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Palaeoworld 25 (2016) 303–317

Dating the origin of the major lineages of Branchiopoda

Xiao-Yan Sun a,∗, Xuhua Xia b, Qun Yang a,∗a LPS, Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, Nanjing 210008, China

b Department of Biology, University of Ottawa, Ontario K1N 6N5, Canada

Received 3 June 2014; received in revised form 30 October 2014; accepted 3 February 2015Available online 14 February 2015

bstract

Despite the well-established phylogeny and good fossil record of branchiopods, a consistent macro-evolutionary timescale for the group remainslusive. This study focuses on the early branchiopod divergence dates where fossil record is extremely fragmentary or missing. On the basis of aarge genomic dataset and carefully evaluated fossil calibration points, we assess the quality of the branchiopod fossil record by calibrating theree against well-established first occurrences, providing paleontological estimates of divergence times and completeness of their fossil record.he maximum age constraints were set using a quantitative approach of Marshall (2008). We tested the alternative placements of Yicaris andujicaris in the referred arthropod tree via the likelihood checkpoints method. Divergence dates were calculated using Bayesian relaxed molecular

lock and penalized likelihood methods. Our results show that the stem group of Branchiopoda is rooted in the late Neoproterozoic (563 ± 7 Ma);he crown-Branchiopoda diverged during middle Cambrian to Early Ordovician (478–512 Ma), likely representing the origin of the freshwateriota; the Phyllopoda clade diverged during Ordovician (448–480 Ma) and Diplostraca during Late Ordovician to early Silurian (430–457 Ma). Byvaluating the congruence between the observed times of appearance of clade in the fossil record and the results derived from molecular data, we

ound that the uncorrelated rate model gave more congruent results for shallower divergence events whereas the auto-correlated rate model givesore congruent results for deeper events.

2015 Elsevier B.V. and Nanjing Institute of Geology and Palaeontology, CAS. All rights reserved.

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eywords: Branchiopoda; Fossil calibrations; Relaxed molecular clock; Likelih

. Introduction

Branchiopods are one of the most diverse groups of crus-aceans with approximately 1200 described species in 28amilies (Adamowicz and Purvis, 2005), occurring in fresh-ater, brackish and marine habitats. The class Branchiopoda

s divided into two subclasses: Sarsostraca and PhyllopodaFig. 1). Sarsostraca contains an extinct order Lipostraca andhe single extant order Anostraca, with some 300 species in 8amilies. Phyllopoda is divided into two subgroups: Calmanos-raca (including the extant order Notostraca and the extinct

rder Kazacharthra) and Diplostraca (= Onychura, includingpinicaudata, Laevicaudata, Cyclestheria, and Cladocera). Thelam shrimps, referring to Spinicaudata, Laevicaudata, and

∗ Corresponding authors. Tel.: +86 25 8328 2103.E-mail addresses: xysun@nigpas.ac.cn (X.-Y. Sun), qunyang@nigpas.ac.cn

Q. Yang).

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ttp://dx.doi.org/10.1016/j.palwor.2015.02.003871-174X/© 2015 Elsevier B.V. and Nanjing Institute of Geology and Palaeontolog

heckpoints; Origin of freshwater biota

yclestherida, were originally included in a single order ‘Con-hostraca’, which later proved to be paraphyletic with respecto the Cladocera (Olesen, 1998; Taylor et al., 1999; Spears andbele, 2000; Braband et al., 2002; Swain and Taylor, 2003;eWard et al., 2006; Stenderup et al., 2006; Sun et al., 2006).ut ‘Conchostraca’ is still commonly used in paleontology. Theigher-level relationships within Branchiopoda based on theorphological characters have been partly confirmed by someolecular analyses, suggesting the monophyly of Phyllopoda,ladocera, and Diplostraca with Laevicaudata as a basal lineage

e.g., Fryer, 1987; Olesen, 1998, 2007, 2009; Negrea et al., 1999;un et al., 2006; Richter et al., 2007; Regier et al., 2010; Regiernd Zwick, 2011).

With the rich and well-studied fossil record, the earliestnown branchiopod Rehbachiella kinnekullensis (Fig. 1), from

he Orsten Lagerstätte of Cambrian Series 3 (Agnostus pisi-ormis Zone of Alum Shale) in Sweden, is a marine crustaceanWalossek, 1993, 1995), interpreted as a stem-group represen-ative of Branchiopoda (Schram and Koenemann, 2001; Olesen,

y, CAS. All rights reserved.

