Re-evaluating species boundaries among members of the Posidonia ostenfeldii species complex (Posidoniaceae) – morphological and genetic variation

Aquatic Botany 66 (2000) 41–56

Re-evaluating species boundaries among members of the Posidonia ostenfeldii species complex (Posidoniaceae) – morphological and genetic variation M.L. Campey a,∗ , M. Waycott b , G.A. Kendrick a a

b

Department of Botany, The University of Western Australia, Nedlands, WA 6907, Australia Department of Tropical Environment Studies and Geography, James Cook University, Townsville, Qld 4811, Australia Received 28 July 1998; accepted 9 February 1999

Abstract A taxonomic re-examination of the Posidonia ostenfeldii complex was undertaken based on field collections in Western Australia. Broad and narrow leaves of seagrass of the Posidonia complex from Success Bank were compared to P. kirkmanii, P. coriacea, P. robertsoniae and P. denhartogii, identified based on their distribution as described by Kuo and Cambridge (1984). The broad and narrow leaf varieties of Posidonia on Success Bank were unable to be identified to species due to the overlap of character ranges between groups. Multivariate analysis of 10 vegetative characters examined indicated a continuous variation of character traits within the complex, suggesting the existence of a morphological continuum. Genetic variation was also examined using starch gel electrophoresis, restricted to Success Bank and Marmion populations. No allozyme polymorphism was observed among all the samples tested across both populations and varying leaf widths. All loci were monomorphic and homozygous. This study implies that the key of vegetative morphological characters, upon which five species of the P. ostenfeldii complex was erected, was not effective for the identification to species of multiple samples from any one location. Presently there is no justification for supporting Posidonia coriacea and Posidonia robertsoniae as separate species and it is further recommended that the whole group be reanalysed. ©2000 Elsevier Science B.V. All rights reserved. Keywords: Seagrass; Taxonomy; Posidonia ostenfeldii complex; Morphometric; Diversity

∗ Corresponding author. Tel.: +61-8-9380-1847; fax: +61-8-9380-1001 E-mail address: [email protected] (M.L. Campey)

0304-3770/00/$ – see front matter ©2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 3 7 7 0 ( 9 9 ) 0 0 0 1 5 - 7

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1. Introduction Seagrasses are clonal angiosperms which produce totally submerged flowers in the marine environment (McComb et al., 1981). Taxonomy of seagrasses, like that of other angiosperms, is based mainly on characters of their reproductive structures i.e. their inflorescences, flowers, fruit and seeds (den Hartog, 1977). However, due to infrequent or rare flowering events (e.g. Posidonia oceanica (L.) Delile; Thelin and Boudouresque, 1985), these structures were not often found, and in some species rarely collected (den Hartog, 1977). Although recent phenological studies of Australian Posidonia species indicate that flowers and fruiting are frequently observed (Kuo and Kirkman, 1996). Fortunately, from thorough morphological and anatomical studies of seagrasses, it is known that all genera and even most of the species are well characterised by their vegetative characters (den Hartog, 1977). However, phenotypic plasticity of vegetative characters resulting from age, genotype, nutrient status of the plant and environmental factors such as light intensity and temperature may be problematic (Sculthorpe, 1967). Posidonia is one of the dominant genera of seagrass occurring on the temperate Australian coastline and in the Mediterranean. A revision of the Australian members of the genus Posidonia (Cambridge and Kuo, 1979; Kuo and Cambridge, 1984) has defined six additional species in two species complexes mainly on the basis of vegetative and ecological characters. The P. australiscomplex contains three species (P. australis Hook.f., P. sinuosa Cambridge and Kuo, P. angustifolia Cambridge and Kuo) and the P. ostenfeldii complex is comprised of five species (P. ostenfeldii den Hartog, P. denhartogii Kuo and Cambridge, P. robertsoniae Kuo and Cambridge, P. coriacea Cambridge and Kuo, P. kirkmanii Kuo and Cambridge) (Kuo and Cambridge, 1984). The P. ostenfeldii complex typically form patchy meadows with mixed species in open ocean or rough water sublittoral habitats (Cambridge, 1975). They are characterised by their long, thick, leathery leaves and long leaf sheaths that are deeply buried. These characters appear to be associated with strong wave movement and mobile sand substratum typical of the environments in which they are found (Kuo and Cambridge, 1984). To investigate the P. ostenfeldii complex, Kuo and Cambridge (1984) examined 150 freshly collected or herbarium specimens, that represented the total collections made of the P. ostenfeldii complex along the Western and South Australian coastlines. They found the best characters to distinguish between the species were leaf morphology and anatomy. However, the range of many of the anatomical characters examined was not discrete but overlapped with other species within the complex (Table 1). Information on reproductive morphology and geographic distribution was also taken into account. Two morphological types of the Posidonia ostenfeldii complex, distinguished by differences in the width of the leaves (broad and narrow), were found to co-occur on Success Bank, Western Australia. We were not able to identify these morphological types to species at this location. Unfortunately both morphological types exhibited characters from three of the five species of P. ostenfeldii complex: P. denhartogii, P. robertsoniae and P. coriacea. This led us to re-evaluate the key characters used to identify species of the P. ostenfeldii complex. In addition, leaves of varying form from Success Bank and Marmion Lagoon were analysed using allozymes to determine if any genetic identity could be ascribed to the different types. The aims of this study were to assess the efficacy of the species iden-

