Submitted 4 October 2017Accepted 29 January 2018Published 16 March 2018

Corresponding authorChristine D Baconchristinebaconbioenvgusechristinedbacongmailcom

Academic editorLuis Eguiarte

Additional Information andDeclarations can be found onpage 14

DOI 107717peerj4388

Copyright2018 Bacon et al

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

Evolutionary persistence in Gunneraand the contribution of southern plantgroups to the tropical Andes biodiversityhotspotChristine D Bacon123 Francisco J Velaacutesquez-Puentes34 Luis Felipe Hinojosa5Thomas Schwartz1 Bengt Oxelman1 Bernard Pfeil1 Mary TK Arroyo5Livia Wanntorp6 and Alexandre Antonelli1278

1Department of Biological and Environmental Sciences University of Gothenburg Gothenburg Sweden2Gothenburg Global Biodiversity Centre Gothenburg Sweden3 Laboratoacuterio de Biologiacutea Molecular (CINBIN) Universidad Industrial de Santander Bucaramanga Colombia4Departamento de Quiacutemica y Biologiacutea Universidad del Norte Barranquilla Colombia5 Institute of Ecology and Biodiversity Facultad de Ciencias Universidad de Chile Santiago Chile6Department of Phanerogamic Botany Swedish Museum for Natural History Stockholm Sweden7Gothenburg Botanical Garden Gothenburg Sweden8Department of Organismic and Evolutionary Biology Harvard University Cambridge MA USA

ABSTRACTSeveral studies have demonstrated the contribution of northern immigrants to theflora of the tropical Andesmdashthe worldrsquos richest and most diverse biodiversity hotspotHowever much less is known about the biogeographic history and diversification ofAndean groups with southern origins although it has been suggested that northern andsouthern groups have contributed roughly equally to the high Andean (ie paacuteramo)flora Here we infer the evolutionary history of the southern hemisphere plant genusGunnera a lineage with a rich fossil history and an important ecological role as anearly colonising species characteristic of wet montane environments Our results showstriking contrasts in species diversification where some species may have persisted forsome 90 million years and whereas others date to less than 2 Ma since origination Theoutstanding longevity of the group is likely linked to a high degree of niche conservatismacross its highly disjunct range whereby Gunnera tracks damp and boggy soils in coolhabitats Colonisation of the northern Andes is related to Quaternary climate changewith subsequent rapid diversification appearing to be driven by their ability to takeadvantage of environmental opportunities This study demonstrates the compositeorigin of a mega-diverse biota

Subjects Biogeography Plant Science TaxonomyKeywords Diversification Climate change Biogeography Neotropics Gondwana Specieslongevity

INTRODUCTIONAmong the 34 biodiversity hotspots currently recognized the tropical Andes is the richestand most diverse comprising some 30000 plant species (Myers et al 2000) This equatesto nearly a tenth of the worldrsquos flora contained in less than one per cent of its land area

How to cite this article Bacon et al (2018) Evolutionary persistence in Gunnera and the contribution of southern plant groups to thetropical Andes biodiversity hotspot PeerJ 6e4388 DOI 107717peerj4388

The tropical Andean hotspot which stretches from western Venezuela to northern Chileand Argentina constitutes an ideal arena for investigating the role of historical migrationsin generating the exceptional plant species diversity found in the American tropics (theNeotropics)

Most evolutionary models proposed to explain Neotropical diversity (Antonelli ampSanmartiacuten 2011b Rull 2011) postulate a major role for in situ diversification eg inAmazonia (eg Haffer 1969 Smith et al 2014 but see Dexter et al 2017) and the Andes(Gentry 1982 Luebert amp Weigend 2014) The relative contribution of immigrant lineagesto modern Neotropical diversity is less understood but has certainly played an importantrole (Villagraacuten amp Hinojosa 1997) For example northern immigrants contributed moreto the species diversity of the high elevation Andean paacuteramo than southern immigrants(eg Sklenaacuteř Duškovaacute amp Balslev 2011) The contribution of immigrant taxa to modernNeotropical diversity may be primarily explained by either continuous range expansionsfrom neighboring regions or long-distance dispersal both from what today are temperatelineages into tropical latitudes (often facilitated by climatic change andmountain building)and from other trans-oceanic tropical regions (Antonelli et al 2015) Modern distributionsreflect ancestral ecological requirements (niche conservatism Wiens et al 2010) but theyalso are affected by biome shifts such as adaptation of cool temperate immigrants intocold tropical areas (Donoghue amp Edwards 2014) More rarely migration events can alsobe directly associated with physiological adaptations intro new environments (Crisp et al2009 Simon et al 2009)

