In situ morphometric study of the Diuris punctata ... - CSIRO Publishing

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Australian Systematic Botany, 21, 289
300

In situ morphometric study of the Diuris punctata species complex (Orchidaceae), with implications for conservation Zoë F. Smith A,B,D, Elizabeth A. James B and Cassandra B. McLean C A

Australian Research Centre for Urban Ecology, School of Botany, The University of Melbourne, Vic. 3010, Australia. B National Herbarium of Victoria, Royal Botanic Gardens Melbourne, Birdwood Avenue, South Yarra, Vic. 3141, Australia. C School of Resource Management, Faculty of Land and Food Resources, Burnley College, The University of Melbourne, 500 Yarra Boulevard, Richmond, Vic. 3121, Australia. D Corresponding author. Email: [email protected]

Abstract. Taxa within the Diuris punctata species complex exhibit high levels of variation at both species and population level. Morphometric data collected in situ were used to investigate species boundaries of four Victorian Diuris species within the Diuris punctata species complex. Morphological characters and taxonomic groups identified in the present study were compared to those described under the current taxonomic treatment. Sixty-five multistate and continuous characters, including seven vegetative and 58 floral characters, were measured in situ across the range of each species within Victoria. The importance of flower colour in distinguishing taxa was highlighted but characters used were generally indiscrete. Certain characters used in current taxonomic descriptions, e.g. floral fragrance, were found to be uninformative. D. fragrantissima was confirmed as a separate taxon within the D. punctata group, justifying its recognition as a unique entity for conservation. Clustering of D. daltonii within D. punctata suggests that the recent elevation of the D. punctata var. daltonii to species level is not justified. The in situ measurement of morphological characters made it possible to incorporate sufficient sampling to encompass intra-specific and intra-population variation and is a feasible method to overcome sampling limitations encountered when herbarium specimens and limited destructive sampling are used.

Introduction Morphological characters are essential tools for identification of orchid taxa in the field and are still relied on in taxonomic studies, although their value is dependent on capturing the range of variation present. Herbarium specimens and dried plant material are often the only types of material examined in studies of orchids (e.g. Kores et al. 1993; Shipunov and Bateman 2005; Shipunov et al. 2005) because the protected status of orchids limits destructive sampling and even the exchange of material (Roberts and Solow 2008). This may result in the exclusion of informative characters, such as flower colour, orientation and phenology. Instead, in situ morphometric studies of species that are threatened or of limited geographic distribution, allows capture of maximum morphological variation by limiting character loss in preserved samples and may enable a larger specimen pool because sampling is non-destructive. Although the genus Diuris Sm. is easily recognised by its distinct floral morphology (Backhouse and Jeanes 1995), extensive morphological variation among and within the species and populations has caused difficulty in defining taxa at species level and below (Bishop 2000; Clements 2001; Kores
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22 October 2008

et al. 2001). The number of recognised species has varied considerably since the circumscription of the genus in 1798 (Kores et al. 2000; Clements 2001), and new species continue to be described (Jones and Clements 2004). Currently, 56 Diuris species are recognised in seven morphological groups. The present study focuses on the following four Victorian members of the D. punctata species complex: D. punctata Sm., D. dendrobioides Fitzg., D. daltonii (C.Walter) D.L. Jones & M.A.Clem. and D. fragrantissima D.L.Jones & M.A. Clem. (Clements 2001; Jones and Clements 2004). The D. punctata species complex is distributed throughout eastern Australia and includes D. alba, D. arenaria, D. oporina, D. parvipetala and D. tricolor outside Victoria (Fig. 1; Clements 2001). Difficulties in identifying morphological species and population boundaries in orchids have been discussed repeatedly in the taxonomic literature (e.g. Andersson 1994; Backhouse and Jeanes 1995; Bernardos et al. 2005; Bytebier et al. 2007); however, morphological characters have been helpful in distinguishing difficult orchid taxa (Bateman and Hollingsworth 2004; Clements et al. 2007). The extent of taxonomic uncertainty owing to morphological diversity 10.1071/SB08014

1030-1887/08/040289

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QLD

NSW

N

VIC 400 km

Victoria 10 9 3 7 8

4

6 5

11

1 2

150 km Fig. 1. Distribution of species of Diuris punctata group in Eastern Australia and Victoria: D. punctata (&), D. daltonii ( ), D. dendrobioides ( ), D. fragrantissima: in situ (^), ex situ (¤) D. tricolor ( ), D. alba ( ), D. arenaria ( ), D. parvipetala ( ), D. oporina ( ). Sampled populations are shaded black and numbered according to Table 1.

within D. punctata itself was highlighted by Dockrill (1964), who stated that some populations are far more variable in flower size and colour than others. He suggested that Walter (1907) may have unnecessarily described D. punctata var. daltonii C.Walter as a distinct variety and felt it may be just a small, isolated,

aberrant D. punctata population that did not warrant a new taxonomic rank. This variety was recently elevated to a species (Jones and Clements 2004), although a recent study by Smith et al. (2007a) on the basis of AFLP data did not support the recognition of D. daltonii at the rank of species.