304 X.-Y. Sun et al. / Palaeoworld 25 (2016) 303–317

Fig. 1. Branchiopod phylogeny sensu Olesen (2009) superimposed on the known stratigraphic record. Geological dates from the IUGS International Stratigraphic Chart(Cohen et al., 2013). 1. Rehbachiella kinnekullensis (Walossek, 1993; Olesen, 2009); 2. Riley Lake taxa (Harvey et al., 2012); 3. Unnamed Silurian Species (Schram,1986); 4. Lepidocaris rhyniensis (Scourfield, 1926, 1940a,b; Walossek, 1993, 1995); 5. Palaeochirocephalus sp. (Shen and Huang, 2008); 6. Palaeochirocephalusrasnitsyni (Trussova, 1971); 7. Branchiopodites vectensis (Woodward, 1879); 8. Archaebranchinecta barstowensis (Belk and Schram, 2001); 9. Artemia salina(Djamali et al., 2010); 10. Castracollis wilsonae (Fayers and Trewin, 2003); 11. Notostracan indet (Garrouste et al., 2012); 12. Triops ornatus (Voigt et al., 2008);13. Notostracan trace fossil (Minter and Lucas, 2009); 14. Lepidurus occitaniacus (Gand et al., 1997); 15. Lepidurus stormbergensis (Townrow, 1966); 16 and 18.Prolynceus (Shen and Chen, 1984; Shen et al., 2006); 17. Paleolynceus (Tasch, 1956); 19. Cyclestherioides pintoi (Raymond, 1946); 20. Cyclestheria detykteica(Novojilov, 1959); 21. Cyclestheria sp. (Gallego and Breitkreuz, 1994); 22. Euestheria sparsa (Zhang et al., 1976); 23. E. atsuensis (Kobayashi, 1952); 24. Cyclestheriawyomingensis (Shen et al., 2006); 25. Ebullitiocaris oviformis (Anderson et al., 2004); 26. E. elatus (Womack et al., 2012); 27. Leptodorosida zherikhini (Kotov,2007); 28. Smirnovidaphnia smirnovi (Kotov, 2007); 29. Leposida ponomarenkoi (Kotov, 2007); 30. Archelatona zherikhini (Kotov and Korovchinsky, 2006). Boldlines indicate relatively higher diversity. Translucent pinkish box indicates the gap of some 68 million years between the earliest Cambrian marine and Devoniannon-marine branchiopod fossils. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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009). Branchiopod-type appendages, particularly the mandibu-ar gnathal edges (autapomorphies of Anostraca), occur inhallow marine deposits of the Cambrian Series 3 and Furongianeries (ca. 488–510 Ma; Harvey et al., 2012).

Non-marine branchiopods abundantly occur in the Devonian,ith representatives of all four extant orders. However, bran-

hiopod fossil record is missing from Early Ordovician to lateilurian when most of the deep divergences most likely haveccurred, highlighting an apparent gap of some 68 million yearsetween the Cambrian marine and Devonian non-marine fos-ils. It has been suggested that the branchiopod major groupsre rooted deep within the Silurian (Tasch, 1969; Negrea et al.,999). These paleontological inferences have been dismissed asnon-evidence’ due to the high preservation potential of Noto-traca and ‘Conchostraca’.

A number of attempts have been recently made in molec-lar dating of the arthropod tree, branchiopods involved (e.g.,ehm et al., 2011; Oakley et al., 2013; Wheat and Wahlberg,013). The reported time estimates for some crustacean lineagesppear to be significantly younger than corresponding fossilates, especially for the divergence time of crown-Branchiopodasee Fig. 2). Critical to molecular dating is the use of fossil infor-ation to calibrate the clock. The incompleteness of the fossil

ecord may cause underestimation of node ages in a phyloge-etic tree (Springer, 1995). Hug and Roger (2007) suggestedhat the best dating strategy was to maximize the number ofeliable and reasonably narrow calibration constraints, ratherhan to maximize the number of gene sequences included. Theotential sources of error in the calibration process generallynclude the incompleteness of the fossil record, erroneous fossilge estimates, and the placement of fossils on the tree (Forest,009).

The age of a lineage’s first appearance in the fossil records generally treated as a minimum constraint in calibration pro-edures; however, the maximum age constraints are difficult tostablish. Marshall (2008) developed a quantitative approach tostimate maximum age constraints of lineages on the basis ofdding a confidence interval onto the end point of the calibrationineage, which is adopted in this study.

The fragmentary nature of the fossil record and the lin-age extinction have important consequences for the accuratelacement of fossil calibration points. For example, two earlyambrian crustaceans, Yicaris dianensis (Zhang et al., 2007)nd Wujicaris muelleri (Zhang et al., 2010) both occurringn the Yu’anshan Formation (Eoredlichia-Wutingaspis Zone,unnan, China), are commonly used as calibration points inivergence dating of Pancrustacea (Oakley et al., 2013; Wheatnd Wahlberg, 2013). Yicaris, compared to branchiopods andephalocarids based on similarities in the endites on its pro-opodites, was assigned to the Entomostraca. Wujicaris, knownrom metanauplius larvae resembling those of copepods andarnacles, is also considered of entomostracan grade. But theubclass Entomostraca is considered as an outdated classifica-

ion that is consistently resolved in molecular phylogenies asolyphyletic (Regier et al., 2010; Regier and Zwick, 2011). Inurrent crustacean classification, the phylogenetic position oficaris and Wujicaris is uncertain. Thus, we used the likelihood

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d 25 (2016) 303–317 305

heckpoints method (Pyron, 2010) to assess alternate placementf Yicaris and Wujicaris in the arthropod tree of Regier et al.2010).

Despite well-established phylogenetic relationships and theirood fossil record, a consistent macroevolutionary time scale forranchiopods has remained elusive. This study focuses on thisime interval aiming to decode the deep time evolution whereossil record is extremely fragmentary or missing, using the pan-rustacean part of the phylogenomic dataset of Regier and Zwick2011). This time interval is also a critical period for the earlyvolution of the freshwater ecosystem.

This is the first attempt to approach the branchiopod phy-ochronology using a comprehensive molecular dataset andarefully devised fossil calibrations. We mainly carried out theollowing: (1) estimating the quality of the branchiopod fossilecord by calibrating this tree against the observed record ofrst occurrences; (2) estimating divergence time using relaxedolecular clock; (3) quantifying the match between the observed

imes of appearance of clade in the fossil record and the resultserived from molecular data.

. Paleontological time for branchiopod early evolution

Traditionally, Branchiopoda comprise four extant orders:nostraca, Notostraca, Cladocera, and ‘Conchostraca’. Becausenostraca has thin and flexible exoskeletons lacking a cara-ace, Cladocera is small and fragile, whereas ‘conchostracans’nd notostracans have hard exoskeletons well-preserved asossils, the branchiopod fossil records are taphonomicallyiased. Here we summarize general stratigraphic occur-ence of major branchiopod groups in order to assess itsompleteness/incompleteness in geological record and tovaluate its congruence with molecular divergence time esti-ates.All four extant branchiopod orders are known from the Paleo-

oic. The small carbonaceous branchiopod appendages recentlyiscovered from the Cambrian of Canada indicate that crown-ranchiopoda may have originated at least 488 Ma (Harvey andutterfield, 2008; Harvey et al., 2012). The ‘conchostracan’ fos-

il records indicate that the crown-Diplostraca at least originatedn the late Silurian (∼420 Ma) (Tasch, 1969) (see Fig. 1).