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Table 1 Comparison of anatomical characters in species of the P. ostenfeldii complex (from Kuo and Cambridge, 1984) P. ostenfeldii

P. denhartogii

P. robertsoniae

P. coriacea

P. kirkmanii

Rhizome Shape in cross-section Diameter (mm) No. of cortical vascular bundles No. of cortical fibre bundles

oval 1–2 4 20–30

oval 2–3 4–6 20–30

rectangular 1.2–3 4–6 15–20

elliptical 2.5 × 4 6–8 >50

elliptical 2.5 × 6 8–10 40–60

Shoot No. of leaves per shoot

1–3

2–3

1–2

2–3

2–3

120–180 5

80–150 5

90–140 7

80–200 5–7

100–250 7–11

abundant

sparse

sparse

abundant

sparse

1–1.5 terete 3–5

1–2 biconvex 5–7

2–4.5 flatten 6–9

2.5–7 biconvex 7–11

6–12 biconvex 9–17

Epidermal cells (surface view) Outer cell wall Shape Cell length × as width

smooth rectangular 1.5–4×

smooth rectangular 1.5–2.5×

corrugated rectangular 1.5–4×

smooth oval 1–3×

smooth rectangular 1.5–3.5×

Epidermal cells (cross-section) Outer surface Height × as wide

smooth 1.5–2.5×

smooth 1.5–2.5×

scalloped 1.5–2×

smooth 3–5×

smooth 1.5–2.5×

Leaf sheath Length (mm) No. of longtitudinal vascular bundles Fibre bundles in parenchyma Leaf blade Width (mm) Shape in cross-section No. of longtitudinal vascular bundles

tifications that Kuo and Cambridge (1984) erected for the P. ostenfeldii complex and, if possible, identify the broad and narrow leaf varieties of the P. ostenfeldii complex common on Success Bank.

2. Materials and methods 2.1. Sampling design This study was based on field collections in southwestern Australia. Posidonia denhartogii occurs along the southwestern and southern coastline of Australia from Cottesloe near Perth (32◦ S, 115◦ 400 E) to Backstairs Passage (35◦ 400 S, 138◦ 100 E) in South Australia at depths of 1–10 m. P. robertsoniae is endemic to the southwestern coastline of Western Australia between Warnbro Sound (32◦ 20 S, 115◦ 430 E) and Israelite Bay (34◦ S, 123◦ E) in water 0.5–20 m deep. However, this may not be a true distributional range as Warnbro Sound specimens were collected from drift material. P. coriacea occurs 1–30 m deep between Shark Bay (25◦ S, 113◦ E) and Geographe Bay (33◦ S, 113◦ E) on the west coast of Australia and between Roe Plain (32◦ S, 128◦ E) and Backstairs Passage (35◦ 400 S, 138◦ 100 E) on the

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Fig. 1. Location of collection sites in Western Australia.

