Proc. R. Soc. B (2005) 272, 1481–1490 doi:10.1098/rspb.2005.3067 Published online 28 June 2005

Repeated evolution of net venation and fleshy among monocots in shaded habitats confirms a priori predictions: evidence from an ndhF phylogeny Thomas J. Givnish1,12,*, J. Chris Pires2, Sean W. Graham3, Marc A. McPherson3, Linda M. Prince4, Thomas B. Patterson1, Hardeep S. Rai3, Eric H. Roalson5, Timothy M. Evans6, William J. Hahn7, Kendra C. Millam1, Alan W. Meerow8, Mia Molvray9, Paul J. Kores9, Heath E. O’Brien3, Jocelyn C. Hall1,10, W. John Kress11 and Kenneth J. Sytsma1 1Department of and 2Department of Agronomy, University of Wisconsin, Madison, WI 53706, USA 3UBC Botanical Garden, Centre for Research, and Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada 4Rancho Santa Ana Botanic Garden, Claremont, CA 91711-3157, USA 5School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA 6Department of Biology, Hope College, MI 49422-9000, Holland 7Georgetown College, Washington, DC 20057-1003, USA 8USDA-ARS-SHRS, National Germplasm Repository and Fairchild Tropical Garden, Miami, FL 33156, USA 9Department of Biology, Moorpark College, Moorpark, CA 93021, USA 10Arnold Arboretum, Harvard University, Cambridge, MA 02138, USA 11National Museum of Natural History, Department of Botany, Smithsonian Institution, Washington, DC 20013-7012, USA 12Research School of Biological Sciences, Australian National University, Canberra 2601, ACT, We present a well-resolved, highly inclusive phylogeny for monocots, based on ndhF sequence variation, and use it to test a priori hypotheses that net venation and vertebrate-dispersed fleshy fruits should undergo concerted convergence, representing independent but often concurrent adaptations to shaded conditions. Our data demonstrate that net venation arose at least 26 times and was lost eight times over the past 90 million years; fleshy fruits arose at least 21 times and disappeared 11 times. Both traits show a highly significant pattern of concerted convergence ( p!10K9), arising 16 times and disappearing four times in tandem. This phenomenon appears driven by even stronger tendencies for both traits to evolve in shade and be lost in open habitats ( p!10K13–10K29). These patterns are among the strongest ever demonstrated for evolutionary convergence in individual traits and the predictability of evolution, and the strongest evidence yet uncovered for concerted convergence. The rate of adaptive shifts per taxon has declined exponentially over the past 90 million years, as expected when large-scale radiations fill adaptive zones. Keywords: adaptation; correlated evolution; molecular systematics;

1. INTRODUCTION development, independent of convergence among dis- Over the past decade, molecular systematics has revolu- tantly related taxa or divergence among close relatives tionized our understanding of higher-level relationships (Givnish 1997; Soltis et al. 1999; Zanis et al. 2003; Davies within the angiosperms, shifting genera among families et al. 2004). and families among orders and leading to the first coherent Within the angiosperms, higher-level relationships view of the phylogeny of flowering as a whole are today perhaps best understood in the monocotyle- (Chase et al. 1993; Soltis et al. 2000; APG 2003). Such dons (Chase et al. 2000, 2005; Michelangeli et al. 2003; analyses provide the basis for rigorous studies of adaptive Davis et al. 2004; Graham et al. 2005). Monocots— radiation, historical biogeography and the evolution of with approximately 60 000 , 92 families and 12 orders—are by far the most species-rich, morphologi- cally diverse and ecologically successful of the early- 1 * Author for correspondence ([email protected]). divergent clades of angiosperms, from which—based

