DOI:10.1093/ sysbio/ syx029
Ad vance Access p u blication Febru ary 27, 2017
A Phylogen etic, Biogeograp h ic, an d Taxon om ic stu d y of all Extan t Sp ecies of Anolis (Squ am ata; Igu an id ae)
STEVENPOE1,∗, ADRIÁNNIETO-MON TES DEOCA2, OMARTORRES-CARVAJAL3, KEVIN DEQUEIROZ4, JULIÁNA. VELASCO5, BRADTRUETT1, LEVIN. GRAY1, MASONJ. RYAN 1, GUN TH ERKÖH LER6, FERNAN DOAYALA-VARELA3,AN DIANLATELLA1
1Department of Biology and Museum of Southwestern Biology, University of New Mexico, Albuquerque, NM 87131, USA;2Departamento de Biología Evolutiva, Facultad de Ciencias, Universidad Nacional Autónoma de México, México, D.F. 04510, México;3Museo de Zoología, Escuela de Biología, Pontificia Universidad Católica del Ecuador, Avenida 12 de Octubre y Roca, Apartado 17-01-2184, Quito, Ecuador;4Smithsonian Institution, National Museum of Natural History, Washington DC 20560-0162, USA;5Laboratorio de Análisis Espaciales, Instituto de Biologia, Universidad Nacional Autónoma
de México, México, D.F., México & Grupo de Investigación en Ecología Animal, Departamento de Biología, Facultad de Ciencias Naturales y Exactas, Universidad del Valle, Cali-Colombia;6Forschungsinstitut und Naturmuseum Senckenberg, Senckenberganlage 25, 60325 Frankfurt a.M., Germany
∗Correspondence to be sent to: Department of Biology and Museum of Southwestern Biology, University of New Mexico, Albuquerque, NM 87131, USA;
E-mail:[email protected]
Received 21 December 2015; reviews returned 9 October 2016; accepted 21 January 2017 Associate Editor: Robb Brumfield
Abstract.——Anolis lizard s (anoles) are textbook stu d y organism s in evolu tion and ecology. Althou gh several top ics in evolu tionary biology have been elu cid ated by the stu d y of anoles, p rogress in som e areas has been hamp ered by lim ited p hylogenetic inform ation on this grou p . H ere, we p resent a p hylogenetic analysis of all 379 extant sp ecies of Anolis, w ith new p hylogenetic d ata for 139 sp ecies inclu d ing new DNA d ata for 101 sp ecies. We u se the resu lting estim ates as a basis for d efining anole clad e nam es u nd er the p rincip les of p hylogenetic nom enclatu re and to exam ine the biogeograp hic history of anoles. Ou r new taxonom ic treatm ent achieves the su p p osed ad vantages of recent su bd ivisions of anoles that emp loyed ranked Linnaean-based nom enclatu re w hile avoid ing the p itfalls of those ap p roaches regard ing artificial constraints imp osed by ranks. Ou r biogeograp hic analyses d em onstrate comp lexity in the d isp ersal history of anoles, inclu d ing m u ltip le crossings of the Isthm u s of Panam a, two invasions of the Caribbean, single invasions to Jam aica and Cu ba, and a single evolu tionary d isp ersal from the Caribbean to the m ainland that resu lted in su bstantial anole d iversity. Ou r comp rehensive p hylogenetic estim ate of anoles shou ld p rove u sefu l for rigorou s testing of m any comp arative evolu tionary hyp otheses. [Anoles; biogeograp hy; lizard s; Neotrop ics; p hylogeny; taxonomy.]
Anolis is a well-stu d ied , ecologically d iverse, sp ecies- rich clad e of Neotrop ical lizard s. Anatom ically, Anolis lizard s (anoles) are characterized by exp and ed toep ad s that facilitate an arboreal lifestyle and a throat fan, or d ewlap , u sed m ainly in intrasp ecific signaling.
Anoles occu py a d iverse range of m icrohabitats w ith m ost sp ecies living on trees, bu shes, or grasses, bu t som e sp ecializing on rocks, stream s, or leaf litter.
Com m u nities of anoles range from u p to 12 symp atric sp ecies (e.g., at Parqu e Om ar Torrijos in Panam a;Poe 2012) to solitary sp ecies. Behaviorally, all sp ecies are d iu rnal except for a few Caribbean form s that m ay be noctu rnally active arou nd artificial lighting (examp les inSchwartz and H end erson 1991). The over 379 sp ecies of Anolis (see below ) natively range from Florid a sou th throu gh Central Am erica and the Caribbean to Bolivia, w ith natu ralized p op u lations as far as Asia.
Anolis lizard s are m od el stu d y organism s in ecology and evolu tion. They have been su bjects of classic stu d ies of com m u nity ecology (e.g., William s 1983), ecom orp hology (e.g., Collette 1961), com m u nication (e.g.,Rand and William s 1970), character d isp lacem ent (e.g.,Schoener 1970), biogeograp hy (e.g., Lazell 1972), ad aptive rad iation (e.g.,William s 1972), and comp etition (e.g.,Pacala and Rou ghgard en 1982), to nam e ju st a few textbook examp les. Recent au thors have incorp orated comp arative m ethod s and anole p hylogeny into stu d ies of these and other imp ortant top ics in evolu tion and ecology (e.g.,Losos et al. 1998;N icholson et al. 2005;Ord and Martins 2006). Many evolu tionary stu d ies of Anolis
wou ld have benefited from better p hylogenies based on m ore comp rehensive taxon samp ling, p articu larly of m ainland form s, and attempts at a comp rehensive taxonomy have also been hamp ered by lim ited samp ling.
For instance, the best-samp led m olecu lar p hylogenetic analysis of anoles to d ate (Gam ble et al. 2014) inclu d ed 216 (<57% of) sp ecies, the m ost recent comp arison of m ainland and island evolu tion in Anolis (Pinto et al. 2008) inclu d ed 35 (<17% of) m ainland sp ecies, and the m ost recent attempt at a comp rehensive taxonomy of anoles (N icholson et al. 2012) analyzed 240 (<63% of) sp ecies.
Etherid ge’s (1959) land m ark stu d y of skeletal m orp hology was the first large-scale p hylogenetic analysis of anoles. This work erected inform al grou p s ranked as “sections” and “series”, w hich were elaborated u p on (e.g., by the ad d ition of “sp ecies grou p s”) in William s’ (1976a,b) influ ential taxonom ic treatm ents that were u tilized by d escribers of sp ecies seeking p ools for taxonom ic comp arison and evolu tionary biologists seeking u nits for comp arative stu d y.Gu yer and Savage (1992; p reced ed byGu yer and Savage 1986) erected new genera w ithin Anolis based on a p hylogenetic analysis of 27 sp ecies. The ad vent of m olecu lar d ata brou ght reorganization of the Etherid ge–William s grou p s (e.g., Gorm an 1973; Shochat and Dessau er 1981; Bu rnell and H ed ges 1990), as well as m olecu lar p hylogenetic analyses of m any su bclad es of Anolis (e.g., Gorm an et al. 1983;H ed ges and Bu rnell 1990;Creer et al. 2001;
Schneid er et al. 2001; Brand ley and d e Qu eiroz 2004;
Glor et al. 2004; Castañed a and d e Qu eiroz 2011).
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Follow ing the p ioneering DNA sequ ence work of Jackm an et al. (1999) and the com bined -d ata stu d y of Poe (2004), the m ost recent large-scale p hylogenetic work (Alföld i et al. 2011;N icholson et al. 2012;Gam ble et al. 2014) has ad d ed taxa and d ata to bu ild on the Etherid ge–William s fram ework.
Progress in anole p hylogeny som etim es has been overshad owed by controversy regard ing the taxonomy of anoles (e.g.,Gu yer and Savage 1986,1992;Cannatella and d e Qu eiroz 1989; William s 1989; N icholson et al.
2012,2014;Poe 2013). Disagreem ents in anole taxonomy owe largely to d ifferences am ong au thors concerning the clad e or clad es to w hich the Linnaean rank of genu s is to be assigned . Becau se trad itional nom enclatu re is based on taxonom ic ranks, those d ifferences have created m ajor d iscrep ancies in the nam es ap p lied to variou s anole clad es d esp ite consid erable agreem ent regard ing their comp osition and p hylogenetic relationship s.
Consequ ently, d ebates have tend ed to focu s on the scientifically m eaningless qu estion of how m any genera ou ght to be recognized , thu s d iverting attention from scientifically germ ane d isagreem ents concerning the relationship s of anole sp ecies and the comp osition of anole clad es.
