Leaf Anatomy of Orcuttieae (Poaceae: Chloridoideae): More Evidence of C 4 Photosynthesis without Kranz Anatomy

2008·7.1 MB·English

by  Laura M Boykin

Madrono, Vol. 55, No. 2, pp. 143-150, 2008
LEAF ANATOMY OF ORCUTTIEAE (POACEAE: CHLORIDOIDEAE): MORE
EVIDENCE OF C4 PHOTOSYNTHESIS WITHOUT KRANZ ANATOMY
Laura M. Boykin' - \ William T. Pockman' and Timothy K. Lowrey'
^Department of Biology, University of New Mexico, Albuquerque, NM 87131
Abstract
C4photosynthesis without Kranzanatomy (single-cell C4 photosynthesis) occurs in only 0.003% of
known species of C4 flowering plants. To add insight into the evolution of C4 photosynthesis, we
studied the tribe Orcuttieae (Poaceae: Chloridoideae), which has species that can grow under both
aquatic and terrestrial conditions, and utilize single-cell C4 photosynthesis when growing submerged.
Carbon isotope ratios from aquatic, floating, and terrestrial leaves were in the range —12.25 to
—14.31, suggesting that all speciescarry out C4 photosynthesis. Using light microscopy, we examined
the anatomy of aquatic, floating and terrestrial leaves from eight of the nine species in the tribe to
assess the pattern of evolution of C4 photosynthesis and Kranz anatomy among these vernal pool
grasses. Kranz anatomy was present in all floating and terrestrial leaves of Orcuttia ca/ijornica, O.
imiequalis, O. pi/osa, O. tenuis, O. viscidcLTuctoria greenei\ T. luucrouata, and Neostapjia colusaua.
Although carbon isotope data indicated C4 photosynthesis, aquatic leaves ofall members ofOrcuttia
lacked Kranz anatomy, while aquatic leaves of Tuctoria and Neostapjia possessed Kranz anatomy.
When considered in a phylogenetic context, these findings support previously proposed hypotheses
suggesting that Orcuttieae are derived from a terrestrial ancestor and are now becoming more
specialized to an aquatic environment.
Key Words: C4 photosynthesis, Chloridoideae, Kranz anatomy, Neostapjia, Orcuttia, Orcuttieae,
Poaceae, Tuctoria, single-cell C4 photosynthesis, vernal pool.
Since the discovery ofC4 photosynthesis in the case activity is restricted to mesophyll cells while
1960's, there has been great interest in document- RUBISCO activity occurs in the bundle sheath
ing the biochemical and anatomical features of cells, in the high CO2 microenvironment created
the process since it has a much greater photo- by the decarboxylation of C4 products.
synthetic capacity as compared to C3 photosyn- It has been generally accepted that Kranz
thesis from which it has been derived (Leegood anatomy is essential for C4 photosynthesis
2002). In the chloroplast, the C3 pathway (Kellogg 1999), but several studies have shown
assimilates CO2 to form phosphoglycerate otherwise for submerged monocots (Keeley
(PGA), catalyzed by the enzyme ribulose bispho- 1998), submerged dicots (Casati et al. 2000; de
sphate carboxylase oxygenase (RUBISCO). The Groote and Kennedy 1977; Holaday and Bowes
C4 pathway, observed in 19 plant fainilies 1980; Lara et al. 2002; Salvucci and Bowes 1983;
(Kellogg 1999; Sage 2004), couples the C3 Spenceret al. 1996), and terrestrial dicots (Freitag
pathway with a prior carboxylation step cata- and Stichler 2002; Sage 2002; Voznesenskaya et
lyzed by phosphoenolpyruvate carboxylase (PEP- al. 2002; Voznesenskaya et al. 2001). With the
case), producing four-carbon organic acids such exception of a few terrestrial dicot taxa, the
as malate and aspartate. Initial carboxylation by majority of species that lack Kranz anatomy are
PEPcase and subsequent decarboxylation of the aquatic (Bowes et al. 2002). The aquatic plants
C4 products, acts as a carbon concentrating that have C4 photosynthesis without Kranz
mechanism that effectively minimizes the oxy- anatomy are: Hydrilla verticilhita (Holaday and
genase activity of RUBISCO and minimizes Bowes 1980; Magnin et al. 1997; Salvucci and
photorespiration, increasing quantum yield at Bowes 1983; Spencer et al. 1996), Egeria clensa
the low CO2 concentrations (Ehleringer et al. (Casati et al. 2000; Lara et al. 2002), Elodea
1991; Sharkey 1988). In most C4 species, the canadensis (de Groote and Kennedy 1977), and
carbon concentrating activity depends upon the the tribe Orcuttieae (Keeley 1998).