Received 9 November 2004 1481 q 2005 The Royal Society Accepted 30 January 2005 1482 T. J. Givnish and others Concerted convergence of net venation and fleshy fruits on molecular data (Bremer 2000)—they appear to have (a) Predicted patterns of concerted convergence diverged more than 160 Myr ago. Cladistic analysis of Some of these patterns of concerted convergence and more than 500 sequences of rbcL plastid DNA sequences plesiomorphy may hold throughout the monocots. In this identified six major monocot clades: the paper, we test the hypotheses that net venation and (including the orders , , , vertebrate-dispersed fleshy fruits frequently evolve and are Dasypogonales and ), , , Pan- retained with each other and life in shaded forest danales, and , with Acorus being understories, and that parallel venation and non-fleshy sister to all other monocots (Chase et al. 1995). Yet, even fruits (dispersed mainly by wind, water or gravity) when these data were augmented with sequences for frequently evolve and are retained with each other and plastid atpB and nuclear ribosomal 18S for a smaller life in open habitats. subset of 140 species (Chase et al. 2000), relationships The functional rationale for these predictions is as among most major clades remained unresolved or weakly follows. First, even though parallel veins are a monocot supported and relationships among several lineages of hallmark, shady conditions should favour the evolution of commelinids and asparagoids remained poorly net (i.e. branching) venation based on biomechanical understood. economy (Givnish 1979). Shady conditions favour thin, To investigate the intriguing phenomena of concerted broad , which cannot support themselves mechani- convergence and plesiomorphy (see below), we cally (especially after small losses in turgor pressure) and produced a well-resolved, highly inclusive monocot thus require longitudinal and lateral reinforcement from phylogeny based on ndhF sequence variation (figure 1). pronounced primary and secondary veins. Because the Plastid-encoded ndhF provides a wealth of data for cost per unit length of such veins scales like their diameter phylogenetic reconstruction: it is a larger gene (ca. squared, while their strength scales like their diameter 2200 bp) than rbcL (ca. 1464 bp) and has a greater cubed, the economics of support in soft, thin, broad leaves fraction of variable sites for a given set of taxa (Gaut favours the coalescence of nearby, nearly parallel veins into et al. 1997; Patterson & Givnish 2002). Our ndhF one or a few branching ribs of lower total cost. The phylogeny provides the best available basis for analysing broader and thinner a or its divisions, the greater patterns of repeated convergence and divergence within should be the advantage of net venation and a single the monocots, involving many more characters, greater midrib. Here we add that if soft, thin, broad leaves are resolution and better support for specific clades than favoured by conditions other than shade—specifically, in rbcL alone (albeit for fewer taxa), and including many fast-growing, emergent aquatic plants with access to more lineages than the existing 3-gene phylogeny based abundant moisture and nutrients (e.g. Sagittaria) and in on rbcL, atpB and 18S (Chase et al. 2000), or the filmy-leaved submersed species adapted for photosyn- 7- and 17-gene trees now in press (Chase et al. in press; thesis underwater (e.g. Aponogeton)—net venation should Graham et al. in press). The latter are about as also evolve under those conditions. Givnish (1979) well resolved as our ndhF phylogeny, albeit better observed that net venation occurs in several monocot supported. groups with thin, broad leaves in forest understories, Concerted convergence (Givnish & Sytsma 1997)is including Arisaema, Smilax, Trillium and various tropical the independent rise of two or more traits that are gingers. Conover (1983) and Chase et al. (1995) noted genetically, developmentally and functionally unrelated similar, qualitative associations of net venation with in different lineages under similar ecological conditions; broad-leaved forest vines; Cameron & Dickison (1998) concerted plesiomorphy involves the evolutionary reten- later noted an association with achlorophyllous vanilloid tion of the same suite of traits in different lineages orchids. under similar conditions (Patterson & Givnish 2002). Second, wind dispersal of is likely to succeed in Both phenomena are examples of correlated evolution open, windy habitats, but animal dispersal of fleshy fruits and can result from the adaptation of unrelated traits to should be more effective below closed canopies (Croat the same environmental conditions or different com- 1978). Indeed, up to 95% of the woody understory species in neotropical rain forests bear fleshy fruits (Gentry 1982). ponents of the same set of conditions. Both may be Phylogenetic reconstructions indicate that fleshy fruits especially challenging to detect and study using have evolved repeatedly in association with forest under- phylogenies based on traditional phenotypic data, stories in commelinids (Givnish et al. 1999) and Liliales given that multiple (and seemingly independent) char- (Patterson & Givnish 2002) among monocots, and in acters would carry the same, misleading signal regard- Lobeliaceae (Givnish 1999), Gesneriaceae (Smith 2001) ing evolutionary relationships. Within monocots, and Urticales (Sytsma et al. 2002) among dicots. Patterson & Givnish (2002) demonstrated that con- certed convergence and plesiomorphy occur in the order Liliales. Phylogenetic reconstruction showed that (i) visually showy flowers, capsular fruits, wind-dis- 2. MATERIAL AND METHODS persed seeds, narrow leaves, parallel venation and To test our hypotheses, we derived a monocot-wide phylo- evolved upon invasion of open seasonal habitats and (ii) geny based on ndhF sequences for 282 species chosen to visually inconspicuous flowers, fleshy fruits, animal- represent as broad a swath of the monocots as possible, dispersed seeds, broad thin leaves, net venation and including members of 78 of 92 families and all 12 orders (see were retained in lineages inhabiting ancestral Electronic Appendix). The unsampled families are generally forest habitats. For each trait, the observed variation in very small and account for only 1.2% of monocot species. phenotype with environment across lineages appears to The data were analysed using maximum parsimony, with be functionally adaptive. Ceratophyllum as the out-group based on its sister position

