Divergence Dates of Demosponges

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Accepted Manuscript
Title: Early divergence dates of demosponges based on
mitogenomics and evaluated fossil calibrations
Author: Jun-Ye Ma Qun Yang
PII: S1871-174X(15)00016-5
DOI: http://dx.doi.org/doi:10.1016/j.palwor.2015.03.004
Reference: PALWOR 291
To appear in: Palaeoworld
Received date: 10-6-2014
Revised date: 27-10-2014
Accepted date: 21-3-2015
Please cite this article as: Ma, J.-Y., Yang, Q.,Early divergence dates of demosponges
based on mitogenomics and evaluated fossil calibrations, Palaeoworld (2015),
http://dx.doi.org/10.1016/j.palwor.2015.03.004
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http://dx.doi.org/doi:10.1016/j.palwor.2015.03.004
http://dx.doi.org/10.1016/j.palwor.2015.03.004
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Early divergence dates of demosponges based on mitogenomics and evaluated 
fossil calibrations
Jun-Ye Ma a, b, Qun Yang b*
a Department of Micropalaeontology, Nanjing Institute of Geology and Palaeontology, 
Chinese Academy of Sciences (CAS), Nanjing 210008, China
b State Key Laboratory of Palaeobiology and Stratigraphy (SKLPS), Nanjing Institute 
of Geology and Palaeontology, Chinese Academy of Sciences (CAS), Nanjing 210008, 
China
*Corresponding author. E-mail address: qunyang@nigpas.ac.cn
Abstract
Demosponges are among the most primitive biomineralized metazoans to appear first 
in the fossil record with hard skeletons; their confirmed earliest fossils are from the 
lower Cambrian rocks about 520 Ma, with putative demosponge biomarkers reported 
from 713~635 Ma sediments. In this study, we use mitogenomic data to approach the 
early divergence timescale of demosponges using relaxed molecular clock techniques 
and likelihood-evaluated fossil calibration strategies. We found that among various 
molecular dating models, the correlated rate model yielded time estimates of 
demosponges in this analysis which is most congruent with the fossil appearance 
dates of demosponges. Our dating analyses show that crown groups of Demospongiae 
appeared at about 704 (674~741) Ma, and the silicification in demosponges 
(divergence of spicular sponges) began about 633 (616~648) Ma indicating a gap of 
over 100 million years between the origin of silicification and their first unequivocal 
appearance of siliceous spicules in the fossil record (520~525 Ma); demosponges with 
tetraxon-type spicules (Tetractinellida) are dated here at about 514 (498~530) Ma, an 
estimate comparable with the earliest tetraxial megasclere fossil records (510~520 Ma, 
Ordian Age, middle Cambrian).
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Keywords: Demospongiae; Mitochondrial genes; Relaxed clock; Silicification 
origination
1. Introduction
Recent molecular studies have contributed substantially to the understanding of 
phylogenetic relationships among sponges; they also raised additional questions; e.g., 
Are the sponges monophyletic (Dohrmann et al., 2008; Philippe et al., 2009; Pick et 
al., 2010; Nosenko et al., 2013) or paraphyletic with Calcarea closer to Eumetazoan 
than other sponges (Borchiellini et al., 2001; Sperling et al., 2007, 2009; Erwin et al.,
2011; Mallatt et al., 2012)? Is the subclass Homoscleromorpha more closely related 
with Calcarea (Dohrmann et al., 2008; Philippe et al., 2009; Gazava et al., 2010; 
Erwin et al., 2011) or Eumetazoa (Sperling et al., 2007; Hejnol et al., 2009), forming 
the fourth class of Phylum Porifera (Gazave et al., 2012)? The phylogeny of sponges 
is critical to understanding the origin of metazoans.
Demospongiae is the most diverse group among Porifera; the earliest 
demosponge fossils with siliceous skeletons have been documented in the lower 
Cambrian (Rigby and Collins, 2004; Xiao et al., 2005), although questionable 
demosponge-related fossils were reported from the Neoproterozoic Doushantuo 
Formation (ca. 580 Ma) in South China (Li et al., 1998) and demosponge biomarkers 
were reported in Cryogenian strata in Oman (713~635 Ma and later sediments) 
(Bowring et al., 2007; Love et al., 2009), which was considered to be accordant with 
molecular dating (Sperling et al., 2010). Considering the huge gap of about 150~200 
million years between the earliest biomarkers and unquestionable fossil sponges 
(mainly archaeocyaths) found in the lower Cambrian Tommotian Age (540~535 Ma; 
Riding and Zhuravlev, 1995), Sperling et al. (2010) provided the following possible 
scenarios: 1) the first appearance of spicules signifies the origination of the 
demosponges during the early Cambrian, and the biomarkers found in Precambrian, 
while the molecular divergence dates are inaccurate and supposedly 
demosponge-related were likely related with non-demosponge organisms; 2) the 
Silicea, including the hexactinellids and the spicule-bearing demosponges, is a 
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monophyletic group, both derived from the aspiculate Keratosa possibly rooted deep 
in the Precambrian as molecular divergence estimates and biomarkers indicate; 3) due 
to taphonomic bias, siliceous sponges are not preserved in Precambrian rocks.