In an attempt to rectify this and sim ilar cou nter- p rod u ctive situ ations in taxonom ies throu ghou t the tree of life som e system atic biologists have been d evelop ing a tree-based ap p roach to biological nom enclatu re in w hich taxon nam es are tied exp licitly and d irectly to clad es (e.g.,d e Qu eiroz and Gau thier 1990,1992;Cantino and d e Qu eiroz 2014). By contrast, the trad itional, rank-based system d oes not necessarily tie nam es to clad es, and even w hen it d oes, the connection is ind irect and tenu ou s. The tree-focu sed ap p roach also has the ad vantage of p rod u cing taxonom ies w ith higher inform ation content, becau se the nam ed clad es are not restricted to a p articu lar taxonom ic level (in this case, the genu s). That is, instead of the nam es all being ap p lied to m u tu ally exclu sive clad es (as wou ld be the case w ith genera), they can be ap p lied to both nested and m u tu ally exclu sive clad es. Althou gh the tree-based ap p roach has been ad opted for som e su bclad es of anoles (N icholson 2002;Brand ley and d e Qu eiroz 2004;Castañed a and d e Qu eiroz 2013), it has not yet been ap p lied across the entire anole clad e.
As w ith taxonomy, rigorou s biogeograp hic treatm ents of anoles m ainly have been confined to su bgrou p s of the clad e, w ith a focu s on Caribbean form s (e.g.,Brand ley and d e Qu eiroz 2004; Glor et al. 2005; Klu tsch et al.
2007;Rod rígu ez-Robles et al. 2007; seePhillip s et al. 2015 for a m ainland examp le). Larger-scale treatm ents have exam ined general Caribbean p atterns (Alföld i et al. 2011;
H elm u s et al. 2014) or sp ecific biogeograp hic hyp otheses (e.g., the “back-invasion” of the m ainland ; N icholson et al. 2005). The one qu antitative attempt at d escribing overall Anolis biogeograp hic history N icholson et al.
(2012) likely su ffers from gross overestim ation of the age of the Anolis clad e (see Tow nsend et al. 2011;
Mu lcahy et al. 2012; Prates et al. 2015; and below ).
Nevertheless, that work erected testable hyp otheses that m ay be assessed w ith m ore realistic d ating. H ere, we test several biogeograp hic hyp otheses to exp lain the p resent-d ay d istribu tion of Anolis in the Neotrop ics. In p articu lar, we exam ine the follow ing historical events:
tim ing and ancestral area of the m ost recent com m on ancestor of anoles; tim ing and frequ ency of transitions of anole lineages between areas (inclu d ing m ainland and island s, and am ong island s); tim ing of biotic exchange of anole lineages between Mid d le Am erica and Sou th Am erica; existence of “sou rces” or “sinks” for anole d iversity.
The goals of this work are to estim ate the p hylogeny of all 379 sp ecies of Anolis and u se this estim ate to d escribe the biogeograp hic history of the clad e and erect a new p hylogenetic taxonomy of anoles.
MATERIALS AN D METH ODS
Taxon Sampling
We end eavored to inclu d e all valid sp ecies of Anolis as of 1 Ju ne 2014 in ou r analysis.
Su p p lem entary Ap p end ix S1 (available on Dryad at http :/ / d x.d oi.org/ 10.5061/ d ryad .s80jq) lists ou r ju d gem ents of sp ecies statu s for form s inclu d ed here;
this ap p roach resu lted in 379 sp ecies inclu d ed for p hylogenetic analysis. We inclu d ed the follow ing ou tgrou p s: Basiliscus plumifrons, Polychrus marmoratus, Pristidactylus scapulatus, Urostrophus gallardoi. These were selected based on m axim izing available d ata for close relatives of Anolis (e.g.,Pyron et al. 2013).
Data
We obtained DNA sequ ence d ata (varying coverage of m itochond rial genes N D2 and COI and the nu clear exon that cod es for end othelin-converting enzym e-like 1 [ECEL1]) for 101 sp ecies of Anolis not p reviou sly scored for DNA and com bined these w ith p u blished DNA d ata (e.g., Jackm an et al. 1999; Castañed a and d e Qu eiroz 2011; Alföld i et al. 2011) to p rod u ce a m atrix w ith varying taxonom ic coverage of 24,879 sites across 50 loci for 317 sp ecies. Ap p end ix 1 shows gene coverage for each sp ecies. Su p p lem entary Ap p end ix S2 gives sp ecim en vou chers and genes for DNA d ata new to this article. Sanger sequ encing was d one in the labs of SP, AN, GK, and OT, and by the Barcod e of Life Initiative (w w w.barcod eoflife.com). Alignm ents of ou r newly generated sequ ence d ata (ECEL1, N D2, COI) were p erform ed u sing Mu scle in Mega (Tam u ra et al. 2011) and checked and imp roved w ith reference to cod on p osition, p reviou s alignm ents of these genes in Anolis (e.g.,Jackm an et al. 1999), and the p u blished Anolis genom e (Alföld i et al. 2011).Alföld i et al.’s(2011) alignm ent was u sed for their d ata, except that we aligned
16S ou rselves after ad d ing ad d itional sequ ences from Genbank.
We collected new m orp hological d ata for 144 sp ecies not p reviou sly scored for m orp hology and com bined these w ith p u blished d ata to p rod u ce a m orp hological p hylogenetic m atrix of 46 characters (Ap p end ix 2) for all 379 sp ecies of Anolis. Su p p lem ental Ap p end ix S3 d escribes ou r cod ings for sp ecies for w hich we were u nable to exam ine sp ecim ens.
Phylogenetics
Phylogenetic m atrices su ch as ou rs that inclu d e large nu m bers of term inals and d iverse kind s of d ata are not straightforward to analyze. In p articu lar, the intent to integrate ord ered and u nord ered m u ltistate m orp hological d ata w ith GTR-m od eled DNA d ata greatly restricts the available ap p roaches and comp u ter p rogram s for analysis. H ere, we u se Bayesian p hylogenetic analysis of ou r com bined m atrix imp lem ented in MrBayes version 3.2.6 (H u elsenbeck and Ronqu ist 2001; Ronqu ist et al. 2012; Su chard and H u elsenbeck 2012). This ap p roach allows integration of comp lex m orp hological d atasets w ith m od el-averaging of GTR-class m od els for DNA d atasets (H u elsenbeck et al. 2004).
We u sed Partitionfind er (Lanfear et al. 2012) to select an optim al p artitioning schem e for the DNA d ata accord ing to the Bayesian Inform ation Criterion u nd er Partitionfind er’s “greed y” algorithm . We hyp othesized sep arate m od els for each cod on p osition for the well- samp led m itochond rial genes (N D2, COI) and for each entire gene for the 48 other analyzed genes.
Becau se MrBayes allows m od el-averaging across the entire GTR m od el sp ace (“nst=m ixed ”), there is little reason to d esignate p articu lar GTR-class m od els in comp arison of p artitioning schem es. Therefore, we comp ared only GTR versu s GTR+G m od els and ignored GTR-class su bm od els (e.g., H KY, F81) for each p artition in Partitionfind er. We agree w ith p reviou s au thors (e.g., Stam atakis 2006; Moyle et al. 2012) that the assu m ed benefit of ad d ing an invariant- sites p aram eter (i.e., “accou nting for” gene regions that cannot change) d oes not ou tweigh the p otential d ow nsid es (i.e., d u p lication of the fu nction of the rate heterogeneity p aram eter; p aram eter interaction;
overp aram eterization) and therefore exclu d ed invariant- sites m od els from consid eration.
The 46 m orp hological characters (Ap p end ix 2) were analyzed w ith the Stand ard m od el for inform ative characters (“cod ing=inform ative”) inclu d ing 42 ord ered (“ctyp e:ord ered ”) and fou r u nord ered (the d efau lt) characters and allow ing gam m a-d istribu ted rate variation w ith six categories (“rates=gam m a ngam m acat=6”). Top ology and branch lengths were linked across p artitions and other p aram eters were u nlinked .
Som e of ou r analyses requ ire a tim etree so we emp loyed a relaxed -clock ap p roach allow ing rate variation across lineages accord ing to the ind ep end ent
gam m a rates m od el (“brlensp r=clock:u niform clockvarp r=igr”) w ith Urostrophus gallardoi constrained as the ou tgrou p in MrBayes.
We exp erim ented extensively w ith MCMC p aram eter settings and settled on the follow ing strategy: two concu rrent ru ns of one cold and five heated chains w ith heating p aram eter T= 0.001, for 10 m illion generations, samp ling every 1000 trees. We exam ined p aram eter estim ates over generations u sing Tracer (Ram bau t and Dru m m ond 2007). We d iscard ed the first 50% of samp led trees as bu rnin. MrBayes analyses were p erform ed on the comp u ters of the Cyberinfrastru ctu re for Phylogenetic Research (CIPRES) Project. We p resent a m ajority- ru le consensu s of p ost bu rnin trees for taxonom ic conclu sions, and u se two fu lly resolved trees selected from the p ost bu rnin samp le for biogeograp hic and d ating analyses: a m axim u m clad e cred ibility tree (hereafter, MCC tree;Ram bau t et al. 2014) and the tree w ith the m inim al sym m etric d istance (Robinson and Fou ld s 1981) from the 50% m ajority ru le consensu s tree (hereafter, MRC tree). The top ology of the MRC tree was also analyzed w ith BEAST (Dru m m ond et al.