Proc. R. Soc. B (2005) Concerted convergence of net venation and fleshy fruits T. J. Givnish and others 1483

Chusquea Dracaena Bambusa Nolina Phyllostachys Polygonatum 1 ndhF monocot phylogeny Olyra Polygonatum 2 Ruscaceae Lithacne Ophiopogon Poa Convallaria L = 16,489 steps Avena Maianthemum Hordeum Asparagaceae CI = 0.211 Phaenosperma Dianella Brachyelytrum Arthropodum Schizachyrium Eustrephus Laxmanniaceae CI' = 0.192 Coix Poaceae Cordyline Sorghastrum Brodiaea 1 Tripsacum Brodiaea 2 Zea Dichelostemma Panicum Triteleia Themidaceae Zoysia Muilla Sporobolus Bessera Arundo Milla Guaduella Lomandra Laxmanniaceae Oryza Aphyllanthes Aphyllanthaceae Pharus Agave 1 Anomochloa Agave 2 Joinvillea Joinvilleaceae Yucca Ecdeiocolea Restionaceae Camassia 1 Elegia Ecdeiocoleaceae Camassia 2 Flagellaria Flagellariaceae Hosta Agavaceae Carex Anthericum Scirpus 61 Chlorophytum Dulichium Herreria Eleocharis Behnia Rhynchospora Anemarrhena Gahnia 1 Mapania Ornithagalum 2 Juncus Albuca 1 Prionium Albuca 2 Thurnia Thurniaceae Ornithagalum 3 Hyacinthaceae Xyris Hyacinthus Orectanthe Xyridaceae Muscari Tonina Scilla Eriocaulon Eriocaulaceae Paramongaia Mayaca Mayacaceae Hymenocallis Stegolepis Eustephia Amphiphyllum Griffinia Epidryos Hippeastrum Schoenocephalium Narcissus Kunhardtia Sternbergia Asparagales Saxofridericia Poales Leucojum Monotrema Ungernia Potarophytum Scadoxus Maschalocephalus Proiphys Spathanthus 1 Cyrtanthus Spathanthus 2 Amaryllis Rapatea Böophone Cephalostemon Allium 1 Canistrum Allium 2 Alliaceae 94 Nidularium Ipheion Aechmea Leucocoryne Ananas Agapanthus Agapanthaceae Bromelia Hensmannia Puya Johnsonia Dyckia Arnocrinum Encholirium Caesia Hemerocallidaceae Deuterocohnia Tricoryne Fosterella Phormium Pitcairnia Geitonoplesium Brewcaria Asphodelus Navia Xanthorrhoeaceae Xeronema Xeronemaceae Tillandsia Iris 1 Iris 2 Iridaceae Sisyrinchium Glomeropitcairnia Gladiolus Brocchinia 1 Cyanastrum Brocchinia 2 Tecophilaceae Typha Typhaceae Tecophilaea Sparganium Sparganiaceae Ixiolirion Ixioliriaceae Riedelia Hypoxis Alpinia Curculigo Hypoxidaceae Globba Lanaria Lanariaceae Zingiber 1 Siphonochilos Collospermum Tapeiospermum Astelia 2 Costus Costaceae 65 Blandfordia Blandfordiaceae Marantachloa Borya Maranta Alania Boryaceae Spiranthes Thaumatococcus Zingiberales Epipactis 100 Canna Cannaceae Diuris Musella Ensete Musaceae Ridleyella Musa Neuwiedia Heliconia Heliconiaceae 1 Ravenala Lilium 2 Phenakospermum Strelitziaceae Strelitzia Orchidantha Lowiaceae 49 1 Cardiocrinum 2 Monochoria Eichhornia Tulipa Lachnanthes Xiphidium Haemodoraceae Lloydia 85 Anigozanthos Amischotolype Spatholirion Commelinales 1 Aneilema Commelinaceae Calochortus 2 Cartonema Calochortus 3 Hanguana 1 1 52 Hanguana 2 Hanguanaceae Tricyrtis 2 Liliales Hanguana 3 1 Philydrum Philydraceae Streptopus 2 Dasypogon Dasypogonaceae Calectasia Dasypogonales 100 Allagoptera Smilax Smilacaceae Beccariophoenix Philesia Philesiaceae Bactris Ripogonum Ripogonaceae Reinhardtia Androcymbium Elaeis Wurmbea Areca Disporum Colchicaceae Drymophloeus Uvularia Leopoldinia Arecales Alstroemeria Alstroemeriaceae 99 Xerophyllum Chamaedorea Trillium Melanthiaceae Ravenea Veratrum Phoenix 100 Campynema Campynemaceae 100 Serenoa Croomia Nypa Stichoneuron Stemonaceae Calamus Stemona Pandanus Pandanaceae Talbotia 85 Vellozia Velloziaceae Tacca Dioscorea Dioscoreaceae 85 Trichopus Dioscoreales Aletris 99 Narthecium Nartheciaceae Japonolirion Petrosaviales Halodule Cymodoceaceae Zostera Zosteraceae Triglochin Juncaginaceae Scheuchzeria Scheuchzeriaceae Aponogeton Aponogetonaceae Alisma Hydrocleys Alismataceae Sagittaria Alismatales Butomus Butomaceae 92 Tofieldia Arisaema 50 changes Spathiphyllum Gymnostachys 100 Acorus 1 Acorus 2 Acoraceae Acorales Figure 1. A phylogram of one of 874 most-parsimonious trees produced by a maximum-parsimony analysis of ndhF sequence variation. Branch lengths are proportional to the number of mutations inferred down each lineage. Arrowheads indicate nodes that collapse in the strict consensus tree; bootstrap values (orders and commelinids only) are indicated above each node. See Electronic Appendix for bootstrap values at other nodes. to the monocots in recent studies (Chase et al. 2000; Soltis correlated evolution using DISCRETE (Pagel 1994). et al. 2000). DISCRETE employs a continuous Markov model to examine We tested whether fleshy fruits, net venation the evolution of binary characters, taking branch length into and occurrence in shaded forest understories show account and weighting gains and losses equally.

