13C12C ratios in seagrasses

Aquatic Botany, 9 (1980) 237--249

237

Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

13C/12C RATIOS IN SEAGRASSES

CALVIN MCMILLAN

Plant Ecology Research Laboratory and Department of Botany, University of Texas at Austin, Austin, TX 78712 (U.S.A.) PATRICK L. PARKER and BRIAN FRY

Marine Science Institute, University of Texas at Austin, Port Aransas, TX 78373 (U.S.A.) (Accepted 1 May 1980)

ABSTRACT McMillan, C., Parker, P.L. and Fry, B., 1980. 13C/12C ratios in seagrasses. Aquat. Bot., 9: 237--249. Seagrasses have a wide range of ~ 'sC values. For 47 species from 12 genera, the values were within the range of --3.0 to --19.0%0. Only two species, Halophila tricostata Greenway and Halophila beccarii Aschers., had lower values, o f - - 2 0 . 8 and --23.8%0, respectively. Among the 12 genera, Syringodium and Enhalus had the highest mean values and Phyllospadix and Amphibolis had the lowest mean values. The 8 zsC values for most seagrasses are within the range usually associated with C4 metabolism, but the status of seagrasses as members of this photosynthetic group has not been confirmed by morphological and physiological studies. The high variability in 8 zsC values may reflect a variable photosynthetic metabolism.

INTRODUCTION

The stable carbon isotopic composition of seagrasses is receiving increasing attention. Some of this interest centers upon discrepancies between the lsC/I~C ratio, measured as 813C, and the apparent mode of photosynthesis (McRoy and McMiUan, 1977; Zieman and Wetzel, 1980). The 5 ~sC values in the range - 3 to - 1 5 ° 0 that have been reported for seagrasses (Craig, 1953; Parker, 1964; Calder, 1969; Parker and Calder, 1970; Smith and Epstein, 1971; Doohan and Newcomb, 1976; Black and Bender, 1976; Benedict and Scott, 1976; Fry, 1977; Andrews and Abel, 1979) are similar to those of C4 plants that fix CO2 during photosynthesis with phosphoenolpyruvate carboxylase {Bender, 1971; Smith and Epstein, 1971; Troughton et al., 1974). Biochemical studies of Thalassia testudinurn Banks ex KSnig seemed to confirm that seagrasses are C4 plants (Benedict and Scott, 1976), but recent studies of four other species, Halodule uninervis (Forsk.) Aschers., Syringodiurn isoetifoliurn (Aschers.) Dandy, Thalassia hernprichii (Ehrenb.) Aschers. and Halophila spinulosa (R. Br.) Aschers. indicate that photosynthesis occurs 0304-3770/80/0000--0000/$02.50 © 1980 Elsevier Scientific Publishing Company

238

via the C3 rather than the C4 pathway (Beer and Waisel, 1979; Andrews and Abel, 1979). Studies of photorespiration (Hough, 1976) and leaf anatomy (Doohan and Newcomb, 1976) have not resolved the problem, and suggest that seagrasses may not be typical C3 or C4 plants. The 513C values of seagrasses, in relation to those of phytoplankton and most other marine plants (Smith and Epstein, 1971), have been of interest in food chain research. Because animals approximate the isotopic composition of their diets, natural food webs based on seagrasses have been distinguished from those based on tropical and temperate phytoplankton, - 1 8 to -240/o0 (Thayer et al., 1978; McConnaughey and McRoy, 1979; Fry and Parker, 1979). The present survey extends the list of seagrass species with known 513C values from 14 to 49. The object of this survey was not to clarify the photosynthetic metabolism of seagrasses, but to determine whether the 5 laC values of seagrasses have taxonomic or ecological implications.