presence of Kranz anatomy in leaves. Kranz Orcuttieae is a small tribe of semi-aquatic
anatomy is the wreath of radially arranged grasses (three genera and nine species) restricted
mesophyll cells surrounding the bundle sheath to vernal pools in California and Baja California.
Haberlandt (1882, 1914). In these species, PEP- Vernal pools result from an unusual combination
of soil conditions, summer-dry Mediterranean
-Current Address: USDA, ARS, USHRL, 2001 climate, topography, and hydrology. These pools
South Rock Road, Fort Pierce, FL 34945. form in small depressions that are filled by winter
''Author for correspondence (E-mail lboykin@ precipitation and retain moisture longer than
macworld.com) surrounding grasslands before evaporation dur-
MADRONO
144 [Vol. 55
Table 1. Locality Information Provided for Orcuttieae Samples Included in the Leaf Anatomy
AND Carbon Isotope Analysis. Voucher specimens are deposited at University of New Mexico Herbarium
(UNM) (except for Titctoviafnigiliswhich islocated at University ofArizona [ARIZ]). Collectorsofplantmaterial
were Laura M. Boykin (LMB), Paula M. Hall (PMH), and John Reeder.
Species Location Voucher Latitude Longitude
Orciittici californica y-dSQy Riverside, Co. LMB & PMH 44 33 43'356" 117 03'3045"
Oi-cuttia imieqiiaUs WooMQV Merced, Co. LMB & PMH 32 37 14.818" 120 13.521"
Ora//r/V/ vwr/V/rt (Hoover) J. Reeder Sacramento, Co. LMB & PMH 42 39 31'291" 121 11'345"
Orcuttiapilosa Hoover Tehama, Co. LMB & PMH 16 39 54.407" 122 58.942"
Orcuttia tenuis A.S.W\ic\\c. Shasta, Co. LMB & PMH 62 40 17'115" 122 07'185"
Tiictoria greenei (Vasey) J. Reeder Tehama, Co. LMB & PMH 28 39 54.124" 121 58.963"
Tuctoriafragilis (Swallen) J. Reeder B.C.S., Mexico J. Reeder 7141 Baja California Sur, Llanos de Hiray
Tiictoria miicromita (Crampton) J. Yolo, Co. LMB & PMH 19 38 29.509" 121 41.157"
Reeder
Ncostcipfia co/usana Davy Yolo, Co. LMB & PMH 21 37 14.852" 120 37.130"
ing the hot, dry summer causes the pools to study had the NADP-ME C4 photosynthetic
shrink and eventually disappear. As a result, subtype based on chloroplast position and bundle
vernal pool habitat represents a continuously sheath cell outline. The exception, A^. colusana,
changing environment, which contains a special- had high activities of both NADP-ME and
ized biota and relatively large numbers of NAD-ME in pulse-chase experiments indicating
threatened and endangered species (Holland a mixed subtype (Keeley 1998).