southeastern coast of Australia including Spencer and St. Vincent Gulf. P. kirkmanii is endemic to the southern Australian coast between Cape Leeuwin and Israelite Bay, Western Australia, occupying the south-western break in the distribution of P. coriacea (Kuo and Cambridge, 1984). Ten replicate shoots of these taxa were sampled from populations throughout the geographical range of species along southwestern Australia as well as from locations across Success Bank. Collections of samples of varying leaf morphology of the genus Posidonia were taken from Success Bank (32◦ 060 S 115◦ 420 E) located near Perth (Fig. 1) in April, 1996. Success Bank is a submerged sandbank comprised mostly of calcium carbonate material and covers an area of 3700 ha. It has a water depth ranging from 2 to 6 m and is exposed to oceanic swells, especially during winter storms. A hierarchical design was used where sampling was nested at regions, between meadows within regions and between replicates. Success Bank was divided into three regions (east, central, west) separated by shipping and dredging channels and were >2 km apart. Within each region, two meadows were chosen approximately 200 m apart. Random collections of 10 replicate broad leafed shoots (>4 mm wide) and 10 replicate narrow leafed shoots (<4 mm wide) were taken from each meadow using SCUBA. The 10 broad shoots collected from east site 2 were subsequently lost and therefore, not included in the analysis. Extra collections were made for genetic analysis at each site of each leaf width. Reference groups of seagrass belonging to the Posidonia ostenfeldii complex were collected from locations within their distributions described by Kuo and Cambridge (1984). Sites were selected to represent the geographic range of the species complex (Fig. 1). All samples were collected from a depth between 5 and 8 m. For P. coriacea, collections were taken from Marmion Lagoon, near Perth (31◦ 500 S 115◦ 450 E) and from Dunsborough

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Table 2 Characters included in the morphological analysis of Posidonia ostenfeldii species complex Shoot characters: 1. Number of leaves Leaf characters: 2. Width (mm) 3. Shape in cross section 1: concave/convex 2: ellipsoidal convex/convex 3: ellipsoidal straight/convex 4: rectangular 4. Number of vascular bundles 5. Shape of epidermal cell wall 1: smooth 2: scalloped 6. Ratio of epidermal cell height by cell width 7. Shape of epidermal cells in surface view 1: elliptical/spherical 2: rectangular Sheath characters: 8. Length (mm) 9. Number of vascular bundles 10. Fibre bundles 1: sparse 2: abundant

(33◦ 360 S 115◦ 06’E). For P. robertsoniae collections were taken from Two Peoples Bay near Albany (34◦ 570 S 118◦ 110 E) and from Wylie Bay near Esperance (33◦ 500 S 122◦ 000 E). For P. kirkmanii shoots were collected from Two Peoples Bay and from Starvation Boat Harbour near Hopetoun (33◦ 540 S 120◦ 340 E). Shoots of P. denhartogii were only collected from a single location, Two Peoples Bay near Albany. A sample of 10 shoots was randomly collected for each species from each location for the analysis. Collections from Marmion, Wylie Bay and Starvation Boat Harbour were made in April, 1996. Collections from Dunsborough and Two Peoples Bay were made in December, 1996. Duplicate material was collected for herbarium specimens (UWA) for future morphological and anatomical examinations. P. ostenfeldii was only collected once from Wylie Bay. This species is apparently rare and although there has been extensive diving along its geographical range, we were unable to collect material to include in this analysis. 2.2. Characters To identify the broad and narrow leafed seagrasses, 10 clearly identifiable vegetative characters were chosen from the 14 vegetative characters used by Kuo and Cambridge (1984) (Table 2). Vegetative characters were more useful in identification of species in the P. ostenfeldii complex because the flowers were usually damaged as they mature and differences between species in shape and size of mature fruits were not obvious in immature fruits or in distorted dried specimens. Therefore, the main characters used by Kuo and