MATERIALS AND METHODS

Plant material was obtained from various sources. Most samples were dried shortly after collection by numerous scientists, a few samples being removed from herbarium sheets (Table I). For five seagrass species, individual leaves from separate plants within the same or adjacent sites were analyzed and compared with four land plants (Table II) for evaluation of infraspecific variability. For most 5 ISc analyses, 2--5 leaves were homogenized, acidified to remove carbonates and then dried for combustion. All samples were combusted using a modified LECO radio-frequency induction furnace (Parker et al., 1972). The resulting CO2 gas was analyzed with a dual collector isotope ratio mass spectrometer (Nuclide Corporation Model RMS). All values are reported relative to the international standard, PDB, where 1aC/12C sample -

~aC- --~aC/~2 C standard

) -1

x

10 a

Based on replicate samples, the average overall error during sample preparation and measurement was +0.5°/00. Because some samples may have been kept in preservative prior to drying, some plant material was examined after storage in formalin. For these tests, fresh seagrass leaves were split lengthwise, and half of the leaf was placed in 5% formalin for 6 h before drying. The sample that had been in the preservative was 0.7%0 more negative than the unpreserved sample.

239

c6

e,

0 o~ .,b.} ~"0 ,.-w C) T.)

0

•-

0

~.o~'~.~T

~ o'~

-~

0

0 {,,)

c~

0

o

I

(Q

(P

I

I

I

I

I

I

oo

oO

~b

I

t

I

0

LO

~C)

,4

,.6

T~

T~

I

0

t

I

I

I

I

II

t

III

I

t--

,'~ {J

o

o

m.< E~

C)

240

=

~

¢

~

~..d

~"

°~

o.~

oo--

.~;

0 0

e~ U 0 r~

III

I

~ I

IIII

I

I1

u~

e4

I

I

11

Illlll

III

~ I

u

P

•4

r~

0

~4

0

o 0

.~

0

0

~e2 <

0

g r~

e~

~

~ ' ~

~

241

&-

~.~ ~

[

[

-~

.~

I I I I I

~

_

I l l l

I

~

[I

I

I

~ ~

~

.

I

[

I

I

~

~.=

I

~

II

,~ ~ , ~ , , ~

I I I I I I

(2~

C',1

0"~

tD

I

0

• o ~

~

o

~

~

r~

~

,~

.~ ~

•

._.

242 N

~ ~, ~

0 u

~- ~

r)

N 0 0 . ~ < ~

.

Gw

0 c~

0

0

I 0 •

°

0 0

Ill

1

tl

I

I

.~

I

II

I

I

I

t

I

oO

~-

I

I

I

I

I

I

I

I

I

O~D

~J

N

~

I

I

I

oO

• o

<

I

~:

243

cO ¢,1

~

,~.

-~

...

',2 ~

•~ ! ~ ~

g~ ~.~

•

.- ~ -

•

Oo

~,-~

~ r . ~

o.~N . ~ , ~ -~

I I ( l l l

Ill

I

,--t

eq

I

I

I

,-.i

,~

O~

I

II

~

a

~3

0

"'2,

~

~.~ ~ - ~

~

0

Q;

244

IIIII

~IILL

IIlll

IIIII

v v v

l l I l l

.6

0 ,.~,~ 0

~

0

| (p

E'-,

.W.

245

RESULTS The seagrasses had a 13C values ranging from - 3 . 0 to -23.8°/oo (Table I). The three genera, Zostera, Phyllospadix and Heterozostera, of the subfamily Zosteroideae of the Potamogetonaceae had a 13C values ranging from - 7 . 8 to - 1 9 . 0 . The other six genera of the Potamogetonaceae had values ranging from - 3 . 0 to - 1 6 . 6 . The three genera of the Hydrocharitaceae had values ranging from - 4 . 9 to - 2 3 . 8 . Although no correlation of a ~3C values with family or subfamily was indicated, there were some trends among the 12 genera. Syringodium and Enhalus had the least negative and Phyllospadix and Amphibolis the most negative values. The other eight genera were variously intermediate (Fig. 1). When tabulated at the species level, the a ~3C distribution clusters about four modes. The species means center around - 6 , - 1 0 , - 1 6 and - 2 0 % 0 (Fig. 1). The genus Zostera showed a wide, 11.2°/0o range (-7.8 to-19.0°/00). In Z. marina, plants from diverse sites in North America showed a range of values from-7.8 to-12.4% 0. Among samples of seven species collected in Australia, New Zealand, Japan and Spain, the range of values was relatively narrow, extending from - 9 . 2 for Z. capricorni Aschers. to - 1 2 . 8 % 0 for Z. asiatica Miki. Values for Z. japonica Aschers. and Graebner, Z. americana den Hartog and Z. capensis Setehell were lower ( - 1 5 . 3 t o - 1 9 . 0 % o ) . In Thalassia, a different range of values was shown by the two species. The Gulf--Caribbean species, T. testudinum, had values ranging from - 8 . 3 to -0