The anostracan fossil records are only sporadically known.s mentioned earlier, Anostraca-related appendages, assignable

o Sarsostraca (Fig. 1), occurred in the Cambrian Series 3nd Furongian Stage (Harvey and Butterfield, 2008; Harveyt al., 2012), at least 488 Ma. After a long gap in the fos-il record, occurred the oldest possible anostracan in Silurianerrestrial sediments of Indiana (Schram, 1986). The extinctrder Lipostraca, interpreted as the stem-Anostraca (Fig. 1),as found from the Devonian Rhynie Chert in Aberdeen-

hire, Scotland (ca. 411 Ma; Walossek, 1993; Schram andoenemann, 2001; Olesen, 2004, 2009). True fairy shrimps

Anostracina) first occurred in the Middle Jurassic Jiulong-

han Formation, Inner Mongolia, China (Shen and Huang,008) (ca. 165 Ma; Gao and Ren, 2006). Cretaceous, Paleogenend Neogene anostracans, assignable to extant genera, includealaeochirocephalus rasnitsyni from Lower Cretaceous of

306 X.-Y. Sun et al. / Palaeoworld 25 (2016) 303–317

Fig. 2. Phylogeny of Pan-crustacea and related groups plotted in a chronostratigraphic framework. Black boxes: reliable earliest fossil occurrences (see Appendix fordetails); blue lines: indicating divergence time deduced from fossil record; dashed lines: the divergence time from Wheat and Wahlberg (2013); red dots: divergencetime estimates significantly younger than corresponding fossil dates, especially for the crown-Crustacea. Clado-gram based on Regier et al. (2010) and geologicaldates from the IUGS International Stratigraphic Chart (Cohen et al., 2013). (For interpretation of the references to color in this figure legend, the reader is referredto the web version of this article.)

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astern Transbaikal, Russia (Trussova, 1971), Branchiopoditesectensis from Eocene of freshwater (Bembridge) Limestonef Gurnet Bay, Isle of Wight (Woodward, 1879; Rolfe,967), Archaebranchinecta barstowensis from Middle Miocenearstow Formation, California (Belk and Schram, 2001), and therine shrimps, Artemia salina, from Pleistocene (Lake Urmia,W Iran; Djamali et al., 2010).Calmanostraca contains Notostraca (extant) and

azacharthra (extinct). Notostraca has two extant genera,riops and Lepidurus, in the family Triopsidae. Castracollisilsonae from the Rhynie Chert (ca. 411 Ma; Parry et al., 2011)

s probably a stem-lineage Calmanostraca (Fayers and Trewin,003; Olesen, 2007, 2009). The first notostracan fossil is foundrom the upper Famennian strata (ca. 360 Ma; Garrouste et al.,012). The oldest confirmed Triops dated back to the latearboniferous (Voigt et al., 2008) and Lepidurus in the Permian

Gand et al., 1997). Kazacharthra is the closest known relativef the Notostraca, with fossils discovered from the Upperriassic to Lower Jurassic (Briggs et al., 1993; Olesen, 2009).ccording to Tasch (1969), the Calmanostraca diverged fromiplostraca during the Silurian.The earliest ‘Conchostraca’ fossils are Early Devonian spini-

audatans with 10 families occurring almost simultaneously,ollowed by 4 periods of rapid radiation in late Paleozoicnd Mesozoic. The rapid diversification of spinicaudatan faunaakes them biostratigraphically useful for subdivision and cor-

elation of non-marine successions (Kozur and Weems, 2010).aevicaudata as a basal lineage of Diplostraca first appeareduring the Middle Jurassic (Shen and Chen, 1984). Accordingo the diversification of Spinicaudata in the Devonian, ‘Con-hostraca’ was presumed to have originated in the late SilurianTasch, 1969; Negrea et al., 1999).

Cladoceromorpha includes Cladocera and Cyclestherida, asuggested by molecular and morphological cladistic analy-is (Crease and Taylor, 1998; Ax, 1999; Spears and Abele,000). The earliest known cladoceran Ebullitiocaris oviformisomes from the Early Devonian Rhynie Chert (ca. 411 Ma)Anderson et al., 2004). Fossils of two cladoceran suborders,nomopoda and Ctenopoda, are found from Mesozoic (Kotov

nd Korovchinsky, 2006; Kotov, 2007, 2009; Kotov and Taylor,011). Cyclestherida ranged from late Permian to Holocene,ith an extended gap in Jurassic and Cretaceous (Shen et al.,006).

. Data and methods

We analyzed the molecular data of Regier et al. (2010; alsoee Regier and Zwick, 2011), focusing on major pancrustaceanlades, including 68 single-copy nuclear protein-coding geneoci of 36 species (29 pancrustacean species, and 6 myriapodslus one onychophoran as outgroups). We realigned each of

he 68 gene fragments by alignments of coding DNA fromligned amino acid sequences using DAMBE (Xia and Xie,001). The reference topology of Regier et al. (2010; also seeegier and Zwick, 2011) was used for molecular dating in this

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d 25 (2016) 303–317 307

.1. Stratigraphic completeness estimate

Relative completeness index (RCI), gap excess ratio (GER)Benton, 1995, 2001), and the stratigraphic consistency indexSCI) (Huelsenbeck, 1994) were used to measure the fit of strati-raphic data to the current branchiopod topology (Fig. 1). Thiseasures the amount of missing range that must be added toake stratigraphic record fit the phylogeny. The geological ages

f the earliest fossil representative of each clade at the suborderevel included in this analysis are listed in Appendix.

