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Evidence for synonymizing Zostera americana den hartog with Zostera japonica Aschers. & Graebn.
Aquatic Botany, 14 (1982) 349--356 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
349
EVIDENCE FOR SYNONYMIZING ZOSTERA AMERICANA DEN HARTOG WITH ZOSTERA JAPONICA ASCHERS. & GRAEBN.
RICHARD E. BIGLEY Department of Botany, University of British Columbia, Vancouver, British Columbia V6T 2B1 (Canada) JEANNETTE L. BARRECA Department of Biology, Western Washington University, Bellingham, WA 98225 (U.S.A.) (Accepted 19 May 1982)
ABSTRACT Bigley, R.E. and Barreca, J.L., 1982. Evidence for synonymizing Zostera americana den Hartog with Zostera japonica Aschers. & Graebn. Aquat. Bot., 14: 349--356. A numerical analysis of four species of the Zostera subgenus Zosterella was conducted to determine the taxonomic position of Zostera americana den Hartog. Results of principal components analysis support the determination that Z. americana on the northeast Pacific coast is synonymous with Zostera japonica Aschers. & Graebn.
INTRODUCTION In 1 9 5 7 , a t h i n - l e a v e d , i n t e r t i d a l seagrass was c o l l e c t e d b y N. H o t c h k i s s in Willapa Bay, W a s h i n g t o n ( H i t c h c o c k et al., 1 9 6 9 ) . This was the first reco r d o f a m e m b e r o f t h e Z o s t e r a s u b g e n u s Zosterella g r o w i n g in N o r t h A m e r i c a . Since t h e n , it has b e e n f o u n d o n m u d a n d sand flats ranging as f a r s o u t h as N e t a r t s Bay, O r e g o n ( 4 5 o 2 4 ' N; 1 2 4 ° 53' W) a n d as far n o r t h as B u r r a r d inlet, V a n c o u v e r , British C o l u m b i a ( 4 9 ° 1 7 ' N ; 123 ° 13'W). H i t c h c o c k et al. ( 1 9 6 9 ) i d e n t i f i e d t h e H o t c h k i s s c o l l e c t i o n as Zostera nana R o t h , a n o w invalid n a m e w h i c h at t h e t i m e i n c l u d e d all o f t h e Zosterella species u s e d in this s t u d y . In a m o n o g r a p h of w o r l d seagrasses, d e n H a r t o g ( 1 9 7 0 ) d e s c r i b e d t h e H o t c h k i s s s p e c i m e n s as a n e w species, b a s e d p r i m a r i l y on m o r p h o l o g y o f r e t i n a c u l a (sterile flaps o n t h e f l o w e r i n g spadix) a n d leaf tips. H e n a m e d t h e species Z o s t e r a americana d e n H a r t o g . Phillips a n d S h a w ( 1 9 7 6 ) c o m p a r e d s p e c i m e n s o f Z o s t e r a americana f r o m f o u r W a s h i n g t o n c o l l e c t i o n s w i t h a v e g e t a t i v e c o l l e c t i o n o f Z. noltii H o r n e m . f r o m E n g l a n d a n d w i t h d e n H a r t o g ' s illustrations o f Z. noltii. T h e y f o u n d t h a t leaf tips a n d r e t i n a c u l a in Z. americana w e r e t o o variable t o d i f f e r e n t i a t e t h e p l a n t f r o m t h e E u r o p e a n species, Z. noltii. T h e y c h a l l e n g e d d e n
0304-3770/82/0000--0000/$02.75 O 1982 Elsevier Scientific Publishing Company
350
Hartog's determination of Z. americana as a new species, and concluded Z. americana should be called Z. noltii until further investigations could be conducted. Harrison (1976), also questioning den Hartog's determination, compared retinacula and leaf tips from British Columbia collections of Z. americana with den Hartog's illustrations of Z. japonica Aschers. & Graebn. Harrison concluded that the North American species was Z. japonica, probably introduced from Japan by oyster growers. Biologists have called the North American Zosterella species any of four names, and are often unsure if other researchers are referring to the same species. Previous investigators, trying to show the relationship between the North American Zosterella and its close relatives, have neither quantified species characteristics nor examined enough material to resolve the issue. The purpose of this study is to clarify the taxonomic position of Zostera americana using numerical t a x o n o m y . METHODS