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Cambridge (1984) to distinguish between species were leaf morphology and anatomy. For the anatomical study, transverse sections were taken from the mid-point of the leaf sheath and from 10 cm above the top of the leaf sheath for the leaf blade. A freezing microtome was used for all sectioning. The surface view of the leaf epidermis was observed after cells were cut from the leaf specimen with a razor blade. All sections were mounted in distilled water, stained with toluidine blue (pH 6) and then examined using a compound microscope. 2.3. Data analysis Patterns of variation in the 10 vegetative characters in the Posidonia ostenfeldii species complex were assessed using multidimensional scaling (MDS) using the PATN pattern analysis package developed by Belbin (1987). To calculate dissimilarity among species, data were first associated using the Gower metric association measure. This measure was considered most appropriate as morphometric data generally displays a continuous response (Belbin, 1987). Ordination was performed on the association matrix using hybrid multidimensional scaling in three dimensions. To clarify patterns, only two of the three dimensions have been presented and the data, although analysed together, have been plotted separately. Loadings of individual vegetative characters have been determined using Principle axis correlation (PCC) (Belbin, 1993). The direction of these loadings was used to determine the axis of influence and the correlation was used to estimate how important each character was in describing the observed pattern. The ordination pattern was considered to be significantly influenced by those characters with a correlation coefficient greater than 0.8. 2.4. Genetic analysis Allozyme electrophoresis was conducted on the meristematic tissue of wide and narrow forms of P. coriacea from two populations, Marmion Lagoon and Success Bank. These populations are separated by 32 km. A total of 24 shoots of different thickness were sampled from the Marmion population and 32 shoots of different thickness were sampled from the six sites across Success Bank. The clean meristematic tissue at the base of leaf shoots was collected from fresh material for morphometric analysis to determine if there were identifiable genetic differences between leaf forms. Approximately 200 mg of the meristematic leaf tissue was ground in a leaf grinding buffer (Waycott, 1995) and then frozen at −80◦ C until analysis. Starch gel electrophoresis was conducted according to Waycott and Sampson (1997) except that 3 loci gave no results, MDH-3 and 4, PER and PGM-2. 3. Results 3.1. Morphological variation The broad and narrow leaf varieties of Posidoniaon Success Bank were unable to be identified to species using the characters described by Kuo and Cambridge (1984). The large range within individual characters for both the broad leaved (Table 3) and the narrow

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Table 3 Character ranges for broad leafed samples from locations E1, C1, C2, W1, W2 on Success Banka Character

Group E1

Group C1

Group C2

Group W1

Group W2

1. Number of leaves/shoot 2. Leaf width (mm)

1–2 4.8–6.0 (5.3)b 2,4 9–11 1 2.5–3.5 2 100–160 (135.9) 9–15 1–2

1–2 4.1–5.3 (4.8) 4 9–11 1 2–3 2 74–118 (102.2) 11–13 1–2

1–2 3.9–4.9 (4.3) 1,2,4 7–9 1 2–4 1,2 48–116 (83.9) 8–11 1–2

1–2 3.4–4.7 (4.0) 1,2,4 7–9 1 2–4 1,2 42–129 (98.2) 7–11 1–2

1–3 4.0–5.0 (4.3) 1,2,4 7–11 1 2–4 2 62–151 (103.8) 9–13 1–2

3. Leaf shape in cross-section 4. No. leaf vascular bundles 5. Shape of epidermal cell wall 6. Epidermal cell height × width 7. Shape of epidermal cells 8. Sheath length (mm) 9. No. sheath vascular bundles 10. Fibre bundles a E1:

east site 1; C1: central site 1; C2: central site 2; W1: west site 1; W2: west site 2. in brackets.

b Means

leaved (Table 4) samples at each location resulted in samples not being able to be defined as only one possible species. For example, the character ranges of the east site 1 (E1) group of broad leaved samples (Table 3) overlapped with those of the reference species P. coriacea, P. kirkmanii and P. robertsoniae, with leaf width the only character excluding the E1 group from the P. denhartogii reference group (Table 5). Some characters, such as fibre bundles in sheath parenchyma, which were definitive for species in the Kuo and Cambridge (1984) study, were found to exhibit all traits within each group for both broad and narrow leaved species (Tables 3 and 4). The leaf and sheath characters examined did not separate the reference species into distinct groups (Table 5). There was much overlap of character ranges between species. Character traits such as fibre bundles, shape of epidermal cells and shape of leaf in cross-section were found to encompass the entire range of the P. ostenfeldii complex within each reference Table 4 Character ranges for narrow leafed samples from locations E1, E2, C1, C2, W1, W2 on Success Banka Character

