CAM Evolution in Bromeliads: Resolving Patterns of Ecological Opportunity, Speciation and Niche Conservatism

C4–CAM–2013: August 6–10, 2013 Champaign–Urbana, Illinois, U.S.A.

CAM evolution in bromeliads: resolving patterns of ecological opportunity, speciation and niche conservatism

J. Andrew C. Smith

Department of Plant Sciences, University of Oxford, U.K.

in collaboration with: Klaus Winter (STRI, Panama) Darren Crayn, Katharina Schulte (Cairns, Australia) Daniele Silvestro, Georg Zizka (Frankfurt, Germany) Steven Heathcote, Nick Brown and Yadvinder Malhi (Oxford) with financial support from the Smithsonian Institution and NERC (postgraduate studentship to S.H.) Families containing species capable of CAM

Major families Minor families Rubiaceae Talinaceae Polypodiaceae Aizoaceae Vitaceae Vittariaceae Apocynaceae Zamiaceae Asparagaceae Welwitschiaceae Bromeliaceae Anacampserotaceae Aquatic plants Cactaceae Araceae Isoetaceae Crassulaceae Asteraceae Alismataceae Didiereaceae Clusiaceae Apiaceae Commelinaceae Euphorbiaceae Crassulaceae Cucurbitaceae Orchidaceae Hydrocharitaceae Geraniaceae Xanthorrhoeaceae Plantaginaceae Gesneriaceae

Lamiaceae Montiaceae Oxalidaceae Total = 36 families Passifloraceae Terminology after Piperaceae APG III (2009) Portulacaceae ≈ 17 000 species CAM photosynthesis and succulence in the semi-desert biome Cactaceae and Ferocactus acanthodes Agavoideae (Asparagaceae) Agave deserti Sonoran Desert, California

Mammillaria sp. Opuntia bigelovii The forest canopy: a highly stratified and heterogeneous ecological niche for epiphytes CAM in the forest canopy: tropical epiphytes – Bromeliaceae and Orchidaceae CAM in the forest canopy: tropical epiphytes – Bromeliaceae and Orchidaceae

Coutinho, L.M. (1963) Boletim no. 288, Faculdade de Filosofia, Ciências e Letra de Universidade de São Paulo, Botânica, 20, 81–98.

Coutinho, L.M. (1969) Boletim no. 331, Faculdade de Filosofia, Ciências e Letra de Universidade de São Paulo, Botânica, 24, 77–102.

Distribution of carbon-isotope ratios in Bromeliaceae

with Klaus Winter and Darren Crayn

45 40 Bromeliaceae A (1873 spp.) 35 30 25 20 15

10 C3 CAM

Number Number species of 5 0 4540 35 30 25 20 15 10 5 40 Bromelioideae13C (‰) B (498 spp.) 35 30 25 20 15 10 5 0 45 40 Pitcairnioideae C (586 spp.) 35

Number of species of Number 30 25 20 15 10 5 0 45 40 Tillandsioideae D 35 (789 spp.) 30 25 20 15 10 5 0

Š 40 Š3535 Š3030 Š2525 Š2020 Š1515 Š1010 Š 5 13C13 C(‰ (ä)) Main topics

● Bromeliaceae as a study-group: biogeography and ecophysiology

● Phylogenetic approaches to understanding the origins of CAM

● Niche partitioning and the adaptive significance of CAM in bromeliads

● Photosynthetic plasticity and the significance of

C3 – CAM intermediates

● Future prospects for understanding the evolution of CAM Neotropical family Bromeliaceae (~ 3200 spp.)

Tillandsia

Hechtia Life-forms in Bromeliaceae After: Schimper (1888); Mez (1904); Tietze (1906); Pittendrigh (1948); Benzing (1990)

Type I Type II Type III Type IV

Ananas comosus Bromelia serra Aechmea fasciata Tillandsia recurvata 1. Roots: Soil Soil + tank Mechanical Mechanical 2. Tank: Lacking Rudimentary Prominent Lacking 3. Trichomes: Simple Rel. simple Absorbent, Absorbent, leaf bases covering shoot Life-forms in Bromeliaceae After: Schimper (1888); Mez (1904); Tietze (1906); Pittendrigh (1948); Benzing (1990)

TypeTERRESTRIAL I Type II Type III EPIPHYTIC Type IV

Ananas comosus Bromelia serra Aechmea fasciata Tillandsia recurvata 1. Roots: Soil Soil + tank Mechanical Mechanical 2. Tank: Lacking Rudimentary Prominent Lacking 3. Trichomes: Simple Rel. simple Absorbent, Absorbent, leaf bases covering shoot Biogeographic regions of bromeliads deduced from species presence–absence data