Flowering Zosterella specimens were collected in 1980 and preserved in 5% (v/v) formaldehyde in seawater. Zostera americana was collected from Willapa Bay on the southwest coast of Washington (46°24 ' N, 123057 ' W) and from Roberts Bank in southern British Columbia (49002 ' N, 123°08 ' W). Zostera noltii was collected from Krabbenkreek,the Netherlands (51 ° 37' N, 04o03 ' E) and from the Tay estuary, on the east coast of Scotland (56°27 ' N, 02°53 ' W). Zostera japonica was collected from Yamada Bay (39°29 ' N, 141°51 ' E) and Odawa Bay (35°12 ' N, 139°39 ' E) in central Japan. A collection of Z. capricorni Aschers. from Smiths Bay, New South Wales, Australia (35°36 ' S, 137°27 ' E) was examined also. Characters of the subgenus Zosterella used in den Hartog's monograph which appeared to differentiate taxa were chosen for quantification (Table I, Fig. 1). Three new characters which varied between species were also measured; these were the thickness of spathal corners, thickness of spathe at midrib and retinaculum length to width ratio (characters 4, 5, and 16 in Table I). To minimize character differences due to age, only specimens in which pollen had dehisced but ovules had n o t elongated were selected. The first 12 specimens from each collection which were complete, non-deformed and met the age criterion were used for microscopic and macroscopic sets of character measurements. To obtain measurements on anatomical characters, specimens were embedded in paraffin (Feder and O'Brien, 1968) and sectioned at 12 pm. From prepared slides, seven characters (4, 5, and 11--15 in Table I) were measured under a calibrated c o m p o u n d microscope. Bulked specimens were grouped by collection site, creating 12 operational taxonomic units (OTUs) per collection site. Characters from each species were compared using analysis of variance
16.
15.
14.
13.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Female flowers within spathe (no.) Retinaculum length (mm) Retinaculum width (mm) Thickness of spathal corners (mm) Thickness of spathe at midrib (ram) Spathal width (mm) Spathal length (mm) Prophyllum length (mm) Generative leaf length (mm) Generative leaf width (ram) Thickness of generative leaf at midrib (mm) Accessory fibre bundles between mid and last lateral nerves, generative leaf (no.) Accessory fibre bundles exterior to marginal nerves, generative leaf ( n o . ) Accessory fibre bundles between mid and last lateral nerves, spathal sheath (no.) Accessory fibre bundles external to marginal nerves, spathal sheath (no.) Length to width ratio of retinaculum
Characters
9.71 2.94
5.33
(2.91) (0.60)
(0.96)
(0.44)
(0.91)
4.71 1.25
(1.03) (0.17) (0.06) (0.02) (0.02) (0.23) (7.08) (33.96) (5.94) (0.50) (0.02)
3.13 0.94 0.33 0.10 0.11 1.58 14.58 22.79 11.87 1.64 0.11
s
Z. noltii
4.21 1.80
4.58
3.42
4.17
6.67 0.74 0.56 0.17 0.14 1.96 23.54 20.04 8.25 1.35 0.13
Z
(1.18) (0.26)
(0.97)
(1.02)
(0.87)
(1.34) (0.42) (0.08) (0.04) (0.02) (0.39) (2.40) (3.68) (2.61) (0.29) (0.03)
s
Z. americana
8.96 1.65
6.37
3.50
(0.25)
(2.53)
(1.44)
3.25 1.44
6.25
1.33
5.00 (1.22) (0.83)
(1.13)
6.58 1.31 0.83 0.37 0.10 2.79 22.17 19.17 6.17 2.42 0.12
(1.78) (o.10) (0.06) (0.03) (0.02) (0.30) (5.27) (4.89) (3.37) (0.27) (0.o2) 5.67 0.97 0.59 0.21 0.12 2.21 18.29 15.42 8.17 1.33 0.14 5.42
(1.00) (o.12) (0.25) (0.02) (0.01) (0.34) (3.56) (3.95) (1.53) (0.26) (0.03)
X
S
X
(0.97) (0.30)
(1.14)
(0.65)
S
Z. capricorni
Z. japonica
Mean character values and standard deviations of four species in the Zostera subgenus Zosterella
TABLE I
352
1 9
B
A
7
<
6
>
11
aoooo
/8
/
f
<
10
>
Fig. 1. Characters used to differentiate Zosterella taxa. A. Flowering shoot showing generative leaf, spathe, and prophyllum. B. Spadix showing the positions of retinacula and female flowers; male flowers have been omitted. C. Retinaculum. D. Spathe crosssection. E. Generative leaf cross-section. Numbers refer to character numbers from Table I. Drawings not to scale. with a significance level o f 5%. C h a r a c t e r d a t a for all specimens were evalu a t e d using principal c o m p o n e n t s analysis (PCA).