.2. Molecular clock tests

To test whether evolutionary rate is constant across the wholehylogeny, we used a likelihood ratio test to compare the likeli-ood of a model that enforces a strict molecular clock to a modelith rates free to vary on each branch implemented in PAUP*

Swofford, 2003). For this global clock test, we assumed theest-fit model of molecular evolution as estimated in ModeltestPosada and Crandall, 1998).

We further tested the rates on each branch via PATHd8Britton et al., 2006) using Mean Path Length (MPL) analyses.

Finally, we tested two clade-specific molecular clockypotheses by multiple pair-wise relative rate tests, implementedn HyPhy (Kosakovsky Pond et al., 2005), assuming the best-fitodel of molecular evolution to be estimated with Modeltest.

.3. Fossil calibration age priors

Paleontological data of fossil Branchiopoda and their rela-ives were reviewed from available summaries and the originaliterature, together with hypotheses about their probable stemineages and evolutionary relationships (Appendix). The evolu-ionary tree combining cladograms with the fossil record wassed to calibrate molecular clock or to constrain estimates ofivergence times (Fig. 2).

.4. Maximum age bracket for divergence time estimates

The maximum age constraints were obtained by adding aonfidence interval onto the end point of the calibration lineage,stimated from the equation given in Marshall (2008):

Ac = FAcalnH√

(1 − C)

here FAc is the maximum age bracket, FAcal is the calibrationate (age of the oldest fossil in the lineage), C is the confidenceevel, and n is the number of lineages with a fossil record, eachnown from an average of fossil localities, which is set to 1 heres recommended by Marshall (2008).

.5. Molecular estimates of divergence times

We performed dating analyses using four different relaxedolecular clock methods, which have complementary advan-

ages and limitations. The Bayesian relaxed molecular methodsere implemented by the program MCMCTree v. 4.4e (Yang,

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007), Multidivtime (Thorne et al., 1998), and BEAST v1.5.4Drummond and Rambaut, 2007). Analyses with penalized like-ihood (PL) method were performed with the r8s softwareSanderson, 2003).

Bayesian relaxed molecular clocks, which assume rates ofolecular evolution are uncorrelated but lognormally distributed

mong lineages (Drummond et al., 2006), as implemented inEAST v1.5.4, were used for dating analyses. The Yule modelas applied to model cladogenesis in all analyses. The effective

ample sizes (ESS, >300) and convergence were summarizedsing Tracer (version 1.5) included in the BEAST programackage.

.6. Likelihood checkpoint test for uncertain fossilalibrations

In order to evaluate the alternative placement of Yicaris andujicaris, we used the likelihood checkpoints method (Pyron,

010). It is a posterior method for an objective assessment of theikelihood of inferred divergence times to evaluate the place-

ent of fossil constraints. Given the fossil constraints Ft, theikelihood of a chronogram T can be assessed by calculating theoint probability densities of the inferred ages for the likelihoodheckpoints, Nt(i):

(T |Ft) =V∏

i=1

P(Nt(i)).

Three fossil dates were used as likelihood checkpoints onabeled nodes (D, E, G) to evaluate the likelihood for the threelternative placements for Yicaris and Wujicaris (C1, C2, C3;ig. 3, Table 2). A lognormal distribution was assumed foralibration points and check points.

.7. Congruence measures of fossil and molecularivergence

On the basis of the paleontological and molecular estimates,e calculated a congruence metric of WSS (weighted sum of

quares; Tinn and Oakley, 2008), using equation

SS = 1 −{∑n

1(Fn − Mn)2/F2n

n

}

here n is each node with independent fossil and molecularivergence estimates. Fn is the fossil divergence estimate at node, and Mn is the molecular divergence estimate at node n.

. Results

.1. Stratigraphic completeness estimate

For the current branchiopod topology (Fig. 1), the duration

f standard range lengths (SRL) observed is 2196 Myr, of whichhost lineages implied at suborder level constitute approxi-ately 31.9% of the total duration. Despite a relatively high

ossil completeness estimate (RCI = 68.1%), the GER (=0.49) is

ld 25 (2016) 303–317

ot significant, indicating that the fossil record may be relativelyoor. The relatively poor SCI (0.4) indicates that the majority ofheir nodes are stratigraphically inconsistent.

.2. Molecular clock tests

A global molecular clock was rejected in a likelihoodatio test. The log likelihood assuming a molecular clockas −10 643.74 compared to the non-clock likelihood of10 564.38, resulting in a likelihood ratio statistic of 158.72

P < 0.005, df = 35).The null hypothesis of constant rate of evolution was rejected

or two a priori clade-specific hypotheses. Branchiopod Artemiaalina showed a significant slow rate of molecular evolution.

ost (28 of 34) of the possible 3-taxon relative rate compar-sons using Peripatus sp. as outgroup and Artemia salina as onengroup rejected the null hypothesis (P < 0.05). In every pair-ise comparison, Artemia salina’s branch was shorter than thether ingroups, indicating a relatively slow rate of evolution.f the six comparisons that did not show significantly slower

volution in Anostraca, four were comparisons with branchio-od species, suggesting that branchiopods also may have a slowvolutionary rate.

Remipedia showed a significantly elevated rate of molecu-ar evolution. Most (31 of 34) of possible 3-taxon relative rateomparisons using Peripatus sp. as outgroup and Speleonectesulumensis as one ingroup rejected the null hypothesis (P < 0.05).

The ucld.stdev parameter estimated by BEAST programan reflect the extent of molecular rate heterogeneity. Theean substitution rate is 2.01% per Myr, and the parameter

cld.stdev = 0.286 (ESS = 739) indicates a slight deviation fromhe constant molecular clock based on this data set.