and Jain 1977) including the Orcuttieae. Orcut- The most unexpected finding of Keeley (1998)
tieae are unusual among C4 plants because they was that aquaticleaves ofOrcuttia californicaand
spend a large portion of their life cycle as O. viscida carry out C4 photosynthesis but lack
submerged aquatics (Ehleringer and Monson Kranz anatomy while terrestrial and floating
1993). The presence of C4 photosynthesis in this leaves ofthese species carry out C4 photosynthe-
group may have been favored because it reduces sis with Kranz anatomy. The other members of
water loss at anthesis when the pools are dry in the tribe included in his study, Tuctoria greenei
late summer and plants are subjected to drought and Neostapfia colusana, carry out C4 photosyn-
and high temperatures (Keeley 1998). thesis but possess Kranz anatomy in both aquatic
C4 plants are divided into three biochemical and terrestrial leaves. The photosynthetic and
subtypes that differ mainly in the C4 acid anatomical diversity that exists among such
transported into the bundle sheath cells (malate closely related taxa provides a unique opportu-
and aspartate) and in the way in which it is nity to study the evolution of single-cell C4
decarboxylated; they are named, based on the photosynthesis. Single-cell C4 photosynthesis in
enzymes that catalyze their decarboxylation, aquatic plants has generated great interest as the
NADP-dependent malic enzyme (NADP-ME) system that may hold promise for genetic
found in the chloroplasts, NAD-dependent malic engineering in C3 plants (Leegood 2002).
enzyme (NAD-ME) found in the mitochondria, To assess the anatomical and photosynthetic
and phosphoenolpyruvate (PEP) carboxykinase pathway variation ofall species in the Orcuttieae
(PCK), found in the cytosol ofthe bundle sheath (excluding Tuctoria fragilis), we surveyed leaf
cells (Edwards and Walker 1983; Ghannoum et anatomy at the aquatic, floating and terrestrial
al. 2001; Hatch 1987; Hatchet al. 1975; Jenkinset stages using light microscopy and carbon isotope
al. 1989). Anatomically, the three subtypes ofC4 ratio of leaf tissue. Leaf anatomy and carbon
photosynthesis differ in chloroplast position and isotope data for four species of Orcuttieae, O.
cell outline ofthe bundle sheath cells (Hattersley inaequalis, O. tenuis, O. pilosa, and T. mucronata
and Browning 1981; Hattersley and Watson are presented for the first time.
1976; Hattersley and Watson 1992; Prendergast
and Hattersley 1987). Both NADP-ME and PCK Methods
have an uneven outline ofthe bundle sheath cells,
while NAD-ME has an even outline. The Seed and Soil Collection
chloroplasts in the bundle sheath cells of
NADP-ME and PCK have a centrifugal position, Seeds, and associated soil, of eight species in
while NAD-ME has a centripetal position. Orcuttieae were collected from vernal pools
Keeley (1998) provided photosynthetic and ana- throughout the geographic distribution of Orcut-
tomical data for four of the nine species of tia, Tuctoria, and Neostapfia (Table 1) (federal
Orcuttieae: Orcuttia cciUfornica, O. viscida, Tuc- permit # TE-029387-0 and California state
toria greenei, and Neostapfia colusana. In addi- permit # 00-04). Collections were made from
tion, he found that all species examined in his different populations of the species {Orcuttia
2008] BOYKIN ET AL.: LEAF ANATOMY OF ORCUTTIEAE 145
californica, O. viscida, Tuctoria greenei, and al). Aquatic leaves were collected three months
Neostapfia coluscma) than those previously sam- after the seeds and soil were submerged. Floating
pled by Keeley (1998). Exact locaHty data for all leaves were collected the first week after they
collections are provided in Stone et al. (1988) and reached the surface of the water. Terrestrial
the California Natural Diversity Database leaves, which developed entirely in air, were
(CNDDB [CDFG 2007]). collected approximately two weeks after the tubs
The ninth species in Orcuttieae, Tuctoria had completely dried down. All leafmaterial was
frcigilis\ could not be sampled due to the lack of cut into small pieces and fixed for one week in
living plants in the only known population in formalin acetic acid (FAA) (Berlyn and Miksche
Baja California Sur. Cold stratification ofthe soil 1976).
