Molecular Clocks Keep Dispersal Hypotheses Afloat: Evidence For

Journal of Biogeography (J. Biogeogr.) (2010) 37, 305–324

ORIGINAL Molecular clocks keep dispersal ARTICLE hypotheses aﬂoat: evidence for trans-Atlantic rafting by rodents Diane L. Rowe1,2*, Katherine A. Dunn3, Ronald M. Adkins4 and Rodney L. Honeycutt5,2

1Pengana Place, Blackmans Bay, TAS, ABSTRACT Australia, 2Department of Wildlife & Fisheries Aim In order to resolve disputed biogeographical histories of biota with Sciences, Texas A&M University, College Station, TX, USA, 3Department of Biology, Gondwanan continental distributions, and to assess the null hypothesis of Dalhousie University, Halifax, NS, Canada, vicariance, it is imperative that a robust geological time-frame be established. As 4Department of Pediatrics, University of an example, the sudden and coincident appearance of hystricognath rodents Tennessee Health Sciences Center, Memphis, (Rodentia: Hystricognathi) on both the African and South American continents TN and 5Natural Sciences Division, has been an irreconcilable controversy for evolutionary biologists, presenting Pepperdine University, Malibu, CA, USA enigmas for both Gondwanan vicariance and Late Eocene dispersal hypotheses. In an attempt to resolve this discordance, we aim to provide a more robust phylogenetic hypothesis and improve divergence-date estimates, which are essential to assessing the null hypothesis of vicariance biogeography. Location The primary centres of distribution are in Africa and South America. Methods We implemented parsimony, maximum-likelihood and Bayesian methods to generate a phylogeny of 37 hystricognath taxa, the most comprehensive taxonomic sampling of this group to date, on the basis of two nuclear gene regions. To increase phylogenetic resolution at the basal nodes, these data were combined with previously published data for six additional nuclear gene regions. Divergence dates were estimated using two relaxed-molecular-clock methods, Bayesian multidivtime and nonparametric rate smoothing. Results Our data do not support reciprocal monophyly of African and South American lineages. Indeed, Old World porcupines (i.e. Hystricomorpha) appear to be more closely related to New World lineages (i.e. Caviomorpha) than to other Old World families (i.e. Bathyergidae, Petromuridae and Thryonomyidae). The divergence between the monophyletic assemblage of South American lineages and its Old World ancestor was estimated to have occurred c. 50 Ma. Main conclusions Our phylogenetic hypothesis and divergence-date estimates are strongly at odds with Gondwanan-vicariance isolating mechanisms. In contrast, our data suggest that transoceanic dispersal has played a signiﬁcant role in governing the contemporary distribution of hystricognath rodents. Molecular- clock analyses imply a trans-Tethys dispersal event, broadly conﬁned to the Late Cretaceous, and trans-Atlantic dispersal within the Early Eocene. Our analyses also imply that the use of the oldest known South American rodent fossil as a calibration point has biased molecular-clock inferences. Keywords *Correspondence: Diane L. Rowe, 16 Pengana Place, Blackmans Bay, TAS 7052, Australia. Dispersal, fossil, historical biogeography, Hystricognathi, molecular clocks, E-mail: [email protected] phylogenetics, rodents, vicariance.

ª 2009 Blackwell Publishing Ltd www.blackwellpublishing.com/jbi 305 doi:10.1111/j.1365-2699.2009.02190.x D. L. Rowe et al.

Although rodents are extraordinarily taxonomically diverse INTRODUCTION and geographically widespread, representing more than one- Whether vicariance or dispersal mechanisms should be third of all extant mammalian lineages and distributed widely invoked to account for biogeographically disjunct distributions across nearly all continents (Wilson & Reeder, 1993), the oldest of terrestrial biota that are widely separated by oceanic barriers undisputed fossil representative is only 55 Myr old (Harten- remains a point of contention (Hunn & Upchurch, 2001; de berger, 1998). However, a Gondwanan vicariance explanation Queiroz, 2005). Inferences about the biogeographical history requires that lineage divergences must pre-date the geological of the Southern Hemisphere, in particular, continue to opening of the Atlantic Ocean, c. 100 Ma (Parrish, 1993). generate intense debate (e.g. Cook & Crisp, 2005; McGlone, Thus, for many, attributing the distribution of a derived 2005; Sparks & Smith, 2005; Gamble et al., 2008). Since the suborder of rodents (i.e. Hystricognathi) to Gondwanan advancement of plate-tectonic theory in the 1960s, Gondwa- geological events seems a remote and unrealistic extrapolation, nan distributions of flora and fauna have been routinely particularly given that the oldest fossils attributable to any ascribed to the geological fragmentation (i.e. vicariance) of the modern Eutherian mammalian order are only around 65 Myr southern continents, supported by concordance of pattern (i.e. old (McKenna & Bell, 1997). However, early molecular-clock similar area-cladograms) across numerous unrelated taxo- estimates upheld this notion, suggesting that the order nomic groups (Nelson & Platnik, 1981; Patterson, 1981; Craw, Rodentia, and more specifically the suborder Hystricognathi, 1982; Morrone & Crisci, 1995; Sparks & Smith, 2004). may have been derived over 100 Ma (Kumar & Hedges, 1998). Unfortunately, such pattern-based conclusions, for which Not surprisingly, inferences about the process by which cladogenesis coincides with the geological sequence of conti- reciprocally monophyletic groups of hystricognath rodents nental fragmentation, neglect to address the problem of came to occupy Africa and South America became the subject diminishing statistical power in inferring concordance as the of intense debate, presenting enigmas to both Gondwanan number of clades and occupied continents declines. For vicariance and dispersal hypotheses (Lavocat, 1969; Patterson instance, the pattern of species cladogenesis has a high & Wood, 1982; George, 1993a,b; Craw et al., 1999; Huchon & probability of matching the geological sequence of fragmen- Douzery, 2001; Marivaux et al., 2002; Martin, 2005). tation by chance alone when only three continents are It is widely accepted that the contemporary distributions of occupied. Intuitively, when this is reduced to only two a few Hystricognathi species in Southeast Asia and North continents, pattern becomes irrelevant, and an independent America are attributable to overland dispersal following inference of the geological time-frame is essential to assessing continental re-connections during the Miocene and Pliocene, the null hypothesis of vicariance (Lavin et al., 2004). However, respectively (Jaeger, 1988; Janis, 1993; Flynn & Wyss, 1998). even for groups with such a restricted distribution, vicariance However, prior to the Miocene, identifiable hystricognath conclusions are persistently given precedence by a priori lineages are known only from the African and South American discounting the possibility of shared dispersal avenues, and continents, being notably absent from fossiliferous beds lent credence irrespective of the establishment of an indepen- elsewhere in the world (George, 1993a; McKenna & Bell, dent temporal framework (e.g. Craw et al., 1999; Sparks & 1997; Hartenberger, 1998; Marivaux et al., 2004). Interestingly, Smith, 2004). the first unambiguous lineages assigned to the group are not Such presumptions are being challenged with the wide- observed in the fossil record until the Late Eocene, emerging spread application of molecular-clock methods for estimating nearly simultaneously on both continents (c. 35 Ma; Wyss divergence dates, resulting in a paradigm shift to transoceanic et al., 1993; McKenna & Bell, 1997). If, by default, a vicariance dispersal as the leading contributor to contemporary biogeo- explanation is invoked then we are forced to contend with a graphical disjunctions (de Queiroz, 2005; Cowie & Holland, conspicuous absence of lineages in the fossil record during the 2006). Problematically, however, molecular-clock estimates 60-Myr interim between the opening of the Atlantic Ocean and are often dramatically inconsistent, reinforcing scepticism the Eocene debut of hystricognath lineages. Alternatively, given about the utility and reliability of such methods (Benton, that the null hypothesis of vicariance cannot be rejected on the 1999; Smith & Peterson, 2002). Furthermore, because infer- basis of pattern alone, owing to a restricted two-continent ences of time may be prone to circularity and bias owing to distribution, a temporal inference would intuitively take an incomplete and/or fragmented fossil record, controversy precedence. In this instance, a strict interpretation of the fossil persists. Nowhere is this disparity more prominently played record indicates a Late Eocene time-frame, requiring oceanic out than across the southern Atlantic, where a number of dispersal of more than 1700 km (Janis, 1993; Holroyd & Maas, unrelated taxonomic groups share a disjunct distribution, on 1994) by a terrestrial organism (Lavocat, 1969). However, the continents of Africa and South America (e.g. Vences fossils themselves generally serve only as indicators of the et al., 2001; Schrago & Russo, 2003; Danforth et al., 2004). minimum ages of lineages (Smith & Peterson, 2002; Renner, Discerning whether these distributions are the product of 2005), with uncertainty in the case of hystricognath rodents vicariance or dispersal mechanisms is fraught with difficulties, compounded by known gaps of 10–15 Myr in the terrestrial as exemplified by the monophyletic rodent suborder Hyst- fossil record immediately preceding the appearance of rodents ricognathi (Hartenberger, 1998; Adkins et al., 2001; Marivaux in both Africa and South America (Flynn & Wyss, 1998; Flynn et al., 2004). et al., 2002; Jaeger, 2003).