(S. Heathcote)

. Based on 31 239 georeferenced herbarium specimens to give presence–absence data for 2° grid cells . Distance matrix constructed using β-similarity index (Kreft & Jetz, 2010) . Regions delimited using non- hierarchical k-means clustering (Hammer et al., 2001) Biogeographic regions of bromeliads deduced from species presence–absence data

(S. Heathcote)

. Based on 31 239 georeferenced herbarium specimens to give presence–absence data for 2° grid cells . Distance matrix constructed using β-similarity index (Kreft & Jetz, 2010) . Regions delimited using non- hierarchical k-means clustering (Hammer et al., 2001) Biogeographic realms of bromeliads deduced from species presence–absence data

Guiana Shield many early-diverging lineages and endemics e.g. Brocchinia Lindmania Connellia Cottendorfia Navia Brewcaria Biogeographic realms of bromeliads deduced from species presence–absence data

Northern South America

Pitcairnia (C3) Many epiphytic Tillandsioideae

(both C3 and CAM), including early-diverging lineages (Glomeropitcairnia and Catopsis) Terrestrial and epiphytic Bromelioideae (CAM)

Biogeographic realms of bromeliads deduced from species presence–absence data

Central America

Hechtia

Large number of Tillandsioideae

and Bromelioideae (both C3 and CAM) incl. endemic Bromelioideae Androlepis Hohenbergiopsis Ursulaea Biogeographic realms of bromeliads deduced from species presence–absence data

Caribbean

many epiphytic Tillandsioideae, especially ‘atmospherics’ (CAM) Terrestrial and epiphytic Bromelioideae (CAM) Biogeographic realms of bromeliads deduced from species presence–absence data

North America

relatively few species, epiphytic Tillandsioideae (CAM) Biogeographic realms of bromeliads deduced from species presence–absence data

Andes and Amazon basin

many representatives of:

Pitcairnioideae (C3)

Puyoideae (C3 and CAM)

Tillandsioideae (C3 and CAM) Biogeographic realms of bromeliads deduced from species presence–absence data

Chile

North: drought-tolerant Puya and Tillandsia

South: notable for early- diverging lineages of: Puyoideae (Puya spp.) Bromelioideae Greigia Fascicularia Ochagavia Biogeographic realms of bromeliads deduced from species presence–absence data

CAM Pitcairnioideae the ‘Dyckia’ clade: Deuterocohnia Dyckia Encholirium ‘Pleistocenic Dry Arc’

many Bromelioideae (CAM), esp. terrestrial

Biogeographic realms of bromeliads deduced from species presence–absence data

Atlantic Forest

many terrestrial and epiphytic Bromelioideae (CAM)

CAM to C3 transition along a climatic gradient in the bromeliad flora of Trinidad

100 % CAM

8 % CAM

After Griffiths & Smith (1983) Distribution of carbon-isotope ratios in Bromeliaceae

with Klaus Winter and Darren Crayn

45 40 Bromeliaceae A (1873 spp.) 35 30 25 20 15

10 C3 CAM

Number Number species of 5 0 4540 35 30 25 20 15 10 5 40 Bromelioideae13C (‰) B (498 spp.) 35 30 25 20 15 10 5 0 45 40 Pitcairnioideae C (586 spp.) 35

Number of species of Number 30 25 20 15 10 5 0 45 40 Tillandsioideae D 35 (789 spp.) 30 25 20 15 10 5 0

Š 40 Š3535 Š3030 Š2525 Š2020 Š1515 Š1010 Š 5 13C13 C(‰ (ä)) 18 16 Epiphytes A (667 spp.) 14 12 10 8 6

4 C3 CAM 2 0

18 16 Lithophytes B (202 spp.) 14 12 10 8 6 4 Relative2 abundance of CAM in terrestrial 0 and epiphytic bromeliads 18 16 Terrestrials C (466 spp.) 14

Number of species C CAM 12 3 10 8

4 52 % 48 % 2 0

30 Epiphytes + D 25 lithophytes (869 spp.)

20 Number species of Number

15 Number ofNumberspecies 10

5 57 % 43 %

0 –40 –35 –30 –25 –20 –15 –10 –5 1313  CC (‰)(‰) C3 – CAM distribution by genus

Phylogenetic niche conservatism (PNC)