One of the characteristic elements of the Andes is the plant genusGunnera (GunneraceaeFig S1) Although eleven Gunnera species are reported in the paacuteramo (Luteyn 1999)most of these are found in montane forests and only one is a strict paacuteramo speciesG magellanicamdashwhich is also found in the southern temperate Andes in wet habitats bothbelow and above treeline (Sklenaacuteř Duškovaacute amp Balslev 2011) Gunnera has been present inmontane forests since at least the Middle Pliocene in Colombia (Hooghiemstra Wijningaamp Cleef 2006) In comparison to paacuteramo species from southern regions fewer montaneforest species such as Gunnera have successfully colonised the northern Andes (Wanntorpamp Wanntorp 2003)

Gunnera includes 58 species primarily of the Southern Hemisphere (Africa NewZealand South America and Tasmania) but also reaches Hawaii Mexico and SoutheastAsia (Fig 1 Table S1 Bader 1961 Mora-Osejo Paboacuten-Mora amp Gonzaacutelez 2011) Despitethis wide geographical distribution encompassing all southern continents apart fromAntarctica the majority of extant species of Gunnera (up to 40 species in subgenus Panke)are distributed in Central and South America (Mora-Osejo Paboacuten-Mora amp Gonzaacutelez2011) most of them within the northern Andean biodiversity hotspot However Gunnerahad an even wider geographic distribution during the Cretaceous as demonstrated bynumerous fossil pollen records from the Antarctic Peninsula Australia the KerguelenPlateau as well as in both North and South America (Jarzen 1980) The oldest of thesedates to the Turonian (ca 90 Ma) of Peru (Brenner 1968) and ten million years laterGunnera became widespread across all landmasses that previously formed Gondwana(Jarzen 1980) Initial evidence suggested that biogeographic patterns in Gunnera are in

Bacon et al (2018) PeerJ DOI 107717peerj4388 221

Figure 1 Map of the extant distribution ofGunnera where high numbers of georeferences are re-flected by darker blue colour The Tricolpites reticulatus pollen fossil was used to calibrate the Gunneraphylogeny in absolute time for this study and is also mapped through major geological time periods Ele-vation is shown in grey scale where the lowest and highest global elevations are found in white and blackrespectively

Full-size DOI 107717peerj4388fig-1

agreement with the sequence of Gondwanan break-up promptingWanntorp amp Wanntorp(2003) to suggest a Gondwanan origin for the genus and vicariance by continental drift asa plausible hypothesis to explain the present-day distribution of most species in the genusHowever these conclusions involved neither formal ancestral area reconstruction normolecular dating The study also did not explicitly investigate the biogeographical historyof the Andean species comprising subgenus Panke

The current widespread distribution of Gunnera its rich fossil record and the many stillunanswered questions regarding its biogeographic history all make Gunnera an ideal targetfor investigating the role of southern immigrants in the assembly of the flora of the Andeanbiodiversity hotspot Here we infer the biogeographic climatic and evolutionary historyof Gunnera to explicitly address the following questions Where did Gunnera most likelyoriginate How and when did it attain its current distribution When did it colonise thenorthern Andes where it is most diverse Are areas of high diversity in the genus associatedwith higher rates of net diversification or is diversity instead due to gradual accumulationof species To what extent have species of Gunnera tracked the ancestral climatic nicheWe also compare Gunnera with other Andean groups Our study sheds further light on thegeographical and temporal origins and composition of the highly diverse Andean flora

MATERIAL AND METHODSPhylogeny and divergence time estimationTaxon sampling was complete at the species level for five of six subgenera of Gunnera (27out of 58 species ca 46) Within subgenus Panke we could only obtain material for 14 ofthe up to 40 species because many species are only known from few collections or localitieshence the difficulty in defining the exact number of species (Mora-Osejo Paboacuten-Mora amp

Bacon et al (2018) PeerJ DOI 107717peerj4388 321

Gonzaacutelez 2011) New DNA sequence data was generated following the respective authorprotocols for the chloroplast regions rps16 (Oxelman Liden amp Berglund 1997) rpoC1and ycf5 from the Consortium for the Barcode of Life (httpwwwbarcodeoflifeorg)psbA-trnH (Pang et al 2012) as well as the nuclear genes ITS (Blattner 1999) and SEX4(Kotting et al 2009) All new sequences generated in this study have been deposited inGenBank (File S1) Nucleotide alignments were obtained independently for each of the lociusing default parameters for MUSCLE in Geneious (Biomatters Auckland New Zealand)Due to poor alignability the ITS sequences of G herteri and G perpensa were removedWe used the Akaike Information Criterion implemented in MrModelTest 22 (Nylander2004) to select the best-fitting nucleotide substitution model(s) and incorporated it in theinference of the species tree