.3. Fossil calibration points

The fossil record of Pancrustacea is extensive. To obtain cali-ration points for the node-dating method, we assigned fossils toarticular well-supported nodes of the tree of Regier et al. (2010)Fig. 2). This study adopted new fossil data (see Appendix) toive a total of 35 fossil points, including 33 within Pancrustaceand 2 myriapod outgroups. Fifteen of the fossil calibration pointsere selected via relative completeness and consistency evalua-

ion (analysis not included herein) (Table 1, Fig. 2). While otheralibration points are used as in previous studies (e.g., Rota-tabelli et al., 2013), the following 7 fossil calibration pointsre newly applied or updated.

Branchiopoda: Spinicaudata-Cladocera (min: 416 Ma);Node E in Fig. 3

Because the fossil record of Spinicaudata is one of the oldestamong Diplostraca, extending at least from the Lower Devo-nian and ‘Conchostraca’ was presumed to have originated

during the late Silurian (Tasch, 1969; Negrea et al., 1999), weadvocate a minimum constraint of 416 Ma as a conservativecalibration point for the divergence time of Spinicaudata-Cladocera.

X.-Y. Sun et al. / Palaeoworld 25 (2016) 303–317 309

Fig. 3. Fifteen fossil calibration points (green circles with letters in them; data in Table 1) on the tree. Alternative placements of Yicaris and Wujicaris labeled as C1,C2, C3. The nodes used as checkpoints are indicated with an asterisk. Numbered nodes (1–5): targets for time estimation in this study (see results in Table 3; Fig. 5).Outgroup (onychophoran species Peripatus) not shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web versionof this article.)

Table 1Selected fossil calibration points with estimated maximum dates.

Nodea Clade Min. calib. (Ma) Max. ageb (Ma)

A Palaeoptera-Neoptera 318 394B Zygentoma-Pterygota 396 490C Archaeognatha-Dicondylia 390 483D Collembola-Diplura 396 490E Cladocera-Spinicaudata 416 515F Hoplocarida-Peracarida 411 509G Eumalacostraca-Phyllocarida 485 601H Pedunculata-Sessilia 306 379I Copepoda-(Malacostraca, Thecostraca) 500 619J Myodocopa-Podocopa 478 592K Sarsielloidea-Cypridinidae 387 479L Pentastomida-Branchiura 500 619M Miracrustacea-Vericrustacea 520 644N Notostigmophora-Pleurostigmophora 420 520O Diplopoda 419 519

a Node letters used here are the same as in Fig. 3.b Maximum age estimated based on Marshall (2008) with confidence interval at 0.95.

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10 X.-Y. Sun et al. / Palae

Ostracoda: Myodocopa (Sarsielloidea-Cypridinoidea,min: 387 Ma), Podocopa (min: 478 Ma); Nodes K and J inFig. 3

The earliest occurrence of ostracods with calcified cara-paces is the Lower Ordovician (Tinn and Meidla, 2004). Theseostracods are multi-lobate forms such as the palaeocopidsNanopsis nanella (Moberg and Segerberg, 1906), podocopsElliptocyprites nonumbonatus (Tinn and Meidla, 2004) andthe binodicopid Kimsella (Salas et al., 2007).

The oldest myodocopids with preserved limbs and in situembryos are from the Upper Ordovician Katian Stage Lor-raine Group of New York State (ca. 450 Ma; Siveter et al.,2014). We advocate a minimum constraint of 478 Ma (youngestdate of Tremadocian) as the divergence time of Myodocopa-Podocopa, on the basis of the events that major diversificationof ostracods occurred after the Tremadocian Age (Williamset al., 2008).

Cypridinid-like myodocopids appeared in the Late Ordovi-cian (Tolmacheva et al., 2003). However, the typical pattern offan-like adductor muscle scar first appeared in the Devonianwith Eocypridina campbelli (Wilkinson et al., 2004). The fossilrecord of Sarsielloidea is scant. On the basis of the finding thatHamaroconcha kornickeri from the Eifelian (Middle Devo-nian) of southern Morocco is morphologically similar to thatof some Mesozoic and Cenozoic philomedidids (Sarsielloidea,Philomedidae) (Olempska and Belka, 2010), we advocate aminimum constraint of 387 Ma (youngest date of Eifelian)as calibration point for the Cypridinoidea-Sarsielloidea diver-gence.Malacostraca: Phyllocarida (min: 485 Ma), Hoplocarida-Peracarida (min: 411 Ma); Nodes G and F in Fig. 3

Phyllocarids are divided into two classes, the extinctArchaeostraca (Cambrian–Permian) and the extant Leptostraca(Permian–Recent). The oldest phyllocarid is Arenosicarisinflata from Elk Mound Group, Cambrian Furongian of Mosi-nee, Wisconsin (Collette and Hagadorn, 2010). Phyllocaridsdiversified substantially in the early Paleozoic with 83–93named species. Thus the minimum constraint for calibratingcrown-Malacostraca is 485 Ma (youngest date of Furongian).