306 Journal of Biogeography 37, 305–324 ª 2009 Blackwell Publishing Ltd Trans-Atlantic dispersal of rodents

In an attempt to resolve this discordance, molecular-clock reliability of molecular-clock estimates for lineages within the methods (Welch & Bromham, 2005) have been applied as a suborder Hystricognathi. In particular, we focus on minimiz- means of ascertaining a more robust temporal framework. ing the impact of two implicated sources of error: insufficient However, age estimates for lineage separation between the two taxonomic sampling and fossil calibration-point limitations continents have varied widely, from 37 to 85 Ma (see Table 1). (Bromham et al., 2000; Conroy & van Tuinen, 2003; Graur & Such inconsistencies have persisted despite the development of Martin, 2004; Near & Sanderson, 2004; Linder et al., 2005). In numerous and increasingly sophisticated methods of dealing addition, we address potential problems associated with the with the inherent complexities of molecular-clock dating seemingly routine incorporation of the geological age of the (Rodrı´guez-Trelles et al., 2002; Bromham & Penny, 2003; oldest known South American rodent fossil as a calibrating Bromham & Woolfit, 2004; Near & Sanderson, 2004; Rutsch- point for inferring the timing of their initial occupation of the mann, 2006). In addition, alternative phylogenetic hypotheses, South American continent. based on molecular data, have brought into question the validity of reciprocal monophyly of the clades occupying the MATERIALS AND METHODS two continents, Africa and South America, and are further suggestive of the possibility of an ancestral presence in Asia Phylogenetic framework (e.g. Adkins et al., 2001; Huchon & Douzery, 2001). Such alternative phylogenetic hypotheses can modify area-clado- Taxon sampling and DNA sequences grams to a three-continent distribution, and, consequently, impact on the plausibility of vicariance based on pattern. Throughout this manuscript, we make reference to three Therefore, at present, phylogenetic and temporal uncertainty reciprocally monophyletic crown-clades, as recognized by prohibit sound inference of the biogeographical history of Wood (1965; see George, 1993a). These include the New hystricognath rodents. World Caviomorpha, the Old World porcupines (i.e. To better understand the mechanism by which hystricog- Hystricomorpha), and a strictly African clade composed of nath rodents first came to occupy South America, we enhance Bathyergomorpha and Phiomorpha (herein designated both the resolution of phylogenetic relationships and the Bathy–Phiomorpha). Taxonomic sampling was inclusive of

Table 1 Estimates for the timing of separation of African and South American hystri- Age (Ma) Data Method SA-cavy Reference cognath rodent lineages derived from African and South American lineage split molecular-clock methods (NPRS indicates 37 (34–40) 1 nuc, 1 mt Bayesian Yes; 37 Ma Opazo, 2005 nonparametric rate-smoothing methods) and 38 (16–54) 2 nuc NPRS No Adkins et al., 2003 utilizing various gene regions (‘nuc’ indicates 38 (34–42) 19 nuc, 3 mt Bayesian No Springer et al., 2003; nuclear gene regions and ‘mt’ refers to (Murphy et al., 2001b) mitochondrial gene regions). 43–54 1 nuc Local clocks Yes; 31 Ma Huchon & Douzery, 2001 45 (41–49) 3 nuc Bayesian No Poux et al., 2006; (Huchon et al., 2002) 55 (42–69) 2 nuc Bayesian No This study 58–66 1 nuc (TTR) NPRS No This study 85 (71–85) 12 mt Rate correct No Mouchaty et al., 2001 South American (Caviomorpha) radiation 31.5–37 Oldest fossil Radiometric 32 Ma Wyss et al., 1993 22–57 3 nuc Local clocks Yes; 31 Ma Douzery et al., 2003; (Huchon et al., 2002) 28–51 1 nuc Linearized Yes; 31 Ma Huchon et al., 2000 32 (29–35) 1 nuc Bayesian Yes; 32 Ma Galewski et al., 2005 34 (32–36) 1 nuc, 1 mt Bayesian Yes; 37 Ma Opazo, 2005 34.5 (33–36) 12 nuc, 1 mt Bayesian Yes; 37 Ma Hasegawa et al., 2003; (Murphy et al., 2001a) 37 2 mt Local clocks No Montgelard et al., 2002 37 (33–41) 3 nuc Bayesian No Poux et al., 2006; (Huchon et al., 2002) 40–46 1 nuc (TTR) NPRS No This study 45 (35–53) 2 nuc Bayesian No This study

The age of the oldest known rodent fossil in South America is provided for comparison, and molecular studies utilizing this fossil as a calibration point (and its estimated geological age) are indicated in the column ‘SA-cavy’.

Journal of Biogeography 37, 305–324 307 ª 2009 Blackwell Publishing Ltd D. L. Rowe et al. all 16 extant families of Hystricognathi (Wilson & Reeder, for each partition was determined using Modeltest (version 1993; Honeycutt et al., 2007). All seven genera of Bathy– 3.0, Posada & Crandall, 1998) and the model substitution Phiomorpha and one of the three extant genera of the family parameters estimated using the program MrBayes (version Hystricidae (Hystricomorpha) represented the Old World 3.1; Ronquist & Huelsenbeck, 2003). Starting from random lineages. Twenty-nine of the 47 extant New World genera were trees, four chains were run simultaneously in each analysis, included, representative of the four Caviomorpha superfam- over 5 · 106 generations. To check for consistency, the runs ilies. Within the species-rich group Caviomorpha, taxon were repeated with 1 · 106 generations. A consensus of post- sampling was designed to include lineages encompassing the burn-in trees, determined empirically from likelihood values, diversity and breadth of body sizes and life-history traits, as sampled every 100 generations was created for each dataset. these attributes appear to be correlated with rates of molecular One-tailed Kishino–Hasegawa tests (KH-tests; Kishino & evolution (Rowe & Honeycutt, 2002), and ultimately influence Hasegawa, 1989) were implemented to assess a priori taxo- the utility of molecular-clock methods. Two outgroup taxa, nomic hypotheses, reflecting the uncertain relationships of Old Ctenodactylus and Pedetes, were incorporated for all phyloge- World and New World porcupines (Hystricidae and Eret- netic analyses, with the former being the probable sister-group hizontidae, respectively; Wood, 1965; Bugge, 1985; Lavocat & to Hystricognathi (Bugge, 1985; Adkins et al., 2001; Huchon Parent, 1985; Woods & Hermanson, 1985). et al., 2002; Marivaux et al., 2002; Veniaminova et al., 2007). Two nuclear gene regions, intron 1 of the transthyretin gene Divergence-date estimates (TTR, 1264 bp) and exon 10 of the growth hormone receptor gene (GHR, 831 bp) were sequenced for use in phylogenetic Detecting among-lineage rate heterogeneity analyses of the 39 sampled taxa. In addition, our data from TTR and GHR were combined with previously published Prior to estimating divergence times from relaxed-molecular- nuclear sequence data of taxonomic subsets held in common, clock methodologies, two approaches were used to assess rate and combined phylogenetic analyses were performed. These homogeneity among taxa. First, adherence to a strict molecular subsets included: (1) an analysis of 25 taxa for TTR, GHR and clock was investigated by comparing ML-derived log-likeli- von Willebrand factor (vWF, 1263 bp; Huchon & Douzery, hood values, using a GTR+C (general time-reversible nucle- 2001); and (2) an analysis of eight taxa for TTR, GHR and otide substitutions with a gamma distribution for rate vWF, plus five nuclear genes (PNOC, RAG1, TYR, CREM, heterogeneity across sites) model of evolution, for a given tree PLCB4) reported by Murphy et al. (2001a). GenBank accession topology with and without enforcement of a molecular clock. numbers for sequences are listed in Table 2. A likelihood ratio test was used to verify the validity of the molecular-clock hypothesis (Tajima, 1993; P < 0.05). In the event of significant rate heterogeneity, two different relaxed- Phylogenetic inference molecular-clock methods were implemented to infer diver- Sequences were aligned using ClustalX (Thompson et al., gence dates. 1997) and modified to conform to amino acid sequence in the The pattern of rate heterogeneity was subsequently exam- case of GHR, vWF, PNOC, RAG1 and TYR. Genes were ined by implementing the Lintre program (Takezaki et al., analysed separately and in combination, following partition- 1995). Use of both the branch-length test and the two-cluster homogeneity tests for combinability (PHT; Farris et al., 1995) test allowed identification of both lineages and clades under- using paup* (version 4.0b10; Swofford, 2002). Maximum going rates of evolution significantly different from the parsimony (MP) and maximum likelihood (ML) analyses were average. Tamura–Nei distances were used, as the GTR model also performed using paup*. MP analyses employed equal was unavailable for this analysis. Assessment of rate patterns weighting and heuristic searches with 10 random additions of allowed us to optimize the implementation of fossil calibration tree bisection–reconnection (TBR) branch-swapping. Prior to points across lineages experiencing a variety of rates of ML analyses, the program Modeltest (version 3.0, Posada & evolution. Crandall, 1998) and hierarchical Akaike information criteria (AIC) were used to select the most appropriate model of Calibration point designation molecular evolution. All ML analyses employed heuristic searches with 10 random additions of TBR branch-swapping. Ideally, fossil calibration points should be applied broadly Support for nodes in resultant phylogenies derived from both across the phylogeny to reduce the magnification of error MP and ML analyses were determined using bootstrap analyses associated with extrapolating to nodes increasingly distant and the same heuristic search options. from the calibration points (Nei et al., 2001; Linder et al., Phylogenetic analyses of the two-gene (i.e. TTR and GHR) 2005). As such, fossils were targeted to span the phylogenetic and eight-gene (i.e. TTR, GHR, vWF, PNOC, RAG1, TYR, breadth and depth of hystricognath rodents (see Tables 2 CREM and PLCB4) combinations were conducted using a and 3). To minimise unforeseen errors associated with among- partitioned mixed-model Bayesian analysis with posterior lineage rate heterogeneity, calibration points were chosen to probabilities estimated using a Markov chain Monte Carlo incorporate the potential range of evolutionary-rate classes (MCMC) procedure. The optimal model of sequence evolution through inclusion of fossil representatives from five different