RESULTS
Table I shows t h e m e a n values o f characters f o r t h e f o u r species. Zostera noltii, t h e m o s t distinctive in t e r m s o f t h e characters m e a s u r e d , was signifi c a n t l y d i f f e r e n t f r o m t h e o t h e r species f o r 9 o f t h e 16 characters (1, 3, 4, 6, 7, 9, 10, 14, and 16 in T a b l e I); Z. americana and Z. japonica were sign i f i c a n t l y d i f f e r e n t f r o m t h e o t h e r species, b u t n o t f r o m each o t h e r , in r e t i n a c u l u m w i d t h ( c h a r a c t e r 3), generative leaf w i d t h ( c h a r a c t e r 10) and n u m b e r o f a c c e s s o r y bundles e x t e r i o r to marginal nerves {character 13).
353
Z. n o l t i i
Z. americana
Z. japonica
Z. capricorni
Fig. 2. Representative spathe cross-sections of Zosterella taxa. Fibre bundles have been omitted.
In the principal components analysis, c o m p o n e n t 1 accounted for 28% of the total variance. The main contributors to c o m p o n e n t 1, in order of decreasing importance, were retinaculum width, the retinaculum length to width ratio and the thickness of the spathal comers. These were followed by spathal sheath width and n u m b e r of female flowers. The three characters with the heaviest loadings on c o m p o n e n t 1 separated Z. japonica and Z. americana from the other species. Zostera noltii retinacula were long and thin, giving them a high length to width ratio (Table I). Zostera capricorni retinacula did not have significantly smaller length to width ratios than Z. japonica retinacula, b u t they were significantly larger. The mean thicknesses of spathal comers were significantly different for all four species, b u t the mean values for Z. americana and Z. japonica spathes were most similar (see Table I, character 4, and Fig. 2). Principal c o m p o n e n t 1 values were plotted against c o m p o n e n t 2 values for individual OTUs (Fig. 3). Together, these components accounted for 45% of the total variance. Zostera noltii and Z. capricorni were clearly separate from the others; Z. americana and Z. japonica overlapped. DISCUSSION
It is our judgment, based on results of principal components analysis and similarities of major characters, that Z. americana and Z. japonica are the same species. The wide spread of points in the Z. ]aponica--Z.americana cluster (Fig. 3) illustrates the plasticity of this taxon; other seagrass taxa also vary morphologically when grown in a~em 4~hich differ in climate (McMillan, 1978). Zostera noltii displayed a similar range of points between its two collecting sites. In his monograph, den Hartog (1970) emphasized differences in the mor-
354 T v
,4 v
Vv V
-4 "IT I
olo
-2 I
o
•
I
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•
•
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%
V
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I
v~
2 I
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0
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00
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Fig. 3. Principal component analysis of Zosterella taxa; projections of characters on components I and II. Inverted open triangle: Zostera noltii from Krabbenkreek, The Netherlands. Inverted solid triangle: Z. noltii from Tay estuary, Scotland. Solid square: Z. americana from Willapa Bay, WA, U.S.A. Open square: Z. americana from Roberts Bank, B.C., Canada. Solid circle: Z. japonica from Yamada Bay, Japan. Open circle: Z. japonica from Odawa Bay, Japan. Solid triangle: Z. capricorni from Smiths Bay, Australia. Characters are listed in Table I.
phologies of leaf tips and retinacula in a key to Zosterella species and through illustration. We f o u n d that leaf tip morphologies were too variable within each of our collections to use as distinguishing characteristics. Phillips (1960) f o u n d that leaf tips, used to distinguish species of the seagrass Diplanthera (= Halodide), varied with the environment. Harrison (personal communication, 1981) observed that retinacular morphology of Zostera japonica varied with the age of the specimen. In the present study, age influence was controlled by selecting plants at the same stage of reproduction; leaf tip characters were not used.