Hoplocarida is represented by one extant order, the Stom-atopoda, and the extinct order, Aeschronectida (MiddlePennsylvanian). Stomatopoda includes Palaeostomatopoda(Late Devonian–Late Mississippian), the Archaeostom-atopodea (Middle Pennsylvanian–Upper Pennsylvanian), andthe Unipeltata (Upper Jurassic–Recent). The oldest hoplocaridis Pechoracaris aculicauda from the Early Devonian (Lochko-vian Age) of northern Russia (Dzik et al., 2004), setting 411 Ma(youngest date of Lochkovian Age) as the minimum constrainton the divergence of Hoplocarida and Peracarida.Lepas-Semibalanus (min: 306 Ma); Node H in Fig. 3

This represents the divergence of Pedunculata and Ses-silia within Cirripedia (Thecostraca). Although the earliestpossible cirripede has been reported from the Burgess Shale(middle Cambrian), its affiliation with cirripedes or even

arthropods is questionable (Briggs et al., 2005). The Silu-rian Cyprilepas holmi is interpreted to be phylogeneticallybetween the cirripede stem and the Thoracica (Høeg et al.,

rNc

ld 25 (2016) 303–317

2009), whereas the Silurian Ramphoverritor reduncus is con-sidered as sister-group to all extant Cirripedia (Høeg et al.,2009). The oldest undisputed crown-Cirripedia is the peduncu-latan Illilepas damrowi (Schram, 1975) from the Carboniferous(359–259 Ma) and Praelepas jaworski from the Middle Penn-sylvanian (311–306 Ma) (Glenner et al., 1995; Høeg et al.,1999). The earliest fossils of Sessilia have been reported fromJurassic and Cretaceous. Thus we set the minimum constraintfor calibrating the Lepas-Semibalanus divergence at 306 Ma(the youngest date of Pennsylvanian).Altocrustacea-Oligostraca: the placement of Yicaris andWujicaris (min: 520 Ma); Node M (C1) in Fig. 3

Wujicaris muelleri and Yicaris dianensis were discoveredfrom an Orsten-type Konservat-Lagerstätte of the lower Cam-brian, Southwest China (Zhang et al., 2007, 2010). Accordingto Zhang et al. (2010), the current species occurs in theEoredlichia-Wutingapsis trilobite Zone, belonging to Cam-brian Stage 3, dated 515–521 Ma.

Yicaris or Wujicaris can be assigned to three different nodesin the phylogeny of Regier et al. (2010) (Fig. 2). The resultsfrom the likelihood checkpoints (Table 2) indicate a signifi-cantly better fit of the C1 calibration set. Thus, the minimumcalibration point for divergence node M is also set at 515 Ma.

.4. Divergence time estimates

The phylogeny obtained with the software BEAST resembleshat of Regier et al. (2010). Fourteen external and one internalossil calibration points were used to estimate the divergenceates within the Branchiopoda. The divergence time estimatesith Multidivtime (MLT), r8s, MCMCTree, based on the super-atrix of 62 genes and multiple calibrations dataset as discussed

bove, are generally concordant, overlapping within the 95%I limits (Table 3), although progressively older time estimatesith broader confidence intervals toward the deeper divergencesroduced by BEAST were observed when compared to resultsf MCMCTree, r8s and MLT (Fig. 4).

. Discussion

.1. Congruence between paleontological and molecularime scales for branchiopod evolution

The application of seven relaxed molecular clock methods inating early divergences of branchiopods yields strikingly con-ruent time scales except for estimates via BEAST (see remarksbove in Section 4.4.). On the basis of the paleontological andolecular estimates, we calculated a metric WSS (weighted sum

f squares, Tinn and Oakley, 2008) to measure the congruenceetween paleontological and molecular time estimates (Table 3).

Our congruence analyses of the molecular estimates obtainedgainst the fossil dates of branchiopods suggest that the MCM-

elatively more congruent with the fossil records (see Table 3).evertheless, congruence measures for individual nodes indi-

ate that for deeper divergences (nodes 5, 3, 2, Table 3; Fig. 5),

X.-Y. Sun et al. / Palaeoworld 25 (2016) 303–317 311

Table 2Parameter and likelihood values for checkpoints used to assess fossil calibrations.

Node of divergence Distribution Mean (SD) −Ln

C1 C2 C3

D: Collembola-Diplura Lognormal 6.088 (0.054) 5.85 5.96 5.92E: Spinicaudata-Cladocera Lognormal 6.137 (0.054) 9.97 10.08 10.04G: Phyllocarida-Eumalacostraca Lognormal 6.295 (0.055) 19.93 42.40 48.97

AICa 74.51 84.80 101.95

a AIC (Akaike Information Criterion, Akaike, 1974): to evaluate the relative goodness of fit of models of evolution.

Table 3Divergence time estimates (Ma) using BEAST, Multidivtime, MCMCTree and r8s for major branchiopod nodes from the supermatrix and multiple calibration dataset and their compatibility with real fossil dates.

Node Fossil BEAST r8s (independent clock)

Ave 95% CI WSSb Ave 95% CI WSSb

2 420 548 469–656 0.91 465 448–480 0.993 488 606 510–713 0.94 495 478–512 1.004 162 216 196–394 0.89 310 218–402 0.415 500 678 485–829 0.87 580 562–598 0.97

WSS = 0.91 WSS = 0.78

Node Fossil Multidivtime MCMCTree-IR-AA

Ave 95% CI WSSb Ave 95% CI WSSb

2 420 491 450–522 0.97 515 475–558 0.953 488 532 509–552 0.99 564 522–600 0.984 162 278 162–384 0.49 210 92–388 0.915 500 562 550–575 0.98 621 595–643 0.94

WSS = 0.86 WSS = 0.94

Node Fossil MCMCTree-CR-AA MCMCTree-IR-Nuc

Ave 95% CI WSSb Ave 95% CI WSSb

2 420 513 488–544 0.95 503 474–537 0.963 488 549 517–583 0.98 562 531–591 0.984 162 277 58–455 0.50 176 108–264 0.995 500 611 584–634 0.95 620 602–638 0.94

N of squ

Mf

3elCc(tO

doati

OWLpsms

mmfbeW

WSS = 0.85

otes: Node numbers used here are the same as in Fig. 3. WSS, weighted sum

CMCTree and BEAST estimates are more congruent with theossil dates (with higher WSS values).