Mitochondrial genomes of Porifera, like Cnidaria, are characterized by 
exceptionally slow evolution, especially for Demospongiae and Homoscleromorpha 
(Lavrov, 2007; Wang and Lavrov, 2008), suitable for phylogenetic analyses and 
divergence dating (Lavrov et al., 2008; Erpenbeck et al., 2009), although some 
lineages experienced relatively faster rate such as Hexactinellida and Calcarea (Haen 
et al., 2007, 2014; Rosengarten et al., 2008; Lavrov et al., 2013). In this study, we 
designed an approach to estimate the origination of biomineralization in demosponges 
in a phylocrhonological framework on the basis of mitogenomics and carefully 
evaluated fossil calibration.
Because the fossil record is generally incomplete and biased among different 
organism groups, the early divergence time of an organism group can be inferred by 
applying molecular clock analysis in the context of the geological timescale and the 
early fossil record (Ayala et al., 1998; Peterson et al., 2004, 2008; Peterson and 
Butterfield, 2005; Yang et al., 2007). By phylochronological analysis, we trace the 
evolutionary history of major demosponge lineages on the basis of their spicules fossil 
record and molecular-based phylogenetic timescale. In this study, we adopted the 
molecular phylogeny of spiculate and aspiculate demosponges to identify the 
important evolutionary event in the history of early silicification in Demospongiae on 
the basis of the mitochondrial gene dataset.
2. Materials and method
Table 1 here
2.1. Sequence
Mitochondrial genomes of 24 demosponges, 2 homoscleromorphs, 11 
eumetazoans (including 9 cnidarians and 2 arthopods), 1 hexactinellid, and 1 outgroup 
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(choanoflagellate Monosiga brevicollis) were downloaded from Genbank for the 
phylogenetic analyses of this study (Table 1). Nucleotide sequences of the 12 coding 
genes are translated into protein sequences and each is individually aligned using the 
MEGA v4.0 (Tamura et al., 2007). Nucleotide sequences are subsequently aligned 
matching the animo acid (AA) sequence alignment. The 12 alignments are 
concatenated into a single matrix with 11526 nucleotide sites (Supplementary 
datafile).
Table 2 here
2.2. Fossil calibration dates
Based on their morphological characters and previous published data, nine
fossil/geological dates are used for calibration in our divergence time estimation (Fig. 
1, Table2). The root age prior, representing the divergence of 
Choanoflagellida-Metazoa, is set to 1000 Ma, which is a quite conservative maximum 
constraint as no true metazoan fossils (body, trace or biomarkers) have been 
confirmed to occur in older sediments; this date is also broadly compatible with 
previous phylochronological estimates, such as those in Peterson et al. (2008) and 
Sperling et al. (2010). In order to test the effects of root age prior, 1200 and 1500 Ma 
were also used in the dating analysis.
The earliest monaxial demosponge spicules are found in the lower Cambrian 
Atdabanian strata worldwide (Bengtson et al., 1990; Gruber and Reitner, 1991; 
Rozanov and Zhuravlev, 1992; Zhang and Pratt, 1994; Xiao et al., 2005; Carrera and 
Botting, 2008). According to Xiao et al. (2005), the lower Cambrian monaxial 
sponges found in Anhui and Yunnan, South China, may be dated to 520 to 535 Ma. 
For calibrating the demosponge tree in this study, we use the minimum age of 520 Ma 
as the first appearance of monaxial demosponges. Because biomineralized animals 
have not been found before the Ediacaran Period (Gaidos et al., 2008), the maximum 
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age control for the first appearance of sponge spicules may be set to the base of 
Ediacaran Period at 635 Ma. The monaxon spicule was considered to be an ancestral 
character for demosponges based on the fossil (Reid, 1970) and molecular 
(Borchiellini et al., 2004) data, suggesting that the appearance of monaxon spicules 
may be represented by the stem lineage (therefore, representing node A, Fig. 1) or the 
crown lineages (therefore, representing node B) of spiculate demosponges (G3+G4, 
Fig. 1). These placements indicate these monaxons represented the earliest 
unquestionable biomineralization event in demosponges. On the other hand, these 
monaxial spicules morphologically resemble that of the crown lineage of G3 and G4 
which made their first appearance in the fossil record synchronously during 
early–middle Cambrian (Rigby, 1986; Xiao et al., 2005), so they could also represent 
the crown lineage of G3 (node C) or G4 (node D). These alternative placements of the 
earliest monaxial sponge date as a calibration point will be subjected to a test via a 
likelihood method proposed by Pyron (2010) (see section 3.2. below).