2012) to p rod u ce a third fu lly resolved tree for analysis (see below ). The MrBayes N EXUS file of DNA and m orp hological d ata are in Su p p lem ental Ap p end ix S4.
Biogeography
Divergence times—Divergence-tim e estim ates were generated u sing a Bayesian ap p roach in BEAST v. 1.8.1 (Dru m m ond et al. 2012). We fixed the tree top ology as the MRC tree and p ru ned sp ecies not scored for at least one gene (i.e., sp ecies scored only for m orp hology).
We ap p lied an u ncorrelated log-norm al relaxed - clock m od el to the DNA d ata u sing the sam e DNA p artitioning schem e d iscu ssed above and two fossil calibrations. The root of ou r tree was calibrated w ith the crow n grou p p leu rod ont igu anian Saichangurvel (Conrad et al. 2007) from the late Camp anian (70.6 +- 0.6 Mya) (Tow nsend et al. 2011). This fossil was u sed by Tow nsend et al. (2011) to constrain the crow n of the Pleu rod onta clad e of the igu anian tree. We assigned this fossil p oint calibration to the root of ou r tree (anoles+ ou tgrou p s) u sing a u niform d istribu tion p rior (70–72 mya). The second fossil calibration p oint was located in the m ost recent com m on ancestor (MRCA) of the Anolis chlorocyanus grou p (A.aliniger, A.
chlorocyanus, A. coelestinus, A. singularis). We u sed a Dom inican am ber anole fossil assigned to this grou p (d e Qu eiroz et al. 1998) w ith a m inim u m age of 23 mya.
We also u sed a u niform p rior d istribu tion for this nod e based on stratigrap hic inform ation from the fossil (17–23 mya). Both fossil calibration p oints u sed in this stu d y were p laced conservatively at the crow n of each clad e. Analyses were p erform ed on the CIPRES clu ster, w ith two ind ep end ent ru ns for 50 m illion generations samp ling every 5000. We checked log files to assu re stationarity in likelihood valu es and convergence u sing Tracer. We u sed 5 m illion generations as a bu rn-in p eriod and generated a m axim u m clad e cred ibility tim e
tree (hereafter, MRCT tree). We su m m arized p osterior d ivergence d ate estim ates for the m ost recent com m on ancestor of anoles and for regional trees in ord er to associate p articu lar historical events (e.g., the u p lift of the And es) w ith anole d ivergence tim es.
Biogeographic regions.—We d efined a set of 14 areas for biogeograp hic analyses based on the p resent-d ay d istribu tion p atterns of Anolis lizard s and the geological history of the Mid d le and Sou th Am erican m ainland and the Caribbean region (i.e., geological barriers and areas of end em ism ;Gregory-Wod zicki 2000;Losos 2011;
Coates and Oband o 1996; Su p p lem entary Fig. S1). We follow p reviou s workers on Mid d le Am erica, Sou th Am erica and the Caribbean island s (Castoe et al. 2009;
Santos et al. 2009;Antonelli et al. 2009;Daza et al. 2010) and u se the follow ing regions in ou r analysis: a) Lesser Antilles; b) Pu erto Rico and satellite island s and banks;
c) Cu ba and satellite island s and banks p lu s Caym an island s; d ) H isp aniola and satellite island s and banks;
e) Jam aica; f) the Baham as; g) sm all Caribbean island s (i.e., San And res and Provid encia island s and Swan island s); h) Nearctic from the Isthm u s of Tehu antep ec to the United States; i) Up p er Central Am erica from the N icaragu an d ep ression to the Isthm u s of Tehu antep ec;
j) Lower Central Am erica from the Panam a Isthm u s to the N icaragu an d ep ression; k) Sou th Am erican Chocó region encomp assing Pacific lowland s from Colom bia and Ecu ad or; l) Caribbean region and inter-And ean valleys in Colom bia and northwestern Venezu ela; m ) And es region from Venezu ela to Bolivia, above 1000 m ; n) Am azonia, inclu d ing Orinoco and Am azon river basins. We assigned each sp ecies to one or m ore regions based on d istribu tional record s comp iled from several sou rces (e.g.,N icholson et al. 2012;Velasco et al.
2015).
Statistical biogeographic methods.—Biogeograp hic analyses were p erform ed u sing the BioGeoBEARS R p ackage (Matzke 2013b) on the MCC tree and the MRC tree (i.e., inclu d ing all taxa) and on the MRCT tree (i.e., on the d ated tree inclu d ing only those taxa scored for m olecu lar d ata).
We p erform ed two biogeograp hic reconstru ctions on the MRC tree and the MCC tree, one focu sed on the m ainland and the other focu sed on the Caribbean.
We p erform ed sep arate reconstru ction analyses rather than a single large analysis d u e to the comp u tational comp lexity of p erform ing likelihood reconstru ctions w ith a large nu m ber of areas as in this case (Matzke 2014).
Becau se ou r exp loratory analyses d etected a low nu m ber of d isp ersal events between the m ainland and Caribbean island s (see below ), this strategy seem s u nlikely to have had a m ajor effect on ancestral range estim ates. For the analysis on the m ainland areas, all Caribbean island s were m erged into two d iscrete regions, a) Lesser Antilles and b) Greater Antilles p lu s the Baham as and Caym an Island s. Thu s, this first analysis was cond u cted on nine regions, inclu d ing seven m ainland and two Caribbean
regions. For the analysis focu sed on the Caribbean region, all m ainland areas were m erged into two regions, Mid d le Am erica and Sou th Am erica. Thu s, this second analysis inclu d ed seven Caribbean and two m ainland regions.
We p erform ed an ad d itional set of tim e-calibrated analyses on the MRCT tree. This tree is better samp led for Caribbean form s relative to m ainland form s, so we u sed the Caribbean-focu sed cod ing d icu ssed above. We p erform ed an analysis w here d isp ersal rate is assu m ed constant between all areas. This scenario costitu tes a nu ll biogeograp hic m od el that ignores the geological history of Caribbean land m asses and the land connections between island s. We also p erform ed a tim e-stratified analysis based on the geological m od el from Itu rrald e- Vinent and MacPhee (1999; see also Itu rrald e-Vinent 2006). For this scenario, we p enalized strongly against d isp ersal across water assigning a very low p robability of traversal (alm ost zero; d= 0.001) w hen land m asses were sep arated and a p robability of 1 w hen land m asses were connected . We bu ilt cost d isp ersal m atrices based on p aleogeograp hical scenarios hyp othesized byItu rrald e- Vinent and MacPhee (1999; see also Itu rrald e-Vinent 2006) for five tim e p eriod s as follows: (1) Early Eocene (55 Ma): all Caribbean land m asses were hyp othesized to be sep arated at this tim e, so we p enalized d isp ersal between island s, assigning d isp ersal p robability of 0.01 for all transitions between land m asses; (2) Late Eocene–Early Oligocene (35-33 Ma): d u ring this narrow tim e fram e all Greater Antilles except Jam aica were hyp othesized to be connected as a single land m ass (GAARland ia), w hich was connected to Sou th Am erica by the Aves Rid ge; thu s, we assigned a d isp ersal p robability of 1 for all transitions between regions (Greater Antilles, Lesser Antilles, and Sou th Am erica) except transitions involving Jam aica; (3) Late Oligocene (27–25 Ma): Cu ba was hyp othesized to be fragm ented in three land m asses, H isp aniola was d ivid ed into northern and sou thern island s w ith Sou thern H isp aniola being connected to Pu erto Rico, and the Lesser Antilles were isolated ; for this p eriod , we assigned a d isp ersal p robability of 1 to transitions between connected land m asses and 0.01 between sep arated land m asses;
(4) Mid d le Miocene (16–14 Ma): Cu ba rem ained fragm ented , H isp aniola m erged again bu t rem ained narrowly connected to Pu erto Rico, and the Lesser Antilles were isolated ; for this p eriod , we assigned a d isp ersal p robability of 0.01 between all land m asses;
(5) Pliocene to p resent (5–0 Ma): cu rrent sep aration of land m asses is assu m ed ; we set a d isp ersal p robability to 0.01 between all island s. We comp ared the fit of nu ll and tim e-stratified biogeograp hic m od els u sing the Akaike inform ation criterion (AIC).
For each set of reconstru ctions, we cond u cted Disp ersal–Extinction–Clad ogenesis (DEC) analyses comp aring m od els w ith and w ithou t a ju mp d isp ersal p aram eter J (Matzke 2014). We u sed ou r ancestral area reconstru ctions to estim ate the nu m ber of d isp ersal events in and ou t of each biogeograp hic region. The cou nts allowed u s to establish w hether an area was a
relative sink (i.e., areas receiving im m igrant lineages from other areas) or sou rce (i.e., areas from w hich lineages d isp ersed to other areas) (Sed ano and Bu rns 2010; Castroviejo-Fisher et al. 2014). We u se the term s sink and sou rce in a m acroevolu tionary context, as in Gold berg et al.(2005).