-3

I

-6

-9

I

Syringodium

I

-12 I

I

-15

-18 I

[

-~'I I

-24

I

Enhalus

I

•

I

Thalass~a Hatophila Thalassodendron

I I

Cymodocea

Posidoniq Halodule

b

----

•

I

Zostera Heterozostera Phyllospadix

I

Amphibolis

C

,

•

--

I

#

Io ]

All species combined

0

°"

,

I

-3

L

t

-6

,

,

I

-9

,

,

I

-12

,

~

I

- 15

t

~

I

-18

~

,

1

-21

,

-24

Fig. 1. C o m p a r i s o n o f m e a n 61~C values ( - ) f o r 4 9 species o f seagrass from 1 2 genera. T h e ranges o f values (~ i) for the genera are arranged in descending order w i t h t h e genus containing the least negative m e a n at t h e top. T h e m e a n values for t h e 4 9 species are s h o w n c o m b i n e d at the b o t t o m o f the figure.

246 --12.5°/0o (Table I). The Indo-Pacific species. T. hemprichii, showed a higher range, - 5 . 2 to -8.0o/00 (Table I). The lowest values recorded among the seagrasses were in Halophila. Halophila tricostata Greenway, a new species from Australia, had a 5 ~3C value o f - 2 0 . 8 and H. beccari Aschers. from Singapore, had a value o f - 2 3 . 8 % o. The other species in the genus showed a range from - 4 . 9 for H. stipulacea (Forsk.) Aschers. from the Red Sea to -15.5°/0o for H. ovalis from Australia (Table I). Among the species that were examined from more than one collection site, the infraspecific variability was usually < 5°]0~ Zostera capensis, Halophila ovalis, H. beccarii, and Syringodium filiforme had wider ranges, 6.5 to 9.1%0 (Table I). The values for individual leaves of a species at the same site varied from 1.9 to 4 . 7 ° o (Table II). This infraspecific variation was approximately twice that of four terrestrial species collected for comparison. The ~3C values of Halophila decipiens collected between - 1 m and - 4 2 m (Table I) did not correlate significantly with depth. DISCUSSION The survey of 49 species of seagrass from 12 genera showed a wide range of ~ 13C values. Earlier surveys of 14 species had shown a range from - 3 . 0 to -15.5u/00 (Fry, 1977; Andrews and Abel, 1979). The extension of the range from - 15.5 to - 23.8% 0 included values for nine species (Phyllospadix iwatensis Makino, Ph. japonicus Makino, Ph. serrulatus Rupr. ex Aschers., Zostera capensis, Z. americana, Thalassodendron pachyrhizum den Hartog, Amphibolis griffithii (J.M. Black) den Hartog, Halophila beccarii, and H. tricostata) that had not been reported previously. Among the 12 genera, the patterns of 513C suggest some systematic and ecological implications. Syringodium and Enhalus have the highest mean values and Phyllospadix and Amphibolis have the lowest. Although additional collections may provide values outside the current range, the present survey shows no overlap in the range of the former more tropical pair of genera and the latter more temperate pair of genera. In general, species of more tropical distribution have higher 513C values and species of more temperate distribution have lower values. The ~3C/12C ratio is influenced by the habitat to some extent. Smith et al. (1976) demonstrated that the 5 ~3C values for seagrasses from Texas were lower under greenhouse conditions than in their native habitats. They recorded ~ ~3C values for Thalassia testudinum, Halophila engelrnanni and Halodule wrightii of - 14.8, - 16.8 and - 18.8, respectively. The lowest values reported in Texas seagrass beds were - 11.0, - 14.0 and - 12.3°/oo, respectively, for the three species (Table II). Values which were lower than those in the native habitats possibly resulted from differences in the isotopic composition of the inorganic carbon (IOC) pools. Fluctuations in the IOC may b e c o m e