308 Journal of Biogeography 37, 305–324 ª 2009 Blackwell Publishing Ltd Trans-Atlantic dispersal of rodents

Table 2 GenBank accession numbers of the hystricognath rodent taxa and nuclear gene regions (GHR, TTR, vWF, PNOC, RAG1, TYR, CREM and PLCB4) included in this study.

Species GHR TTR vWF

Outgroups Pedetidae Pedetes capensis AF332025/H551 FJ865447/H551 AJ238389 Ctenodactylidae Ctenodactylus gundi (vali*) AF332042/H2202 FJ865448/H2202 AJ238387* Bathy–Phiomorpha Bathyergidae Bathyergus suillus FJ855201/BS AF159321/BS AJ238384 Cryptomys hottentottus FJ855202/H688 AF159314/CHH1 AJ251132 Georychus capensis FJ855203/GPPH3 AF159319/GPPH3 Heliophobius argenteocinereus FJ855204/H066 AF159323/H066 AJ251133 Heterocephalus glaber AF332034/unk AF159324/H004 AJ251134 Petromuridae Petromus typicus FJ855205/H550 AF159313/H550 AJ251144 Thryonomyidae Thryonomys swinderianus AF332035/unk AF159312/H571 AJ224674 Hystricomorpha Hystricidae Hystrix africaeaustralis AF332033/unk AF159311/SP7702 Atherurus macroura* AJ251131* Caviomorpha Cavioidea Agoutidae Agouti taczanowski (paca*) AF433929/H6192 AF433882/H6192 AJ251136* Caviidae Cavia tschudii (porcellus*) FJ855206/H5601 FJ865449/H5601 AJ224664* Dolichotis patagonum AF433939/H6193 AF433893/H6193 Galea musteloides AF433933/AK13818 AF433886/AK13818 Kerodon rupestris AF433938/H5835 AF433892/H5835 Microcavia australis AF433937/AK13309 AF433889/AK13309 Dasyproctidae Dasyprocta aguti FJ855207/NZG6227 FJ865450/NZG6227 U31607 Myoprocta acouchi AF433945/H5837 AF433899/H5837 Hydrochaeridae Hydrochaeris hydrochaeris FJ855208/NK13155 FJ865451/NK13155 AJ251137 Chinchilloidea Chinchillidae Chinchilla laniger AF520660/NK13161 FJ865452/HZG AJ238385 Lagidium viscacia FJ855209/NK14538 FJ865453/NK14538 Lagostomus maximus FJ855210/UF571 FJ865454/UF569 Dinomyidae Dinomys branickii AF520659/K8 FJ865455/K8 AJ251145 Erethizontoidea Erethizontidae Coendou bicolour AF520663/K5 FJ865456/K5 Erethizon dorsatum FJ855211/H5828 FJ865457/H5828 AJ251135 Sphiggurus mexicanus (melanurus*) FJ855212/H5830 FJ865458/H5830 AJ224664* Octodontoidea Abrocomidae Abrocoma bennetti FJ855213/H5613 FJ865459/H5613 AJ251143 Capromyidae Capromys piliroides AF433949/H575 AF433903/H575 AJ251142 Ctenomyidae Ctenomys boliviensis (maulinus*) FJ855214/NK15277 FJ865460/NK15277 AJ251138* Echimyidae Echimys chrysurus FJ855215/LMP27 FJ865461/LMP27 AJ251141

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Table 2 Continued

Species GHR TTR vWF

Isothrix bistriata FJ855216/M1273 FJ865462/M1273 AJ849308 Proechimys longicaudatus (oris*) FJ855217/NK15758 FJ865463/NK15758 AJ251139* Myocastoridae Myocastor coypus AF520662/H584 AF520669/H584 AJ251140 Octodontidae Aconaemys fuscus AF520657/K38 FJ865464/H4468 Octodon lunatus AF520650/H4463 FJ865465/H4463 AJ238386 Octodontomys gliroides AF520649/AK15686 FJ865466/AK15686 Octomys mimax AF520665/AK13474 FJ865467/AK13474 Spalacopus cyanus AF520653/H5626 FJ865468/H5626 Tympanoctomys barrerae AF520655/AK13811 FJ865469/AK13811 Data from Murphy et al. (2001a) Species PNOC RAG1 TYR CREM PLCB4 Pedetes capensis AY011824 AY011882 AY012000 AY011642 AY011765 Heterocephalus glaber AY011830 AY011889 AY012005 AY011649 AY011772 Hystrix (brachyura*) AY011827* AY011886* AY012003* AY011646* AY011769* Cavia tschudii AY011831 AY011890 AY012006 AY011650 AY011773 Hydrochaeris hydrochaeris AY011832 AY011891 AY012007 AY011651 AY011774 Dinomys branickii AY011834 AY011893 AY012009 AY011653 AY011776 Erethizon dorsatum AY011828 AY011887 AY012004 AY011647 AY011770 Myocastor coypus AY011833 AY011892 AY012008 AY011652 AY011775

Numbers in bold identify the GHR and TTR sequences generated in this study, with accession numbers followed by specimen identiﬁcation numbers (GenBank accession number/species identiﬁcation number). Unknown specimens are labelled ‘unk’. An asterisk (*) is indicative of the concatena- tion of sequence information from congeneric species, when identical species were not available. Specimens are listed in accordance with taxon- omy, with the outgroup taxa and members of Hystricomorpha ranging from Africa to Southeast Asia, the Bathy–Phiomorpha being restricted to Africa, and Caviomorpha occurring primarily within South and Central America.

families that encompass various life-history attributes (Wilson ately prior to the clade within which they exhibit synapomor- & Reeder, 1993; Rambaut & Bromham, 1998; Rowe & phies (Smith & Peterson, 2002; Renner, 2005). Honeycutt, 2002; Dobson & Oli, 2007). In effect, we employed An upper bound (i.e. maximum age) for multidivtime only calibration points that collectively fitted the criteria of analyses was most reliably designated at the node associated falling within the group of interest (i.e. Hystricognathi), with the diversification of caviomorph lineages (i.e. Cavio- spanning the range from recent to more basal nodes, and morpha), given the more widespread geographical distribution representing both slow and rapid rate classes (i.e. the range of and comparative abundance of fossil information (i.e. both observed rates of molecular evolution). presence and absence of fossils) available from South America. Designation of an upper bound for the root node (Hystric- ognathi–Ctenodactylidae) would have been ideal, but was Bayesian MULTIDIVTIME estimations prohibitive in terms of the potential for misleading conclu- multidivtime (Thorne & Kishino, 2002), a Bayesian meth- sions. Given the current limitations of the palaeontological odology that incorporates a probabilistic model to describe record, particularly the rarity of Late Cretaceous–Palaeocene changes in rates of molecular evolution through time, was fossil records outside Laurasia (Savage & Russell, 1983; selected because it allows for the simultaneous use of multiple Novacek, 1992; Lucas, 2001; Smith et al., 2001; Jaeger, 2003; calibration points, can accommodate multiple genes or data Marivaux et al., 2004), a reliable age estimate for this node partitions with different evolutionary characteristics, and could not be established. In particular, there is a lack of provides credibility intervals for estimated divergence times substantial information from the Late Cretaceous–Palaeocene (Rodrı´guez-Trelles et al., 2002; Thorne & Kishino, 2002). of Southeast Asia and Africa, the probable areas of origin for Because fossils are not considered to be precise representations ancestral ctenodactyloid and hystricognath lineages, respec- of a true lineage and their ages are not known without error, tively (Hartenberger, 1998; Marivaux et al., 2002; Jaeger, 2003; the use of lower and upper bounds to constrain node ages, Seiffert, 2006). rather than the definition of fixed ages, provides a more The combined dataset, with independently modelled gene realistic approach. When employed as lower bounds, fossils partitions (TTR-all + GHR-all), was used to estimate diver- were used to establish a minimum age for the node immedi- gence times. For comparison, the GHR gene region was further