Majority of genera are

either entirely C3 or entirely CAM …

… with the exception of Puya and Tillandsia Environmental influences on carbon-isotope ratios . Light intensity . Aridity . Elevation Decreasing Increasing 4545 4040 BromeliaceaeBromeliaceae AA (1873 spp.) (1873(1873 spp.spp.)) 3535 3030 2525 2020 1515 Higher Lower pi /pa pi /pa 1010 C3 CAM C3

55 Number species of 00 454540 35 30 25 20 15 10 5 13 4040 BromelioideaeBromelioideaeδ C (‰)13 C (‰) BB (498(498 spp.spp.)) 3535 3030 2525 2020 1515 1010 55 00 4545 4040 PitcairnioideaePitcairnioideae CC (586(586 spp.spp.)) 3535

Number of species Number of species 3030 Number of species of Number 30 2525 2020 1515 1010 55 00 4545 4040 TillandsioideaeTillandsioideae DD (789(789 spp.spp.)) 3535 (789 spp.) 3030 2525 2020 1515 1010 55 00

ĞĞ 4040 Ğ35Ğ35 Ğ30Ğ30 Ğ25Ğ25 Ğ20Ğ20 Ğ15Ğ15 Ğ10Ğ10 ĞĞ 55 Š 40 Š3535 Š3030 Š2525 Š2020 Š1515 Š1010 Š 5 1313 13C13 CC(‰ (ä)(ä)) Environmental influences on carbon-isotope ratios . Light intensity Kohn (2010) Aridity: . Aridity . Elevation Decreasing Increasing 4545 4040 BromeliaceaeBromeliaceae AA (1873 spp.) (1873(1873 spp.spp.)) 3535 3030 2525 2020 Puya chilensis complex 1515

1010 C3 CAM C3

55 Number species of 00

454540 35 30 25 20 15 with D.10 Silvestro, 5 13 K. Schulte & G. Zizka 4040 BromelioideaeBromelioideaeδ C (‰)13 C (‰) BB MAP (mm) (498(498 spp.spp.)) 3535 3030 2525 2020 1515 1010 55 00 4545 4040 PitcairnioideaePitcairnioideae CC (586(586 spp.spp.)) 3535

ĞĞ 4040 Ğ35Ğ35 Ğ30Ğ30 Ğ25Ğ25 Ğ20Ğ20 Ğ15Ğ15 Ğ10Ğ10 ĞĞ 55 Š 40 Š3535 Š3030 Š2525 Š2020 Š1515 Š1010 Š 5 1313 13C13 CC(‰ (ä)(ä)) Environmental influences on carbon-isotope ratios . Light intensity . Aridity . Elevation Elevation: Decreasing Increasing 4545 −8 −10 4040 BromeliaceaeBromeliaceae AA (1873 spp.) −12 (1873(1873 spp.spp.)) 35 −14 3535 −16 N.S. 3030 −18 (n = 390) −20 2525 −20 −22 2020 −24

) −26

1515 ‰ −28

C ( C −30 1010 C 13 CAM 3  C −32 3 p < 0.001 55 Number species of −34 y = 0.00147x − 28.8 0 −36 (n = 824) 0 −38 0 1000 2000 3000 4000 5000 454540 35 30 25 20 15 10 5 Altitude (m) 13 4040 BromelioideaeBromelioideaeδ C (‰)13 C (‰) BB (498(498 spp.spp.)) 3535 3030 2525 2020 1515 1010 55 00 4545 4040 PitcairnioideaePitcairnioideae CC (586(586 spp.spp.)) 3535

ĞĞ 4040 Ğ35Ğ35 Ğ30Ğ30 Ğ25Ğ25 Ğ20Ğ20 Ğ15Ğ15 Ğ10Ğ10 ĞĞ 55 Š 40 Š3535 Š3030 Š2525 Š2020 Š1515 Š1010 Š 5 1313 13C13 CC(‰ (ä)(ä)) The quantitative relationship between δ13C and CAM

Winter & Holtum (2002) 45 100 Bromeliaceae 40 A (1873 spp.) 35 30

100 % C as fixation

25 100 % CAM 2 −26.9 ‰ −8.7 ‰

20 hour fixation - 15

10 C3 CAM % of 24 of %

Number Number species of 5 Daytime CO Daytime 0 0 4540 35 30 25 20 15 10 5 40 Bromelioideae13C (‰) B (498 spp.) 35 30 25 20 15 10 5 0 45 40 Pitcairnioideae C (586 spp.) 35

Number of species of Number 30 25 20 15 10 5 0 45 40 Tillandsioideae D 35 (789 spp.) 30 25 20 15 10 5 0

