyses by Wang and Li [1] and Regalado et al. [5] to show that Hypodematiaceae is not positioned at the base of Polypodiineae but rather at the base of a clade within Polypodiineae (Figure 2(a)). The primary cause of these topological discrepancies appears to be differences in dataset sizes. Further investigation with expanded datasets is necessary to assess how these topological differences influence divergence age estimates compared to previous studies.
5. Conclusion
This is our second attempt to combine morphological data from both extinct and extant taxa with DNA sequence data to estimate the diversification ages of eupolypods, the most species-rich fern lineage. The results further support previous hypotheses of Jurassic diversification across all eupolypods, indicating that both suborders, Polypodiineae and Aspleniineae, began diversifying during this period. However, estimates of Polypodiineae’s diversification ages rely heavily on its earliest fossil records, underscoring the fossil record’s critical role in calibrating clade origins. Tip-dating has once again proven to be an effective tool in a phylogenetic context. Ongoing research using tip-dating methods, along with new fossil discoveries, aims to shed more light on fern evolutionary history.
Acknowledgments
The authors wish to thank the editors and anonymous reviewers for constructive suggestions and comments that have improved the paper. This research was supported by the Basic Frontier Scientific Research Program of the Chinese Academy of Sciences (No. ZDBS-LY-DQC021-02). The authors deeply appreciate the support.