The oldest tetraxon demosponges appeared in middle Cambrian (van Kempen, 
1985). This fossil provides a minimum age of about 515 Ma (Kruse, 2009) for a 
calibration of the crown-group Tetractinellida (Node M). Alternatively, we assigned 
the fossil to the stem lineage of Tetractinellida (Node N), considering the possibility 
that the tetraxon spicules could also represent a stem lineage, which will also be tested 
via the likelihood method of Pyron (2010).
Table 3 here
For other calibration points, we only provide the conservative age constraints for 
their stem lineages (Table 2) because of few sampled data, most of them having been
discussed and used in previous studies (Benton and Donoghue, 2007; Park et al., 2012;
shown in Fig. 1 and Table 2). All dates of calibration points were treated as softbound 
in this study.
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2.3. Evaluation of fossil calibration settings
The lineages with well-established first appearances could serve as reference 
nodes (checkpoints) to probabilistically assess divergence time estimates and fossil 
calibration placement in the tree of life (Pyron, 2010). To evaluate which calibration 
sets were more internally consistent than others (Table 3), we implemented the 
likelihood method of Pyron (2010) to assess the placement of the sponge calibration 
sets using program BEAST (Drummond and Rambaut, 2007), with the calibrations of 
I, J, K, L, S, and Y (cnidarians and arthropods) used as the checkpoints in this study 
(Fig. 1), their fossil records being set as priors to calculate the joint probability 
densities under different calibration sets. Two parameters of lognormal distribution, 
Ñt(i) and σt(i), were calculated based on the equation 2a,b in Pyron (2010, p. 187), 
and the probability distributions (P(Ñt(i)) of checkpoints were calculated based on the 
equation of 3 and 4 (Pyron, 2010, p. 187). Finally, the Akaike information criterion 
(AIC; Akaike, 1974) was used to compare the relative fit of different calibration sets.
2.4. Phylogenetic and molecular dating analyses
The program MrBayes 3 (Huelsenbeck and Ronquist, 2001; Ronquist and 
Huelsenbeck, 2003) and RaxML (Stamatakis, 2014) were used for phylogenetic 
reconstruction. A general time reversible (GTR) model with gamma-distributed rate 
heterogeneity and a proportion of invariant sites (GTR+I+G) inferred from Modeltest 
(Posada and Crandall, 1998) was used for the mitochondrial concatenated sequence. 
Based on previous studies (Benton and Donoghue, 2007; Park et al., 2012), the 
monophyletic constraints on the Eumetazoa and Cnidaria were forced in these 
analyses.
Program BEAST v1.6.1 (Drummond and Rambaut, 2007) was used to estimate 
the divergence times under the uncorrelated lognormal relaxed-clock model with a 
Yule process prior on speciation (Drummond et al., 2006). Fossil calibrations were 
enforced as lognormal priors on the divergence node in concern. Analyses were run 
for 20 million generations, with the first 5 million discarded after calculating burn-in. 
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Convergence and chain length were assessed by assuring that the estimated sample 
sizes for all parameters were greater than 100 (Drummond et al., 2006). Runs were 
replicated several times to ensure that global stationarity had been reached and that 
individual analyses were not merely converging on local optima.
Molecular dating analyses also were carried out via the program MCMCTREE 
(Yang, 2007). In MCMCTREE, the independent and auto-correlated rates models 
(clock = 2, 3) were used to specify the prior of rates under the GTR+I+G of 
substitution model.
In order to test the effects of heterogeneous evolutionary rate in codon sites of 
mtDNA, different partitioning strategies of mtDNA were used in molecular dating 
analyses, including unpartitioned, 1+2 partitioned (the 3rd codon sites separate from 
other two) and codon partitioned analyses.
3. Results
3.1. Phylogenetic relationships
Same topological trees of demosponge phylogeny with high support were 
obtained based on different strategies of sequence partition, and accord with previous 
studies (Borchiellini et al., 2004; Nichols, 2005; Lavrov et al., 2008), as shown in Fig.