310 Journal of Biogeography 37, 305–324 ª 2009 Blackwell Publishing Ltd Trans-Atlantic dispersal of rodents partitioned into individual codons (GHR-123), and TTR was ancestral and descendant nodes), and 1.5 (150 Ma) for bigtime reanalysed after removing gaps from the dataset (TTR-gap). In (the largest value of time between the root and the tips). all instances, model parameters were estimated using an F84+C model of evolution (the most complex model available in the Nonparametric rate-smoothing estimations program) on a defined tree topology that was derived from the combined ML analysis of the TTR and GHR data The nonparametric rate-smoothing (NPRS) method was (TTR + GHR). Model parameters were estimated using the utilized primarily to allow direct comparison with previously program baseml (paml v.3.14; Yang, 1997). These parameters published studies. This approach, like Bayesian methods, does were then used to estimate the branch lengths in the rooted not assume globally or locally constant rates of molecular evolutionary tree, in conjunction with a variance–covariance evolution, but uses a stochastic method to optimize rate matrix of the branch lengths estimated using the program changes across all lineages (Sanderson, 1997). It is based on the estbranches (Thorne et al., 1998). After pruning the out- assumption that rates are auto-correlated and attempts to group (Pedetes), the program multidivtime (Thorne & maximize covariance of rates over the entire tree according to Kishino, 2002) was used to approximate the posterior distri- an optimal least-squares smoothing criterion. bution of substitution rates and divergence times. An MCMC Analyses based on the NPRS method used fixed fossil- analysis was run for 100 million generations and sampled calibration points, independently assigned to particular nodes, every 100 generations after the initial burn-in period of 50,000 in order to estimate absolute ages for all other nodes on the cycles. To ensure convergence, multiple chains of the MCMC phylogeny. Given the greater uncertainty in placing the most analyses were performed. Priors specified included an upper primitive hystricognath lineages in the context of a molecular bound for the origination of Caviomorpha (55 or 90 Ma) and phylogeny of extant taxa, we applied the more derived lower bounds as indicated in Table 3. Other prior distribution Hydrochaeridae and Octodontidae as fixed calibration points settings included: 0.70 (70 Ma) for rttn (mean of the distri- (F1 and F2, respectively, in Table 3). In addition, the oldest bution for the time separating the in-group root from the South American rodent fossil (‘dasyproctid indet.’; F3 in present), 0.35 (35 Ma) for rttnsd (the standard deviation), 0.15 Table 3) was assigned to a more derived node, in contrast to for rtrate (mean distribution for the rate of molecular previous molecular studies, but in accordance with palaeonto- evolution at the in-group root node), 0.075 for rtratesd (the logical identification as a dasyproctid (Wyss et al., 1993, 1994). standard deviation), 0.75 for Brownmean (Brownian motion No constraints were directly placed on the node associated with parameter v, determining the permitted rate change between the colonization of South America. However, in all instances,

Table 3 Relevant palaeontological information and life-history traits of African and South American hystricognath rodents are summarized for the calibration points implemented in the molecular-clock analyses.

Calibration point designations

Family Hydrochaeridae Octodontidae Capromyidae Chinchillidae Thryonomyidae Dasyproctidae

Designations F1, L1 F2, L2 L3 L4 L5 F3

Palaeontological information Genera Prodolichotis/ Chasicomys/ Zazamys Eoviscaccia/ Gaudeamus ‘dasyproctid Procardiatherium Palaeoctodon chinchillid indet.’ Fossil age (Ma) 16 11 19 30 34 31 Life-history traits Body size (g) 40,000–70,000 60–300 500–8500 500–4500 4000–9000 600–4000 Litter size 2–8 2–10 1–6 1–6 1–2 1–2 Gestation (d) 149–156 77–109 110–140 111–166 137–172 99–120 Maturity (mo) 15 1.5–6 10 5–12 12 8–12 Longevity (yr) 12 7 11 20 4 17 References 3, 4, 5, 11, 13 4, 5, 8, 13 11, 13 2–5, 7, 8, 13 1, 4, 10, 12, 13 2, 3, 8, 9, 13

References: 1, Lavocat (1973); 2, Wyss et al. (1993); 3, Flynn & Swisher (1995); 4, McKenna & Bell (1997); 5, Walton (1997); 6, Hartenberger (1998); 7, Kay et al. (1999); 8, Vucetich et al. (1999); 9, Flynn et al. (2002); 10, Antonãnzas et al. (2004); 11, MacPhee (2005); 12, Seiffert (2006); 13, Wilson & Reeder (1993) Calibration points are referred to by familial alignment and are additionally designated numbers (F1–F3, L1–L5) for assignment to specific phylogenetic nodes. A calibration point designation beginning with the letter F signifies use as a fixed point, whereas an L signifies use as a lower bound (minimum age). The oldest named genera representing the families are listed, along with geological age estimates of the fossil beds from which they were derived. For the life-history traits, age at maturity reflects documented ages for first reproduction by females, and longevity is the maximum number of years documented in captivity.

Journal of Biogeography 37, 305–324 311 ª 2009 Blackwell Publishing Ltd D. L. Rowe et al. the date of the basal Hystricognathi divergence was constrained fractional numbers to whole numbers, as done by Adkins et al. to lie between 34 Ma (i.e. the oldest hystricognath rodent fossil) (2003). To determine branch lengths near the base of the tree and 110 Ma (i.e. accounting for hiatuses in the fossil records of more accurately, an additional non-hystricognath rodent both Africa and South America, and in accordance with previous (Pedetes) was used to root the tree. For the estimation of molecular studies of mammalian diversification; Kumar & divergence dates, Pedetes was pruned from the rooted tree. This Hedges, 1998; Penny et al., 1999). These ages were broadly allowed determination of the length of branches on each side of defined in order to increase the likelihood of including the true the root (i.e. Hystricognathi and the Ctenodactylidae out- age of that node. Independent divergence-date estimates were group) of the phylogeny. Divergence times were then estimated obtained for the TTR and GHR datasets. using the NPRS methodology, as implemented in the program Using the designated calibration points in conjunction with r8s (version 1.60 for Unix; Sanderson, 1997, 2003). estimates of branch lengths, absolute ages for nodes could be inferred. To estimate branch lengths, the tree topology was first RESULTS constrained to match that of the ML analysis of the concat- enated TTR + GHR dataset. The appropriate model of Phylogenetic relationships molecular evolution previously designated for each independent dataset (i.e. TTR and GHR) was then used to obtain ML There were minor topological differences observed between the branch-length estimates, as implemented in paup* (Swofford, independent TTR and GHR phylogenetic estimations, partic- 2002). Branch lengths were scaled by a factor of 1000 to convert ularly concerning the placement of Abrocoma, Capromys and

Figure 1 Summary of the phylogenetic relationships among African and South American hystricognath rodent lineages, with taxonomic designations and general distributions as outlined in Table 2. (a) Phylogenetic topology derived from the two-gene (TTR + GHR) dataset, consistent with maximum parsimony (MP), maximum-likelihood (ML) and Bayesian reconstruction methods. Bootstrap values are indicated above the branches subtending nodes, with ML preceded by MP values (i.e. MP/ML). Posterior probabilities are given below the branches. In all instances, an asterisk (*) indicates a bootstrap or posterior value of 100% or 1.00, respectively. An ‘X’ signiﬁes bootstrap support of less than 50%. (b) Maximum-likelihood phylogram derived from the eight-gene dataset, inclusive of a reduced number of representative taxa. The MP and Bayesian phylogenies are identical to this topology. MP and ML (i.e. MP/ML) bootstrap support values are given above the nodes, and the Bayesian posterior probabilities are displayed below the branches.