We used STACEY 104 (see httpwwwindriidcomsoftwarehtml) in BEAST 230 andthe DISSECT method (Jones Aydin amp Oxelman 2014) to infer a multispecies coalescenttree The method uses a version of the birthdeath branching model for the species treewhich assigns high probabilities for branching events close to time zero how close is definedby the lsquolsquocollapse heightrsquorsquo parameter which should be set as small as possible (see JonesAydin amp Oxelman 2014) The approach thus enables simultaneous exploration of speciestree and species delimitation space Individuals or groups of individuals known to belongto the same single species are operationally defined as minimal species Clusters below thecollapse height are considered to belong to the same species as defined by the multispeciescoalescent model Here we defined all sequences from the same individual as minimalspecies A lognormal (mean 46 standard deviation 2) growth rate prior distribution wasused for the species tree The growth rate is roughly 1 divided by the branch length so that95 of the distribution falls within the interval [2 5000] with median e46 asymp 100 Betapriors with shape parameters 1 1 (resulting in uniform distributions) were used on collapseweight and relative death rates A lognormal (minus7 2) prior was used for popPriorScalewhich should approximate the average time to coalescence between two gene copiesIn order to scale branch lengths in substitutions per site the ITS rate was set to 1 andlognormal (0 1) priors for the relative rates of the cpDNA and SEX4 trees were usedCollapse height was set to 00001 Ploidy was set to 1 for cpDNA and 2 for the two nucleargenes The substitution model was GTR with a gamma prior distribution (005 10) on eachsubstitution type with rate variation among sites was modeled with four rate categories forall three loci Each locus also had branch rates constrained to an uncorrelated lognormalclock The MCMC was run for 100 million generations and all parameters had effectivesample sizes gt180 after removing the first 10million generations as burn-in Themaximumclade credibility species tree was generated by sampling trees every 50000th generationin the stationary phase (the last 90 million generations) where the heights are commonancestor heights scaled in substitutionssite

A fossil Gunnera pollen grain was used to calibrate the phylogeny Tricolpites reticulatusfrom the Turonian of Peru (Brenner 1968) represents the first unambiguous appearanceof the genus Based on this calibration point the TuronianConiacian boundary (LateCretaceous) was used to provide a crown age ofGunnera by scaling the root of the STACEYtree (see above) using a mean age of 90 Ma Its placement on the crown of Gunnera is

Bacon et al (2018) PeerJ DOI 107717peerj4388 421

based on a morphological review of extant and fossil pollen of Gunnera species as assessedwith scanning electron microscopy to define morphological characters of the exine and itsstructure to support its placement on the topology (Wanntorp Dettmann amp Jarzen 2004)

Biogeographic analysesDistribution data were compiled from Mora-Osejo Paboacuten-Mora amp Gonzaacutelez (2011)and Wanntorp amp Wanntorp (2003) together with records from the Global BiodiversityInformation Facility (httpwwwgbiforg) and regional herbaria (CONC and MEL)that were vetted by the authors Using the extant distribution of Gunnera we definednine operational areas for ancestral area estimation (Fig 2) (A) northern Andes fromVenezuela and Colombia to northernmost Peru corresponding to the paacuteramos northof the Huancabamba Depression (B) central Andes from northern Peru (south of theHuancabamba Depression) south to the Tropic of Capricorn and including the AltiplanoJalca and Puna (C) southern Andes from northern Chile south to Patagonia including theislands off the coasts of Chile and Argentina (D) southeastern South America includingthe lowlands of southeastern Brazil and the Rio Paranaacute drainage (E) Mesoamericafrom southern Mexico to southernmost Panama (F) the Hawaiian islands (G) Africaincluding Madagascar (H) the Malay archipelago including New Guinea (I) Tasmaniaand New Zealand

We inferred ancestral biogeographic ranges using the package BioGeoBEARS 021(Matzke 2014) in the R platform (R Development Core Team 2010) BioGeoBEARSimplements widely used models of range evolution (eg Ree amp Smith 2008) but itincludes an additional parameter of cladogenetic speciation mediated by founder eventsthe jump parameter lsquolsquojrsquorsquo This parameter allows daughter species to instantaneouslylsquolsquojumprsquorsquo outside the geographical range of parental species We considered this modelappropriate since several Gunnera species occur on oceanic islands (eg the Hawaiianand Juan Fernandez Islands) We inferred ancestral ranges across the Gunnera phylogenyusing the Dispersal Extinction Cladogenesis (DEC) model with the J parameter (+j) Theamong-area connectivity was constrained in the following time slices as follows northernand central Andean co-distributions were not permitted before 40 Ma (Garzione et al2008 Hoorn et al 2010) Hawaiian distributions were not permitted before 30 Ma (Clagueet al 2010) lower connectivity (01 rate of dispersal) was set between Africa and SouthAmerica throughout the last 90 Ma as well as between South America and New Zealandplus Tasmania throughout the last 30 Ma (McLoughlin 2001) (Table S2)

Diversification rate analysisTo test for diversification rate shifts we used the software BAMM 20 (Bayesian Analysis ofMacroevolutionary Mixtures Rabosky 2014) BAMM implements a Bayesian frameworkto estimate evolutionary rate parameters and explore candidate models of lineagediversification to quantify and detect heterogeneity in evolutionary rates We ran BAMMusing default priors for 1000000 generations sampling every 20 steps and accountingfor incomplete taxon sampling We analysed the output in R using the BAMMtoolspackage 202 (Rabosky 2014) We discarded the first 25 estimates as burn-in based on