Š 40 Š3535 Š3030 Š2525 Š2020 Š1515 Š1010 Š 5 13C13 C(‰ (ä)) Potential dark CO2 fixation 0 – 30 % of 24-hour total

Winter – Holtum zone

45 100 Bromeliaceae 40 A (1873 spp.) 35

30 fixation as fixation

25 2

20 hour fixation - 15

10 C3 CAM % of 24 of %

Number of species of Number 30 25 20 15 10 5 0 45 40 Tillandsioideae D 35 (789 spp.) 30 25 20 15 10 5 0

Š 40 Š3535 Š3030 Š2525 Š2020 Š1515 Š1010 Š 5 13C13 C(‰ (ä)) Bromeliad species showing stress- inducible CAM – relatively few

Guzmania monostachia (many authors) incl. P.N. Pereira et al.: Poster P21

Ronnbergia explodens Pierce, Winter & Griffiths (2002)

Tillandsia cretacea Pierce, Winter & Griffiths (2002)

Werauhia sanguinolenta Pierce, Winter & Griffiths (2002)

( 3 of 50 species tested) Bromeliad phylogeny Monocot ndhF chronogram (part)

Bromeliaceae as earliest- diverging lineage of Poales

Last common ancestor of Bromeliaceae ~100 Ma

Relatively recent crown- group radiation (~20 Ma)

Long branch subtending crown group, implying much extinction

Givnish et al. (2011) Amer. J. Bot. 98, 872–895. Combined matK + rps16 intron phylogeny in Bromeliaceae

Multiple origins of epiphytism … … and CAM photosynthesis

Crayn, Winter & Smith (2004) PNAS 101, 3703–3708. Chronogram for 8-locus ML tree Minimum 5 independent of Bromeliaceae origins of CAM : Givnish et al. (2011) Amer. J. Bot. 98, 872–895. Core Bromelioideae 8 Ma

Puya 10 Ma

‘Dyckia’ clade 9 Ma

Hechtia ~ 12 Ma

Tillandsia 6 Ma Distribution of basally diverging lineages in Bromeliaceae

Guiana Highlands

Ayensua Cottendorfia All C3 Brewcaria Lindmania Brocchinia Navia CAM lineage 1: Hechtia

66 spp.: all CAM

GAARlandia (?) 35 – 33 Ma

Hechtia Stem node 15 – 11 Ma Panamanian Isthmus (?) ≥ 3.5 Ma CAM lineage 1: Hechtia

66 spp.: all CAM

LDD Hechtia Stem node 15 – 11 Ma CAM lineage 2: Puya

Andean distribution of

Puya ~ 10 Ma Puya 100100 Puya evolution % CAM species

5050

0 0-499 500-999 1000- 1500- 2000- 2500- 3000- 3500- 4000- 0 1499 20001999 2499 2999 349940003999 4500 m

Puya raimondii NorthNorth AndesAndes Puya aequatorialis Puya mima WAPWAP Puya castellanosii Puya laxa CentralCentral Puya alpestris AndesAndes Puya chilensis Puya venusta CAM lineage 3: the ‘Dyckia’ clade

Stem-group node with

Fosterella (C3) ~ 10 Ma

Deuterocohnia F Dyckia Encholirium

Crown-group radiation < 6 Ma CAM lineage 4: radiation of the Bromelioideae

Puya Bromelioideae

(91 % CAM) C3 taxa of: Puya Fascicularia Stem group Ochagavia 8 Ma Greigia CAM lineage 5: epiphytic Tillandsia Tillandsia > 560 spp. All epiphytic (or lithophytic)

Both C3 and CAM ● Stem group age ~16 Ma ● Crown-group radiation of ‘higher’ tillandsioids ~ 6 Ma 13 Tillandsia (364 spp.) Life-forms and δ C 0 CAM as a values in Tillandsia -5 ‘key innovation’

-10 in evolution of the

C atmospheric life-form

13 -15 c



-20 b

-25 a

n = 97 n = 134 n = 133 -30

100

80 Tank species 60

Proportion of species with CAM (%) CAM of with Proportion species 0

Tank

Lithophytes IntermediateAtmospheric Atmospherics Drivers of CAM evolution in five independent lineages of Bromeliaceae