Taxonomy
In the interest of d evelop ing a taxonomy of anoles that is stable w ith resp ect to taxonom ic ranks bu t allows for ap p rop riate changes in the hyp othesized comp osition of taxa u nd er new hyp otheses abou t p hylogenetic relationship s, we ap p ly the m ethod s of p hylogenetic nom enclatu re to the m ajor clad es of anoles. Sp ecifically, we ap p ly nam es to both nested and m u tu ally exclu sive anole clad es u sing exp licit p hylogenetic d efinitions (e.g., d e Qu eiroz and Gau thier 1990, 1992; Cantino and d e Qu eiroz 2014). Ou r cu rrent selection of anole clad es to nam e is su bjective and based on trad itionally recognized grou p s of anoles. We d o not nam e every clad e d u e to sp ace constraints, and we d o not nam e only well-su p p ored clad es becau se som e p oorly su p p orted clad es have recu rred in several analyses and seem likely to w ithstand fu rther scru tiny. In the interest of nom enclatu ral stability, we ap p ly existing nam es to the clad es w ith w hich they have the longest associations.
H owever, becau se not all of the clad es have existing nam es, and becau se som e nam es have p reviou sly been ap p lied to m ore than one clad e, we resu rrect fou r long- u nu sed nam e and coin two new ones. For the p u rp ose of assessing trad itional u se, we ad opt a criterion sim ilar to that ad opted by the ICZN (1999) for m aintaining p revailing u se (Art. 23.9.1.2). That is, the nam e m u st have been ap p lied to the taxon in qu estion (ju d ged on the basis of comp osition and d iagnostic characters) in at least 25 works, p u blished by at least 10 au thors, in the im m ed iately p reced ing 50 years, and encomp assing a sp an of not less than 10 years. This ap p roach corresp ond s rou ghly w ith treating the (form al) nam es recognized by Etherid ge (1959), w ho is com m only consid ered to have initiated the m od ern era of anole system atics, as those having trad itional u se. The m ain exception is Tropidodactylus, w hich was recognized by Etherid ge bu t d oes not m eet all the criteria for trad itional u se.
RESULTS
The Partitionfind er analysis su ggested a 15-p artition schem e for the 50 gene, 54 p otential p artition m olecu lar d ata (Table1). The MrBayes analysis took 138 h on the CIPRES XSEDE clu ster (m axim u m allowed is 168 h). The average stand ard d eviation of sp lit frequ encies (ASDSF) was 0.059. The two ru ns ap p ear to have p lateau ed and converged on sim ilar likelihood s (Su p p lem entary Fig.
S2), and MCC trees calcu lated sep arately for each ru n are nearly id entical. These resu lts are consistent w ith the id ea that MCMC m ixing was su fficient.
TABLE1. Partitioning schem e estim ated in Partitionfind er analysis
Partition Mod el Genes
1 GTR+G COI p osition 1, KCN V2
2 GTR+G COI p osition 2, u nnam ed
# 146
3 GTR+G COI p osition 3
4 GTR+G ECEL1, H OXB1, KIF24
5 GTR+G N D2 p osition 1
6 GTR+G N D2 p osition 2
7 GTR+G N D2 p osition 3
8 GTR+G SOCS5 12, PCDH 10,
FN IP2, KIAA2018, IRS2, RAPGEF2, u nnam ed # 50, u nnam ed # 57, TMTC4, RAG1
9 GTR+G 16S
10 GTR+G u nnam ed # 127,
EN SACAG00000014694, STRN 4, PDE4D
11 GTR+G EN SACAG00000011799,
PDS5A, u nnam ed #183,
PPP2R5C, SFRS18,
u nnam ed # 59
12 GTR+G FRYL, DH X15, KIAA,
TLK2, TJP2, ATP2B1, u nnam ed # 53, TRPA1
13 GTR u nnam ed #60
14 GTR+G ELAVL2, u nnam ed #10,
FBXW7, u nnam ed #152, GLRB, BNC2, N FIB, PTPRD
15 GTR+G RXF3, EXPH 5, "C10 or
F71", BRCA1, u nnam ed
#177
Note: “Unnam ed ” gene sequ ences refer to regions listed in Alfold i et al.
(2011: Su p p lem entary Table 20).
Relationships
A m ajority-ru le consensu s (inclu d ing ad d itional comp atible grou p ings) of p ost-bu rnin trees from the MrBayes analysis is show n in Figu res 1–4. This tree d ep icts m any relationship s inferred in p reviou s analyses (e.g., Etherid ge 1959; Jackm an et al. 1999; Alföld i et al. 2011). In p articu lar, the overall stru ctu re of two p red om inantly m ainland rad iations, two Lesser Antillean clad es, and m u ltip le Greater Antillean rad iations is evid ent. We consid er ou r estim ate to su p ersed e p reviou s attempts at reconstru cting anole p hylogeny d u e to ou r greater character and sp ecies samp ling. Fu rtherm ore, comp arison w ith earlier analyses is not straightforward in cases w here taxonom ic coverage d iffers. For these reasons, we d o not m ake grou p -by-grou p comp arisons w ith all p reviou s stu d ies, bu t instead m ainly d iscu ss p reviou sly recognized grou p s that bear on ou r form ally nam ed clad es d iscu ssed below. We note that several well-su p p orted sm all grou p s recognized by Etherid ge (1959) and William s (1976a,b) and su bsequ ent au thors were m ostly or comp letely m onop hyletic (e.g., Beta anoles, bimaculatus series, roquet series, cristatellus series, sagrei series, grahami series), and that five of the eight genera ofN icholson et al.(2012) were corroborated as m onop hyletic. In the su m m ary
FIGURE1. Consensu s p hylogenetic estim ate for the Dactyloa clad e of Anolis based on Bayesian analysis of m orp hological and DNA d ata.
Nu m bers on clad es are p osterior p robabilities×100.
below, we refer to nam ed grou p s in Figu res 1–4. The nam es of these grou p s are d efined p hylogenetically in Ap p end ix 3.
A large clad e of p red om inantly Sou th Am erican sp ecies (ap p roxim ately equ al to Etherid ge’s [1959]
latifrons series and N icholson et al. [2012] Dactyloa) is
FIGURE2. Consensu s p hylogenetic estim ate for Digilimbus (m inu s Ctenonotus and Norops) clad e of Anolis based on Bayesian analysis of m orp hological and DNA d ata. Nu m bers on clad es are p osterior p robabilities×100.
FIGURE3. Consensu s p hylogenetic estim ate for Norops (m inu s Draconura) and Ctenonotus clad es of Anolis based on Bayesian analysis of m orp hological and DNA d ata. Nu m bers on clad es are p osterior p robabilities× 100.
sister to the rest of Anolis (Fig. 1). This Dactyloa clad e inclu d es lineages that extend north into the sou thern Lesser Antilles (m em bers of the roquet series) and into Central Am erica. Many of these sp ecies are large-bod ied
w ith high nu m bers of toe lam ellae. The clad e inclu d es great variation in head scale size w ith som e sp ecies (e.g., sp ecies form erly recognized as Phenacosaurus) d isp laying as few as two scales across the snou t w hereas
FIGURE4. Consensu s p hylogenetic estim ate for Draconura clad e of Anolis based on Bayesian analysis of m orp hological and DNA d ata.
Nu m bers on clad es are p osterior p robabilities× 100.
TABLE2. Comp arison of DEC (Disp ersal–Extinction–Clad ogenesis) and DEC+J (Disp ersal–Extinction–Clad ogenesis p lu s ju mp d isp ersal) m od els of historical biogeograp hy of Anolis lizard s
Tree Region Mod el LnL P d e j AIC d AICc Weights
MCC tree
Mainland DEC −597.3 2.0 1.2 0.0 0.0 1198.6 38.1 0.0
D EC+J −577.3 3.0 1.0 0.0 0.0 1160.5 0.0 1.0
Caribbean DEC −335.6 2.0 0.5 0.0 0.0 675.1 91.0 0.0
D EC+J −289.1 3.0 0.3 0.0 0.0 584.1 0.0 1.0
MRC tree
Mainland DEC −591.1 2.0 1.3 0.3 0.0 1186.1 36.2 0.0
D EC+J −572.0 3.0 1.0 0.2 0.0 1149.9 0.0 1.0
Caribbean DEC −347.4 2.0 0.3 0.0 0.0 698.8 108.5 0.0
D EC+J −292.1 3.0 0.3 0.1 0.0 590.3 0.0 1.0
Notes: Ln: Ln likelihood ; P: nu m ber of p aram eters; d : d isp ersal; e: extinction; j: ju mp -d isp ersal; AIC: Akaike Inform ation Criterion; d AICc: d elta AICc; weights: m od el weights. MCC tree: Maxim u m Clad e Cred ibility tree from MrBayes analysis; MRC tree: Consensu s-like tree from MrBayes analysis (see text). Mainland : refers to biogeograp hic analysis focu sed on continental areas. Caribbean: refers to biogeograp hic analysis focu sed on Caribbean areas. Best m od el in each comp arison is highlighted in bold .
others (e.g., William s’ 1976b aequatorialis grou p ) have m ore than 20.