The time estimate for the origin of crown branchiopods (node) at about 495 ± 17 Ma (r8s, WSS = 1.0, Table 3) during thearliest Ordovician to late Cambrian is consistent with the ear-iest confirmed anostracan fossil (Riley Lake taxa) from theambrian Furongian (Harvey et al., 2012). The origin of therown group Phyllopoda (node 2) dated at about 465 ± 16.2 Mar8s, with highest WSS = 0.99, Table 3) possibly indicates theime of origin of freshwater phyllopods during late Cambrian tordovician interval.Although the fossil record so far established of Laevicau-

ata only dates back to the Middle Jurassic and Spinicaudatariginated during the Early Devonian (Shen et al., 1982; Shen

nd Chen, 1984), the divergence between Laevicaudata andhe ancestor of (Spinicaudata + Cladocera) (node 1) is datedn this study at about 430–457 Ma (early Silurian to the Late

vTw

WSS = 0.97

ares.

rdovician). This result supports the hypothesis put forward byalossek (1993, 1995) that the two suborders of ‘Conchostraca’,

aevicaudata and Spinicaudata, separated from the ancestralhyllopod probably in late Silurian. This implies that the fos-il record for ‘Conchostraca’ (especially for Laevicaudata) hasissed a substantial part of the evolutionary history, potentially

ignificant for future paleontological investigation for the group.It is noted that the molecular based divergence time esti-

ation for Branchiopoda by various techniques of relaxedolecular clock shows reasonably good congruence with the

ossil record (Table 3). It is interesting to note that on theasis of the overall WSS values for the various dating mod-ls, MCMCTree estimates are most congruent (highest modelWS = 0.97); however, when comparing the individual WSS

alues for the deep time estimates, we found that MCMC-ree produced more congruent dates for shallower divergences,hereas Multidivitime and r8s produced more congruent dates

312 X.-Y. Sun et al. / Palaeoworld 25 (2016) 303–317

Fig. 4. Comparison of divergence time estimates across four dating analyses (MCMCTree, Multidivtime, r8s, BEAST) with variable models. Each bar shows meanand 95% confidence intervals. Node numbers are the same used in Fig. 3. CR: correlated rate model; Nuc: nucleotide sequence; IR: independent rate model; AA:amino acid sequence; PL: penalized likelihood model.

F es at mD

famr

5d

l

(efdcR

ig. 5. Comparison between fossil dates and molecular divergence time estimatot line: y = x.

or deeper divergences (Table 3). We suggest that without othervailable criteria for selecting the dating models, the congruenceeasures could be adopted for choosing among varying dating

esults from the different models.

.2. Comparison with previous estimates of branchiopod

ivergence times

Our divergence time estimate shows that branchiopod stemineage may be rooted deep in the Ediacaran Period at 562.9 Ma

dotd

ajor branchiopod divergence nodes (node numbers refer to Fig. 3 and Table 3).

mean time estimate, node 5, Table 3; Fig. 3), which is appar-ntly too early from the view point of identifiable fossil recordor all arthropods; however, the crown group branchiopods likelyiverged at about during late Cambrian to earliest Ordovician,onfirming the fossil findings (see remarks above in Section 2.).ehm et al. (2011) utilized a large multiple sequence alignment

erived from EST (Expressed Sequence Tags) and genomes,nly including four representative crustaceans, resulting in aime estimate for the branchiopod-hexapod divergence in mid-le Cambrian (∼520 Ma) with no inference for branchiopod

oworl

ca6tbaeaiagfmRgfgceigtclad

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X.-Y. Sun et al. / Palae

rown group divergence timing. Wheat and Wahlberg (2013)nalyzed a large phylogenomic dataset (122 panarthropod taxa,2 genes) to reconstruct the arthropod time tree, resulting inhe branchiopod-other vericrustacea divergence in the Cam-rian (∼500 Ma) and the branchiopod crown group divergencet 410 Ma. Rota-Stabelli et al. (2013) presented a timescale ofcdysozoan evolution using a total of 402 gene partitions acrossll major lineages of ecdysozoans, with 78 calibration pointsnvolved, indicating the Late Ordovician radiation of crustaceannd an Ordovician–Silurian divergence of branchiopod crownroup at about 443 Ma. As discussed earlier, the branchiopodossils assignable to crown lineages are much older than theolecular time estimates by Wheat and Wahlberg (2013) andota-Stabelli et al. (2013), which probably need further investi-ation. Our preliminary analysis suggests that a possible causeor such under-estimates may have been derived from a two strin-ent fossil calibration constraints near the divergence notes inoncern. The fragmentary nature of the fossil record and lineagextinction may also lead to the underestimation of node agesn a phylogenetic tree (Springer, 1995). Different branchiopodroups have significantly different preservation potential, thushe fossil record is biased towards groups and structures moreonducive to fossilization, producing false signals of clusteredineage origins that could mislead divergence time studies. Thentiquity of branchiopods and the tempo of early branchiopod

iversification remain open questions in evolutionary biology.

In conclusion, this study shows that the crown groups of Bran-hiopoda originated in late Cambrian–earliest Ordovician time

Ntw

d 25 (2016) 303–317 313

nd freshwater phyllopods originated in the Middle Ordovician.hese estimated time interval fills the gap in the terrestrial fos-il record which is normally very poor, probably signifying thenitial phase of invertebrate animal’s invasion into the terrestrialnvironment.

Although studies of molecular clock are still in their infancy,t could be shown that this interdisciplinary study can contributeo a better understanding and reconstruction of evolutionaryrocesses. We suggest that fossil calibration evaluation andtrategies are critical for phylochronological analyses based onarge genomic datasets and the congruence measures shouldlways be used as a reference for choosing among results byifferent dating models.

cknowledgments

This work was supported by the National Natural Sci-nce Foundation of China (40902004, 40572070, 41272008,SXK0801), Chinese Academy of Sciences (KZCX2-YW-C104), the CAS/SAFEA International Partnership Programor Creative Research Teams and the State Key Laboratory ofalaeobiology and Stratigraphy at Nanjing Institute of Geologynd Palaeontology, Chinese Academy of Sciences. We thankan-Bin Shen (NIGP, CAS) and Di-Ying Huang (NIGP, CAS)

or valuable discussion and comments; Jia-Sheng Hao (Anhui

ormal University) and Gang Li (NIGP, CAS) for reviewing

he manuscripts with encouragement and important suggestionshich helped improving the manuscript.