Bacon et al (2018) PeerJ DOI 107717peerj4388 521

Figure 2 Biogeographic history based on the BioGeoBEARS optimization of theGunnera topologycalibrated in absolute time using Tricolpites reticulatus pollen Coloured boxes at the terminals of thephylogeny show the extant geographic distribution of species with the colour legend defining the areasAll nodes over 08 PP are marked and node bars representing the 95 HPD time interval are shown fornodes of interest Clades discussed in the text are marked with numerals IndashVII Uncertainty in ancestralrange is shown where distributions with lt5 probability of occurrence are combined into white portionsin pie charts Inset Operational areas used (A) northern Andes (B) Central Andes (C) southern Andes(D) southeastern South America (E) Central America (F) Hawaii (G) Africa (H) the Malay Archipelago(I) Tasmania and New Zealand and other biogeographic areas based on combinations of those defined apriori

Full-size DOI 107717peerj4388fig-2

Bacon et al (2018) PeerJ DOI 107717peerj4388 621

the convergence of our data (effective sampling size of parameters greater than 200) Wegenerated plots of net diversification and speciation rates through time and inferred theoccurrence and position of rate shifts accounting for the 95HPD of BAMM-inferred rateshifts based on a Bayes factor cut-off of 5

Climatic niche evolutionWe performed ecological niche modeling for all 27 species of Gunnera included in thephylogenetic analysis using Maxent (Phillips Anderson amp Schapire 2006) with eightWorldClim bioclimatic variables at a resolution of approximately 1 km2 (Hijmans et al2005) following themethodology ofEvans et al (2009) The bioclimatic variables associatedwith temperatureweremean annual temperature (MAT)minimum temperature of coldestmonth (MTCM) mean temperature of warmest quarter (MTWQ) and mean temperatureof coldest quarter (MTCQ) Bioclimatic variables associated with precipitation were meanannual precipitation (MAP) precipitation of wettest quarter (PWETQ) precipitation ofdriest quarter (PDQ) and precipitation of warmest quarter (PWARMQ) Climatic variableswere chosen because of their biological meaning and by removal of the other 11 correlatedWorldClim variables after a principal components analysis We used a total of 882 vettedspecies occurrence points (see (b) above) From the ecological niche models we obtainedthe mean value for each of the eight bioclimatic variables weighted by the cumulativeprobability of each value (weighted mean Wmean) to be used in comparative analysesusing the R package phyloclim (Heibl 2011) To infer the climatic history of Gunnera weused the projection of our phylogenetic tree in environmental (each bioclimatic variable)and temporal space assuming Brownian motion evolution (BM Evans et al 2009 Schluteret al 1997) using the R package phytools (Revell 2012)

Phylogenetic niche conservatism (PNC) defined as the retention of ecological traits eachbioclimatic variable (Wmean values) over time among related species (Wiens et al 2010)was estimated using Pagelrsquos lambda (Pagel 1994) in the R package GEIGER (Schluter et al1997) Lambda ranges from one when trait evolution is strongly influenced by phylogenyand a species niche-to-phylogeny correlation is equal to the Brownianmodel expectation tozero when trait evolution is independent of phylogeny (Pagel 1999) We used a likelihoodratio test (Neyman amp Pearson 1928) to determine whether the observed values differedsignificantly from zero and one In general phylogenetic signal indicates a statisticalnon-independence among species trait values due to relatedness consistent with PNC(Wiens et al 2010) To examine PNC explicitly we used the Akaike Information Criterion(wAIC) to compare the relative fit of three models of evolution to each bioclimatic variable(Wmean values) The models include (i) a Brownian motion (BM) model of gradual andcontinuous drift (ii) a stabilizing selection OrnsteinndashUhlenbeck (OU) model with oneoptimum and (iii) a white noise (WN) model of random variation in which the similarityof species is independent of their phylogenetic relationships (Hansen Pienaar amp Orzack2008) We performed this comparison using GEIGER The phylogenetic dependence ofthe realized climatic variation between related species combined with the comparison ofBM OU and WN models provides an assessment for testing PNC (Losos 2008 Wiens etal 2010) and was thus suitable for our study

Bacon et al (2018) PeerJ DOI 107717peerj4388 721

RESULTSMultispecies coalescent treeThe multispecies coalescent tree reconstructed here is consistent with the topology of themaximum parsimony tree based on molecular and morphological data of Wanntorp ampWanntorp (2003) All major clades were resolved with moderate to high support (gt080posterior probabilities Fig 2) but some recent species-level relationships received poorbranch support