Sister to the Dactyloa clad e is the Digilimbus clad e, w hich inclu d es Deiroptyx as sister to the rem aind er of Anolis (Fig. 2). Deiroptyx is comp osed of the Cu ban crow n-giant anoles (e.g., A. equestris) as sister to a clad e of H isp aniolan form s inclu d ing A. darlingtoni, the H isp aniolan green anoles, and the hendersoni and monticola series of William s (1976a). The d ewlap less Cu ban sp ecies A. bartschi and A. vermiculatus are sister to this clad e, and the u nu su al Pu erto Rican tw ig anole A. occultus is sister to the rest of Deiroptyx.
Also w ithin Digilimbus, a weakly su p p orted clad e inclu d es five nam ed grou p s as sister to a clad e comp osed of Ctenocercus, Ctenonotus, and Norops (Fig.2). This clad e inclu d es the cybotoid anoles (Audantia), the d istinctive terrestrial form Anolis (Chamaelinorops) barbouri, and the grass-bu sh anoles (Schmidtanolis) from H isp aniola, as well as a clad e of m ostly large-bod ied Greater Antillean anoles (Xiphosurus). The Cu ban cham aeleon-like anoles (Chamaeleolis) form a strongly su p p orted clad e w ithin Xiphosurus.
Ctenocercus is an ecom orp hologically d iverse clad e of p red om inantly Cu ban sp ecies that is sister to Ctenonotus and the beta anoles (Norops) ofEtherid ge(1959) (Fig.2).
Ctenocercus inclu d es m ini-rad iations of grass anoles (e.g., Anolis alutaceus), tw ig anoles (e.g., A. angusticeps), and green tru nk-crow n anoles (e.g., A. carolinensis), as well as the weakly su p p orted p lacem ent (p osterior p robability 0.41) of Cu ban A. argenteolus and A. lucius as sister to the rem aining Ctenocercus.
Ctenonotus is a chrom osom ally d iverse clad e (Gorm an 1973) that inclu d es well su p p orted Lesser Antillean (William s’ [1976a] bimaculatus series), H isp aniolan (distichus series), and Pu erto Rican Bank (cristatellus series) su bclad es (Fig. 3). Ctenonotus anoles tend to be abu nd ant and highly visible, and som e are am ong the best-stu d ied anole sp ecies (e.g.,Rand 1964;Pacala and Rou ghgard en 1982;Losos 1990;Dobson et al. 1992;H ertz 1992;Fleishm an et al. 1997).
Sp ecies of the Norops clad e of “beta” anoles (Etherid ge 1959) share the anatom ical trait of anteriorly d irected
transverse p rocesses on the p osterior cau d al vertebrae (Figs. 3 and 4). This well-established clad e inclu d es three geograp hically coherent su bclad es. Trachypilus is a Cu ban clad e of sp ecies m ainly belonging to the tru nk- grou nd ecom orp h. Placopsis is the Jam aican rad iation of m u ltip le ecom orp hs. Draconura is a m ainland rad iation, w ith several form s that have becom e established on offshore island s (e.g., A. townsendi, A. concolor, A.
villai). Trachypilus and Placopsis are very well-stu d ied clad es (e.g.Und erwood and William s 1959;Ru ibal 1961;
Bu nd y et al. 1987; Losos 1990; Jackm an et al. 2002;
Vanhooyd onck et al. 2005;Knou ft et al. 2006;Cád iz et al.
2013), w hereas Draconura rem ains the p rop ortionately least-know n anole clad e (bu t see, e.g., And rews 1971;
Fitch et al. 1976;N icholson 2002;Vitt et al. 2002).
Biogeography
Biogeographic model selection and anole diversification.—
In all analyses, DEC+J m od els were favored over DEC m od els accord ing to both AIC and m od el weights, su ggesting that fou nd er-event d iversification has been p revalent d u ring the anole rad iation (Table 2).
Fu rtherm ore, biogeograp hic m od els incorp orating p aleogeograp hic inform ation were favored (Table 3).
These biogeograp hic m od els incorp orate inform ation abou t historical connections between Caribbean land m asses by red u cing d isp ersal p robabilities between areas that were not connected over tim e. Several instances of fou nd er-event d iversification were inferred d u ring the colonization of the Caribbean (Fig. 5;
Su p p lem entary Figs. S3–6). Divergence d ate estim ates in ou r BEAST analysis (MRCT tree; Su p p lem entary Fig.
S6; Fig.5) p rovid ed evid ence of an origin of the anole rad iation at the Paleocene–Eocene bou nd ary (64.4–46.3 Ma). Posterior d ensity p lots su m m arizing d ivergence d ate estim ates (Fig. 6) show that m ost clad ogenetic events occu rred d u ring the Miocene (20–5 Ma). For Sou th Am erican clad es, the m ajority of clad ogenetic events seem not to be associated w ith And ean u p lift (m ain And ean u p lift events occu rred between 10–3 Ma
TABLE3. Comp arison of DEC (Disp ersal–Extinction–Clad ogenesis) and DEC+J (Disp ersal–Extinction–Clad ogenesis p lu s ju mp d isp ersal) m od els of historical biogeograp hy of Anolis u sing MRCT tree (BEAST version of MRC, consensu s-like tree; see text) w ith focu s on Caribbean areas
Typ e Mod el LnL P d e j AIC d AICc Weights
Tim e stratified DEC −290.9 2.0 0.0 0.0 0.0 585.9 78.1 0.0
D EC+J −250.9 3.0 0.0 0.0 0.0 507.8 0.0 1.0
Nu ll DEC −298.6 2.0 0.0 0.0 0.0 601.3 93.5 0.0
D EC+J −260.5 3.0 0.0 0.0 0.0 527.0 19.2 0.0
Notes: Nu ll m od el assu m es equ al d isp ersal p robability between island s or land m asses. Tim e stratified m od el allows d ifferences in d isp ersal rates in the m od el form u lation based on Caribbean p aleogeograp hic m od els (Itu rrald e-Vinent and MacPhee 1999;Itu rrald e-Vinent 2006; see m ain text for d etails). LnL: Ln likelihood ; P: nu m ber of p aram eters; d : d isp ersal; e: extinction; j: ju mp -d isp ersal; AIC: Akaike Inform ation Criterion;
d AICc: d elta AICc; weights: m od el weights. Best m od el in each comp arison is highlighted in bold .
[Antonelli et al. 2009; H orn et al. 2010]). In contrast, m ost clad ogenetic events for Mid d le Am erican clad es ap p ear correlated w ith intense tectonic activity d u ring the m id -Miocene (15–10 Ma) (Fig.6;Castoe et al. 2009;
Daza et al. 2010).
Early evolution and mainland-island transitions.—
Analysis of the MRCT tree (Fig. 5) estim ated a comp osite ancestral area for all Anolis. This estim ate m ay be interp reted either as am bigu ity or that the ancestor of all Anolis occu p ied a large area inclu d ing Caribbean and Sou th Am erican regions.
Other reconstru ctions u sing the fu ll comp lem ent of taxa (MCC, MRC trees; Su p p lem entary Figs. S3-6) id entify Sou th Am erica as the origin of Anolis. If the anole ancestor was only p resent in Sou th Am erica, p articu larly the Am azonia region (Su p p lem entary Figs.
S4, S6), at least two d isp ersal events are necessary to exp lain the cu rrent d istribu tion of Caribbean clad es.
The first d isp ersal event, to the Northern Caribbean, likely occu rred d u ring the Paleocene–Eocene bou nd ary (42.4– 61.7 Ma; Su p p lem entary Fig. S7; Fig. 5). The tim ing of this event p red ates the em ergence of the Aves rid ge land brid ge (Itu rrald e-Vinent and MacPhee 1999;
Itu rrald e-Vinent 2006). Thu s, u nd er this scenario, an overwater d isp ersal event likely exp lains the d istribu tion of all Northern Caribbean clad es (i.e., a ju mp d isp ersal event p rom oting fou nd er-event d iversification in the Caribbean Digilimbus clad e). The second d isp ersal event is the invasion of the roquet series to the Lesser Antilles, w hich likely occu rred near the Eocene–Oligocene bou nd ary (23.9–40.1 Ma; Su p p lem entary Fig. S7; Fig.5) w hen the Aves Rid ge is hyp othesized to have been p resent.
All biogeograp hic reconstru ctions ind icate a West Ind ian ancestry for the Draconura invasion of Mid d le Am erica (N icholson et al. 2005) w ith later d isp ersal to Sou th Am erica. One of the ancestral range estim ates for Draconura inferred a scenario involving a d isp ersal event from Jam aica to Mid d le Am erica, w hich wou ld have occu rred d u ring the Eocene–Oligocene bou nd ary (29.9–41.Ma; Fig.5; Su p p lem entary Fig. S7).
Caribbean dispersal events.—In the biogeograp hic m od els that inclu d ed m axim al taxonom ic coverage and d id not
incorp orate geological inform ation (i.e., MCC and MRC analyses) the ancestral range estim ated for Caribbean Anolis was H isp aniola (Su p p lem entary Figs. S3, S5).