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Agouti, and genera within the family Batheyergidae (data not lineages identified as deviating from rate homogeneity shown). However, when the two datasets were combined (i.e. (P < 0.05), for both the TTR and the GHR dataset. TTR + GHR), the inconsistencies were resolved, and all associated nodes received strong bootstrap support and Divergence-date estimates posterior probabilities (see Fig. 1). There was strong support for the placement of two taxonomically problematic genera, Representative node ages, estimated using the multidivtime with Dinomys aligning with the superfamily Chinchilloidea and and r8s programs, are summarized in Table 4. Dates derived Abrocoma within Octodontoidea (Woods & Hermanson, 1985; from the two different methodologies are generally consistent. Martin, 1994; McKenna & Bell, 1997; Ko¨hler et al., 2000). All Divergence times estimated using multidivtime and their 95% data unequivocally supported monophyly of the South Amer- confidence intervals, derived from five lower-bound calibration ican Caviomorpha and its component superfamilial groupings points and an upper bound of 55 Ma, are depicted by the (Cavioidea, Chinchilloidea, Erethizontoidea and Octodontoi- phylogram in Fig. 2. The Bayesian prior date assumption, when dea), consistent with previous studies (e.g. Vucetich et al., increased from 55 to 90 Ma for the initial diversification 1999; Huchon & Douzery, 2001). However, the placement of of Caviomorpha (node 70), resulted in a roughly 2–7 Myr New World porcupines (Erethizontoidea) within the Cavio- increase (c. 15%) in node ages across the phylogeny (Table 4). morpha remained problematic (Bugge, 1985; Woods & Our phylogenetic and temporal framework implicates a Hermanson, 1985; Huchon & Douzery, 2001). Placement trans-Tethyan dispersal event of the Hystricognathi ancestor varied with both taxonomic and character sampling and the from Asia to Africa, occurring in the interim between the method of phylogenetic reconstruction (data not shown). ancestral hystricognath splitting from the ctenodactyloid Although most analyses suggested a sister-group relationship ancestor and the onset of hystricognath lineage diversification between the superfamilies Cavioidea and Erethizontoidea, the within Africa (i.e. between nodes 73 and 72, Fig. 2 and Table 4), node was considered unresolved in all subsequent analyses. between c. 92 and 59 Ma. Our estimated Palaeocene divergence This study provides the strongest support to date for the of the ancestral Hystricomorpha from the Bathy–Phiomorpha relationship among the three reciprocally monophyletic (c. 59 Ma; node 72 in Fig. 2 and Table 4), within Africa, is crown-group clades of hystricognath rodents, Bathy–Phio- closely followed by the derivation of the ancestor to Caviomor- morpha (Wood, 1965; see George, 1993a), Hystricomorpha pha near the Palaeocene–Eocene boundary (c. 55 Ma; node 71). (sensu Wood, 1965) and Caviomorpha (sensu Wood, 1955; Diversification of Caviomorpha lineages within South America Lavocat, 1973). Overwhelmingly, the data are consistent with a appears to have commenced by c. 45 Ma (node 70). This places basal placement for the strictly African lineages of Bathy– their African ancestor well before the Late Eocene–Early Phiomorpha, outside the sister-group association between the Oligocene appearance in the fossil record (c. 34 Ma; McKenna South American Caviomorpha and the Old World Hystrico- & Bell, 1997), but long after the rifting of Africa from South morpha (Asian and African porcupines). In contrast to America (c. 100 Ma; Parrish, 1993). The interim between the previous studies, we rejected both a monophyletic assemblage divergence from an African ancestor and the descendant of all Old World lineages (Bathy–Phiomorpha + Hystrico- radiation of the South American crown-group implies an Early morpha; e.g. Lavocat, 1973; Jaeger, 1988; Murphy et al., Eocene trans-Atlantic dispersal event (c. 45–55 Ma). The 2001b) and a basal placement for Hystricomorpha (e.g. Adkins subsequent diversification of Caviomorpha lineages on the et al., 2001, 2003; Eizirik et al., 2001; Huchon & Douzery, South American continent is estimated to have commenced 2001; Douzery et al., 2003; Huchon et al., 2007). Monophyly roughly 10–20 Myr prior to their first appearance in the fossil of the Old World hystricognath lineages was rejected using record, preceding estimates derived from the majority of both the three-gene (TTR + GHR + vWF) and the eight-gene molecular studies by c. 20 Myr (see Table 1). Extension of the dataset (KH-tests, P < 0.05). Likewise, a basal placement for Bayesian age prior (e.g. upper bound) at this node (i.e. node 70) the Hystricomorpha, with a sister-group relationship between to 90 Ma, allowing for the possibility of Gondwanan isolating the African (Bathy–Phiomorpha) and South American (Cav- mechanisms, resulted in a Palaeocene–Early Eocene posterior iomorpha) clades, was rejected using the eight-gene dataset age estimate (c. 62–51 Ma) for the African–South American (P < 0.05). divergence. Placing no constraints directly at this node, utilizing the NPRS dating method for the TTR gene region, gave similar estimates of c. 66–46 Ma for the divergence of African and Rate heterogeneity South American lineages (see Tables 1 and 4). Relative-rate tests (RRT; Tamura-Nei+C model, a = 3.2), Although our estimates are overwhelmingly consistent across including branch-length and two-cluster tests (Lintre; Take- our chosen fossil calibration points, between the two methods zaki et al., 1995), indicated a substantial amount of rate (multidivtime and NPRS) and, in general, among gene heterogeneity among lineages and clades for both the TTR and partitions, dates derived from the GHR gene region for the the GHR datasets. Over one-third of all terminal taxa for TTR NPRS method did give some highly stochastic and anomalous and more than three-quarters of taxa for GHR were evolving at results (Table 4). In one instance, for the Hydrochaeridae rates significantly different from the average rate (P < 0.05; calibration point (F1, Table 3), derived dates tended to be data not shown). Nearly half of all nodes had descendant much younger than other inferences, with the GHR estimates

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Table 4 Representative divergence-date estimates for given nodes (‘Node no.’) of African and South American hystricognath rodents, as derived from both Bayesian multidivtime and nonparametric rate-smoothing (NPRS) molecular-clock methods using the growth hormone receptor (GHR) and transthyretin (TTR) datasets.

Bayesian multidivtime age estimates (Ma)

Gene partitions, U = 55 Ma U = 90 Ma Calibration points GHR TTR GHR/TTR GHR/TTR

Node no. Cal. Age All 123 All Gap 123/all All/gap All/gap

42 – – 24 23 43 39 32 34 (22–49) 38 43 L5 30 41 46 68 62 54 55 (41–70) 61 44 – – 12 13 07 09 07 09 (05–16) 11 45 – – 18 20 17 18 17 19 (13–27) 22 46 L1 16 21 23 21 23 20 23 (17–32) 26 51 – – 32 38 37 38 33 37 (28–47) 42 53 – – 09 12 19 19 13 15 (08–23) 17 58 – – 07 07 12 10 09 08 (05–12) 09 59 L2 11 23 21 24 24 22 22 (17–30) 25 63 L3 19 17 14 19 18 16 15 (11–21) 17 68 – – 28 34 28 26 26 29 (20–38) 33 69 L4 30 40 45 42 40 39 43 (33–52) 48 70 U 55 42 47 46 44 42 45 (35–54) 51 71 – – 47 53 65 58 55 55 (42–69) 62 72 – – 51 55 71 64 58 59 (44–74) 66 73 – – 87 82 94 89 94 92 (68–121) 101

Nonparametric rate-smoothing (NPRS) age estimates (Ma)

F1 F2 F3

Calibration points Hydrochaeridae Octodontidae Dasyproctidae

Node no. Cal. Age GHR TTR GHR TTR GHR TTR

42 – – 18 38 44 38 23 34 43 – – 30 61 74 62 39 54 44 – – 08 07 20 07 11 06 45 F1 16 16 16 40 16 21 14 46 – – 18 19 43 20 23 17 51 F3 31 25 36 61 36 32 32 53 – – 09 17 22 17 11 15 58 F2 11 05 11 11 11 06 10 59 – – 14 25 35 25 18 22 63 – – 09 19 24 19 12 17 68 – – 24 29 60 29 31 26 69 – – 32 42 79 43 41 38 70 – – 33 46 80 46 42 40 71 – – 36 65 89 66 46 58 72 – – 39 68 97 69 51 61 73 – – 55 93 138 94 72 83

For multidivtime analyses, gene regions were analysed unpartitioned (‘all’) as well as partitioned by codon positions (‘123’) for GHR and without gaps (‘gap’) for TTR. Node ages with 95% posterior probability estimates, shown in parentheses, are provided for the GHR/TTR-all/gap analysis. This combined dataset (GHR/TTR, all/gap) was reanalysed with the upper bound (‘U’) for Caviomorpha extended from 55 to 90 Ma, as displayed in the far right column. Nodes with calibrating lineages are identiﬁed in bold, where the node number refers to those designated in Fig. 2. The calibration points (‘Cal.’) and assigned ages (‘Age’) are concordant with those listed in Table 3. sometimes less than half that of the TTR age estimates. Perhaps the majority of previously published molecular studies (see coincidentally, this combination yielded an estimated Africa– Table 1). In much the same manner, but at the other extreme, South America divergence of c. 36–33 Ma, similar to results in the GHR data with the Octodontidae calibration point (F2,

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Figure 2 Chronogram of divergences in African and South American hystricognath rodents, as estimated from the two-gene dataset (GHR/TTR-all/gap) using five internal calibration points set as minimal ages (designated in bold as L1–L5) and a maximum age of 55 Ma (U = 55 Ma) for the diversification of Caviomorpha (node 70). The nodes at which fixed calibrations were applied for nonparametric rate-smoothing analyses are included for reference purposes, identified here as F1, F2 and F3. Grey horizontal bars indicate the 95% posterior probabilities for key nodes (labelled with numbers between 42 and 73, as presented in Table 4), as derived from the Bayesian multidivtime analysis. Approximate positions of continents at 105 and 45 Ma are given below the figure, and vertical bars (black, white, grey) at the right of the figure indicate contemporary geographical distributions of taxa. Node 71, marked with a star, identifies a transoceanic disjunction that post-dates the associated continental break-up of Africa and South America, inferring a long-distance dispersal event. Key to epochs on the timeline: L-CR, Late Cretaceous; PAL, Palaeocene; OLIG, Oligocene; Pl, Pliocene; and P, Pleistocene, with K-T identifying the Cretaceous–Tertiary boundary.