355 Because Phillips and Shaw (1976) lacked flowering specimens of Z. noltii, they did not see cross-sections of the spathes, which are very different from those of Z. americana (Fig. 2). Lack of age control could explain the variability which they found among Z. americana retinacula from different collections. Immature Z. americana retinacula are as narrow as mature Z. nol tii retinacula. Harrison (1976) suggested that Z. japonica had been introduced from Japan through the oyster industry. Oyster growers in Washington and British Columbia began importing Pacific oysters from Japan in the early 1900s. By the 1930s, successful industries had developed in Puget Sound and especially in Willapa Bay on the Washington Coast (Elsey, 1933). Sayce (1976) listed eight animal species which were accidentally introduced into Willapa Bay and Puget Sound from Japan. Most of those listed are parasites or predators of the Pacific oyster. Scagel (1956) reported that Sargassum muticum (Yendo) Fensholt had been introduced from Japan to Washington, Oregon, and southern British Columbia waters. Sargassum, a c o m m o n intertidal alga in Japan, appeared in the northeast Pacific by the 1940s, often in or near Pacific oyster beds. By 1954, Sargassum was abundant in northern Willapa Bay, attached to oyster shells. Zostera japonica was probably introduced to the northeast Pacific as dormant seed, either in sediment associated with Pacific oysters (in southwest British Columbia dormant seed of Z. japonica is present within the sediment year-round (Bigley, 1981)) or attached to adult plants. There is anecdotal evidence that Japanese oysters were packed and shipped in an u n k n o w n species of seagrass (Harrison, 1976). In this study species characteristics which appeared to differentiate taxa have been quantified. We agree with Phillips and Shaw (1976) that den Hartog based the Z. americana designation more on a geographical than a morphological basis. After evaluating PCA results and observing the similarities of major characteristics we conclude, as did Harrison (1976), that Z. americana is s y n o n y m o u s with Z. japonica.
ACKNOWLEDGEMENTS The authors thank E.A. Drew of the Gatty Marine Laboratory, St. Andrews, Scotland, R.P.M.W. Jacobs, of the Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands, V. Holland of the Dept. of Botany, University of New South Wales, Kensington, N.S.W., Australia, and K. Aioi, of the Ocean Research Institute, University of T o k y o , Japan, for collecting and sending Zostera specimens. We also thank P.G. Harrison and R.C. Phillips for encouragement throughout this study and C. den Hartog for his valuable comments.
356 REFERENCES Bigley, R.E., 1981. The population biology of two intertidal seagrasses, Zosterajaponica and Ruppia maritima, at Roberts Bank, British Columbia. M.Sc. Thesis, University of British Columbia, Vancouver, 205 pp. Den Hartog, C., 1970. The sea-grasses of the world. Verh. K. Ned. Akad. Wet. Afd. Natuurk., Reeks 2, 59: 1--275. Elsey, C.R., 1933. Oysters in British Columbia. Biol. Board Can. Bull. 34, 34 pp. Feder, N. and O'Brien, T.P., 1968. Plant microtechnique: some principles and new methods. Am. J. Bot., 55: 112--142. Harrison, P.G., 1976. Zostera japonica Aschers. & Graebn. in British Columbia, Canada. Syesis, 9: 359--360. Hitchcock, C.L., Cronquist, A., Ownbey, M. and Thompson, J.W., 1969. Vascular Plants of the Pacific Northwest. Part 1. University of Washington Press, Seattle, 914 pp. McMillan, C., 1978. Morphogeographic variation under controlled conditions in five seagrasses, Thalassia testudinum, Halodule wrightii, Syringodium filiforme, Halophila engelmannii, and Zostera marina. Aquat. Bot., 4: 169--189. Phillips, R.C., 1960. Environmental effect on leaves of Diplanthera du Petit-Thouars. Bull. Mar. Sci. Gulf Carib., 10: 346--353. Phillips, R.C. and Shaw, R.F., 1976. Zostera noltii in Washington, U.S.A. Syesis, 9: 355--358. Sayce, C.S., 1976. The oyster industry of Willapa Bay. Proc. Symp. Terrestrial Aquat. Ecol. Studies of Northwest. E. Wash. State College Press, Cheney, WA, pp. 347--356. Scagel, R.F., 1956. Introduction of a Japanese alga, Sargassum muticum, into the northeast Pacific. Fish. Res. Papers, Washington Dept. Fisheries, Vol. 1 No. 4, 10 pp.