H owever, in the MRCT tree analysis (Fig. 5), the ancestral area was comp osite inclu d ing Cu ba and H isp aniola. This latter inference is consistent w ith ancestral Caribbean anole occu p ation of the comp osite area know n as GAARland ia (Itu rrald e-Vinent and MacPhee 1999;Itu rrald e-Vinent 2006). The MRCT tree ind icated at least 18 transitions am ong Caribbean island s. Many biogeograp hic m ovem ents occu rred w hen island s were connected as either GAARland ia or w hen H isp aniola was connected to Pu erto Rico. Both Cu ba and H isp aniola were m ajor sou rces of Northern Caribbean anole lineages, w ith at least 12 d isp ersal events to other island s. Som e Caribbean transitions were from Greater Antilles to sm all Caribbean island s (e.g., Baham as and Caym an island s) and likely occu rred across water. At least three d isp ersals are inferred to exp lain the anole d iversity in each of Cu ba, H isp aniola, and Pu erto Rico.
Exclu d ing A. sagrei, w hich m ay not be native to Jam aica (Und erwood and William s 1959), a single overwater d isp ersal event exp lains cu rrent anole d iversity in Jam aica (Su p p lem entary Table S1).
Faunal exchange through the Isthmus of Panama.—From 18-20 crossings of the Isthm u s of Panam a were reconstru cted (Su p p lem entary Table S2). Dep end ing on w hich tree is analyzed , we inferred m ore d isp ersal events from Mid d le Am erica (MA) to Sou th Am erica (SA) than the reverse (MRC, MRCT trees), or an ap p roxim ately equ al nu m ber of MA to SA and SA to MA d isp ersals (MCC tree; Su p p lem entary Table S2). Based on the MRCT tree, at least two d isp ersal events from MA to SA were very early (~30 Ma; Su p p lem entary Fig. S7; Fig.5), one for the sm allest clad e containing Anolis auratus and A. brasiliensis and another for the sm allest clad e containing A. notopholis and A. gracilipes. Accord ing to recent geological evid ence, the em ergence of the Sou th- Mid d le Am erican land brid ge started between 25 and 23 Ma (Farris et al. 2011), and the final closu re occu rred by 10 Ma (Farris et al. 2011;Bacon et al. 2015). Other d isp ersal events inferred in the trees involved range exp ansions d u ring the Miocene.
FIGURE5. Biogeograp hical reconstru ction for Anolis lizard s w ith emp hasis in Caribbean areas u sing the d ated MRCT tree from MrBayes and BEAST analyses (see text). Vertical d otted lines rep resents the tim ing of p aleogeograp hical reconstru ctions based onItu rrald e-Vinent and MacPhee(1999; see alsoItu rrald e-Vinent 2006). Scale is m illions of years. SLA=Sou thern Lesser Antilles; SA= Sou th Am erica; CA= Central Am erica; C=Cu ba; H=H isp aniola; N LA=Northern Lesser Antilles; PR=Pu erto Rico; J= Jam aica.
Dispersals among other mainland areas.—Ou r m ainland analyses estim ated at least 77 d isp ersal events either as range exp ansions or long-d istance events between m ainland areas (Su p p lem entary Table S3;
Su p p lem entary Figs. S4, S6). Disp ersal events were estim ated to occu r evenly across the tim esp an of anole history. This resu lt m ay ind icate som e constancy of d isp ersal rates, bu t we note that som e very ancient d isp ersal events m ay not be inferred d u e to lineage extinction, w hich cou ld bias the overall p attern to u nd erestim ate the relative rate of earlier d isp ersals. Mainland areas w ith the highest nu m ber of im m igrations were Nearctic, Up p er Central Am erica,
Lower Central Am erica and the Chocó. Invasions to these areas occu rred in several instances and involved m u ltip le clad es. Up p er Central Am erica, Lower Central Am erica, the Chocó and the And es exhibited the highest nu m ber of lineage em igrations. Finally, the Caribbean coast of Colom bia and Am azonia exhibited the highest nu m ber of d isp ersals into versu s ou t of the area. In other word s, anole d iversity in these two areas is a com bination of im m igration of lineages from nearby regions and in situ sp eciation. In general, m ainland areas can be characterized as both sou rce and sink areas of fau nal d iversity (Su p p lem entary Table S3).
FIGURE6. Posterior d ensity p lots of d ivergence tim es for Anolis based on the d ated MRCT tree from MrBayes and BEAST analysis (see text). a) All sp ecies; b) only Caribbean sp ecies. The vertical lines rep resent the tim ing of the form ation of the Aves Rid ge; c) Sou th Am erican sp ecies. The vertical lines rep resent the tim ing of the And ean u p lift in Sou th Am erica; d ) Mid d le Am erican sp ecies. The vertical lines rep resent the tim ing of the form ation of the Isthm u s of Panam a.
DISCUSSION
Anolis lizard s are classic stu d y organism s in evolu tion, p hysiology, and ecology. The p hylogenetic estim ate p resented here shou ld enable novel and m ore comp rehensive comp arative analyses of this well- stu d ied clad e. Many su bjects that cou ld be ad d ressed only weakly or p artially w ith lim ited samp ling, su ch as m ainland -Caribbean comp arisons, comp arative com m u nity evolu tion, and rates of sp eciation, m ay now be tested rigorou sly.
The ou tstand ing asp ect of ou r p hylogenetic estim ates is their comp leteness. Their p rincip al fau lt is the weak su p p ort for m any nod es, esp ecially d eep in the trees.
Sixty-three p ercent of clad es are su p p orted at less than 95% p robability in the comp rehensive estim ate (Figs.1- 4). We su ggest this weak su p p ort is d u e to two factors.
First, ap p rop riately evolving nu clear genes have not yet been su fficiently taxonom ically samp led to p rovid e su p p ort for the d eep sp lits in the anole tree (e.g., Ap p end ix 1). Second , the m atrix inclu d es several taxa scored for only a few characters of external m orp hology (e.g., Anolis vicarius, A. pseudotigrinus) that are likely to be
weakly p laced in the tree. It wou ld be p ossible to imp rove su p p ort valu es by rem oving these taxa, as is som etim es d one (e.g., Sand erson and Shaffer 2002; Moyle et al.
2012). For examp le, a RaxML (v 1.5, Stam atakis 2006) analysis of all DNA d ata inclu d ing the 294 sp ecies scored for N D2 had only 41% of clad es su p p orted at less than 95% bootstrap (sam e p artitioning schem e as above, “ML+ thorou gh bootstrap ” com m and ; resu lts not show n). Bu t this p ractice obviou sly wou ld resu lt in a less comp rehensive estim ate of anole p hylogeny and taxonomy, and accu racy m ight be red u ced as well (see e.g.,Gau thier et al. 1988). Most imp ortantly, su ch an ap p roach wou ld gu arantee an incomp lete (i.e., inaccu rate) biogeograp hic reconstru ction for the anole clad e, as transitions to areas of m issing sp ecies m ight not be rep resented . For examp le, rem oval of p oorly scored sp ecies A. concolor and A. pinchoti wou ld p reclu d e estim ation of an imp ortant biogeograp hic event—the d isp ersal of the Draconura (i.e., m ainland Norops) clad e to oceanic Caribbean island s San And res and Provid encia, w here these anoles are solitary end em ic sp ecies.
Many inferred relationship s in Figu res 1–4 m ake sense in light of p reviou s stu d ies and exp ectations
based on m orp hology. Ou r p hylogenetic estim ates for m u ch of the anole clad e are heavily d eterm ined by p reviou sly p u blished d ata, and ou r resu lts for these well- stu d ied form s are largely congru ent w ith p reviou s work on Caribbean (e.g., Jackm an et al. 1999) and Dactyloa (e.g., Castañed a and d e Qu eiroz 2011) anoles. Bu t relationship s of m ainland beta anoles (Draconura; Fig.4) were largely u nknow n before this stu d y (seeN icholson [2002],Poe[2004], andN icholson et al.[2012] for analyses inclu d ing a few Draconura). Draconura relationship s that are u nsu rp rising inclu d e the m onop hyly of anoles sim ilar to Anolis fuscoauratus (the clad e sp anning A.
tenorioensis to A. bocourtii in Fig. 4), the m onop hyly of tropidolepis-like anoles (clad e sp anning A. pachypus to A. pseudopachypus), the geograp hic coherence of the Mexican form s (the A. cobanensis to A. pygmaeus clad e and the A. dunni to A. subocularis clad e), and the m onop hyly of anole sp ecies p reviou sly referred to or associated w ith A. limifrons (clad e sp anning A. apletophallus to A. zeus) and A. lemurinus (clad e sp anning A. bicaorum to A. vittigerus). Nu m erou s sm aller clad es likew ise reassu ringly align w ith exp ectation (e.g., geograp hically p roxim al island form s A. concolor–A. pinchoti; Sou th Am erican sem iaqu atic anoles A. macrolepis–A. rivalis–A.
lynchi; Central Am erican sem iaqu atic anoles A. lionotus–
A. oxylophus–A. poecilopus; form erly consp ecific sp ecies p airs like A. biporcatus–A. parvauritus, A. tropidogaster–
A. gaigei, and A. cupreus–A. macrophallus). In sp ite of the lim ited d ata u sed to reconstru ct these relationship s, these grou p ings seem likely to w ithstand fu rther scru tiny.