Table 3) yielded exceptionally old date estimates of c. 89–80 Ma employing the same molecular-clock dating methods (i.e. for the same event, similar to ages reported by Mouchaty et al. Bayesian multidivtime and NPRS), our relaxation of tem- (2001). These extreme estimates are reminiscent of the original poral constraints otherwise imposed at the node deﬁning controversy over inferences derived from a strict interpretation South American origins (i.e. node 70, Fig. 2) appears to have of the fossil record and presumptions of Gondwanan vicariance. allowed for older divergence-date estimates to be obtained. Nonetheless, age estimates never coincided with or preceded the geological fragmentation of relevant continents and, thus, DISCUSSION vicariance isolating mechanisms can be rejected. Evidence for dispersal Phylogenetic pattern Both phylogenetic and temporal inferences rejected the notion of Gondwanan vicariance as a driving force in hystricognath Our data strongly supported a monophyletic New World clade rodent diversiﬁcation. In comparison to previous studies (i.e. Caviomorpha) nested within a paraphyletic group of Old

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World taxa, rejecting the traditionally held reciprocal mono- In support of our proposed age estimates, Palaeogene faunal phyly of Old World and New World lineages. It has generally exchanges between south Asia and the Arabo-African conti- been assumed that the suborder Hystricognathi descended nent, occurring well after Pangean fragmentation and long from an Asian ctenodactyloid ancestor (George, 1993a; Flynn & before the well-known Miocene collision of Africa with Eurasia Swisher, 1995; Marivaux et al., 2002) and that the widespread (Janis, 1993), have been proposed for numerous other distribution of contemporary Hystricomorpha lineages (i.e. mammalian groups, such as anthracotheres, proboscideans, Asia and Africa) is attributable to overland dispersal following primates and anomalurid rodents (Ducrocq, 1997, 2001; Jaeger the Miocene collision of the African and Asian continental et al., 1999; Marivaux et al., 2002, 2004). Although there is plates (Janis, 1993). As such, reciprocal monophyly of the Old growing support for concerted transoceanic dispersal events World and New World hystricognath clades generates an area- (e.g. Givnish & Renner, 2004; Pennington & Dick, 2004; cladogram not inconsistent with vicariance biogeography (i.e. Sanmartıń & Ronquist, 2004; de Queiroz, 2005), without a [Asia (Africa–South America)]; Nelson & Platnik, 1981; Craw need to invoke stepping-stone modes of dispersal, the range of et al., 1999). However, our non-traditional placement of the proposed dates of divergence presented here does, nonetheless, Old World porcupines (i.e. Hystricomorpha) allows for the encompass a major marine regression (c. 63–68 Ma; Haq et al., possibility of an ancient Hystricomorpha lineage originating in 1987). Although such marine low-stands have been hypoth- Asia. This leaves the sequential pattern of continental occu- esized to facilitate island-hopping dispersal via emergent island pation open to speculation and various area-cladogram chains, the temporal coincidence of favourable palaeocurrents interpretations plausible, some of which would be inconsistent and palaeowinds could conceivably have aided either stepping- with vicariance biogeography. Nevertheless, the testing of stone or chance (i.e. long-distance) dispersal in a westerly vicariance and dispersal mechanisms based on these area- direction during the Late Cretaceous–Palaeocene time period cladograms alone is subject to untenable assumptions con- (Holroyd & Maas, 1994). Likewise, a major marine regression cerning the complicated myriad of plausible lineage duplica- with exceptionally low sea levels also occurred c. 88 Ma, tion and continental extinction events, each incurring a pre- potentially facilitating dispersal between the continents at this determined cost that is inversely related to some preconceived time. Such a scenario is consistent with our molecular data, likelihood of occurring (e.g. Ronquist, 1997). Owing to such but more strongly at odds with the current status of uncertainty in pattern, reliable age estimates remain an palaeontological information, with the oldest known fossil of essential component of a full understanding of the evolution- relevance dating only to the Late Eocene. ary history of hystricognath rodents. Narrowing the gap in trans-Atlantic dispersal Inference of trans-Tethys dispersal Our age estimate for divergence between African and South The proposed Late Cretaceous–Palaeogene split (c. 92–59 Ma) American lineages (c. 45–55 Ma) falls between the traditionally between an Asian ctenodactyloid ancestor and the origin of inferred extremes of Gondwanan geological events (c. 100 Ma) Hystricognathi clearly post-dates the continental fragmentation and Late Eocene fossil records (c. 30–35 Ma), requiring a less of Pangea (e.g. > 165 Ma; Scotese et al., 1988), leading to the substantial c. 25-Myr absence of detection in the fossil record rejection of vicariance in association with the divergence of and favouring transoceanic dispersal across a somewhat Asian and African clades. A more precise biogeographical narrower Atlantic Ocean (Holroyd & Maas, 1994). Likewise, interpretation, however, of transoceanic dispersal and Hystric- our estimates contradict previously published molecular-clock ognathi origins, is untenable at present owing to the exception- studies that support a Late Eocene–Early Oligocene dispersal ally large standard error observed at the root node of the event (see Table 1 and references therein), even when the same phylogeny (node 73, Fig. 2 and Table 4). Both simulated and molecular-clock methodologies were employed (e.g. Bayesian empirical evidence has indicated that the estimation of ancient multidivtime and NPRS). Our more comprehensive taxo- divergence times using recent calibration points is prone to nomic sampling is likely to have improved the effectiveness of increasing error with increasing distance from the calibration correcting for among-lineage rate heterogeneity (Sanderson, points (Nei et al., 2001; Linder et al., 2005), and our observa- 1998; Welch & Bromham, 2005) and expanded the number tions are consistent with this finding. Unfortunately, given the and breadth of calibrating nodes, providing for more robust biogeographical and temporal biases in the rodent fossil record, divergence-date estimates from relaxed-clock methods (Near & the nearest calibration point that could reliably be applied to Sanderson, 2004; Linder et al., 2005). infer the age of the Hystricognathi–Ctenodactylidae split (i.e. Perhaps most importantly, and in contrast to our own node 73) was a much more recent divergence (the African approach, previous studies attempting to infer divergence Thryonomyidae, c. 34 Ma; McKenna & Bell, 1997). Further- times of hystricognath rodents from molecular data have more, we cannot dismiss the possibility of limited taxonomic routinely incorporated the oldest known South American sampling of the outgroup (i.e. ctenodactylids) confounding our rodent fossil (c. 32 Ma, ‘dasyproctid indet.’; Wyss et al., 1993, ability to infer accurate patterns of rate change near the root 1994) as a fixed calibration point or as an upper bound (e.g. node, potentially leading to some degree of error in age estimates Huchon et al., 2000; Huchon & Douzery, 2001; Hasegawa at the base of the phylogeny (Yoder & Yang, 2000). et al., 2003; Galewski et al., 2005; Opazo, 2005). In some cases,