Proc. R. Soc. B (2005) 1484 T. J. Givnish and others Concerted convergence of net venation and fleshy fruits

We conducted separate tests of correlated evolution between anatomically but not functionally fleshy fruits. Other features (i) fleshy fruits and shady habitats, (ii) net venation and shady of Acorus morphology and its geographical pattern of genetic habitats, (iii) fleshy fruits and net venation, (iv) net venation variation strongly implicate water dispersal (Liao & Hsiao and shady habitats, emergent broad-leaved aquatics or 1998). Consequently, neither ant-dispersed seeds nor Acorus submersed broad-leaved aquatics and (v) fleshy fruits and berries were scored as fleshy fruits. Finally, species were net venation, excluding emergent and submersed broad- categorized based on their occurrence in primarily open leaved aquatics (see Electronic Appendix). We ran each test sunny habitats (e.g. tundra, grasslands or chaparral) or on four fully resolved trees, chosen at random from each of primarily closed shady habitats (forest under-stories). For the four resolutions of the polytomy at the base of the deciduous forests, the timing of leaf activity and commelinids. The other unresolved nodes in the strict production relative to canopy closure was used to classify consensus tree appear to have few, if any, effects on inferences the habitats occupied (see Electronic Appendix). regarding the evolution of fleshy fruits or net venation. To calculate the timing of the origins of fleshy fruits and net venation, we transformed one of the most-parsimonious 3. RESULTS trees into ultrametric form using cross-verified penalized (a) ndhF phylogeny likelihood (PL), calibrating the tree against the ages of six Maximum parsimony produces one island of 874 trees, Cretaceous fossils and the inferred divergence of Acorus from each 16 489 steps in length based on 1727 variable other monocots 134 Myr ago (see Electronic Appendix). characters, of which 1408 are phylogenetically informative Penalized likelihoods (Sanderson 2002) average local differ- (figure 1). Across monocots, ndhF supports each of the 12 ences in the rate of DNA evolution on different branches, orders identified by prior molecular research (65–99% taking into account branch lengths and branching topology. bootstrap values, excluding Commelinales) and clarifies PL differs from non-parametric rate smoothing (NPRS; several nodes that were previously unresolved or weakly Sanderson 1997) in assigning a penalty for rate changes supported. Our data show that (i) Asparagales is sister to among branches that are too rapid or frequent, based on a the commelinids, (ii) both groups are sister to Liliales and smoothness parameter. If the smoothness parameter is large, Pandanales, (iii) Japonolirion (Petrosaviales), then then PL approaches NPRS. NPRS behaves well in trees with Dioscoreales are sister to all preceding groups and substantial rate variation, but suffers when rates are clock-like (iv) Alismatales is sister to the strongly supported (100% or nearly so (Sanderson 2002, personal communication). We bootstrap) clade of the preceding orders, followed by employed the cross-verification algorithm in r8s to find the Acorus (Acorales). Bootstrap support based on ndhF optimal value of the smoothness parameter, minimizing the exceeds or equals that based on rbcL, 18S and atpB for sum of the squared deviations between observed and 10 of the 12 nodes at or above the ordinal level reported by expected branch lengths, derived through jack-knifing each Chase et al. (2000). Nine nodes are unresolved in the strict individual branch (Sanderson 2002). The smoothness consensus. Of these, only two—involving a four-way parameter was varied from 100 to 103 by steps of 0.25 in polytomy at the base of the commelinids and a trichotomy the exponent. involving four families of Zingiberales—involve substan- We overlaid net venation, fleshy fruits and life in shady tial numbers of species (figure 1). Our analysis strongly habitats on this phylogeny using accelerated transformation supports each of four major commelinid clades (Poales, (to minimize the number of parallel gains inferred), Commelinales, plus Zingiberales, Arecales and Dasypo- tabulated the number of origins of each character every gonales). The lack of resolution of relationships among 10 Myr and then normalized these rates against the number these clades may simply reflect a rapid initial diversifica- of clades present at the midpoint of each 10 Myr period. For tion of commelinids; analyses based on 17 genes (but our purposes, ‘net venation’ includes all leaves with many fewer taxa) also fail to conclusively resolve this node branching support networks, including (i) reticulate vena- (Graham et al. in press). Among the most-parsimonious tion, (ii) simple leaves in which the veins diverge from a ndhF trees, four different relationships among the massive central rib regardless of whether they branch major commelinid clades emerge: ((Poales, Arecales), anatomically (e.g. Costus, Musa) and (iii) compound leaves (Commelinales–Zingiberales, Dasypogonales)); (Poales, with branching rachis (palms). Occasional cross-veins in (Commelinales–Zingiberales, Arecales,Dasypogonales)); leaves with otherwise rigorously parallel venation, with ((Poales, (Commelinales-Zingiberales, Dasypogonales)), multiple major veins, were considered instances of ‘parallel Arecales) and ((Poales, Dasypogonales), Arecales), venation’, given that such a pattern is essentially equivalent Commelinales–Zingiberales). A detailed description of to a purely parallel venation on biomechanical grounds and the systematic implications of our ndhF phylogeny will be is inconsistent with the expected biomechanical optimization presented elsewhere. on to a strongly branching network (see above). ‘Fleshy fruits’ include berries, drupes and seeds with showy and (b) Concerted convergence massive arils dispersed by vertebrates, all with moist, sweet Based on our ndhF phylogeny, fleshy fruits appear to have (or oily) tissue. Brightly coloured seeds or capsules that arisen at least 21 times and been lost 11 times, while net mimic fleshy fruits (Griffinia, Hippeastrum, Proiphys) were venation has arisen at least 26 times and been lost 8 times scored as such. Seeds dispersed by ants, bearing small arils (table 1, figure 2). As predicted, these traits have under- (elaiosomes), occur in forest and non-forest plants and can gone repeated concerted convergence. They have done so serve as adaptations not directly related to dispersal (e.g. in highly significant fashion ( p!10K9, log-likelihood emplacement in nutrient-rich ant nests or shelter from test), with both traits arising together 16 times and frequent fire; Beattie 1985). The fruits of Acorus are berries disappearing together four times (tables 1 and 2, figure 2). but have dry coats and lack the sweet or oily composition Fleshy fruits and net venation arose together in Join- usually associated with vertebrate dispersal—that is, they are villeaceae, Flagellariaceae, Hanguanaceae, Arecales,

Proc. R. Soc. B (2005) Concerted convergence of net venation and fleshy fruits T. J. Givnish and others 1485

Table 1. Evolutionary origins of net venation, fleshy fruits and life in shady microsites, and of parallel venation, passively dispersed seeds and life in sunny microsites. (Most origins of the former character-states represent initial transitions from the latter, while most origins of the latter represent reversals from the former. Transitions on the same line occurred at the same or (in a few cases) adjacent nodes. Taxa in which all three character-states underwent transition at the same or adjacent nodes—entailing concerted convergence—are indicated in bold. All calls are based on overlaying characters on a single most-parsimonious tree using accelerated transformation (see Electronic Appendix).) net venation fleshy fruits shade