1. Four strong supported major groups (G1-4) in Demospongiae were obtained: G1 is 
composed of keratose sponges (Dictyoceratida, Dendroceratida and Verticillitida), G2 
is the Myxospongiae including Verongida, Chondrosida, and Halisarcida, G3 is 
marine Haplosclerida, and G4 is the remaining demosponges including Halichondria, 
Hadromerida, Poecilosclerida, and freshwater sponges (Haposclerida: Spongillina: 
Ephydatia). The phylogenetic position of Hexactinellida is not resolved; however, its 
phylogenetic position in the analysis does not affect the phylogenetic relationships 
and molecular dates within Demospongiae (result not shown).
Fig. 1 here
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3.2. Test result of alternative placements of demosponge fossil calibrations (Table 
3)
Based on the fixed topology (Fig. 1), divergence times and the likelihood values 
of the checkpoints (i.e., the well-established reference fossil appearance dates) were 
estimated separately based on alternative calibration combination sets. As shown in 
Table 3, the alternative placements of the first appearance of monaxial demosponge 
spicules (for node A, B, C or D; Table 3) and of the tetraxon demosponges were 
evaluated against the checkpoints. The resulting likelihood and AIC values suggest 
that B for the first monaxial demospongeand M for the first tetraxon demosponge (-ln
= 36, AIC = 74) are the best calibration combination among all other probable 
calibration plans in these analyses.
3.2. Effects of heterogenic molecular rates
P- vs. ML-distance plots (Fig. 2) suggest that the whole mitochondrial coding 
sequences and codon positions 1 and 2 show no significant saturation, as compared 
with codon position 3 which may have experienced a degree of substantial saturation. 
Based on the correlated rate model, the molecular dates inferred from the 
unpartitioned dataset were different from those inferred from partitioned datasets, 
whereas no obvious difference is found between the different partitioned sets 
(including 3 codon partitions and 1+2 partitions) for most of the nodes; furthermore, 
the deletion of 3rd-codon did not yield outstanding effects on the results of divergence 
time estimation (Fig. 3). Likewise, the dating results for shallow nodes between 1+2 
partition and 3 codon-partition datasets were not obviously different (Fig. 3). 
Therefore, it appears that, in the demosponge mitogenomic dataset, the effect of 
heterogenic rates on divergence time estimation is not outstanding.
Fig. 2 here
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3.4. Phylochronology of demospongiae
Estimates of divergence times with 95% confidence intervals (CIs) for the 
demosponge tree were obtained on the basis of various data partitions and molecular 
models via BEAST and MCMCTREE programs as shown in Fig. 3 and Table 4. 
Generally speaking, our divergence time estimates for demosponges are compatible 
with previously published dates in Peterson et al. (2008) and Sperling et al. (2010). 
For example, the divergence time of crown-group Demospongiae is estimated here to 
be 704 (674~741) Ma (Node A, MCMC-correlated model of 3 codon partition 
dataset), compared to ca. 692 Ma of Sperling et al. (2010); the spiculate demosponges 
(G3+G4; node B) diverged about 633 (616~647) Ma in this analysis versus 623 Ma in 
Sperling et al. (2010). We also found that demosponges with tetraxon-type spicules 
(Tetractinellida) originated at about 514 (498~530) Ma, an estimates comparable with 
the earliest tetraxial megasclere fossil records from Australia (van Kempen, 1985) 
(510~520 Ma, Ordian Age, middle Cambrian). Therefore, it may be postulated that, 
although Precambrian fossil demosponges are extremely sporadic or questionable, 
demosponge ancestors are deeply rooted in Ediacaran Period (Neoproterozoic) or 
earlier (Fig. 4).
Fig. 3 here
It is noted that different dating models yielded slightly different dating results 
(Table 4). Although we are not assessing the relative advantages of these models to 
our datasets, we may compare the congruence of the molecular time estimates with 
the earliest appearance dates of related fossils. For example, our dating results from 
MCMCTREE (Table 4) indicate that divergence times of crown groups Verongida 
(node H) and Dictyoceratida (node G) are pointed to earlier than 522 Ma during or 
before early Cambrian, which are compatible with their fossil appearances (Verongida 
in Cambrian according to Rigby, 1986; Dictyoceratida in Ordovician according to 
Botting, 2005); while corresponding time estimates via BEAST models point to dates 
younger than the their fossil ages, which is considered unlikely. Unless the cited fossil 
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designations are in error, we suggest that the MCMCTREE estimates in this case 
should be adopted, especially the MCMC-correlated model that yielded narrower 
confidence intervals for divergence nodes G and H (Fig. 3, Table 4).
Table 4 here
Time estimates via MCMC-correlated model also show that Keratosa (G1, Node 
G) appeared most likely during Cambrian Period at about 524 (426~587) Ma, 
compatible with their fossil records found in early Paleozoic (Rigby, 1986; Botting, 
2005), rather than in late Paleozoic as suggested by estimates of Sperling et al. (2010) 
at 288 (194~377) Ma.