316 Journal of Biogeography 37, 305–324 ª 2009 Blackwell Publishing Ltd Trans-Atlantic dispersal of rodents the age of this fossil has been directly applied or used to concluded that ‘the fossil record of hystricognathous rodents enforce a limit on the age of the node (i.e. node 70, Fig. 2) for (virtually unknown before the Late Eocene) is still inadequate which the inferred posterior age estimates are subsequently for proposing a realistic palaeobiogeographical model to taken as evidence for the timing of the arrival of rodents to explain the subsequent arrival in South America’. However, South America. Constraining a node in a phylogenetic tree most biogeographical interpretations based on molecular in this way runs the risk of circularity in biogeographical clocks have relied on the most ambiguous of fossils (e.g. the interpretation. In this instance, by limiting the window of oldest known South American rodent) that contradict this opportunity and precluding vicariance, it enforces the assump- statement. This example reinforces the importance of a tion that a Late Eocene dispersal event accounts for the arrival thorough investigation of palaeontological knowledge prior of ancestral lineages to South America, rather than indepen- to the designation of calibration points for molecular clocks dently assessing the temporal coincidence of this fossil with (e.g. Conroy & van Tuinen, 2003; Sanderson et al., 2004). molecular age estimates. Not surprisingly, in cases where this Use of the ‘oldest known’ fossil in this way is also fossil has been used to represent the age of Caviomorpha, the inappropriate on other grounds. The primitive morphological posterior age estimates for the node tend to converge on the characteristics and fragmentary nature of this particular fossil imposed limit (e.g. Hasegawa et al., 2003; Galewski et al., 2005; specimen render its phylogenetic placement uncertain (Wyss Opazo, 2005), further indicating that it may be an unrealistic, et al., 1993, 1994; Flynn et al., 2002). The default, however, has hard upper bound (Yang & Rannala, 2006). Additional been placement at the base of the crown-group, Caviomorpha, problems with this particular fossil calibration point will be as a means of calibrating molecular clocks. This has been done discussed in more detail in the following section. repeatedly, despite the original descriptions of the fossil inferring alignment with the caviomorph superfamily Cavioi- dea (Wyss et al., 1993, 1994), and subsequent identification of Concordance with palaeontological information an additional rodent from the same fossil bed, probably It is conceivable that colonization of South America by rodents belonging to the caviomorph superfamily Chinchilloidea could have commenced up to c. 55 Ma, as our data suggest, (Flynn et al., 2002). Two such divergent lineages at the same without being detected in the fossil record for some time, locale suggest that rodents must have originated and diversi- particularly if caviomorph lineages originally occupied areas of fied in South America well before the Eocene–Oligocene northern South America where palaeontological deposits of boundary. Thus, imposing a date of c. 32 Ma as a calibration an appropriate age are presently unknown. The theoretical point or as an upper bound at this node is unsubstantiated and counter-clockwise rotation of Africa from South America, likely to bias molecular-clock estimates. Concordant with this during the Late Cretaceous, resulted in tropical western Africa suggestion, our derived placement of this fossil (i.e. placed at being the last part of Africa in proximity to north-eastern the node immediately preceding the diversification of the South America, and fossil beds are exceptionally rare or absent family Dasyproctidae; node 51 in Fig. 2) as a fixed calibration in both of these biogeographical regions (Parrish, 1993; Flynn point, in accordance with its palaeontological identification et al., 2002; Jaeger, 2003; MacFadden, 2006). If dispersal did (‘dasyproctid indet.’; Wyss et al., 1993, 1994), yields diver- indeed occur from western Africa to north-eastern South gence dates (e.g. NPRS method, TTR gene region) consistent America, then we might expect a significant gap in time to with our independent multidivtime and NPRS (e.g. TTR exist from the earliest stages of colonization and diversification gene) estimates based on other calibration points. in these lowland tropical areas to presence being detectable (i.e. numerous and geographically dispersed enough to increase the Considering a southern dispersal route likelihood of being preserved) in the Late Eocene of central Chile, where the oldest South American rodent fossils are The plausibility of a southern dispersal route should not be presently recorded (c. 31–37 Ma; Flynn et al., 2002). An earlier precluded, in the light of evidence of trans-Antarctic dispersal colonization, prior to the Eocene–Oligocene debut in the fossil playing a predominant role in southern faunal and floral record, is also consistent with intercontinental habitat simi- exchange (Sanmartıń & Ronquist, 2004). Furthermore, the larity facilitating the survival of new immigrants. From the distribution of fossil and extant Caviomorpha lineages is Late Cretaceous through to the Early Eocene (c. 50 Ma), global concordant with the observation of South America being climatic belts were more tropical than they are today, with clearly divisible into two biotic provinces with different warm and humid tropical-temperate forests existing through- biogeographical affinities: a Southern Biome and a northern out South America and Africa from c. 50 to 80 Ma (Janis, Amazon Basin Biome (Vucetich et al., 1999; Sanmartıń& 1993; Flynn & Wyss, 1998; MacFadden, 2006). Ronquist, 2004). Following from these observations, the first The fossil record is not necessarily inconsistent with a appearance of rodents in the Southern Biome of South significantly earlier colonization of the continent than the America (i.e. central Chile; Wyss et al., 1993) might seem to oldest known rodent fossil might suggest. Many palaeontol- lend credibility to a southern route of colonization. ogists accept an older date of origin for Caviomorpha (Wyss Although the common ancestors of ctenodactylid and et al., 1994; Hartenberger, 1998; Flynn et al., 2002; Vucetich hystricognath rodents appear to be Asian in origin, inferring et al., 2004; MacPhee, 2005). In fact, Marivaux et al. (2002) the geographical origin of the suborder Hystricognathi itself

Journal of Biogeography 37, 305–324 317 ª 2009 Blackwell Publishing Ltd D. L. Rowe et al. has been controversial. Both Asian (e.g. Marivaux et al., 2002) an African origin for Hystricognathi and a trans-Atlantic and African (e.g. Lavocat, 1969) origins have been supported, dispersal of lineages to South America during the Late Eocene. as have multiple hypotheses for the subsequent origin and dispersal of Caviomorpha lineages to South America. Although Reliability of age estimates North America is viewed as an unlikely source for the South American radiation (Martin, 1994; Marivaux et al., 2002), a Factors influencing estimates of divergence time southern route via Antarctica was not ruled out in a previous study (Huchon & Douzery, 2001). As such, we consider the Although rejecting Gondwanan vicariance biogeography by a compatibility of our temporal data with a proposed Asian wide margin, the time-frame inferred for dispersal is likely to origin for Hystricognathi and a southern route of dispersal to be refined as methodological approaches improve. At present, South America. it appears that the younger estimates derived from molecular- If an Asian ancestor gave rise to the suborder Hystricognathi clock studies may be largely attributable to two important on the same continent, then multiple dispersal events must be factors: taxon sampling and calibration point designation. Our invoked to account for their contemporary distribution in minimization of the potential errors associated with limited Asia, Africa and South America. Under this scenario, the sub- taxonomic sampling, erroneous fossil calibration points, and Saharan-restricted Bathy–Phiomorpha clade would have arisen distant extrapolations is likely to provide a more robust as a consequence of an ancestral hystricognath lineage interpretation of the evolutionary history of hystricognath dispersing from Asia to Africa – some time between the origin rodents. Nonetheless, much remains to be elucidated about the of the crown-group Hystricognathi (node 72) and the utility of relaxed-molecular-clock methods. subsequent radiation of Bathy–Phiomorpha lineages within Certainly, Bayesian methods are known to be sensitive to the Africa (node 43). According to our molecular-clock estimates, designation of priors (Bell & Donoghue, 2005; Ho et al., 2005; this would have occurred in the interim of roughly 59–55 Ma. Renner, 2005; Welch & Bromham, 2005; Welch et al., 2005; Regardless of the precise timing of this proposed dispersal Smith et al., 2006). In particular, a prior assumption of a event across the Tethys Sea, a minimum of one additional lognormal distribution of rates has been shown to lead to an transoceanic dispersal event from an Asian ancestor would also underestimation bias for nodes, particularly for those older need to be inferred to account for the presence of rodents in than the calibration points (Ho et al., 2005). Alternatively, South America if they arrived via Antarctica. NPRS has been shown to over-fit the data, leading to rapid A transoceanic dispersal event from Southeast Asia to fluctuations in rates where there are short internodes, and Australia would be required in the interim of c. 45–55 Ma hence to overestimated ages near the root (Bell & Donoghue, (i.e. between nodes 70 and 71; Fig. 2 and Table 4), according 2005; Rutschmann, 2006). In this regard, it is encouraging to to our molecular-clock estimates, a time when Australia see a general agreement between our Bayesian and NPRS remained geographically isolated from Southeast Asia estimates, particularly at the root nodes. (Scotese et al., 1988). Subsequent to arriving in Australia, a The stochastic nature of dates derived from our GHR terrestrial dispersal route could have been facilitated, albeit dataset using the NPRS method may be attributable to known requiring coverage of a vast terrestrial expanse within a inadequacies of this method in terms of over-fitting of the data relatively narrow time-frame, as well as widespread extinction (Bell & Donoghue, 2005). Overestimation with NPRS has been (or lack of detection) from the Australian continent. To shown to be particularly severe when only a shallow, recent, accommodate a terrestrial expansion, dispersal across the node is used as a calibration point. In such cases, estimates for Australian continent would have been required within a the root age are ‘equally likely to move off to infinity or to 10-Myr time-frame, as some degree of separation of Antarc- retain more realistic values’ (Sanderson et al., 2004). Qualita- tica from Australia was attained by 40 to 50 Ma (Lee & tively, in our dataset, the degree to which GHR age estimates Lawver, 1995). Likewise, dispersal across the Antarctic do not conform is magnified as more shallow calibrating nodes continent in a similar time-frame would have been required are implemented. For example, extreme ‘overestimation’ is to allow for the colonization of South America before the observed for the Octodontidae calibration point (F2 in geological interruption of the Antarctic corridor connection Table 3), designated at 11 Ma. Intermediate ‘underestimation’ (with South America) at c. 35 Ma (Lawver et al., 1992). This is observed for the Hydrochaeridae calibration point (F1) at timing is surprisingly concordant with the initial Late Eocene 16 Ma, and no ‘deviant age estimates’ are obtained from the emergence of rodents in the South American fossil record, dasyproctid calibration point (F3) at 32 Ma. However, this which first appeared in the Southern Biome (c. 31–37 Ma; pattern was observed only for the GHR gene region, and not Wyss et al., 1993). for TTR. Nonetheless, we might have expected greater However, because this southern route requires either one stochasticity in age estimates to be observed with the GHR additional transoceanic dispersal event or a rapid and extensive dataset given that there were fewer variable characters and, geo-dispersal across Australia and Antarctica, as well as thus, a greater likelihood of inaccurate branch-length estimates continental extinctions or lack of detection in the fossil record, (Sanderson et al., 2004). In particular, exceptionally short it is deemed the less parsimonious explanation for the presence internodes were observed near the base of the tree, and some of lineages in South America. Therefore, at present, we favour branches were assigned a length of zero (data not shown).