Group E1

Group E2

Group C1

Group C2

Group W1

Group W2

1. Number of leaves/shoot 2. Leaf width (mm)

1–2 2.3–4.0 (3.0)b 1,2,4 7–9 1 2.5–3.8 1 30–117 (92.2) 9–11 1–2

1–2 2.7–4.2 (3.7) 1,2,4 7–9 1 3–4 1–2 40–112 (78) 9–11 1–2

1–2 2.1–2.9 (2.4) 2,4 7 1 3–4 1 50–79 (61.9) 7–11 1–2

1–2 2.2–3.4 (2.7) 1,2,4 7–9 1 2–4 1–2 72–111 (90.2) 7–11 1

1–2 1.9–2.7 (2.2) 1–4 5–9 1 3–4 1–2 47–89 (66.8) 5–13 1–2

1–2 2.7–3.4 (3.0) 2,4 7–9 1–2 2–4.5 1 69–95 (79.4) 11 1–2

3. Leaf shape in cross-section 4. No. leaf vascular bundles 5. Shape of epidermal cell wall 6. Epidermal cell height × width 7. Shape of epidermal cells 8. Sheath length (mm) 9. No. sheath vascular bundles 10. Fibre bundles a E1:

east site 1; E2: east site 2; C1: central site 1; C2: central site 2; W1: west site 1; W2: west site 2. in brackets.

b Means

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Table 5 Character ranges for P. denhartogii, P. robertsoniae, P. coriacea, P. kirkmanii collected for this study Character

P. denhartogii

P. robertsoniae

P. coriacea

P. kirkmanii

1. Number of leaves/shoot 2. Leaf width (mm)

2–3 1.6–1.9 (1.8)a 2,4 5–7 1–2 2–3 2 71–120 (94.5) 9–11 1–2

1–2 2.7–4.5 (3.6) 2,4 7–9 1–2 1.5–4 1,2 92–177 (133.5) 5–13 1–2

2 3.5–6.4 (4.9) 2,4 9–11 1–2 2–3 2 34–183.5 (110.0) 11–15 1–2

1–2 5.5–7.5 (6.7) 1,2,4 11–13 1 2–4 2 102–216 (149.8) 11–16 1–2

3. Leaf shape in cross–section 4. No. leaf vascular bundles 5. Shape of epidermal cell wall 6. Epidermal cell height × width 7. Shape of epidermal cells 8. Sheath length (mm) 9. No. sheath vascular bundles 10. Fibre bundles a Means

in brackets.

species group. This is not consistent with Kuo and Cambridge’s (Kuo and Cambridge, 1984) study (Table 1), despite the seagrass being collected from sites chosen from their paper and those they recommended to us. Some plants were found to have character traits, such as spherical and rectangular shaped epidermal cells, identified with separate species as defined by Kuo and Cambridge (1984), on the same shoot. 3.2. Ordination The MDS ordination of reference species of seagrass indicates that they do not form distinct groups but rather a morphological continuum with narrow leaved Posidonia denhartogiiat one end, broad leaved Posidonia kirkmaniiat the other, and species with intermediate leaf widths occurring in between (Fig. 2(a)). There was much overlap in P. kirkmanii and P. coriaceain both populations. There was also overlap with P. coriaceafrom both locations and P. robertsoniae from Wylie Bay but not Two Peoples Bay. The two populations of P. robertsoniae formed separate groups. The furthest separation of species was between P. kirkmanii and P. denhartogii (Fig. 2(a)). Although the narrow leaved species of Posidonia collected from Success Bank were grouped mainly with the narrow leaved reference species P. denhartogii and P. robertsoniae (Fig. 2(b)) and the broad leaved species were mainly found at the broad leaved end of the continuum with P. kirkmanii and P. coriacea (Fig. 2(c)), there was much overlap between the broad and narrow leaved species from Success Bank. There was also no distinct pattern between sites on Success bank for both the broad and narrow leaf varieties of Posidonia. 3.3. Principal axis correlation (PCC) Four characters were shown to significantly influence the ordination pattern (Table 6.). These were leaf shape (R = 0.943), number of fibre bundles within sheath parenchyma (R = 0.901), leaf width (R = 0.928) and number of leaf vascular bundles (R = 0.877). Both leaf width and number of leaf vascular bundles had a direction of influence similar to

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Fig. 2. Ordination using hybrid MDS showing relationship between (a) reference species and (b) broad and (a) narrow leaved samples from Success Bank. 2PB: Two Peoples Bay; WB: Wylie Bay; DUN: Dunsborough; MARM: Marmion; SBH: Starvation Boat Harbour. Stress = 0.145.