4. Discussions
4.1. Evaluation of fossil calibrations
Accurate time estimation depends on reliable fossil calibrations as well as 
adequate models of molecular evolution. In order to evaluate the uncertainties in 
placing two early demosponge fossil appearances (the oldest monaxial sponge 
spicules and the oldest tetraxial sponge spicules; Table 2 and Fig. 1) on the tree in 
concern, we applied the checkpoint method of Pyron (2010), on the basis of six fossil 
appearance dates (nodes L, I, J, K, Y, S; Table 3), to compare the maximum likelihood 
of the joint probability of the checkpoints. According to this evaluation (age estimate 
and likelihood results in Table 3), we identified the optimum placements of the fossil 
calibration points among alternative schemes. Although we chose B-M calibration 
placement combination (Table 3) as most likely the best strategy on the basis of AIC 
values, i.e., to treat the earliest monaxial sponge spicules as the calibration point for 
the divergence node between demosponge groups G3 and G4 (node B; see Fig. 1) and 
to treat the first appearance of tetraxial sponge spicules as the calibration point for the 
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divergence of crown group Tetractinellida (node M), we note that the time estimates 
in Table 3 bear high level of uncertainties (wide CIs), probably due to low resolution 
of the tree in the deep phylogeny.
4.2. Effects of prior parameters of root age and molecular rate
High heterogeneity of mitochondrial genomic evolutionary rate was found among 
different lineages, with significantly lower evolutionary rates in nonbilaterians 
including Demospongiae (except Keratosa), Homoscleromorpha, and Cnidaria (Fig. 
5A). Fig. 5B-D shows the rate variation of the first and secondary codon sites of 
mitochondrial protein-coding genes, from 0.001 to 0.04 substitution per site per 100 
million years (Fig. 5A, B), while the third codon sites have relative similar 
evolutionary rates among different lineages, 0.013 to 0.087 substitution per site per 
100 million years (Fig. 5D), most likely indicating substitution saturation at this 
codon position.
In order to evaluate the effects of rate heterogeneity, we applied rate priors at 1, 
10 and 100 times of the rate estimates (by PAML, Yang, 2007), and found no 
significant effects on the divergence times (Table 5). We also experimented with 
changing root age priors at 1200 and 1500 Ma, and found no significant changes in 
the divergence date estimates (Table 5), either.
Table 5 here
4.3. Biomineralization in demosponges
Although there have been reports of earliest siliceous spicules in Precambrian, 
e.g., the Doushantuo Formation of China (Li et al., 1998), the Kuibis Formation of 
Namibia (Reitner and Wörheide, 2002) and the Tsagaan Oloom Formation of 
Mongolia (Brasier et al., 1997), and putative demosponge biomarkers in 
Neoproterozoic Cryogenian strata, their relation with true sponge spicules or 
demosponges is in question (Zhou et al., 1998; Yin et al., 2001). Well-confirmed 
sponge spicules appear in the lower Cambrian strata about 520 Ma (Bengtson et al., 
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1990; Xiao et al., 2005; Carrera and Botting, 2008), and these earliest spicule fossils 
are thought to reflect the origination of demosponges in the fossil record (Brocks and 
Butterfield, 2009), likely representing the minimal time estimate for the origin of 
siliceous biomineralization.
Spongin, the fibrous skeleton composed of modified collagen, is considered to be 
the primitive character in demosponges (Borcheilline et al., 2004; Nichols, 2005; Ma 
and Yang, 2007). The divergent timescale established in this study indicates that the 
crown-group of Demospongiae appeared about 700 Ma (node A, Fig. 1, Table 4), as 
also suggested by Sperling et al. (2010), which apparently agrees with the Cryogenian 
biomarkers (Loveet al., 2009). However, this early date could represent the presence 
of sponges without siliceous biomineralization. Thus, combining phylochronological 
and fossil analyses, we suggest that siliceous biomineralization in demosponges 
originated during Neoproterozoic, most likely derived from non-siliceous 
demosponge ancestors. If this is the true scenario for the origin of siliceous 
biomineralization in demosponges, it would suggest that the origin of siliceous 
mineralization in hexactinellids is a parallel, nonhomologous event, which is probable 
because the two groups possess different spicular morphological characters (tetraxial 
and monoaxial or triaxial), secretion mechanisms (sclerosyncytium or not), and shape 
of axial filaments in cross section (quadrangonal or tri- and hexagonal); in addition, 
demosponges and hexactinellids involve different molecular mechanisms in 
silicification (silicatein-based vs. chitin- and collagen-based) (Ehrlich, 2011).