Wide time intervals (95 highest posterior density HPD values) were inferred forearly nodes on the phylogeny (Nodes I and III Fig 2) but more recent nodes had littlevariation around the mean inferred age (eg Nodes VI and VII Fig 2) Overall the crownnode of Gunnera was inferred at a mean age of 90 Ma (95 HPD 165ndash40 Ma) Followingthe origin and diversification of the genus in the Late Cretaceous the two major clades ofGunnera originated between 31 (46ndash17 Ma Node III Fig 2) and 10 Ma (15ndash5 Ma NodeII Fig 2) Contrasting results were resolved with regard to the time of origin of specieswhere Gunnera herteri had a mean age of 90 Ma and many others particularly the Andeanspecies are younger than 2 Ma (Fig 2)

Biogeographic and diversification historyThe BioGeoBEARS analysis resulted in broadly distributed ancestral nodes at the backboneof the tree reflected in the uncertainty in ancestral range (distributions with lt5probability of occurrence combined are thewhite portions in pie charts Fig 2) Despite thisinternal nodes ca 12 Ma and younger were inferred with less ambiguity in ancestral areaThese results reveal an ancient lineage that began extending its distribution significantlyas of the Oligocene (ca 30 Ma node III Fig 2) BioGeoBEARS results show ambiguity inthe biogeographic origin of the genus (node I Fig 2) but early lineages were distributedin the Southern Hemisphere (areas C G I)

Results from the BAMM diversification rate through time analysis shows thatdiversification inGunnera generally increased over the last ca 20Ma (Fig 3) However thisincrease is almost exclusively due to the Pliocene diversification of the Panke clade whenthe genus colonized the Andes This is shown by a significant increase in net diversificationrate in the Panke clade either at its crown (Node VI probability of 25) or at its stem(Node V probability of 22) The highest rates of diversification were found during thePleistocene in the Panke clade for the lineage distributed in the northern Andes

Climatic niches and historyExtant species of Gunnera are inferred to occupy cool (microthermal) and moderate(mesothermal) climatic conditions according to the classification of Nix (1991) with aWmean for mean annual temperature (MAT) ranging from 64 ndash203 C and a Wmeanmean annual precipitation (MAP) ranging from 813ndash3588 mm (Fig S2 Table S2)Microthermal species are distributed mainly in temperate latitudes of the SouthernHemisphere with the exception ofG talamancana which is found at high altitudes (1900ndash3400 m) in Central America (Mora-Osejo Paboacuten-Mora amp Gonzaacutelez 2011) Mesothermalspecies are distributed in tropical latitudes in South and Central America Hawaii New

Bacon et al (2018) PeerJ DOI 107717peerj4388 821

Figure 3 Diversification rate through time analysis using BAMM for all species sampled withinGun-nera (A) The results show a single positive diversification rate shift either at the stem (with 022 PP) orthe crown (with 025 PP) node of the Andean Panke clade (B) Results also support an increase in net di-versification rate through time (C) Some Gunnera species are aggressive colonisers here showing success-ful colonisation and persistence in the margins of a landslide in Costa Rica (image fromWikicommons)

Full-size DOI 107717peerj4388fig-3

Bacon et al (2018) PeerJ DOI 107717peerj4388 921

Guinea and Africa with the exception of G arenaria that occurs in temperate areas inNew Zealand

According to the estimation of ancestral climatic variables the most recent commonancestor (MRCA) of Gunnera lived under a mesothermal climate sensu Nix (1991) witha MAT of 153 C and MAP of 1577 mm Phylogenetic signal using Pagel λ was detectedfor MAT (λ= 093) and mean temperature of coldest quarter (BIO11 λ= 092) andprecipitation of wettest quarter (BIO16 λ= 1) and precipitation of warmest quarter(BIO18 λ= 093 Table S3) w AIC analyses showed that the evolution of the climaticniche is best described by the OU model suggesting that selection pulls the climaticvalues toward an optimum Minimum temperature of coldest month (Bio 6) and meanannual precipitation (Bio 12) showed no difference from a white noise model of evolutionindicating that these variables are independent of phylogenetic relationships in Gunneraspecies (Table S3)

DISCUSSIONBased on the divergence times and relationships of the Gunnera multispecies coalescenttree we examined the contribution of a southern hemisphere taxon to the mega-diversetropical Andean flora of South America Our results show the tempo of range expansionand lineage diversification

Biogeographic history of GunneraA question that has long intrigued biogeographers about widespread southern hemispherelineages such as Gunnera is whether present-day disjunctions are the result of vicariance ordispersal events Given the geographically extensive fossil record dating to the Cretaceous(Jarzen 1980 Wanntorp Dettmann amp Jarzen 2004) and the current distribution in allsouthern continents except Antarctica (Bader 1961) Gunnera has long been considered atypical Gondwanan element (Mora-Osejo Paboacuten-Mora amp Gonzaacutelez 2011 Fuller amp Hickey2005) with vicariance proposed as the main driver of its current geographic distribution(Wanntorp amp Wanntorp 2003) Here we do not find support for unequivocal vicarianceevents in the biogeographical history of Gunnera but instead interpret a general pattern oflong distance dispersal from our results