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25 (2016)

303–317Appendix. Oldest occurrences of Pancrustacea and part of Myriapoda in the fossil record

Taxon Oldest record Source reference(s) Numbersa

Branchiopoda

Branchiopod stem group Rehbachiella kinnekullensis Cambrian, Series 3, >499 Ma Walossek (1993) and Olesen(2009)

1

SarsostracaAnostraca stem group

Riley Lake taxa Cambrian, Furongian, >488 Ma Harvey et al. (2012) 2Lepidocaris rhyniensis Devonian, Pragian, >411 Ma Scourfield (1926, 1940a) 3

Anostraca crown groupAn abdominal fragment Silurian, Ludlow, >421 Ma Schram (1986) 4Palaeochirocephalus sp. Jurassic, Bajocian–Bathonian, >164 Ma Shen and Huang (2008) 5

CalmanostracaNotostraca stem group Castracollis wilsonae Devonian, Pragian, >411 Ma Fayers and Trewin (2003) 6

Notostraca indet. Devonian, upper Famennian, >360 Ma Garrouste et al. (2012)Kazacharthra Almatium, Ketmenia, Kungejia Late Triassic to Early Jurassic (220–187 Ma) Tasch (1969)

Diplostraca

Laevicaudata Prolynceus beipiaoensis Jurassic, Tithonian, >145 Ma Shen and Chen (1984) 7Spinicaudata Palaeolimnadiopseidae Devonian, Lochkovian to Permian, >416 Ma Shen et al. (1982) 8Cyclestherida Cyclestherioides pintoi Middle Permian, >272 Ma Raymond (1946)

CladoceraEbullitiocaris oviformis Devonian, Pragian, >407 Ma Anderson et al. (2004) 9Ebullitiocaris elatus Carboniferous, Pennsylvanian, >318 Ma Womack et al. (2012)Archelatona zherikhini Jurassic/Cretaceous boundary, >145 Ma Kotov and Korovchinsky (2006)

OstracodaPodocopa Metacopa Elliptocyprites nonumbonatus Ordovician, Tremadocian, >478 Ma Hessland (1949) 10

MyodocopaSarsielloidea Hamaroconcha kornickeri Middle Devonian, >387 Ma Olempska and Belka (2010) 11Cypridinoidea Eocypridina campbelli Late Devonian, >358 Ma Tinn and Meidla (2004) 12

MalacostracaPhyllocarida Arenosicaris inflata Cambrian, Furongian, >485 Ma Collette and Hagadorn (2010) 13

EumalacostracaHoplocarida Pechoracaris aculicauda Devonian, Lochkovian, >411 Ma Dzik et al. (2004) 14Peracarida Aciculopoda mapesi Devonian, Famennian, >359 Ma Feldmann and Schweitzer (2010) 15

Copepoda Podoplea Canthocamptidae Fragments Carboniferous, Pennsylvanian, >303 Ma Selden et al. (2010) 16

ThecostracaStem group

Heteralepadomorph Priscansermarinus barnetti Middle Cambrian, >505 Ma Collins and Rudkin (1981) 17Lepadomorpha Ramphoverritor reduncus Silurian, Wenlock, >425 Ma Briggs et al. (2005) 18

Crown groupSessilia Brachylepascretacea Cretaceous, Berriasian, >140 Ma Newman et al. (1969) 19Pedunculata Praelepas jaworski Carboniferous, Middle Pennsylvanian, >306 Ma Høeg et al. (1999) 20

Pentastomida Stem group Bockelericambrian pelturae Cambrian, Furongian, >500 Ma Walossek and Müller (1994) andWalossek et al. (2006)

21

Hexapoda

EntognathaCollembola Rhyniella praecursor Devonian, Pragian, >411 Ma Hirst and Maulik (1926) and

Whalley and Jarzembowski(1981)

22

Diplura Testajapyx thomasi Carboniferous, Westphalian, >307 Ma Kukalová-Peck (1987) 23

Insecta

Archaeognatha Two Gaspé fragments Devonian, Givetian, >390 Ma Labandeira et al. (1988) 24Dicondylia Rhyniognatha hirsti Devonian, Pragian, >407 Ma Engel and Grimaldi (2004)Zygentoma Unnamed Lepismatidae Cretaceous, Santana Formation, >94 Ma Sturm (1998) 25Ephemeroptera Lithoneura Carboniferous, Pennsylvanian, >307 Ma Kukalová-Peck (1987) 26Odonata Various species of Protodonata Carboniferous, Pennsylvanian, ∼318 Ma Riek and Kukalová-Peck (1984) 27Neoptera Species of Paoliidae Carboniferous, Pennsylvanian, ∼315 Ma Prokop and Nel (2007) 28

Eucrustacea EntomostracaYicaris dianensis Cambrian, Series 3, >515 Ma Zhang et al. (2007)Wujicaris muelleri Cambrian, Series 3, >515 Ma Zhang et al. (2010)

MyriapodaDipopoda Paleodesmus tuberculatus Devonian, Lochkovian, >419 Ma Wilson and Anderson (2004) 29

ChilopodaNotostigmophora Crussolum spp. Silurian, Ludlow, >420 Ma Shear et al. (1998) 31Pleurostigmophora Devonobius delta Middle Devonian, >385 Ma Shear and Bonamo (1988) 32

a Corresponding to those in Fig. 2.

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