349
EVIDENCE FOR SYNONYMIZING ZOSTERA AMERICANA DEN HARTOG WITH ZOSTERA JAPONICA ASCHERS. & GRAEBN.
RICHARD E. BIGLEY Department of Botany, University of British Columbia, Vancouver, British Columbia V6T 2B1 (Canada) JEANNETTE L. BARRECA Department of Biology, Western Washington University, Bellingham, WA 98225 (U.S.A.) (Accepted 19 May 1982)
ABSTRACT Bigley, R.E. and Barreca, J.L., 1982. Evidence for synonymizing Zostera americana den Hartog with Zostera japonica Aschers. & Graebn. Aquat. Bot., 14: 349--356. A numerical analysis of four species of the Zostera subgenus Zosterella was conducted to determine the taxonomic position of Zostera americana den Hartog. Results of principal components analysis support the determination that Z. americana on the northeast Pacific coast is synonymous with Zostera japonica Aschers. & Graebn.
INTRODUCTION In 1 9 5 7 , a t h i n - l e a v e d , i n t e r t i d a l seagrass was c o l l e c t e d b y N. H o t c h k i s s in Willapa Bay, W a s h i n g t o n ( H i t c h c o c k et al., 1 9 6 9 ) . This was the first reco r d o f a m e m b e r o f t h e Z o s t e r a s u b g e n u s Zosterella g r o w i n g in N o r t h A m e r i c a . Since t h e n , it has b e e n f o u n d o n m u d a n d sand flats ranging as f a r s o u t h as N e t a r t s Bay, O r e g o n ( 4 5 o 2 4 ' N; 1 2 4 ° 53' W) a n d as far n o r t h as B u r r a r d inlet, V a n c o u v e r , British C o l u m b i a ( 4 9 ° 1 7 ' N ; 123 ° 13'W). H i t c h c o c k et al. ( 1 9 6 9 ) i d e n t i f i e d t h e H o t c h k i s s c o l l e c t i o n as Zostera nana R o t h , a n o w invalid n a m e w h i c h at t h e t i m e i n c l u d e d all o f t h e Zosterella species u s e d in this s t u d y . In a m o n o g r a p h of w o r l d seagrasses, d e n H a r t o g ( 1 9 7 0 ) d e s c r i b e d t h e H o t c h k i s s s p e c i m e n s as a n e w species, b a s e d p r i m a r i l y on m o r p h o l o g y o f r e t i n a c u l a (sterile flaps o n t h e f l o w e r i n g spadix) a n d leaf tips. H e n a m e d t h e species Z o s t e r a americana d e n H a r t o g . Phillips a n d S h a w ( 1 9 7 6 ) c o m p a r e d s p e c i m e n s o f Z o s t e r a americana f r o m f o u r W a s h i n g t o n c o l l e c t i o n s w i t h a v e g e t a t i v e c o l l e c t i o n o f Z. noltii H o r n e m . f r o m E n g l a n d a n d w i t h d e n H a r t o g ' s illustrations o f Z. noltii. T h e y f o u n d t h a t leaf tips a n d r e t i n a c u l a in Z. americana w e r e t o o variable t o d i f f e r e n t i a t e t h e p l a n t f r o m t h e E u r o p e a n species, Z. noltii. T h e y c h a l l e n g e d d e n
0304-3770/82/0000--0000/$02.75 O 1982 Elsevier Scientific Publishing Company
350
Hartog's determination of Z. americana as a new species, and concluded Z. americana should be called Z. noltii until further investigations could be conducted. Harrison (1976), also questioning den Hartog's determination, compared retinacula and leaf tips from British Columbia collections of Z. americana with den Hartog's illustrations of Z. japonica Aschers. & Graebn. Harrison concluded that the North American species was Z. japonica, probably introduced from Japan by oyster growers. Biologists have called the North American Zosterella species any of four names, and are often unsure if other researchers are referring to the same species. Previous investigators, trying to show the relationship between the North American Zosterella and its close relatives, have neither quantified species characteristics nor examined enough material to resolve the issue. The purpose of this study is to clarify the taxonomic position of Zostera americana using numerical t a x o n o m y . METHODS