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Consequently, this combination of parameters may have FUTURE STUDIES contributed to the anomalous nature of estimates derived from the GHR dataset using the NPRS method. In addition, Terrestrial and freshwater animals, as well as plants tradition- more severe rate shifts are observed among clades within the ally considered to be poor dispersers, appear to have crossed GHR dataset (data not shown), potentially violating the the Atlantic Ocean more regularly than previously appreciated, underlying assumption of autocorrelation of rates. with an ever-growing number of studies proposing transoceanic dispersal of biota (e.g. Danforth et al., 2004; Givnish & Renner, 2004; Pennington & Dick, 2004; de Queiroz, 2005; Taxonomic sampling pitfalls Renner, 2005). Among them, molecular-clock studies indicate The detection of variable rates of evolution is known to be that primates (e.g. Schrago & Russo, 2003) and freshwater sensitive to taxonomic sampling (Sanderson, 1998; Bromham cichlid fishes (e.g. Vences et al., 2001) may have shared a et al., 2000), and, more importantly, a recent empirical study dispersal avenue with hystricognath rodents, from Africa to suggests that poor taxonomic sampling leads to erroneous South America, during the Middle to Late Eocene (c. 35– estimations of divergence dates even when relaxed-clock 58 Ma). In addition to transoceanic voyagers to the New methods are employed (Linder et al., 2005). There appears World, it appears that wind-dependent dispersalists, such as to be a logarithmic relationship between the proportion of insects, could have been contemporary colonists as well (e.g. under-sampling and the degree of age underestimation, Danforth et al., 2004). Whether such long-distance transoce- particularly for the NPRS methodology (Linder et al., 2005). anic dispersal events exhibit regularity, as an identifiable and In this instance, sampling less than 10% of extant species predominant biogeographical pattern, or whether they are resulted in estimates that were half the ages of those obtained simply a random and coincident sampling of trans-Atlantic under full taxonomic sampling. In this respect, our inferences dispersalists certainly warrants further investigation. For are unlikely to be vastly underestimated (e.g. to the degree of instance, could these concomitant events be a product of half of their true age), as we have incorporated about 18% of biogeographically and temporally biased fossil records and/or extant Hystricognathi lineages. In addition, Bayesian methods uncertainties in the processes of molecular evolution under- have been shown to be more resilient to under-sampling lying molecular clocks, errors of which are compounded when effects, and we obtain similar age estimates using this method. biased fossil records are used to calibrate irregularly ticking Sparse taxonomic sampling might, however, account for molecular clocks? some of the younger age estimates reported in previous Combining geological, oceanographic and climatic data with molecular studies, particularly in cases where the NPRS temporal inferences has the potential to provide important relaxed-clock method has been implemented. However, this clues to unravelling evolutionary history. It is certainly evident is unlikely to be a universal explanation, given that equally that wind and water circulation systems are not randomly young age estimates have been reported using the Bayesian distributed in space and time and, it seems, may have persisted multidivtime method (see Table 1). for geologically significant and relevant time periods (Morley & Dick, 2003; Sanmartıń & Ronquist, 2004; Renner, 2005). Given that wind and ocean currents are known to influence Compounding effects of under-sampling and calibration contemporary dispersal, we might predict that the observation distance of biased patterns in the timing and direction of historical Both simulated and empirical evidence has indicated that the transoceanic dispersal is inevitable. While a biogeographical estimation of ancient divergence times by using recent calibra- bias in pattern might be detectable, although potentially tion points is prone to increasing error with increasing distance confounded by lineage-specific differences in dispersal and from calibration points (Nei et al., 2001; Linder et al., 2005). establishment capabilities, discernment of the predominant Linder et al. (2005) further reported a positive linear relation- pattern may require the examination of a large number of ship between degree of age underestimation and distance from taxonomic groups (Pennington & Dick, 2004; Sanmartıń& the calibration point, for both NPRS and Bayesian methods. In Ronquist, 2004; Renner, 2005). Most critically, reliable tem- other words, the greater the distance from the calibrating node, poral time-frames (i.e. divergence-date estimates) must be the more sensitive the age estimates become to under-sampling established. To achieve this goal, both palaeontological and (i.e. leading to the underestimation of true ages). This is an molecular-clock estimates of time must be scrutinized, as both important relationship, and fits with the observation of young types of analyses can be biased or mislead interpretations of age estimates being obtained when sparse taxonomic sampling evolutionary history. Studies of this nature should include a has accompanied the use of distant calibration points in previous comprehensive evaluation of the fossil record and identify molecular studies employing Bayesian methods (e.g. Adkins potential errors associated with molecular analyses in order to et al., 2003; Hasegawa et al., 2003; Springer et al., 2003). Our establish the reliability of the temporal component and divergence-date estimates are likely to be more resilient to these facilitate combination of data into large meta-analyses (Con- effects, given our greater taxonomic sampling and the use of roy & van Tuinen, 2003; Sanderson et al., 2004). Only then multiple calibration points within the study group, in close may we begin to elucidate the mechanisms by which contem- proximity to the node ages being estimated. porary distribution patterns of biota have been assembled.

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Craw, R.C. (1982) Phylogenetics, areas, geology and the bio- ACKNOWLEDGEMENTS geography of Croizat: a radical view. Systematic Zoology, 31, The authors thank the many individuals and institutions that 304–316. generously provided tissue samples for this study: R. J. Baker, Craw, R.C., Grehan, J.R. & Heads, M.J. (1999) Panbiogeo- Texas Tech University Museum; Cincinnatti Zoo, Ohio; L. P. graphy: tracking the history of life. Oxford University Press, Costa, Museum of Vertebrate Zoology, University of California, Oxford. Berkeley; R. C. Dowler; L. Emmons; M. Gallardo; A. Harlin; C. Danforth, B.N., Brady, S.G., Sipes, S.D. & Pearson, A. (2004) W. Kilpatrick, University of Vermont; E. Louis, Henry Doorley Single-copy nuclear genes recover Cretaceous-age diver- Zoo, Nebraska; J. Patton; Smithsonian National Zoological gences in bees. Systematic Biology, 53, 309–326. Park; and T. L. Yates, Museum of Southwest Biology, University Dobson, F.S. & Oli, M.K. 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Woods, C.A. & Hermanson, J.W. (1985) Myology of hystri- BIOSKETCHES cognath rodents: an analysis of form, function, and phylogeny. Evolutionary relationships among rodents: a Diane L. Rowe has a wide range of systematic research multidisciplinary analysis (ed. by W.P. Luckett and J.-L. interests, including molecular evolution, historical biogeogra- Hartenberger), pp. 515–548. Plenum Press, New York. phy and patterns of species diversiﬁcation. Current projects Wyss, A.R., Flynn, J.J., Norell, M.A., Swisher, C.C., III, Char- are focused on the geological and palaeontological calibration rier, R., Novacek, M.J. & McKenna, M.C. (1993) South of molecular clocks, life-history correlates of rates of molecular America’s earliest rodent and recognition of a new interval evolution, and ecological correlates of adaptive radiations. of mammalian evolution. Nature, 365, 434–437. Wyss, A.R., Flynn, J.J., Norell, M.A., Swisher, C.C., III, Katherine A. Dunn is a postdoctoral research fellow in the Novacek, M.J., McKenna, M.C. & Charrier, R. (1994) Department of Biology at Dalhousie University. Her research Paleogene mammals from the Andes of central Chile: currently focuses on comparative prokaryote genomics and a preliminary taxonomic, biostratigraphic, and geochro- statistical models of molecular evolution. nologic assessment. American Museum Novitates, 3098, 1– Ronald M. Adkins is Assistant Professor in the Department 31. of Pediatrics at the University of Tennessee Health Sciences Yang, Z. (1997) PAML: a program package for phylogenetic Center. His primary research interests are in the genetics of analysis by maximum likelihood. Computer Applications in foetal growth regulation, the genomics of the growth hormone the Biosciences, 13, 555–556. locus, and mammalian systematics. Yang, Z. & Rannala, B. (2006) Bayesian estimation of species divergence times under a molecular clock using multiple Rodney L. Honeycutt is Professor of Biology at Pepperdine fossil calibrations with soft bounds. Molecular Biology and University. His focal areas of research are in mammalian Evolution, 23, 212–226. evolution and population genetics. Yoder, A.D. & Yang, Z. (2000) Estimation of primate specia- tion dates using local molecular clocks. Molecular Biology Editor: Robert McDowall and Evolution, 17, 1081–1090.

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