5. Conclusion
In this study, we used the relaxed molecular clock with tested fossil calibrations 
to estimate the early divergence time of demosponge crown groups and the 
silicification event in demosponges. Our dating result suggests that among all the 
models applied, the MCMCTREE-correlated model produced time estimations (cited 
in the following discussion) which are most congruent to the fossil appearance ages, 
as discussed under Phylochronology of Demospongiae. It is found that the crown 
group demosponges originated about 704 Ma (node A, Fig. 1, Table 4, 
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MCMCTREE-correlated), probably representing non-mineralized sponges that may 
be hardly preserved as body fossils; spiculate sponges (G3 and G4) diverged about 
633 Ma during the latest Neoproterozoic Ediacaran Period with the oldest confirmed 
fossil record in the lower Cambrian strata (about 520 Ma). This time interval 
(633~520 Ma) probably represents the time of origin of siliceous biomineralization in 
demosponges. Whether this apparent gap of 100+ Ma in fossil record is real, to be 
narrowed by future paleontological discoveries, or it may include over-estimation of 
time due to inadequacy in current molecular models and fossil calibration schemes, 
will be a subject for future investigations through interdisciplinary approaches.
Acknowledges
We thank Erik Sperling and Xuhua Xia for review with very constructive comments 
and expert suggestions that much helped to improve the manuscript. This work is 
partially supported by Chinese Academy of Sciences (KZCX2-YW-JC104), National 
Natural Science Foundation (40806055, 40572070 and 41472008), and State Key 
Laboratory of Palaeobiology and Stratigraphy at NIGPAS.
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Figure captions
Figure 1. Phylogenetic tree of Demospongiae based on nucleotide sequences of 12 
mitochondrial genes, for divergence time estimation. Bayesian posterior probabilities 
are all ≥ 0.96 (not shown). Node letters in black dots (I, J, K, L, S, Y) are nodes with 
fossil calibration dates; in black stars (A, B, C, D; M, N) are nodes with uncertain 
placements for fossil calibration dates. See Table 2 for details.
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entire genes sequences; B, first codon position only; C, second codon position only; D, 
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third codon position only.
Figure 3. Divergence time estimates based on different partitioning strategies and rate 
models (independent or correlated) via MCMCTREE (Yang, 2007) and BEAST 
(Drummond and Rambaut, 2007). Length of each vertical bar = 0.95 confidence 
interval (ML) or 0.95% highest posterior density interval (BI) with the mean (short 
horizontal bars).
Figure 4. Phylochronology of Demospongiae based on 12 protein-coding genes of 
mitochondrial genomes estimated via MCMCTREE correlated rate model. NP, 
Neoproterozoic; To., Tonian; Cr., Cryogenian; Ed., Ediacaran; Є, Cambrian; O, 
Ordovician; S, Silurian; D, Devonian; C, Carboniferous; P, Permian; T, Triassic; J, 
Jurassic; K, Cretaceous; Cz, Cenozoic.
Figure 5. ML tree of selected groups of Porifera, Cnidaria, and Arthropoda 
(choanoflagellate as outgroup) with branch length estimated based on the substitution 
model of GTRCAT+Γ(A); B-D, rate plots of mitochondrial genomes (all lineages in A) 
for 1st, 2nd, and 3rd codon positions, respectively.
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Table 1. Species and genomes used in this study. 
Species Classification GenBank Accession# 
Agelas schmidti Agelasida EU237475 
Ampphimedon compressa Haplosclerida/Haplosclerina EU237474 
Amphimendon queenslandica Haplosclerida/Haplosclerina NC_008944 
Aplysina fulva Verongida EU237476 
Axinella corrugate Halichondrida NC_006894 
Callyspongia plicifera Haplosclerida/Haplosclerina EU237477 
Chondrilla aff. nucula Chondrosida EU237478 
Cinachyrella kuekenthali Spirophorida EU237479 
Ectyopla siaferox Poecilosclerida/Microcionina EU237480 
Ephydatia muelleri Haplosclerida/Spongillina EU237481 
Geodia neptuni Astrophorida NC_006990 
Halisarca dujardini Halisarcida EU237483 
Hippospongia lachne Dictyoceratida EU237484 
Hyattella sinuosa Dictyoceratida JX535019 
Ircinia sp. Dictyoceratida KC510273 
Ircinia sp. Dictyoceratida KC510274 
Ircinia strobilina Dictyoceratida GQ337013 
Igernella notabillis Dendroceratida EU237485 
Iotrochota birotulata Poecilosclerida/Myxillina EU237486 
Ptilocaulis walpersi Halichondrosida EU237488 
Tethya actiraphidites Hadromerida NC_006991 
Topsentia ophiraphidites Halichondrida EU237482 
Oscarella carmela Homoscleromorpha NC_009090 
Plakortis angulospiculatus Homoscleromorpha EU237487 
Vaceletia sp. Verticillitida EU237489 
Xestospongia muta Haplosclerida/Petrosian EU237490 
Aphrocallistes vastus Hexactinellida; Hexactinosida NC_010769 
Astrangia sp. Cnidaria; Sclleractinia NC_008161 
Montastraeaan nularis Cnidaria; Sclleractinia NC_007224 
Acropora tenuis Cnidaria; Sclleractinia NC_003522 
Agaricia humilis Cnidaria; Sclleractinia NC_008160 
Pavona clavus Cnidaria; Sclleractinia NC_008165 
Savalia savaglia Cnidaria; Zoanthidea NC_008827 
Calicogorgia granulosa Cnidaria; Holaxonia GU047880 
Hydra oligactis Cnidaria; Hydroida NC_010214 
Aurelia aurita Cnidaria; Semaeostomeae NC_008446 
Anopheles darlingi Arthopoda; Diptera; Nematocera NC_014275 
Drosophila littoralis Arthopoda; Diptera; Brachycera NC_011596 
Monosiga brevicollis Choanoflagellida; Codonosigidae AF538053 
 
Table 1
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Table 2. Fossil calibrations used in this study. 