Flowering Zosterella specimens were collected in 1980 and preserved in 5% (v/v) formaldehyde in seawater. Zostera americana was collected from Willapa Bay on the southwest coast of Washington (46°24 ' N, 123057 ' W) and from Roberts Bank in southern British Columbia (49002 ' N, 123°08 ' W). Zostera noltii was collected from Krabbenkreek,the Netherlands (51 ° 37' N, 04o03 ' E) and from the Tay estuary, on the east coast of Scotland (56°27 ' N, 02°53 ' W). Zostera japonica was collected from Yamada Bay (39°29 ' N, 141°51 ' E) and Odawa Bay (35°12 ' N, 139°39 ' E) in central Japan. A collection of Z. capricorni Aschers. from Smiths Bay, New South Wales, Australia (35°36 ' S, 137°27 ' E) was examined also. Characters of the subgenus Zosterella used in den Hartog's monograph which appeared to differentiate taxa were chosen for quantification (Table I, Fig. 1). Three new characters which varied between species were also measured; these were the thickness of spathal corners, thickness of spathe at midrib and retinaculum length to width ratio (characters 4, 5, and 16 in Table I). To minimize character differences due to age, only specimens in which pollen had dehisced but ovules had n o t elongated were selected. The first 12 specimens from each collection which were complete, non-deformed and met the age criterion were used for microscopic and macroscopic sets of character measurements. To obtain measurements on anatomical characters, specimens were embedded in paraffin (Feder and O'Brien, 1968) and sectioned at 12 pm. From prepared slides, seven characters (4, 5, and 11--15 in Table I) were measured under a calibrated c o m p o u n d microscope. Bulked specimens were grouped by collection site, creating 12 operational taxonomic units (OTUs) per collection site. Characters from each species were compared using analysis of variance
16.
15.
14.
13.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Female flowers within spathe (no.) Retinaculum length (mm) Retinaculum width (mm) Thickness of spathal corners (mm) Thickness of spathe at midrib (ram) Spathal width (mm) Spathal length (mm) Prophyllum length (mm) Generative leaf length (mm) Generative leaf width (ram) Thickness of generative leaf at midrib (mm) Accessory fibre bundles between mid and last lateral nerves, generative leaf (no.) Accessory fibre bundles exterior to marginal nerves, generative leaf ( n o . ) Accessory fibre bundles between mid and last lateral nerves, spathal sheath (no.) Accessory fibre bundles external to marginal nerves, spathal sheath (no.) Length to width ratio of retinaculum
Characters
9.71 2.94
5.33
(2.91) (0.60)
(0.96)
(0.44)
(0.91)
4.71 1.25
(1.03) (0.17) (0.06) (0.02) (0.02) (0.23) (7.08) (33.96) (5.94) (0.50) (0.02)
3.13 0.94 0.33 0.10 0.11 1.58 14.58 22.79 11.87 1.64 0.11
s
Z. noltii
4.21 1.80
4.58
3.42
4.17
6.67 0.74 0.56 0.17 0.14 1.96 23.54 20.04 8.25 1.35 0.13
Z
(1.18) (0.26)
(0.97)
(1.02)
(0.87)
(1.34) (0.42) (0.08) (0.04) (0.02) (0.39) (2.40) (3.68) (2.61) (0.29) (0.03)
s
Z. americana
8.96 1.65
6.37
3.50
(0.25)
(2.53)
(1.44)
3.25 1.44
6.25
1.33
5.00 (1.22) (0.83)
(1.13)
6.58 1.31 0.83 0.37 0.10 2.79 22.17 19.17 6.17 2.42 0.12
(1.78) (o.10) (0.06) (0.03) (0.02) (0.30) (5.27) (4.89) (3.37) (0.27) (0.o2) 5.67 0.97 0.59 0.21 0.12 2.21 18.29 15.42 8.17 1.33 0.14 5.42
(1.00) (o.12) (0.25) (0.02) (0.01) (0.34) (3.56) (3.95) (1.53) (0.26) (0.03)
X
S
X
(0.97) (0.30)
(1.14)
(0.65)
S
Z. capricorni
Z. japonica
Mean character values and standard deviations of four species in the Zostera subgenus Zosterella
TABLE I
352
1 9
B
A
7
<
6
>
11
aoooo
/8
/
f
<
10
>
Fig. 1. Characters used to differentiate Zosterella taxa. A. Flowering shoot showing generative leaf, spathe, and prophyllum. B. Spadix showing the positions of retinacula and female flowers; male flowers have been omitted. C. Retinaculum. D. Spathe crosssection. E. Generative leaf cross-section. Numbers refer to character numbers from Table I. Drawings not to scale. with a significance level o f 5%. C h a r a c t e r d a t a for all specimens were evalu a t e d using principal c o m p o n e n t s analysis (PCA).