On the other hand , som e novel Draconura resu lts seem qu estionable given the d egree of m orp hological convergence they entail. For examp le, the nonm onop hyly of the humilis grou p (Anolis humilis, A. compressicauda, A. marsupialis, A. notopholis, A.
quaggulus, A. tropidonotus, A. uniformis, A. wampuensis) is su rp rising. These sp ecies share a d eep axillary p ocket, strongly keeled d orsal and ventral scales, an enlarged band of m id d orsal scales, and leaf-litter habitat niche.
Som e m em bers of this grou p that were fou nd to be nonm onop hyletic p reviou sly have been consid ered consp ecific (e.g., A. humilis and A. marsupialis). Ou r d ata for m ost of these form s is m ainly m itochond rial and m orp hological, and it is tempting to su sp ect a m islead ing m itochond rial signal or m ishand led tissu e. H owever, Philllip s et al. (2015) fou nd nonm onop hyly of this grou p u sing greater samp ling of ind ivid u als and a nu clear gene (ITS-1), so p erhap s ou r estim ate is correct. Another seem ingly qu estionable resu lt is the nonm onop hyly of the pentaprion grou p (A. beckeri, A. charlesmyersi, A.
cristifer, A. fungosus, A. ortonii, A.pentaprion, A. salvini, A.
sulcifrons, A. utilensis). These sp ecies are d istingu ished from each other only su btly (e.g.,Köhler 2010) and share an u nu su al p ale lichenou s coloration, short lim bs, and large sm ooth head scales. The sep arate m onop hyly of Sou th (A. sulcifrons, A. ortonii) and Central Am erican pentaprion anoles show n in the tree is geograp hically reasonable, and A. fungosus (w hich seem s ou t of p lace, if p oorly su p p orted , in a clad e of nond escript brow n
m ainland anoles like A. trachyderma and A. tropidogaster [Fig.4]) is u nu su al enou gh that few p lacem ents w ithin Draconura seem comp letely imp lau sible for this sp ecies.
Bu t the sep aration of A. salvini, w hich seem s essentially to be a high elevation version of A. pentaprion (Myers 1971), from the other Central Am erican pentaprion grou p anoles strains cred ibility. Sim ilar to the pentaprion and humilis grou p anoles, the anoles sim ilar to A.
laeviventris (A. laeviventris, A. cusuco, A. kreutzi) are nearly ind istingu ishable from each other bu t they are not m onop hyletic in ou r trees. In this case, the resu lt is likely d u e to lim ited character inform ation for som e “p roblem ” taxa that are sim ilar to the sp ecies of the laeviventris grou p . We lack m olecu lar d ata and p ossess only scant m orp hological d ata for sp ecies su ch as A. wermuthi that d isru pt the m onop hyly of the laeviventris-like form s, and we exp ect that m ore comp rehensive scoring of these sp ecies w ill rend er the laeviventris-like sp ecies m onop hyletic. Ad d itional DNA d ata w ill illu m inate all the u nexp ected resu lts noted above. It w ill be interesting to see w hich of the su rp rising resu lts in Figu res 1–4 are “corrected ” w ith ad d itional d ata and w hich, if any, ind icate convergence to the d egree seen in the anoles of the Greater Antilles (see Losos et al. 1998).
Biogeography
In this stu d y, we have p rovid ed the m ost comp rehensive biogeograp hic analysis of anoles to d ate. Inclu sion of all know n Anolis sp ecies allowed elu cid ation of a comp lex biogeograp hic history that involved m u ltip le vicariance and d isp ersal events. As in other neotrop ical lineages (Miller et al. 2008;Sm ith et al. 2014), both simp le range exp ansions and long d istance d isp ersals were fou nd to be imp ortant asp ects of d iversification in anoles. The best su p p orted DEC+J (d isp ersal–extinction–clad ogenesis p lu s ju mp d isp ersal) m od els incorp orated a series of range evolu tion m od els (Matzke 2013a, 2014) that allow d istinction of biogeograp hic scenarios based on m axim u m likelihood . Ou r analyses allowed u s to corroborate or contrad ict som e p reviou s biogeograp hic hyp otheses regard ing the p resent-d ay d istribu tion of anoles. Below we d iscu ss the origin of the anole clad e. More d etailed d iscu ssion of ou r resu lts w ith regard to d isp ersal w ithin Caribbean and m ainland regions and the role of geologic events in anole biogeograp hy is in ou r Su p p lem entary Ap p end ix 5.
The origin of anoles.—Based on DEC+J analyses u sing all anole sp ecies (Su p p lem entary Figs. S3–S6), we were able to p rovid e an u nam bigu ou s ancestral area of the m ost recent com m on ancestor (MRCA) of Anolis (analysis of the m olecu lar-scored taxa alone p rod u ced an am bigu ou s root state; Fig.5). We inferred a Sou th Am erican ancestor for Anolis. Ou r hyp othesized tim ing of the origin of Anolis (46.3–64.4 Ma; Su p p lem entary Fig. S7, Fig. 5) contrad icts p reviou s stu d ies (e.g.,N icholson et al. 2012)
that su ggested anoles originated ap p roxim ately 95 Ma.
The resu lts of ou r stu d y are concord ant w ith recent work byPrates et al.(2015) w ho fou nd sim ilar d ivergence d ates for the MRCA of Anolis u sing m ore fossil calibration p oints. We su sp ect thatN icholson et al.’s(2012) estim ates of d ivergence tim es were biased to old er d ates by an incorrect assignm ent of the Anolis electrum am ber fossil (Lazell 1965) to the fuscoauratus clad e. As Castañed a et al.(2014) showed , this fossil lacks synap om orp hies that wou ld allow it to be assigned to any anole clad e w ith confid ence. Several stu d ies have fou nd evid ence that incorrect fossil assignm ent m ay d ram atically affect estim ates of d ating (Magallón 2004, 2010; Mello and Schrago 2014).
Taxonomy
Based on ou r resu lts concerning the p hylogeny of the Anolis clad e (Figs. 1–4), we here p rop ose a revised taxonomy of anoles. To p rom ote stability in the associations between nam es and clad es by d issociating the references of nam es from consid erations abou t taxonom ic ranks, clad e nam es are d efined follow ing the m ethod s of p hylogenetic nom enclatu re (Cantino and d e Qu eiroz 2014). Listed synonym s are all consid ered ap p roxim ate (as they are not p hylogenetically d efined ) and are inferred p rim arily on the basis of comp osition.
Inferred comp osition is stated in term s of crow n su bclad es and know n, extant sp ecies only, althou gh it also inclu d es extinct m em bers of the corresp ond ing total clad es. Ou r p hylogenetic taxonomy of anoles is d escribed in Ap p end ix 3.
SUPPLEMEN TARYMATERIAL
Su p p lem entary m aterial, inclu d ing ad d itional d iscu ssion, MrBayes m atrix, and online ap p end ices, figu res and tables, can be fou nd in the Dryad d ata rep ository:http :/ / d x.d oi.org/ 10.5061/ d ryad .s80jq.
ACKNOWLEDGMEN TS
MrBayes analyses were u nd ertaken on the XSEDE clu ster of CIPRES (w w w.p hylo.org). Thanks to the International Barcod e of Life Project at the Biod iversity Institu te of Ontario for p erform ing som e of the m tDNA sequ encing.
For help in the field and / or the lab we thank Eric Schaad , Norm a L. Manríqu ez-Morán, Uri O. García- Vázqu ez, H eather MacInnes, Ju lian Davis, Jenny H ollis, Erik H u lebak, Carlos Vásqu ez Alm azán, Sofia Nu ñez, Gu stavo Cru z, Fed erico Bolaños, Roberto Ibañez, Martha Cald eron, And rew Craw ford , And rés Qu intero-Angel, Ju an Carlos Chap arro, Christian Yañez-Mirand a, Carlos Pavón, Devon Graham , Ju lie Ray, Alvaro Agu ilar, Jam es Ap aricio, and Liliana Jaram illo.
For loan of sp ecim ens, we thank Jose Rosad o (MCZ), Jonathan Losos (MCZ), Joe Martinez (MCZ),
Alan Resetar (FMN H ), Chris Phillip s (IN H S), Dan Wylie (IN H S), Nefti Cam acho (LACM), Greg Pau ly (LACM), Toby H ibbits (TCWC), Lee Fitzgerald (TCWC), Rafe Brow n (KU), Rob Wilson (USN M), Darrel Frost (AMN H ), Margaret Arnold (AMN H ), David Kizirian (AMN H ), Jam es McCranie, QCAZ, and MSB. We also thank Ju an Diego Palacio Mejia and Institu to Alexand er von H u m bold t and And rew Craw ford and the University of the And es for u se of sp ace and laboratory equ ip m ent to cond u ct m olecu lar work in Colom bia.