The mean crown age of 90 Ma for Gunnera is consistent with previous findings andthe variation around the mean age (95 HPD 165ndash40 Ma) reflects what has been foundin earlier work (Bell Soltis amp Soltis 2010 Magalloacuten et al 2015 Tank et al 2015) Gunnerais inferred to have been widespread in the former Gondwanan territories including thesouthern Andes (area C) southeastern South America (area D) and Africa (area G atNode I in Fig 2) during the Cretaceous A Gondwanan distribution is also supportedby several fossils from southernmost South America southwest Africa the AntarcticPeninsula Australia and Tasmania (Macphail 2007) Gunnera herteri from South Americais sister to the remaining Gunnera species in agreement with previous studies (Wanntorpamp Wanntorp 2003 Mora-Osejo Paboacuten-Mora amp Gonzaacutelez 2011 Fuller amp Hickey 2005)which supports a long history in the southern portions of the continent

Bacon et al (2018) PeerJ DOI 107717peerj4388 1021

A vicariance event could be interpreted at the node where the Australasian lineages(Node II) diverge from the South American and African lineages (Node III) based on thephylogenetic pattern However the divergence time for that event dates to the Late Eocene(ca 40 Ma) and geological evidence does not support division of these continents at thattime (Sanmartiacuten amp Ronquist 2004) Suitable areas for Gunnera in the tropical latitudes ofNew Guinea were available when this region emerged above sea level and Australia reachedits current latitudinal position both of which occurred at the end of the Cenozoic (12 MaFuller amp Hickey 2005) In agreement with and according to our estimations the tropicallineage G macrophylla distributed from the Philippines and Sumatra east to New Guineaand the Solomon Islands split from the southern Australia and Tasmania lineages at ca 10Ma This result suggests that the arrival of extant Gunnera in tropical regions (eg area H)occurred via long distance dispersal as has been inferred for other Gondwanan taxa suchas Nothofagus (eg Hinojosa et al 2016)

Another long distance dispersal is inferred at Node III where the African lineage Gperpensa split from rest of the genus at ca 30 Ma at the time when Africa and SouthAmerica were completely separated (Sanmartiacuten amp Ronquist 2004) Node IV joins theAndean species (areas A B C) with subgenus Panke (areas A B C D E F) Node V infersa colonisation event to Hawaii (area F) and Node VI shows a division between southernSouth American species (areas B C and D) and those from the north of South Americaand Central America (areas A and E respectively)

Range expansion in the AndesGunnera is inferred to have been present in southern South America since the Paleogene(Fig 2) By the Mid-Miocene the stem node of the Panke clade (15 Ma Node IV) wasdistributed in Central America andor the Andes and began to diversify both north intoCentral America and south again into the southern Andes (Fig 2) The estimated meanage overlaps with that proposed for the formation of the Isthmus of Panama (Montes et al2015) a primarily terrestrial lowland region that has connected North and South Americasince ca 15 Ma Bacon et al (2015a) and Bacon et al (2015b) proposed that closure of theisthmus enabled taxa to expand their distributions both north and southwards duringpulses of migration (ca 23 8 and 5Ma) Range expansion at Node IV occurred after a longstasis (ca 15 Ma) where there was a dearth of speciation or substantial extinctionmdashtwoalternative explanations that are generally difficult to distinguish (Crisp amp Cook 2009)

The colonisation of the northern Andes (area A at Node VII Fig 2) is inferred to havelikely taken place from Central American ancestors sometime in the Early Pleistocene (ca227 Ma) Although the Andes began to rise as early as the Early Paleogene (Garzione etal 2008) it is often difficult to disentangle the roles of Andean uplift and climate changeon Neotropical diversification (but see Lagomarsino et al 2016) because they occurredcontemporaneously (Gentry 1982) Gunnera is primarily a wet montane rather thanpaacuteramo lineage and does not require high elevations for successful dispersal

North American fossils crown or stem relatives of PankeA sister relationship between the northernmost species of Gunnera (G mexicana) andall other species in subgenus Panke combined with the fact that there are several North

Bacon et al (2018) PeerJ DOI 107717peerj4388 1121

American fossil localities from the Late Cretaceous to the Eocene (80ndash50 Ma Jarzen1980) led Wanntorp amp Wanntorp (2003) to suggest that the South American species ofPanke represented a recolonisation of South America from the north This result is furthersupported by morphological similarities of leaf impressions and pollen size between theLate Cretaceous fossils and modern Panke species (Jarzen 1980 Wilkinson 2000) and theplacement of the Hawaiian species G petaloidea and G kauaiensis as the next branchinglineage after G mexicana