247 locally important where river inflows substantially influence IOC concentrations (Sackett and Moore, 1966; Strain and Tan, 1979) or in shallow oceanic lagoons under calm conditions (Parker and Calder, 1970). The most negative 5 '3C value for a seagrass,-23.8% 0 for Halophila beccarii, may be due to an IOC 513C value more negative than that of seawater (ca. 0°/0o). The specimen was collected along a creek in a mangrove swamp 6 km distant from the open mud-flats along Strait Johore (C. den Hartog, personal communication, 1980). The IOC values for this collection may have been strongly influenced by CO2 from decomposing mangroves (513C values ca. - 2 5 to - 3 0 , Smith and Epstein, 1971; B. Fry, unpublished data). A collection of H. beccarii from mangrove areas near Ratnagiri, Maharashtra, India, has been shown to have a 513C value of - 1 7 . 0 (G.V. Joshi and K.S. Ghevade, personal communication, 1980). Within the same seagrass meadow, 513C values may vary between individuals, among different biochemical fractions and through time. Thayer et al. (1978) showed a 2.1%0 annual variation among composite samples of Zostera marina collected from the same seagrass bed. Benedict and Scott (1976) reported a 4°/00 variation among different biochemical fractions of Thalassia testudinum leaves. The differential storage of biochemical fractions on an annual or short term basis could lead to variation in seagrass 5 ~3C values. The wide variability of 513C values among seagrasses may suggest problems for future research. The clustering of values around four modes, - 6 , - 1 0 , - 1 6 and - 2 0 (Fig. 1) may suggest an inquiry into the possible significance of these values. Investigations are needed to determine if the 5 ~3C diversity involves the operation of one or both of the C3 and C4 photosynthetic pathways and if it is related to kinetic isotope effects related to external or internal IOC pool sizes (Andrews and Abel, 1979). The wide infraspecific variability in seagrasses (Table II) is similar to the +4%o variability observed in some crassulacean acid metabolism plants that employ both C3 and C4 photosynthesis (Winter et al., 1978), and it is possible that the higher variability in seagrasses may also reflect a variable photosynthetic metabolism. Future research comparing seagrass photosynthesis in species having widely different 513C mean values, such as Syringodium filiforme (ca. - 5 ) and Halophila beccarii (ca. - 2 0 ) may provide an improved undelstanding of the photosynthetic pathways operative in seagrasses. ACKNOWLEDGEMENTS This research has been supported from National Science Foundation grants: OCE 74-24357, OCE 76-84298 and OCE 77-26399 to McMillan and OCE 76-01306 and OCE 77-27009 to Parker. We thank the numerous collectors (see Table I) who provided plant material.

248

N o t e added in proof. Thalassia hemprichii f r o m Torres Strait, Qld., Australia, has a range o f 5 ~aC values f r o m - 8 . 0 t o - 8 . 9 , overlapping the range o f values f o r T. testudinum (B. Fry, u n p u b l i s h e d data). REFERENCES