Perm its were p rovid ed by the Secretaría d e Med io Am biente y Recu rsos Natu rales, Dirección General d e Vid a Silvestre (Mexico); Institu to Nacional d e Conservación y Desarrolo Forestal, Áreas Protegid as y Vid a Silvestre (H ond u ras); MinisterioMinistereo d e Am biente y Energía (Costa Rica); Au torid ad Nacional d el Am biente (Panam a); Institu to Nacional d e Recu rsos Natu rales (Peru ); Ministerio d e Am biente (Ecu ad or);
Corp oración Au tónom a Regional d e Risarald a – CARDER (Colom bia)
FUN DING
Fu nd ing was p rovid ed by the National Science Fou nd ation (DEB-0844624 to S.P.); SEN ESCYT and Pontificia Universid ad Católica d el Ecu ad or (to O.T.
and F.A.); DGAPA, UNAM (PAPIIT no. 224009) and CONACYT (no. 154093) (to A.N.M.O.).
APPEN DIX1: SPECIESLIST AN DGEN ECOVERAGE FORDNA DATA
See Alföld i et al. (2011) for varying coverage of 46 genes (the d ifference in nu m ber of sites between ou r p ap er and Alfold i et al.’s [2012]—19,878 versu s 19,987–
is d u e to ou r u se of a shorter segm ent of the 16S gene).
Nu m ber in p arentheses is nu m bers of sp ecies scored for that gene/ d ataset. og = nu m ber of ou tgrou p sp ecies.
All sp ecies were scored for som e or all characters of m orp hology.
APPEN DIX2. MORPH OLOGICALCH ARACTERS Continu ou s qu antitative characters were cod ed u sing the ap p roach ofThiele(1993).Wiens’s(1995) frequ ency cod ing was u sed in cases w herein there ap p eared to be a m orp hological break between recognizable states. States were “ord ered ” if change between m orp hologically ad jacent states seem ed evolu tionarily m ore likely than change between nonad jacent states.
1. Maxim u m snou t to vent length (SVL; m m ; ord ered ). 0: <61; 1: 61–86; 2: 87–112; 3: 113–138; 4:
139–164; 5: >165.
2. Maxim u m fem ale SVL/ m axim u m m ale SVL (ord ered ). 0: <0.60; 1: 0.60–0.69; 2: 0.70–79; 3: 0.80–
0.89; 4: 0.90–0.99; 5: >1.00.
Sp ecies CO1 (142+ 1 og) 734 sites
N D2 (294 + 4 og) 1039 sites
ECEL1 (111) 474 sites
RAG1 (62+ 4 og) 2754 sites
Alfold i et al.
(89+ 1 og) 19878 sites
acutus X X
aeneus X X X X
aequatorialis X X X
agassizi X X X
agueroi X
ahli X X
alayoni X
alfaroi X
aliniger X X
allisoni X X X X
allogus X X
altae X X X X
altavelensis
altitudinalis X
alumina X
alutaceus X X
alvarezdeltoroi X X X
amplisquamosus X X
anatoloros X X X
anchicayae X X
anfiloquiae
angusticeps X X
annectens X
anoriensis X X X
antioquiae X
antonii X X
apletophallus X
apollinaris X X
aquaticus X X X
argenteolus X X
argillaceus X
armouri X X
auratus X X X X
aurifer X
bahorucoensis X X X
baleatus X X
baracoae X
barahonae X X
barbatus X
barbouri X X
barkeri X X X
bartschi X X
beckeri X X X X
bellipeniculus
benedikti X X
bicaorum X X X
bimaculatus X X X
binotatus X X
biporcatus X X X
birama
biscutiger X
blanquillanus
bocourtii X
boettgeri
bombiceps X X
bonairensis X
(continued)
Sp ecies CO1 (142+ 1 og) 734 sites
N D2 (294 + 4 og) 1039 sites
ECEL1 (111) 474 sites
RAG1 (62+ 4 og) 2754 sites
Alfold i et al.
(89+ 1 og) 19878 sites
boulengerianus X X X
brasiliensis X
bremeri X X
breslini X
brevirostris X X
brunneus X
calimae X X X
campbelli X
capito X X X X
caquetae carlostoddi
carolinensis X X X X X
carpenteri X
casildae X X X X
caudalis X
centralis X X
chamaeleonides X X
charlesmyersi X X X
chloris X X X X
chlorocyanus X X
chocorum X X X X
christophei X X
chrysolepis X
chrysops
clivicola X
cobanensis X
coelestinus X X
compressicauda X
concolor
confusus X
conspersus X
cooki X X
crassulus X X X
cristatellus X X X X X
cristifer X X X
cryptolimifrons X X X
cupeyalensis X
cupreus X X X X
cuprinus X
cuscoensis
cusuco X X
cuvieri X X X X X
cyanopleurus X
cybotes X X
cymbops X
danieli X X X
darlingtoni X
datzorum X X
desechensis X X
desiradei dissimilis
distichus X X X
dolichocephalus X
dollfusianus X X X
dominicensis X
duellmani X
(continued)
Sp ecies CO1 (142+ 1 og) 734 sites
N D2 (294 + 4 og) 1039 sites
ECEL1 (111) 474 sites
RAG1 (62+ 4 og) 2754 sites
Alfold i et al.
(89+ 1 og) 19878 sites
dunni X X X
equestris X X X
ernestwilliamsi X X
etheridgei X X
eugenegrahami X
eulaemus X X
euskalerriari X X X
evermanni X X X X
extremus X X X X X
fairchildi
fasciatus X X X
favillarum X
ferreus X
festae X X X
fitchi X X X X
forbesi X
forresti
fortunensis X X
fowleri X X
fraseri X X X X
frenatus X X X X X
fugitivus
fungosus X
fuscoauratus X X X X
gadovii X X X
gaigei X X X
garmani X X X
garridoi X
gemmosus X X X
ginaelisae X X
gingivinus X X
gorgonae X
gracilipes X X
grahami X X
granuliceps X X
griseus X X X
gruuo X X X
guafe X X
guamuhaya X
guazuma X
gundlachi X X X X
haetianus X
hendersoni X
heterodermus X X X
heteropholidotus
hobartsmithi X
homolechis X X
huilae X X X
humilis X X X X
ibanezi X X
ignigularis X
imias X X
inderenae X X X
inexpectatus X
insignis
(continued)
Sp ecies CO1 (142+ 1 og) 734 sites
N D2 (294 + 4 og) 1039 sites
ECEL1 (111) 474 sites
RAG1 (62+ 4 og) 2754 sites
Alfold i et al.
(89+ 1 og) 19878 sites
insolitus X X
isolepis X
jacare X X X
johnmeyeri X X
juangundlachi
jubar X X
kahouannensis
kemptoni X X
koopmani X
kreutzi
krugi X X X X X
kunayalae X X X
laevis
laeviventris X X X
lamari
latifrons X X
leachii X X
lemurinus X X X X
limifrons X X X X
limon
lineatopus X X
lineatus X X
liogaster X X X
lionotus X X X X
litoralis
lividus X
longiceps X
longitibialis X
loveridgei X
loysianus X X
luciae X X X
lucius X X X
luteogularis X
luteosignifer
lynchi X X
lyra X X
macilentus X
macrinii X X
macrolepis X X
macrophallus X X X
maculigula X X X
maculiventris X X
magnaphallus X
marcanoi X X X X X
mariarum X X
marmoratus X X
marron X
marsupialis X X X
matudai X
maynardi X X
medemi X
megalopithecus X
megapholidotus X X X
menta
(continued)
Sp ecies CO1 (142+ 1 og) 734 sites
N D2 (294 + 4 og) 1039 sites
ECEL1 (111) 474 sites
RAG1 (62+ 4 og) 2754 sites
Alfold i et al.
(89+ 1 og) 19878 sites
meridionalis X
mestrei X X
microlepidotus X X X
microtus X
milleri X X
mirus
monensis X X
monteverde X X
monticola X
morazani muralla nasofrontalis
naufragus X
neblininus X X X
nebuloides X X X
nebulosus X X
nelsoni
nicefori X
noblei X
notopholis X X
nubilus X
occultus X X X
ocelloscapularis X X X
oculatus X
oligaspis
olssoni X X
omiltemanus X X
onca X
opalinus X X
ophiolepis X X
oporinus X
orcesi X X
ortonii X X X
otongae X X X
oxylophus X X
pachypus X X X
paravertebralis
parilis X X X
parvauritus X X X
parvicirculatus X X
paternus X X X
pentaprion X X X
peraccae X X X X
petersii X X X
peucephilus X
philopunctatus phyllorhinus pigmaequestris pijolense pinchoti
placidus X X
planiceps X
podocarpus X X
poecilopus X
poei X X X
pogus X X X
(continued)