This scenario implies that the North American fossils belong to the crown or stem groupof Panke ie they would have been derived either from the branch connecting Misandra(G lobata and G magellanica) to Panke (Node IV Fig 2) or from the branch leading toG mexicana Although this is a possible conclusion based on the topology of our Gunneraphylogeny the divergence times estimated here suggest otherwise The splits connectingMisandra to the MRCA of Panke (where the dispersal to North America would have takenplace) are estimated at ca 15 Ma which is almost 65 Ma later than the first documentedNorth American fossils Our results suggest that the North American fossils do not belongto the crown group of Panke Instead we suggest they likely represent a lineage that reachedNorth America during the Cretaceous but did not leave any living descendants A similarscenario was found in the inconsistency between DNA-based divergence times and pollenfossils of Nothofagus where lsquoincongruentrsquo fossils might have been erroneously assigned tocrown Nothofagus whereas they in fact represented extinct stem relatives (Hinojosa et al2016 Cook amp Crisp 2005)

Stasis versus rapid speciationA remarkable aspect of our results is the striking difference in the stem ages of Gunneraspecies Gunnera herteri is inferred to have originated 90 Ma during the Late Cretaceous(Node I) whereas 18 species in the phylogeny are inferred to be younger than 2 Ma(Fig 2) The contrast between stasis and rapid speciation is also seen in the BAMM results(Fig 3) where low net diversification rates are shown at ancestral branches and a shift indiversification rate detected in the Andean Panke clade is followed by a steady rate increase(Fig 3C)

It is puzzling why some lineages have experienced long evolutionary stasis whereasothers underwent rapid speciationmdashall within the genetic constraints of a single clade Ourinterpretation of stasis relies on an accurate inference of the dated tree and wide credibleintervals were detected for some nodes Further this result could be an artefact of extinctionif the lineages leading to the ancient species in fact diversified but all lineages exceptone went extinct (Crisp amp Cook 2009 Antonelli amp Sanmartiacuten 2011a) However there ispalaeontological support for these exceptionally old stem ages Fossil pollen on the VegaPeninsula of Antarctica dated from the CampanianLate Maastrichtian have a distinctiveexine that is nearly indistinguishable from that found in pollen grains of extant speciesof Australia New Zealand and Southeast Asia (Wanntorp Dettmann amp Jarzen 2004)

The persistence of Gunnera for a longer time (ca 90 Ma) than most other angiospermgenera is remarkable We interpret our results with caution but consider them as indicativeof strong niche conservatism across the highly disjunct range of Gunnera for wet montane

Bacon et al (2018) PeerJ DOI 107717peerj4388 1221

forest environments Indeed our climatic reconstruction shows a mesothermal nichepreference for the crown node of Gunnera (Fig S2) similar to that identified in otherCretaceous lineages (DeConto et al 1999) Further our estimate of phylogenetic signalshows high values (Table S3) particularly those associated with both mean annual andcoldest quarter temperature variables and an OrnsteinndashUhlenbeck model which togetherindicate that the climatic history of Gunnera underwent selection pressure (eg stabilizingselection) that favoured the ancestral niche over time (Wiens et al 2010) RecentlyHinojosaet al (2016) suggested that lineages of Gondwanan origin expanded into the tropics bytracking mesothermal climates Dispersal towards current tropical zones has been possiblebecause species have tracked ancestral climatic niches from high or mid-latitudes intolower latitudes sometimes facilitated by climatic and geological changes

It is interesting that Gunnera a montane forest clade of Gondwanan origin successfullydispersed to the northern tropical Andes where few other similar plant clades could A keyelement to this may be due to its colonising nature Gunnera comprises species of forestedges and marginal habitats (Greimler et al 2013) appear after landslides in wet forests(Fig 3C Vanacker et al 2003) underwent long distance dispersal events to Hawaiianand the Juan Fernandez Islands and is persistent in the seed bank (Arroyo Cavieres ampHumana 2004 Fesq-Martin et al 2004) Gunnera species also quickly colonise glacialforelands (Perez et al 2014) and their pollen is commonly found in glacial and post-glacialsediments in both southern South America and in Tasmania (Heusser Heusser amp Hauser1992 McKensie amp Kershaw 2000) Lastly and potentially most convincingly the fact thatsome Gunnera species are invasive (Fennell et al 2013 Skeffington amp Hall 2011) clearlyshows their aggressive colonising abilities that likely differentiate them from other montaneplant groups

Comparison with other Andean groupsThere are multiple examples of plant clades that have colonised the Andes from the northas we suggest for the Panke clade of Gunnera Some are lsquoboreotropicalrsquo elements thatprobably reached South America around the Palaeocene-Eocene Thermal Maximum (sim55Ma Zachos Dickens amp Zeebe 2008) when a large belt of tropical forest covered muchof southern Laurasia thus functioning as a biotic corridor for Palaeotropical lineages(eg Bacon Baker amp Simmons 2012 Antonelli et al 2009) Later dispersals southwardsfrom North to South America may have been facilitated by the Greater Antilles and theAves Ridge around the EoceneOligocene boundary (Iturralde-Vinent amp MacPhee 1999)and finally through the Panama Isthmus after its uplift ca 15 Ma (Montes et al 2015)Examples of northern taxa colonising the Andes with local radiations include Hedyosmum(Antonelli amp Sanmartiacuten 2011a Zhang Feild amp Antonelli 2015) and Lupinus (Hughes ampEastwood 2006) Many of these northern lineages have undergone significant radiation inthe paacuteramo