the main axis of variation indicating that these characters are important in differentiating between the reference groups. P. kirkmanii had a wide leaf (Fig. 3(a)) with a large number of leaf vascular bundles (Fig. 4(a)). P. coriaceahad a medium to wide leaf (Fig. 3(a)) with a medium to large number of leaf vascular bundles (Fig. 4(a)). P. robertsoniaehad a narrower leaf (Fig. 3(a)) with less vascular bundles (Fig. 4(a)). P. denhartogii had the narrowest leaf (Fig. 3(a)) with the least number of vascular bundles (Fig. 4(a)). All species had both ellipsoidal convex/convex and ellipsoidal straight/convex shaped leaves with P. coriacea and P. kirkmanii having concave/convex shaped leaves also (Fig. 5(a)). Both abundant and sparse sheath fibre bundles were found in all species (Fig. 6(a)). The range of leaf widths (Fig. 3(b)and number of leaf vascular bundles (Fig. 4(b)) of broad leaf Posidonia from Success Bank was found to include those of P. kirkmanii, P. coriacea

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Table 6 Principal axis correlations (PCC) between individual characters and the ordination space based on 180 samples from the Posidonia ostenfeldii complex Character

Loadings

1. Number of leaves per shoot 2. Leaf width 3. Leaf shape 4. Number of leaf vascular bundles 5. Shape of epidermal cell wall 6. Ratio of epidermal cell height by width 7. Shape of epidermal cells in surface view 8. Sheath length 9. Number of sheath vascular bundles 10. Number of sheath fibre bundles

Correlation coefficient (R)

Axis 1

Axis 2

+ − − − + + − − − −

+ − + − + − + − + −

0.329 0.928 0.901 0.877 0.571 0.494 0.518 0.597 0.782 0.943

and P. robertsoniae from Two Peoples Bay. Narrow leaf Posidonia from Success bank was found to have leaf widths (Fig. 3(c)) and number of vascular bundles (Fig. 4(c)) similar to those of P. denhartogii, P. robertsoniae and P. coriacea. Both the broad and narrow leaf Posidonia from Success Bank had abundant and sparse sheath fibre bundles (Fig. 6(b) and (c)). There was as much variation in leaf shape for both broad and narrow leaf Posidonia species at one site on Success bank as there was between sites, with narrow leaf samples from west site 1 having all possible leaf shapes (Fig. 5(c)).

3.4. Genetic variation Starch gel electrophoresis resolved 12 loci on two buffer systems with P. coriacea leaf material (Table 7). No allozyme polymorphism was observed among all the samples tested across populations from Success Bank and Marmion Lagoon and leaf widths from broad to narrow within and between populations. All loci were monomorphic and homozygous at all loci. Table 7 Enzyme systems examined and the number of loci detected in P. coriacea from Success Bank and Marmion Lagoon. Electrophoretic conditions were as per Waycott and Sampson (1997) Enzyme

Abbreviation

E.C. code

Buffer system

Number of loci scored

Glucose phosphate isomerase Isocitrate dehydrogenase Malate dehydrogenase Phosphoglucomutase Phosphogluconate dehydrogenase Shikimic acid dehydrogenase Malic enzyme Fluorescent esterase

GPI IDH MDH PGM PGD SDH MEN EST

5.3.1.9 1.1.1.42 1.1.1.37 5.4.2.2 1.1.1.44 1.1.1.25 1.1.1.40 3.1.1.–

SAC/TC SAC/TC MC and SAC/TC SAC/TC MC MC MC SAC/TC

2 1 2 2 2 1 1 1

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Fig. 3. Median leaf width showing 25–75% interquartile range (box) and 95% range (wiskers) of (a) reference species and (b) broad and (c) narrow leaved samples from Success Bank. DEN: P. denhartogii; ROB: P. robertsoniae; CORI: P. coriacea; KIRK: P. kirkmanii; 2PB: Two Peoples Bay; WB: Wylie Bay; DUN: Dunsborough; MARM: Marmion; SBH: Starvation Boat Harbour.