Earliest fossil representative 
Node position 
Minimum 
(Ma) 
Maximum 
(Ma) References 
Spiculatedemosponge: Choia? 
striata 
Possible 
positions: A or 
B or C or D 
520 635 Xiao et al., 2005 
Tetractinellida: Geodia Possible 
positions: M 
or N 
515 - van Kempen, 1985; 
Reitner and Wörheide, 
2002 
Agelasidae: Ropalospongia fluegeli L 270 - Finks et al., 2004 
Homoscleromorpha F 318 - Reitner and Wörheide, 
2002 
Pavona J 33 - Veron, 1995; Medina et 
al., 2006; Park et al., 
2012 
Acropora K 55 - Veron, 1995; Medina et 
al., 2006; Park et al., 
2012 
Astrangia I 70 - Veron, 1995; Medina et 
al., 2006; Park et al., 
2012 
Hexacorallia: Eolympia pediculata Y 540 635 Gaidos et al., 2008; 
Han et al., 2010; Park 
et al., 2012 
Diptera: Grauvogelia arzvilleriana S 235 295 Krzeminski et al., 1994; 
Peterson et al., 2004; 
Benton and Donoghue, 
2007 
 
Table 2
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Table 3. Time estimation and likelihood values of checkpoints for evaluation of alternative fossil calibration combinations. 
Checkpoints 
(Min, Max) (Ma) 
 
Alternative calibration combinations 
A+M B+M C+M D+M A+N B+N C+N D+N 
L (270, -) Time 
Estimates 
(95% CI) 
(Ma) 
141 (86, 229) 139 (37, 308) 204 (22, 403) 167 (27, 386) 180 (64, 363) 124 (8, 331) 116 (5, 343) 189 (59, 383) 
I (70, -) 49 (34, 98) 73 (16, 180) 72 (8, 202) 83 (23, 192) 53 (14, 153) 139 (32, 200) 56 (7, 239) 64 (10, 164) 
J (33, -) 19 (11, 38) 40 (8, 138) 27 (8, 70) 28 (7, 85) 25 (8, 76) 20 (4, 56) 20 (2, 61) 31 (8, 68) 
K (55, -) 55 (36, 101) 124 (34, 257) 88 (17, 248) 107 (32, 256) 96 (28, 208) 104 (26, 183) 95 (10, 244) 94 (31, 264) 
Y (540, 635) 608 (482, 710) 512 (185, 785) 488 (106, 736) 580 (357, 813) 561 (319, 768) 501 (129, 767) 537 (237, 817) 482 (265, 747) 
S (235, 295) 177 (112, 344) 245 (58, 686) 207 (9, 416) 193 (85, 522) 171 (58, 306) 211 (59, 460) 267 (105, 852) 208 (86, 339) 
L Likelihoo
d (-ln) 
6.12 6.1 6.35 6.2 6.3 6.1 6.03 6.3 
I 4.9 5.3 5.26 5.43 5 6.2 5.05 5.16 
J 4.07 4.7 4.3 4.35 4.25 4.1 4.1 4.43 
K 5.0 6.3 5.6 6 5.8 5.9 5.74 5.52 
Y 4.6 9.25 13.64 7.5 21.2 44 21.84 15 
S 26.7 4.3 12.35 17.7 30.9 10.7 3.7 11.67 
 AIC 102.9 74 95.3 98.45 148.05 156.4 95.16 98.68 
See Fig. 1 and Table 2 for information of the calibration points (A, B, C, D; M, N) and checkpoints: divergence nodes with fossil calibrations. CI: confidence interval. 