collected from the Llanos de Hiray, the only
known location for T. fragi/is, produced no seed Dehydration, Infiltration, and Sectioning of
germination. Terrestrial leaves forcarbon isotope LeafTissue
analysis were obtained from herbarium speci-
mens provided by J. Reeder. Fixed leaf material was removed from the
FAA and placed in 70% ethanol overnight and
Seed Germination changed five times in the 24 hr period. The leaf
material was put through an ethanol dehydration
Leaves for anatomy and carbon isotope series: 95% ethanol for 20 min, twice; and 100%
measurements were obtained for eight species ethanol for 20 min, three times. Ethanol dehy-
by growing plants from seed modifying the seed dration was followed by three washes of pure
germination procedures of Griggs (1974, 1980). propylene oxide for 20 min each. Leaf samples
Orcuttia tenuis, O. pilosa, and Tuctoria greenei were then infiltrated with Epon LX112 resin
germination requires an initial period of cold (Ladds Scientific, Williston). An epon mixture of
stratification in cool, wet conditions followed by 4:6 (epon A:epon B) was used in all the
a gradual increase in daily temperature to a infiltration steps after mixtures of 3:7 and 2:8
maximum of 32°C and a minimum of 15 C produced unsatisfactory hardness for cross-sec-
(Griggs 1974). Experiments with all Orcuttia tioning. The epon mixture of4:6 was diluted for
species and Tuctoria greenei showed that naked the first infiltration step in a 1:1 ratio with
seeds cannot be germinated in the laboratory, propylene oxide for three hours. The tissue was
regardless of treatment (Griggs 1980). A germi- then transferred to a 3:1 mixture ofepon (4:6) to
nation rate of 90-100% is possible when sub- propylene oxide and infiltrated overnight. The
merged inflorescences containing ripe seeds are samples were then placed in molds (Pelco, 105)
allowed to become completely engulfed by an filled with only epon 4:6 and left for ten hours.
aquatic fungus (Griggs 1980). After unsuccessful The final step was to transfer the leaf material
attempts using calcium carbonate-rich local clay into a new mold filled with epon 4:6 and incubate
soil in New Mexico, germination of seeds of all at 60°C for 18 hr. Sections 7 |am thick were cut
speciesexcept Tuctoriafragiliswas achieved using with an ultra-microtome (Sorvall MT2-B) and
soil collected from natural vernal pools at the stained with a 1:1 mixture of 1% azure II and 1%
time of seed collection. methylene blue. Light microscopy was used to
Whole inflorescences from field-collected ma- determine presence of Kranz anatomy. All leaf
terial were placed in petri dishes containing sections were photographed at final magnifica-
50 mL of deionized (DI) water and placed at tions of20X and 40x with a Nikon Coolpix 990
20 C for three weeks. Soil was placed in 4-inch attached to a Nikon Eclipse E400 microscope.
pots, saturated with Dl water, and placed at 20 C
forthreeweeks. Whole inflorescences were buried
'^C/'-C Isotope Values
in thecold-stratified soil after the three-week cold
stratification. The pots were submerged in tubs '''C/'-C isotope ratios were determined in leaf
filled with DI waterand placed in a greenhouse at tissue to assess the photosynthetic pathway.