Andrews, T.J. and Abel, I~M., 1979. Photosynthetic carbon metabolism in tropical seagrasses. Plant Physiol., 63: 650--656. Beer, S. and Waisel, Y., 1979. Some photosynthetic carbon fixation properties of seagrasses. Aquat. Bot., 7: 129--138. Bender, M.W., 1971. Variations in the lsC/x~C ratios in plants in relation to the pathway of photosynthetic carbon dioxide fixation. Phytochemistry, 10: 1239--1244. Benedict, C.R. and Scott, J.R., 1976. Photosynthetic carbon metabolism of a marine grass. Plant Physiol., 57: 876--880. Black, C.C. and Bender, M.W., 1976. ~ xsC values in marine organisms from the Great Barrier Reef. Aust. J. Plant Physiol., 3: 25--32. Calder, J.A., 1969. Carbon isotope effects in biochemical and geochemical systems. Ph.D. Dissertation, Univ. Texas, Austin, TX, 132 pp. Cambridge, M.L. and Kuo, J., 1979. Two new species of seagrasses from Australia, Posidonia sinuosa and P. angustifolia (Posidoniaceae). Aquat. Bot., 6: 307--328. Craig, H., 1953. The geochemistry of the stable carbon isotopes. Geochim. Cosmochim. Acta, 3: 53--92. Den Hartog, C., 1970. The Sea-Grasses of the World. North-Holland, Amsterdam, 275 pp. Doohan, M.E. and Newcomb, E.H., 1976. Leaf ultrastructure and 82sC values of three seagrasses from the Great Barrier Reef. Aust. J. Plant Physiol., 3: 9--23. Eiseman, N.J. and McMillan, C., 1980. A new species of seagrass, Halophila johnsonii, from the Atlantic coast of Florida. Aquat. Bot., 9: 15--19. Fry, B., 1977. Stable carbon isotope ratios -- a tool for tracing food chains. M.A. Thesis. Univ. Texas, Austin, TX, 125 pp. Fry, B. and Parker, P.L., 1979. Animal diets in Texas seagrass meadows: ~ ~sC evidence for the importance of benthic plants. Estuarine Coastal Mar. Sci., 8: 499--509. Greenway, M., 1979. Halophila tricostata (Hydrocharitaceae), a new species of seagrass from the Great Barrier Reef region. Aquat. Bot., 7: 67--70. Hough, R.A., 1976. Light and dark respiration and release of organic carbon in marine macrophytes of the Great Barrier Reef region. Aust. J. Plant Physiol., 3: 63--68. McConnaughey, T. and McRoy, C.P., 1979.61 sC label identified eel grass (Zostera marina) carbon in an Alaskan estuarine food web. Mar. Biol., 53: 263--270. McMillan, C., 1980. Isozymes of tropical seagrasses from the Indo-Pacific and the Gulf of Mexico--Caribbean. Aquat. Bot., 8: 163--172. McMillan, C. and Williams, S.C., 1980. Systematic implications of isozymes in Halophila section Halophila. Aquat. Bot., 9: 21--31. McRoy, C.P. and McMillan, C., 1977. Production, ecology and physiology of seagrasses. In: C.P. McRoy and C. Helffench (Editors), Seagrass Ecosystems, Dekker, New York, NY, pp. 53--87. Parker, P.L., 1964. The biogeochemistry of the stable isotopes of carbon in a marine bay. Geochim. Cosmochim. Acta, 28: 1155--1164. Parker, P.L. and Calder, J.A., 1970. Stable carbon isotope ratio variation in biological systems. In: D.W. Hood (Editor), Proc. Univ. Alaska Symp., Organic Matter in Natural Waters, Inst. Mar. Sci., Univ. Alaska, Fairbanks, AK, pp. 107--127. Parker, P.L., Behrens, E.W., Calder, J.A. and Schultz, D., 1972. Stable carbon isotope ratio variations in the organic carbon from Gulf of Mexico sediments. Contr. Mar. ScL, 16: 139--147. Sachet, M.I-L and Fosberg, F.R., 1973. Remarks on Halophila (Hydrocharitaceae). Taxon, 22: 439--443.

249 Sackett, W.M. and Moore, W.S., 1966. Isotopic variations of dissolved inorganic carbon. Chem. Geol., 1: 323--328. Smith, B.N. and Epstein, S., 1971. Two categories of 1sC/12C ratios for higher plants. Plant Physiol., 47: 380--384. Smith, B.N., Oliver, J. and McMillan, C., 1976. Influence of carbon source, oxygen concentration, light intensity, and temperature on 13C/~2Cratios in plant tissues. Bot. Gaz., 137: 99--104. Strain, P.M. and Tan, F.C., 1979. Carbon and oxygen isotope ratios in the Saguenay Fjord and the St. Lawrence estuary and their implications for paleoenvironmental studies. Estuarine Coastal Mar. Sci., 8: 119--126. Thayer, G.W., Parker, P.L., LaCroix, M.W. and Fry, B., 1978. The stable carbon isotope ratio of some components of an eelgrass, Zostera marina, bed. Oecologia, 35 : 1--12. Troughton, J.H., Card, ILA. and Hendy, C.H., 1974. Photosynthetic pathways and carbon isotope discrimination by plants. Carnegie Inst. Washington Yearb., 73: 768--780. Winter, K., Liittge, U. and Winter, E., 1978. Seasonal shift from C3 photosynthesis to crassulacean acid metabolism in Mesembryanthemum erystallinum growing in its natural environment. Oecologia, 34: 225--237. Zieman, J.C. and Wetzel, R.G., 1980. Productivity in seagrass: methods and rates. In: R.C. Phillips and C.P. McRoy (Editors), A Handbook of Seagrass Biology. Garland, New York, NY, pp. 67--116.