Bambusoideae Bambusoideae JoinvilleaCbasal Poaceae Joinvillea JoinvilleaCbasal Poaceae Flagellaria Flagellaria Flagellaria Monotremeae BromelioideaeK Hanguanaceae Hanguanaceae (HanguanaceaeCCommelinaceae–) Amischolotype ZingiberalesCPhilydraceaea Zingiberales Zingiberales Arecaceae Arecaceae Arecaceae Hosta Hosta Behnia Behnia Behnia Chlorophytum Polygonatum Polygonatum RuscaceaeCLaxmanniaceae basal RuscaceaeC Laxmanniaceae Griffinia GriffiniaCHippeastrum GriffiniaCHippeastrum Hymenocallis Hymenocallis ProiphysCScadoxus ProiphysCScadoxus ProiphysCScadoxus Geitonoplesium Geitonoplesium Geitonoplesium Cyanastrum Cyanastrum Curculigo Curculigo Curculigo AsteliaceaeCBlandfordiaceae AsteliaceaeCBlandfordiaceae Neuwiedia Neuwiedia Epipactis Tropidia Cardiocrinum Cardiocrinumb Cardiocrinum higher Liliales (OAlstroemeria) higher Liliales (ORipogonum) higher Liliales (ORipogonum) DisporumKUvularia Disporum DisporumKUvularia Trillium Trillium Trillium Stemonaceae Stemonaceae Pandanaceae Dioscoreaceae Tacca Dioscoreaceae Alismataceaea Alismataceaea Zosteraa Aponogetona Araceae Araceae Araceae parallel venation passively dispersed fruits sun higher Poaceae Poaceae higher Poaceae Cartonema Costaceae Cannaceaea Cannaceae Strelitziaceae Lowiaceae Nypa Nypa Phoenix DracaenaCNolina Nolina DracaenaCNolina Asparagus Asparagus Arthropodium Arthropodium Arthropodium HypoxisKLanaria HypoxisKLanaria HypoxisKLanaria Lilioideae Lilioideae Lilioideae Calochortus CalochortusCTricyrtisb Calochortus Scoliopus Androcymbium–Wurmbea a Associated with broad-leaved aquatic habit. b Associated with origin or retention of passively dispersed fruits adapted to dispersal in autumn under open canopies in deciduous forests, with net venation adapted for leaf activity in summer under closed canopies. Zingiberales, Behnia, two groups of Amaryllidaceae, fruits slightly lagging that of net venation among close Geitonoplesium, Curculigo, the core Liliales, Trillium and relatives and inferred ancestors in the latter three lines Araceae, and are associated with each other in Tacca, (figure 2). Fleshy fruits and net venation were lost together Disporum and Polygonatum, with the evolution of fleshy in Arthropodium, Hypoxis–Lanaria, Lilioideae and Nolina,

Proc. R. Soc. B (2005) 1486 T. J. Givnish and others Concerted convergence of net venation and fleshy fruits

net venation fleshy fruits

Poales

Commelinales

Zingiberales

Dasypogonales

Arecales

Asparagales

Liliales

Pandanales Dioscoreales Petrosaviales

Alismatales Acorales 134120 100 80 60 40 20 00 20 40 60 80 100 120 134 million years (Myr) ago million years (Myr) ago Figure 2. Concerted convergence of net venation (green), fleshy fruits (red), shaded habitats (sand boxes) and broad-leaved aquatic habitats (blue boxes). Note that almost all transitions to net venation and fleshy fruits occur upon invasion of shaded habitats, and that almost all reversals to parallel venation and dry, passively dispersed seeds or fruits occur upon re-invasion of open, sunny habitats. Branching has been calibrated against time based on fossil data, so that the tempo and taxonomic distribution of phenotypic transitions can be visualized.

Proc. R. Soc. B (2005) Concerted convergence of net venation and fleshy fruits T. J. Givnish and others 1487

Table 2. Log-likelihood ratios (LR) and significance levels from five tests of correlated evolution across monocots, involving net venation, fleshy fruits, life under shaded conditions and their converses. (MeanGs.e. log-likelihood ratios reflect the outcome of five analyses per test per tree; grand standard errors are based on the tree-based means, minimums and standard errors displayed).