the Department of Biology, University of New '^C/'-C isotope ratios of Orcuttieae aquatic and
Mexico. DI water was added to the tubs daily. terrestrial leaves were obtained using a Delta E
Approximately four months after the soil and mass spectrometer in the Earth and Planetary
seeds were submerged, the DI water was allowed Sciences Department at the University of New
to evaporate completely and the plants were Mexico. 6'^C values, a measure of the carbon
exposed to the air. composition, were determined on plant samples
using standard procedure relative to PDB (Pee
Collection of Leaf Material Dee Belemnite) limestone as the carbon isotope
standard (Bender et al. 1973). Isotope studies
Once plants wereestablished in the greenhouse, have demonstrated that C4 has less negative 6'^C
leaves were sampled as the different stages values than those found in C3 plants (Bender
became available (aquatic, floating and terrestri- 1968; Bender 1971; Smith and Epstein 1971). This
I
Fig. 1. Transverse sectionsot aquatic, floatingand terrestrial leaves ofOrcuttieae. Aquaticleavesweretaken ata
20X magnification while floating and terrestrial leaves are a 40x magnification.
difference in isotopic composition has become a Considerable morphological and anatomical
standard mechanism for distinguishing plant variation was present in the aquatic leaves of
tissues from these two groups, with C3 plants Orcuttia. Orcuttia viscida, O. califomica, and O.
having a ratio of —20 to —35% and C4 plants pilosa had linear leaves, while O. inaequcdis and
having values of -9 to -14% (Bender 1971). O. tenuis had terete leaves, due to folding of the
leaves so the margins nearly touch (Fig. 1).
Results Extensive lacunae were evident in the Orcuttia
aquatic leaves while they were lacking in aquatic
LeafAnatomy leaves of Tuctoria and Neostapfia species.
Chloroplast arrangement was variable in the
Amongtheeight species ofOrcuttieae sampled, terrestrial leaves of Orcuttia (Fig. 1). Orcuttia
Kranz anatomy was absent in aquatic leaves of inaequcdis, O. viscida (not shown), and O. tenuis
O. pilosa, O. tenuis, and O. imiequalis but present exhibited centrifugal arrangement ofchloroplasts
in floating and terrestrial leaves of these species in terrestrial leaves, while O. califomica (not
(Fig. 1). In addition, we confirmed this pattern, shown) and O. pilosa had chloroplasts arranged
previously observed in aquatic and terrestrial around the outer edge ofthe cell. Floating leaves
leaves by Keeley (1998), for the remaining two of Orcuttia all have centrifugal chloroplast
species in the genus, O. viscida and O. califomica distribution in the bundle sheath cells (Fig. 1
(data not shown, see Keeley 1998 for photos). and Keeley 1998). Neostapfia colusana and T.
Kranz anatomy was present in aquatic and greenei have centrifugal distribution of chloro-
terrestrial leaves of T. niucronata (Fig. 1), and plasts in the bundle sheath cells (Keeley 1998).
we also confirmed this pattern in Tuctoriagreenei Tuctoria niucronata aquatic leaves have the
and Neostapfia colusana (data not shown, see chloroplasts in the bundle sheath cells distributed
Keeley 1998 for photos). centripetally (Fig. 1).
2008] BOYKIN ET AL.: LEAF ANATOMY OF ORCUTTIEAE 147
Table 2. Stable Carbon Isotope Ratios in terrestrial environment. Aquatic single-cell C4
Aquatic, Floating and Terrestrial Leaves of photosynthesis has previously been documented
Orcuttieae. Values shown are the mean of five from several aquatic species in addition to
replicates ± 0.3 standard error. All dried material Orcuttieae: Hyclri//a verticil/ata (Holaday and
excluding T. fragi/is was obtained from greenhouse Bowes 1980; Salvucci and Bowes 1983; Spencer
material. Tuctoriafragi/is material was obtained from et al. 1996; Magnin et al. 1997), Egeria densa
voucherspecimenssupplied byDr.John Reeder. Values (Casati et al. 2000; Lara et al. 2002), and E/odea
represent 6^^C values (%o). canadensis (de Groote and Kennedy 1977).