4. Discussion The analysis of variation in morphology in species within the P. ostenfeldii complex indicates a continuous variation of character traits within the taxa, suggesting the existence of a morphological continuum. The species were previously described as broad geographical types but upon detailed inspection at a single location, we found at least three species descriptions fitting two morphological types. The patterns of morphological character overlap indicate a population-based diversity among these currently described taxa i.e. species have a wide range between population variability. Creed’s (Creed, 1997) study of morphological

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Fig. 4. Mean number of leaf vascular bundles (+/− SD) of (a) reference species and (b) broad and (c) narrow leaved samples from Success Bank. DEN: P. denhartogii; ROB: P. robertsoniae; CORI: P. coriacea; KIRK: P. kirkmanii; 2PB: Two Peoples Bay; WB: Wylie Bay; DUN: Dunsborough; MARM: Marmion; SBH: Starvation Boat Harbour.

variation in Halodule wrightii Aschers. from nine isolated populations in Rio de Janeiro found that each population featured at least one morphological attribute which differed from others. Similar to this study, he found that one population was characterised by two morphs of the species, with one subset of plants having thicker longer rhizomes, longer leaves and sheaths and the other having thin, shorter rhizomes, very short leaf sheaths and narrow leaves. Morphological variation within many seagrasses is apparently the result of an adaptive response to the environment. Creed (1997) suggests that the dimorphism within one population of H. wrightii may have been a result of the two discrete levels in the sediment at which the plants grew. Phillips (1980) found when sediments are highly mobile or input

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Fig. 5. Leaf shape frequency of (a) reference species and (b) broad and (c) narrow leaved samples from Success Bank. DEN: P. denhartogii; ROB: P. robertsoniae; CORI: P. coriacea; KIRK: P. kirkmanii; 2PB: Two Peoples Bay; WB: Wylie Bay; DUN: Dunsborough; MARM: Marmion; SBH: Starvation Boat Harbour.

is constant H. wrightii responds with new rhizome growth upwards resulting in a longer leaf sheath. Light intensity has also been shown to affect the morphology of many seagrass species with higher intensities producing narrower, thicker leaves with greater leaf lacunal area (Abal et al., 1994; Grice et al., 1996). Seagrass has also been found to have reduced leaf length and width in hyper-saline conditions (McMillan and Moseley, 1967). The P. ostenfeldii complex samples examined in this study were collected over a wide range of habitat types and over large distances. The difference in their morphologies may be an adaptive response to different environments or simply due to their age and genotype. The lack of allozyme polymorphism in this study was unexpected given the variety of leaf form assayed and the comparison of material from two, well separated localities,

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Fig. 6. Frequency of sparse and abundant sheath fibre bundles from (a) reference species and (b) broad and (c) narrow leaved samples from Success Bank. DEN: P. denhartogii; ROB: P. robertsoniae; CORI: P. coriacea; KIRK: P. kirkmanii; 2PB: Two Peoples Bay; WB; Wylie Bay; DUN: Dunsborough; MARM: Marmion; SBH: Starvation Boat Harbour.

Success Bank and Marmion Lagoon. In other species of Posidonia, observable allozyme polymorphism is readily detected (Waycott, 1995; Waycott and Sampson, 1997; Waycott et al., 1997). Given that the leaf forms described in this paper are putative species, the lack of allozyme polymorphism is a further support that the flat leaved varieties of the P. ostenfeldii complex do not represent four species. Further genetic studies are presently underway for all species across their entire geographic range. It should be noted that all loci were homozygous and that this may have implications for the ability of this study to detect variability. However, previous analysis has identified allozyme polymorphism among seedlings of P. coriacea from Marmion Lagoon (Waycott and Sampson, unpublished data). Wider sampling with this technique may reveal population genetic variability but no species

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level differentiation was shown in this study as would be expected with this class of markers. This study implies that the key of vegetative morphological characters, upon which five species of the P. ostenfeldii complex were erected, is not effective for the identification of species from multiple samples from any one location. Further research is necessary to clarify species. More genetic work is required to incorporate all groups within the complex. Characters, specifically flowers, need to be examined in more detail. Although insufficient material of P. ostenfeldii was collected to include in our study, thus making our analysis incomplete for the whole group, we suggest that P. robertsoniae is synonymous with P. coriacea and P. coriacea has priority as a species name. Based on the data presented in this paper, there is no justification for supporting P. coriacea and P. robertsoniae as separate species. We also recommend the whole complex be reanalysed and that further morphological and genetic work is carried out on the P. ostenfeldii complex.

Acknowledgements We wish to thank John Kuo, Marion Cambridge and Di Walker for their helpful comments and advice on this study and third year Botany students at the University of Western Australia for their assistance in processing the samples.

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