In contrast there is relatively little evidence of Andean plant clades that are derivedfrom the south Some well-known southern Hemisphere (lsquolsquoGondwanarsquorsquo) groups such asNothofagus and Araucaria simply do not enter the Andean tropical zone but reach tropicalareas in Australasia (Iturralde-Vinent amp MacPhee 1999) Fuchsia which is considered to

Bacon et al (2018) PeerJ DOI 107717peerj4388 1321

have a southern origin based on a rich Antarctic Cenozoic fossil record does not show aclear biogeographic pattern from molecular phylogenies (Berry et al 2004) One robustexample is the Andean wax palms that have moved up the Andean cordilleras from thecentral portion of the range northwards in a stepping stone fashion (Ceroxylon see Saniacutenet al 2016 for a review of the pattern)

CONCLUSIONSFor many decades Gunnera has attracted the attention of botanists and biogeographersconcerned with southern hemisphere disjunctions and the break-up of Gondwana Herewe have shown that it also constitutes a model taxon to study biogeography in general aswell as the colonisation and diversification of southern elements in the tropical Andes inparticular The extraordinary species longevity inferred here for species in Southeast AsiaAfrica and eastern Brazilmdashca 90Ma according to our resultsmdashismost likely due to climaticand potentially morphological conservatism despite the massive geotectonic and climaticchanges that took place during its history In contrast the recent and rapid diversificationof Andean lineages could be explained by the massive increase in the area of suitablehabitats and opportunities for allopatric speciation as a consequence of the Andean upliftand late Neogene climatic changes Understanding the evolution of Andean mega-diversitythus requires identifying and tracing the diversification of southern northern and locallyderived taxa

ACKNOWLEDGEMENTSWe would like to thank C Hughes and U Swenson for reading and commenting on earlyversions of the manuscript and S Razafimandimbison for attempts to sequence ITS forMyrothamnus We are also thankful to HerbariumGB for allowing us to extract samples forDNA analyses Vivian Aldeacuten provided laboratory assistance The analyses were performedon the bioinformatics computer cluster Albiorix at the Department of Biological andEnvironmental Sciences University of Gothenburg

ADDITIONAL INFORMATION AND DECLARATIONS

FundingFinancial support was provided by the Swedish ResearchCouncil (B0569601) the EuropeanResearch Council under the European Unionrsquos Seventh Framework Programme (FP2007-2013 ERCGrant Agreement n 331024) the Swedish Foundation for Strategic Research andthe Knut and Alice Wallenberg Foundation (through a Wallenberg Academy Fellowship)to Alexandre Antonelli Luis F Hinojosa was funded by FONDECYT 1150690 MillenniumInstitute of Ecology and Biodiversity (IEB) Grant P05-002 from MIDEPLAN (Chile)PFB-023 from CONICYT (Chile) The funders had no role in study design data collectionand analysis decision to publish or preparation of the manuscript

Bacon et al (2018) PeerJ DOI 107717peerj4388 1421

Grant DisclosuresThe following grant information was disclosed by the authorsSwedish Research Council B0569601European Research Council 331024Swedish Foundation for Strategic ResearchKnut and Alice Wallenberg FoundationFONDECYT 1150690Millennium Institute of Ecology and Biodiversity (IEB) P05-002CONICYT PFB-023

Competing InterestsThe authors declare there are no competing interests

Author Contributionsbull Christine D Bacon conceived and designed the experiments analyzed the data preparedfigures andor tables authored or reviewed drafts of the paperbull Francisco J Velaacutesquez-Puentes and Luis Felipe Hinojosa performed the experimentsanalyzed the data prepared figures andor tables authored or reviewed drafts of thepaperbull Thomas Schwartz performed the experiments authored or reviewed drafts of the paperbull Bengt Oxelman and Bernard Pfeil analyzed the data authored or reviewed drafts of thepaperbull Mary TK Arroyo authored or reviewed drafts of the paperbull Livia Wanntorp performed the experiments contributed reagentsmaterialsanalysistools authored or reviewed drafts of the paperbull Alexandre Antonelli conceived and designed the experiments contributedreagentsmaterialsanalysis tools authored or reviewed drafts of the paper

DNA DepositionThe following information was supplied regarding the deposition of DNA sequences

DNA sequences have been deposited in GenBank A list of the accession numbers canbe found in File S1

Data AvailabilityThe following information was supplied regarding data availability

Raw data is provided in the Supplemental Information

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj4388supplemental-information

Bacon et al (2018) PeerJ DOI 107717peerj4388 1521

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