Species Aquatic Floating Terrestrial Single-cell C4 photosynthesis also occurs in
several terrestrial species most notably Bienertia
Orcuttia californica -12.7 -12.7 -12.7 cyc/optera and Borszczowia ara/ocaspica (Cheno-
Orcuttia inaequalis -13.0 -12.8 -13.0
Orcuttiapilosa -12.7 -12.6 -12.8 podiaceae) (Voznesenskaya et al. 2001; Vozne-
Orcuttia tenuis -12.6 -12.4 -12.3 senskaya et al. 2002; Edwards et al. 2004).
Orcuttia viscida -13.6 -13.7 -13.7 Orcuttieae is the only documented group of
Tuctoria greenei -13.1 n/a -13.4 species that is both aquatic and terrestrial in a
Tuctoriafragi/is -13.0 n/a -13.2 given year with attendant photosynthetic and
Tuctoria mucronata -13.6 n/a -13.6 anatomical diversity.
Neostapfia co/usana -14.3 n/a -14.2 The variation observed in chloroplast arrange-
ment (Fig. 1) suggests that the biochemical C4
Carbon Isotope Ratio subtype according to C4 acid decarboxylases
(NADP-malic enzyme, NAD-malic enzyme,
The carbon isotope values for all Orcuttieae phosphoenolpyruvate carboxykinase) may differ
from aquatic, floating (only present in Orcuttia) within Orcuttieae. Such variation, which is
and terrestrial leaves (Table 2) are within the common in plant families and genera (Kellogg
range exhibited by C4 plants (—9 to —14%). 1999), has been documented previously in terres-
Terrestrial, floating and aquatic leaves of all trial leaves of Orcuttia viscida (Keeley and
species of Orcuttia had 6'''C values between Rundel 2003) and O. tenuis (Sage et al. 1999)
—12.3 and —13.7%o. Tuctoria aquatic and terres- which have a centrifugal (NADP-ME) and
trial leaves had 6'^C values between —13.0 and centripetal (NAD-ME) chloroplast arrangement,
—13.6%o. Neostapfia colusana aquatic and terres- respectively. Additionally, our data showed
trial leaves had the most negative values (—14.3 centrifugal (NADP-ME) and centripetal (NAD-
and —14.2%o respectively). ME) chloroplast arrangement in terrestrial leaves
of Tuctoria greenei and T. mucronata, respective-
Discussion ly. Further research on Orcuttieae should include
analysis for type of C4 acid decarboxylases
Our findings revealed that C4 photosynthesis (enzyme assays and western blots) to determine
occurs throughout Orcuttieae (Table 2) but with the C4 photosynthetic subtypes (Edwards et al.
considerable leaf anatomical variation (Fig. 1). 2004).
Aquatic leaves in all species of Orcuttia carry out Although there are no apparent performance
C4 photosynthesis (Table 2) in the absence of advantages between NAD-ME and NADP-ME
Kranz anatomy (Fig. 1 and Keeley 1998). The (Voznesenskaya et al. 1999), the chloroplast
aquatic environment might result in less negative variation and inferred C4 subtype variation in
isotope ratios because of diffusion limitations Orcuttieae (Fig. 1) indicates that there may have
(Osmond et al. 1981; Raven et al. 1982). been multiple independent origins of the NAD-
However, the consistency of the isotopic ratios ME and NADP-ME sub-types in the tribe. A
between aquatic and terrestrial leaves provide switch in subtype could occur if the C4 pathway
strong circumstantial evidence that the underly- were controlled by few regulatory genes, which
ing biochemical pathway is C4. Floating and would make evolutionary changes in the pathway
terrestrial leaves of these same species utilize the expression relatively easy (Ehleringer and Mon-
C4 pathway with Kranz anatomy. On the other son 1993; Ku et al. 1996; Monson 1996; Monson
hand, both aquatic and terrestrial leaves of 1999). Variation in environmental conditions
Tuctoria mucronata, T. greenei and Neostapfia such as, depth and length of inundation of the
carry out C4 with Kranz anatomy (Fig. 1). vernal pools might also have led to variation in
Orcuttieae is unique among plants exhibiting chloroplast position. However, this is not a likely
single-cell C4 photosynthesis because it produces explanation for our results because all samples in
both an aquatic and a terrestrial phase ofgrowth. this study were exposed to the same conditions
A
single plantcan be submerged forthreemonths during growth and development.