RESULTS
Table I shows t h e m e a n values o f characters f o r t h e f o u r species. Zostera noltii, t h e m o s t distinctive in t e r m s o f t h e characters m e a s u r e d , was signifi c a n t l y d i f f e r e n t f r o m t h e o t h e r species f o r 9 o f t h e 16 characters (1, 3, 4, 6, 7, 9, 10, 14, and 16 in T a b l e I); Z. americana and Z. japonica were sign i f i c a n t l y d i f f e r e n t f r o m t h e o t h e r species, b u t n o t f r o m each o t h e r , in r e t i n a c u l u m w i d t h ( c h a r a c t e r 3), generative leaf w i d t h ( c h a r a c t e r 10) and n u m b e r o f a c c e s s o r y bundles e x t e r i o r to marginal nerves {character 13).
353
Z. n o l t i i
Z. americana
Z. japonica
Z. capricorni
Fig. 2. Representative spathe cross-sections of Zosterella taxa. Fibre bundles have been omitted.
In the principal components analysis, c o m p o n e n t 1 accounted for 28% of the total variance. The main contributors to c o m p o n e n t 1, in order of decreasing importance, were retinaculum width, the retinaculum length to width ratio and the thickness of the spathal comers. These were followed by spathal sheath width and n u m b e r of female flowers. The three characters with the heaviest loadings on c o m p o n e n t 1 separated Z. japonica and Z. americana from the other species. Zostera noltii retinacula were long and thin, giving them a high length to width ratio (Table I). Zostera capricorni retinacula did not have significantly smaller length to width ratios than Z. japonica retinacula, b u t they were significantly larger. The mean thicknesses of spathal comers were significantly different for all four species, b u t the mean values for Z. americana and Z. japonica spathes were most similar (see Table I, character 4, and Fig. 2). Principal c o m p o n e n t 1 values were plotted against c o m p o n e n t 2 values for individual OTUs (Fig. 3). Together, these components accounted for 45% of the total variance. Zostera noltii and Z. capricorni were clearly separate from the others; Z. americana and Z. japonica overlapped. DISCUSSION
It is our judgment, based on results of principal components analysis and similarities of major characters, that Z. americana and Z. japonica are the same species. The wide spread of points in the Z. ]aponica--Z.americana cluster (Fig. 3) illustrates the plasticity of this taxon; other seagrass taxa also vary morphologically when grown in a~em 4~hich differ in climate (McMillan, 1978). Zostera noltii displayed a similar range of points between its two collecting sites. In his monograph, den Hartog (1970) emphasized differences in the mor-
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Fig. 3. Principal component analysis of Zosterella taxa; projections of characters on components I and II. Inverted open triangle: Zostera noltii from Krabbenkreek, The Netherlands. Inverted solid triangle: Z. noltii from Tay estuary, Scotland. Solid square: Z. americana from Willapa Bay, WA, U.S.A. Open square: Z. americana from Roberts Bank, B.C., Canada. Solid circle: Z. japonica from Yamada Bay, Japan. Open circle: Z. japonica from Odawa Bay, Japan. Solid triangle: Z. capricorni from Smiths Bay, Australia. Characters are listed in Table I.
phologies of leaf tips and retinacula in a key to Zosterella species and through illustration. We f o u n d that leaf tip morphologies were too variable within each of our collections to use as distinguishing characteristics. Phillips (1960) f o u n d that leaf tips, used to distinguish species of the seagrass Diplanthera (= Halodide), varied with the environment. Harrison (personal communication, 1981) observed that retinacular morphology of Zostera japonica varied with the age of the specimen. In the present study, age influence was controlled by selecting plants at the same stage of reproduction; leaf tip characters were not used.