Clade Driver Hechtia Emergence of semi-desert biomes in Central America in mid- to late Miocene Puya High-elevation Andes, esp. final uplift in Pliocene and Pleistocene climate fluctuations Dyckia clade Arid central and eastern S. America (‘Pleistocenic Dry Arc’) Core Bromelioideae Terrestrial and epiphytic forms, almost all CAM; centre of dispersal Brazil, late Miocene/Pliocene Tillandsia Occupation of exposed epiphytic niches; rampant speciation since Pliocene Dissecting the influence of climatic factors

Best predictors of photosynthetic pathway (C3 vs. CAM) from logistic regression model Mixed model Terrestrial species Epiphytic species gives best fit

Temperature-related Precipitation-related variables are best single variables are best single predictors for epiphytes predictors for terrestrials

S. Heathcote Partitioning of 13C ratios along environmental axes of precipitation and temperature

5000 species 1‰ bins

4000 -36

-35 3000 Potential -34 -32 C3 – CAM -30-33-28-31 -29 intermediates -27 2000 -26 -24 -25 -16 -15 -22 -18 -14 Annual(mm) precipitation -23 -17 -13 -19 -11 -12 -21 -20 -10 -9 1000

16 18 20 22 24 Annual mean temperature (°C) S. Heathcote Photosynthetic plasticity

Photosynthetic plasticity ‘In praise of Dr. Pangloss’

• Osmond (1978) “a photosynthetic option …” • Benzing (2000) “an oversimplified paradigm …” • Cushman (2001) “a plastic photosynthetic adaptation …” • Dodd, Griffiths “… plastic, fantastic” et al. (2002) • Zotz (2002) “categories and CAM – blurring divisions …” • Lüttge (2004) “… phenotypic plasticity constitutes the ecophysiological advantage of CAM” • Silvera, Cushman, Winter et al. (2010) “evolution along the CAM continuum” • Borland et al. (2011) “the photosynthetic plasticity of CAM …”

Distribution of carbon-isotope ratios in Bromeliaceae

A bimodal distribution with two adaptive peaks

45 40 Bromeliaceae A (1873 spp.) 35 30 25 20 15

10 C3 CAM

Number Number species of 5 0 4540 35 30 25 20 15 10 5 40 Bromelioideae13C (‰) B (498 spp.) 35 30 25 20 15 10 5 0 45 40 Pitcairnioideae C (586 spp.) 35

Number of species of Number 30 25 20 15 10 5 0 45 40 Tillandsioideae D 35 (789 spp.) 30 25 20 15 10 5 0

Š 40 Š3535 Š3030 Š2525 Š2020 Š1515 Š1010 Š 5 13C13 C(‰ (ä)) Evolution of discrete traits from continuously varying phenotypes

Lande (2009) J. Evol. Biol.; Chevin & Lande (2013) Am. Nat.

Factor in model Evidence from Bromeliaceae

● Intrinsic costs of plasticity Relative rarity of intermediate phenotypes – a corollary of PNC ● Variance in genetic and phenotypic plasticity is proportional to variability CAM species are adapted to of heterogeneity of environment semi-arid habitats with seasonal predictability of water deficits, but are not characteristic of ● Plasticity will tend to be at a minimum driest environments with in the optimal environment hypervariable precipitation (phenotypic canalization)

● Steepness of physiological threshold Need to define bioclimatic between optimal environments envelopes delimiting the ‘optimal’

determines the reaction norm environments for C3 and CAM (evolutionary trajectory) Habitat suitability for C3 vs. CAM bromeliads defined in terms of MAP and temperature

Terrestrial bromeliads

More suitable for C3 4000 More suitable for CAM

3000

2000

AnnualPrecipitation(mm) 1000

5 10 15 20 25 Annual Mean Temperature (C) Habitat suitability for C3 vs. CAM bromeliads defined in terms of MAP and temperature

Terrestrial bromeliads

More suitable for C3 4000 More suitable for CAM

3000 C3

2000

AnnualPrecipitation(mm) 1000

CAM 0

5 10 15 20 25 Annual Mean Temperature (C) Perspectives and prospects

● Recognizing photosynthetic adaptations

“Galapagos finch bills provide one of the few examples of a trait in a natural population where one can say, with any confidence, what the selective agents are ….” A.D.C. MacColl (2011) TREE Perspectives and prospects

● Recognizing photosynthetic adaptations

● Define more precisely the thresholds for C3 – CAM transitions in multiple phylogenetically independent lineages ● Intensify efforts to identify novel intermediate forms as new model systems for evolutionary studies ● Identify recurrent patterns of gene expression (modularity) of CAM lineages to unravel the genetic basis of trait evolution ► ► Bridging the gap between microevolution and macroevolution