with the aquatic leaves lacking Kranz anatomy The vernal pool environment in which Orcuttia
while three months later (after the water in the thrives, has apparently favored different patterns
pool has evaporated), the same plant produces of leaf development across leaf types (aquatic,
leaves with Kranz anatomy after exposure to the terrestrial and floating) while preserving the
MADRONO
148 [Vol. 55
carbon-concentrating effects of C4 photosynthe- were derived from upland C4 ancestors, and the
sis (Keeley 1998). Aquatic plants that utilize the monophyly of the Orcuttieae are well supported
C4 pathway are adapted to reduced levels ofCO2 (Boykin 2003; Boykin et al. in revision; Hilu and
(Keeley 1998; Keeley and Rundel 2003) that are Alice 2001). Since Orcuttia has many aquatic
largely unrelated to long-term patterns of atmo- specializations, including the loss of Kranz
spheric CO2 (Keeley and Rundel 2003). For anatomy, Reeder (1982) and Keeley (1998)
example, Hydrilla verticillata switches between C3 suggest that it is the most derived genus in the
and C4 photosynthesis in response to changes in tribe. In a study of phylogenetic relationships in
CO2 in the surrounding water driven by biogenic the tribe, our data support the derived status of
depletion of carbon in dense mats of vegetation Orcuttia (Boykin 2003). With the data presented
(Salvucci and Bowes 1983). Similarly, the occur- here together with that from previous studies
rence of Kranz anatomy in floating and terres- (Boykin 2003; Keeley 1998; Reeder 1982) we
trial leaves but not in aquatic leaves suggests that believe Orcuttieae originated on land and has
leaf anatomical development responds to the subsequently become more specialized to an
relative abundance ofCO2 and O2 during growth aquatic environment.
of individual leaves. Besides the relative amount
of CO2 and O2 around aquatic versus terrestrial Acknowledgments
leaves, the high diffusive resistance to gases in the TheauthorsthankPaulaHallforvaluablelaboratory
aquatic environment may affect leaf anatomical and field assistance, Jon Keeley and John Reeder for
development. Manipulating the CO2 and O2 plant material, Tom Turner and Gerry Edwards for
concentrations in water or air during develop- valuable comments on the manuscript, Joy Avritt and
ment ofaquatic and terrestrial leaves, respective- Jeffrey Lucero for greenhouse assistance, Sergio Flores
ly, could test this hypothesis. for field assistance in Baja California. Steve Strieker,
Although the advantages of C4 in aquatic Toni Smythe, and Anna Tyler were instrumental in
helping process leafmaterial for cross-sectioning. Zach
leaves are known, the relative benefits associated Sharp and Viorel Autoderi analyzed leaf samples for
with the shift in leaf anatomy from aquatic to carbon isotopedata. Thisstudywasfunded bya P.E.O.
floating and terrestrial leaves of Orcuttia remain fellowship, California Native Plant Society, American
unclear. The densevegetation, high light and high Society of Plant Taxonomists, Botanical Society of
temperatures in the shallow seasonal pools America and various sources at University of New
inhabited by Orcuttieae lead to C02-depletion Mexico (Springfield and Grove fellowships, GRAC,
and O2 supersaturation (Keeley and Busch 1984). SRAC, and RPT). This research represents a partial
C4 photosynthesis, with or without Kranz fulfillment ofthe requirements forthedegreeofDoctor
anatomy, is favored because its CO2 pumping ofPhilosophy in Biology at University ofNew Mexico.
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