355 Because Phillips and Shaw (1976) lacked flowering specimens of Z. noltii, they did not see cross-sections of the spathes, which are very different from those of Z. americana (Fig. 2). Lack of age control could explain the variability which they found among Z. americana retinacula from different collections. Immature Z. americana retinacula are as narrow as mature Z. nol tii retinacula. Harrison (1976) suggested that Z. japonica had been introduced from Japan through the oyster industry. Oyster growers in Washington and British Columbia began importing Pacific oysters from Japan in the early 1900s. By the 1930s, successful industries had developed in Puget Sound and especially in Willapa Bay on the Washington Coast (Elsey, 1933). Sayce (1976) listed eight animal species which were accidentally introduced into Willapa Bay and Puget Sound from Japan. Most of those listed are parasites or predators of the Pacific oyster. Scagel (1956) reported that Sargassum muticum (Yendo) Fensholt had been introduced from Japan to Washington, Oregon, and southern British Columbia waters. Sargassum, a c o m m o n intertidal alga in Japan, appeared in the northeast Pacific by the 1940s, often in or near Pacific oyster beds. By 1954, Sargassum was abundant in northern Willapa Bay, attached to oyster shells. Zostera japonica was probably introduced to the northeast Pacific as dormant seed, either in sediment associated with Pacific oysters (in southwest British Columbia dormant seed of Z. japonica is present within the sediment year-round (Bigley, 1981)) or attached to adult plants. There is anecdotal evidence that Japanese oysters were packed and shipped in an u n k n o w n species of seagrass (Harrison, 1976). In this study species characteristics which appeared to differentiate taxa have been quantified. We agree with Phillips and Shaw (1976) that den Hartog based the Z. americana designation more on a geographical than a morphological basis. After evaluating PCA results and observing the similarities of major characteristics we conclude, as did Harrison (1976), that Z. americana is s y n o n y m o u s with Z. japonica.
ACKNOWLEDGEMENTS The authors thank E.A. Drew of the Gatty Marine Laboratory, St. Andrews, Scotland, R.P.M.W. Jacobs, of the Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands, V. Holland of the Dept. of Botany, University of New South Wales, Kensington, N.S.W., Australia, and K. Aioi, of the Ocean Research Institute, University of T o k y o , Japan, for collecting and sending Zostera specimens. We also thank P.G. Harrison and R.C. Phillips for encouragement throughout this study and C. den Hartog for his valuable comments.
356 REFERENCES Bigley, R.E., 1981. The population biology of two intertidal seagrasses, Zosterajaponica and Ruppia maritima, at Roberts Bank, British Columbia. M.Sc. Thesis, University of British Columbia, Vancouver, 205 pp. Den Hartog, C., 1970. The sea-grasses of the world. Verh. K. Ned. Akad. Wet. Afd. Natuurk., Reeks 2, 59: 1--275. Elsey, C.R., 1933. Oysters in British Columbia. Biol. Board Can. Bull. 34, 34 pp. Feder, N. and O'Brien, T.P., 1968. Plant microtechnique: some principles and new methods. Am. J. Bot., 55: 112--142. Harrison, P.G., 1976. Zostera japonica Aschers. & Graebn. in British Columbia, Canada. Syesis, 9: 359--360. Hitchcock, C.L., Cronquist, A., Ownbey, M. and Thompson, J.W., 1969. Vascular Plants of the Pacific Northwest. Part 1. University of Washington Press, Seattle, 914 pp. McMillan, C., 1978. Morphogeographic variation under controlled conditions in five seagrasses, Thalassia testudinum, Halodule wrightii, Syringodium filiforme, Halophila engelmannii, and Zostera marina. Aquat. Bot., 4: 169--189. Phillips, R.C., 1960. Environmental effect on leaves of Diplanthera du Petit-Thouars. Bull. Mar. Sci. Gulf Carib., 10: 346--353. Phillips, R.C. and Shaw, R.F., 1976. Zostera noltii in Washington, U.S.A. Syesis, 9: 355--358. Sayce, C.S., 1976. The oyster industry of Willapa Bay. Proc. Symp. Terrestrial Aquat. Ecol. Studies of Northwest. E. Wash. State College Press, Cheney, WA, pp. 347--356. Scagel, R.F., 1956. Introduction of a Japanese alga, Sargassum muticum, into the northeast Pacific. Fish. Res. Papers, Washington Dept. Fisheries, Vol. 1 No. 4, 10 pp.