tree A tree B tree C tree D mean grand s.e. significance

fleshy fruits and net venation mean LR 53.0 50.1 53.0 51.2 51.8 0.7 s.e. 0.5 0.5 0.8 0.6 0.7 0.08 minimum 51.4 48.6 51.3 49.8 50.3 0.7 p!10K9 fleshy fruits and shade mean LR 70.8 70.8 72.0 71.0 71.1 0.3 s.e. 0.6 0.4 0.5 0.6 0.5 0.05 minimum 68.9 69.8 70.7 69.4 69.7 0.4 p!10K13 net venation and shade mean LR 115.2 114.6 116.0 115.4 115.3 0.3 s.e. 0.5 0.4 0.5 0.6 0.5 0.05 minimum 113.8 114.0 115.1 113.6 114.1 0.4 p!10K22 net venation and shadeCbroad-leaved aquatics mean LR 146.4 144.3 146.4 146.2 145.8 0.5 s.e. 0.4 0.9 0.3 1.1 2.5 0.19 minimum 145.7 141.2 145.4 142.6 143.7 1.1 p!10K29 fleshy fruits and net venation, excluding broad-leaved aquatics mean LR 63.5 64.7 63.8 60.4 63.1 0.9 s.e. 0.4 0.4 0.8 1.9 0.9 0.36 minimum 62.3 63.9 60.9 56.7 61.0 1.6 p!10K10 with the loss of fleshy fruits in the latter lagging that of net groups but have also invaded open savannas. Aroids are venation by one node. These conclusions would be mainly herbs, vines and epiphytes of densely shaded unaltered if Acorus were instead scored as having tropical rain forests, together with some temperate forest fleshy fruits. herbs (e.g. Arisaema) and broad-leaved submersed Both fleshy fruits and (especially) net venation show aquatics (e.g. Anubias and Cryptocoryne). The most recent even stronger patterns of correlated evolution with shady instances of concerted convergence in fleshy fruits and net conditions. In almost every case, the evolution of net venation occurred in Curculigo of Hypoxidaceae within the venation and fleshy fruits is associated with life in forest last 5 Myr. Fleshy fruits evolved at least three times in understories, while their loss is associated with open Poales, twice in Commelinales, once in Zingibereales, habitats. Specifically, 19 of 21 gains of fleshy fruits are once in Arecales, eight times in Asparagales, three times in associated with life in shady sites, while eight of 11 losses Liliales, once in Pandanales, once in Dioscoreales and are associated with life in sunny sites (figure 2). For net once in Alismatales. Net venation evolved at least three venation, 22 of 26 gains are associated with shady times in Poales, once in Commelinales, once in Zingiber- conditions and all eight losses are associated with sunny ales, once in Arecales, ten times in Asparagales, four times conditions. These patterns of origin and maintenance are in Liliales, once in Pandanales, once in Dioscoreales and K13 K22 four times in Alismatales (figure 2). The frequency of highly significant ( p!10 –10 )whentestedin adaptive change (both gains and losses) in both characters DISCRETE (table 2). These results support our adaptive per clade declines exponentially with time (figure 3), as hypotheses and establish the existence of an extraordinary, expected—but rarely documented—for adaptive radi- highly significant pattern of concerted convergence across ations as they ‘fill’ ecological space. the monocots. Only net venation evolved in some understory groups, Net venation shows an even stronger association with including the bambusoids, basal grasses, Costaceae, shade ( p!10K29) if we factor out the four lineages Hosta, and Stemonaceae. Cardiocrinum (Alismataceae, Aponogetonaceae, Philydraceae and and Tricyrtis of temperate deciduous forests both have net Zosteraceae) in which it arose in broad-leaved aquatic veins only, but are photosynthetically active under shady plants, mostly near the base of the monocots in conditions in summer, releasing seeds after the canopy Alismatales (figure 2). All origins of net venation are re-opens in autumn. Net veins also occur in the absence of associated with shady conditions or broad-leaved emer- fleshy fruits in four lineages of broad-leaved aquatics. gent or submersed aquatics (tables 1 and 2). As expected, Fleshy fruits arose without net venation under shady fleshy fruits show a stronger association with net venation conditions in the bromelioid bromeliads, Amischotolype, if we exclude net-veined, broad-leaved aquatic plants, for Asteliaceae and relatives, and the apostasioid orchid, which we have no a priori reason to expect the evolution of Neuwiedia. fleshy fruits (table 2). The numerous origins of fleshy fruits and net venation are distributed rather evenly across lineages and time 4. DISCUSSION (figure 2). Both traits first arose 80–90 Myr ago in In many ways, the contrast between Trillium and its closest Arecaceae and Araceae. Palms include many rainforest relatives in Melanthiaceae (represented by Xerophyllum)

Proc. R. Soc. B (2005) 1488 T. J. Givnish and others Concerted convergence of net venation and fleshy fruits