 
Table 3
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Table 4. Divergence time estimates (Ma) of major clades of Demospongiae. 
 
Node 
BEAST MCMCTREE-Independent MCMCTREE-Correlated 
3 partition 0 partition 3 partition 0 partition 3 partition 0 partition 
A 675 (584, 794) 657 (571, 778) 730 (682, 778) 722 (644, 836) 704 (674, 741) 670 (626, 725) 
B 608 (558, 683)606 (556, 678) 628 (604, 643) 623 (591, 641) 633 (616, 647) 623 (591, 641) 
C 483 (268, 612) 486 (286, 616) 566 (505, 607) 564 (476, 614) 604 (581, 622) 602 (567, 626) 
D 584 (543, 646) 582 (544, 634) 602 (576, 623) 600 (559, 628) 615 (599, 629) 610 (581, 629) 
E 504 (452, 649) 528 (453, 672) 689 (628, 724) 675 (558, 802) 680 (648, 714) 653 (606, 706) 
F 356 (312, 565) 354 (312, 479) 512 (355, 624) 477 (328, 627) 537 (338, 639) 498 (317, 634) 
G 316 (157, 455) 373 (190, 563) 524 (427, 597) 482 (304, 613) 524 (426, 587) 564 (486, 625) 
H 337 (85, 532) 399 (220, 590) 542 (414, 619) 522 (323, 630) 576 (453, 634) 594 (517, 644) 
M 524 (508, 549) 523 (508, 546) 516 (500, 534) 527 (509, 556) 514 (498, 530) 528 (510, 563) 
O 127 (28, 256) 167 (37, 329) 111 (62, 192) 166 (43, 414) 215 (067, 411) 383 (82, 566) 
P 75 (31, 178) 94 (43, 172) 63 (40, 93) 101 (53, 225) 121 (84, 154) 247 (153, 372) 
Q 46 (20, 75) 62 (22, 108) 41 (28, 59) 63 (29, 108) 68 (49, 74) 138 (88, 201) 
X 773 (643, 940) 761 (639, 932) 862 (786, 933) 811 (711, 929) 776 (737, 835) 712 (660, 777) 
Note: BEAST estimates are in the form of mean (95% highest posterior density); MCMCTREE: 
mean (95% credibility interval). 
 
Table 4
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Table 5. Divergence times (Ma) of major clades of Demospongiae based on alternative priors of root age, 
evolutionary rate, and placement of AN. 
 
Node Root age (BM) Evolutionary rate (BM) AN 
1200 Ma 1500 Ma ×10 ×100 
A 702 (672, 736) 702 (671, 735) 706 (673, 739) 703 (671, 735) 626 (602, 642) 
B 632 (615, 645) 631 (612, 645) 631 (613, 645) 631 (614, 645) 569 (548, 594) 
C 603 (581, 621) 604 (581, 622) 604 (582, 622) 604 (582, 622) 526 (496, 554) 
D 615 (599, 629) 615 (597, 629) 615 (598, 629) 615 (598, 629) 539 (522, 562) 
E 679 (648, 712) 680 (648, 712) 682 (649, 714) 681 (648, 712) 602 (575, 623) 
F 547 (343, 642) 543 (337, 641) 535 (335, 642) 543 (344, 642) 498 (361, 636) 
G 527 (418, 593) 532 (431, 596) 535 (432, 602) 527 (418, 596) 422 (333, 494) 
H 593 (514, 639) 593 (514, 640) 595 (512, 645) 595 (515, 643) 486 (386, 546) 
M 515 (499, 530) 515 (499, 531) 515 (499, 531) 515 (500, 532) 201 (062, 373) 
O 152 (043, 389) 159 (050, 393) 158 (042, 379) 146 (051, 381) 172 (059, 340) 
P 100 (032, 187) 116 (031, 224) 64 (028, 141) 128 (036, 213) 76 (030, 142) 
Q 56 (17, 106) 68 (17, 129) 34 (015, 075) 74 (19, 124) 40 (16, 72) 
X 770 (733, 817) 769 (730, 812) 773 (732, 818) 770 (728, 812) 731 (691, 775) 
Note, BM, AN: calibration placement in Table 3. 
 
Table 5
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Agelas 
Axinella
Iotrochota 
Tethya
Ectyopla 
Ptilocaulis 
Topsentia
Cinachyrella
Geodia
Ephydatia
A. compressa
A. queenslandica
Callyspongia
Xestospongia 
Aplysina
Chondrilla
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Figure 1
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Figure 2
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A: MCMCTREE-Correlated
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1st+2nd codon site partitions
Figure 3
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Figure 4
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Figure 5
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