the chances of contacting a suitable fungal partner. Some –0.018x discords, like the absence of fleshy fruits in the net-veined 0.4 y = 0.052e basal grasses and bambusoids, remain puzzling. The 2 r = 0.78*** persistence of net venation and fleshy fruits in some groups of open habitats, notably Ravenala and Strelitzia of 0.3 Zingiberales, appear to be best explained by phylogenetic inertia. Beyond the taxa surveyed, a few additional cases 0.2 may represent joint origins of fleshy fruits and net venation under shady conditions. Examples include Vanilla (Orch- idaceae), Palisota (Commelinaceae), Eucharis (Amarylli- daceae) and Cyclanthaceae of tropical rainforest 0.1 changes per clade 10 Myr understories. However, one of the most striking cases may involve the tropical vine Gnetum, which has fleshy fruits and broad, net-veined leaves that strongly resemble 0 those of Coffea and other understory angiosperms— 90 80 70 60 50 40 30 20 10 0 despite the fact that it is a gymnosperm! The strong Myr before present resemblance of Gnetum to certain angiosperms inspired the hypothesis that angiosperms were derived from Figure 3. Rate of adaptive transitions in venation and fruit Gnetales (Doyle & Donoghue 1986); molecular data characters (gains and losses per clade per 10 Myr) in the have largely laid that hypothesis to rest, placing gymno- monocots over the past 90 Myr. The rate of such transitions was highest early in monocot evolution and sperms sister to angiosperms and indicating that Gnetales subsequently dropped exponentially with time ( yZ0.052 is well-nested within the conifers (Chaw et al. 2000; Soltis eK0.018x; r 2Z0.78, p!0.001 for 7 d.f.). Dashed curve et al. 2002; but see Rydin et al. 2002). represents the regression without data for the earliest The fact that fleshy fruits and net venation have each interval (outlier for mean, variance) included. This analysis arisen more than 20 times in the monocotyledons raises underestimates the decline in the rate of adaptive change, the key question of whether the same developmental because our sample (ca. 0.5% of all monocot species) is pathways and underlying genes were involved in each case, biased towards including families and groups in which net or whether these adaptations arose in different ways in venation and fleshy fruits have evolved, and away from the different groups. It seems unlikely that more than 20 most recent, phenotypically repetitive diversification within origins of fleshy fruits and net venation over 90 Myr all families and genera. Inclusion of the latter would greater inflate the number of recent clades and add few new shifts tapped into the same genes and developmental pathways, in venation or fruit type. and several lineages show obvious differences in the fine details of venation pattern or use different tissues to attract crystallizes the pattern of concerted convergence dis- dispersers. Determining the genetic and developmental cussed here. Trillium of forest understories has broad, bases for these shifts should be a major goal of new studies thin, soft leaves, net venation and berry-like capsules, at the interface of ecology, evolution and development while Xerophyllum usually grows in more open habitats (‘eco-evo-devo’ (Givnish 2003)). An especially promising and possesses narrow, thick, hard leaves, parallel venation dicot group to investigate in this regard might be the and tiny, wind-dispersed seeds. It would be difficult, based Hawaiian silversword alliance, a recently derived (!6 Myr on gross morphology, to infer that these taxa are, in fact, ago) dicot group with a relationship between leaf thickness very close relatives. and venation pattern similar to that seen across monocots. Across the monocots surveyed, mode of dispersal Parallel venation occurs in Argyroxiphium, Wilkesia and and leaf venation pattern show a phylogenetically uncor- species of Dubautia with narrow, thick leaves adapted to rected correlation (r) of 0.58. Mode of seed dispersal and dry or extremely wet, boggy conditions, while net venation light availability (sun versus shade) show a stronger occurs in Dubautia species (especially D. latifolia) with correlation (rZ0.66), and leaf venation and light avail- broad, thin leaves adapted to moist, shady conditions; ability are even more strongly correlated (rZ0.74). The crosses can be made among many species in this complex last value rises to 0.79 if we include the origin of net (Carlquist et al. 2003). venation in broad-leaved aquatic plants (see above). The evolution of fleshy fruits and net venation is not lock-step: by no means is every invasion of forest understories 5. CONCLUSIONS associated with a gain of both traits, nor is every invasion The repeated, coupled evolution of net venation and fleshy of open sites associated with a loss of both traits. fruits by monocots in forest understories, and the loss of Nevertheless, the patterns seen are highly significant these traits in open sites, is one the strongest patterns ever and explanatory and some of the apparent exceptions demonstrated for the evolutionary convergence of indi- are illuminating. Bromelioid bromeliads evolved fleshy vidual traits, and the strongest evidence yet for concerted fruits but not net venation—perhaps understandable, convergence. The stronger associations of net venation given that they possess Crassulacean acid metabolism and fleshy fruits to shade than to each other suggests that photosynthesis and thick, succulent leaves. Vanilloid each is primarily an independent adaptation to light orchids (not included in our survey) evolved net venation regime (and its correlates) than a co-adaptation to the but not fleshy fruits (except in Va n i l l a itself )—also other trait. This finding adds to a growing set of studies understandable, given that mycotrophy favours tiny, demonstrating the repeatability—and indeed, the predict- numerous, independently dispersable seeds to maximize ability—of evolution, including the repeated evolution of

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