In situ morphometric study of Diuris punctata

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The variable morphology found in the D. punctata species complex also has implications for the conservation of the State and Federally listed Critically Endangered species D. fragrantissima, whose taxonomic rank and therefore conservation status, has been questioned, raising the possibility that allocated management resources do not reflect the true conservation priority of this species. The species was locally abundant in the grassland plains west of Melbourne, Victoria, but largely as a result of habitat destruction, it is now reduced to a few individuals (23 were recorded in 2006) at a single remnant population. Extensive time and monetary resources have been allocated to D. fragrantissima for in situ management, ex situ cultivation and reintroduction efforts (Smith 2006). The objectives of the present study were to (1) utilise an in situ morphometric approach to investigate species boundaries in the Victorian D. punctata group, (2) evaluate the usefulness of morphological data collected in situ in the delimitation of those species, and (3) assess the current taxonomy of the State and Federally listed Critically Endangered D. fragrantissima and the recently described D. daltonii. To achieve these objectives the research focused on the following questions: (1) what are the phenetic relationships between D. punctata, D. daltonii, D. dendrobioides and D. fragrantissima, (2) how informative are morphometric data in defining species boundaries within the D. punctata species complex, and (3) how does floral morphology within individual plants influence phenetic relationships? Methods Sampling strategy Populations of Diuris punctata, D. dendrobioides, D. daltonii and D. fragrantissima were sampled from October to December 2005 across their geographic range in Victoria. In all, 65 morphological characters were measured from 247 plants representing 11 in situ (Diuris punctata [5], D. dendrobioides [2], D. daltonii [3] and D. fragrantissima [1]) and 2 ex situ (D. fragrantissima)

populations (Table 1, Fig. 1). Eight of the sampled populations were identical to those in an earlier molecular study conducted by Smith et al. (2007a). At each site, an informal survey was conducted of the number and geographic distribution of the flowering plants. A centre point was estimated and a transect was established through the population. At each 1-m interval the nearest plant to the transect line was selected for measurement. At least 20 (and up to 30) plants were measured in each population except D. punctata (located at Mornington and Mt Eliza), D. daltonii (located at McCutcheon’s Road) and D. fragrantissima (located at Tottenham and the Royal Botanic Gardens Melbourne) because there were not enough plants in these populations (see Table 1). For floral morphology, only open basal flowers were measured in all populations in an attempt to eliminate ontogenetic variation. In a subset of plants, we investigated the levels of within-plant variation, and potential differences because of flower age. All flowers on six D. punctata (located at Inverleigh) and six D. daltonii (located at Victoria Point Road) individuals were measured. Seven vegetative and 58 floral multistate and continuous characters (Table 2) based on previous descriptions and keys (Backhouse and Jeanes 1995; Bishop 2000) were measured on each plant. Digital photographs of measured flowers were calibrated and used to re-score binary characters. Leaf length was not measured because of damage from predation and natural dieback. Characters 60
65 showed no variation and were excluded from analysis. Voucher specimens are lodged at MEL and MELU (Table 1). We did not collect voucher specimens from five populations because they were considered too small to justify collection. Digital images of flowering plants at these populations have been deposited at the National Herbarium of Victoria library and submitted to Morphbank (http://www.morphbank.net). Phenetic analysis Morphometric data were analysed phenetically by using the PATN pattern analysis program (Belbin 2005). All characters

Table 1. Diuris species, site location in Victoria and sample number Species

Site no.

Site

Grid reference

D. punctata

1 2 3 4 5 6 7 8 9 10 11 12 13

Mt Eliza Mornington Glenrowan Bonegilla Inverleigh Victoria Point Road McGiverns Road McCutcheons Road Bonegilla Boorhaman Tottenham (in situ) RBG (ex situ) Zoo (ex situ)

38110 S, 145050 E 38130 S, 145020 E 36270 S, 146130 E 36080 S, 147000 E 38060 S, 144030 E 37320 S, 142190 E 37320 S, 142190 E 37310 S, 142100 E 36080 S, 147000 E 36120 S, 146170 E 37480 S, 144510 E 37470 S, 144590 E 37500 S, 144570 E

D. daltonii

D. dendrobiodes D. fragrantissima

A

291

Specimens are lodged at MEL and MELU. B Populations were considered too small for voucher specimen collection.

No. of plants sampled 16 8 30 20 30 20 20 10 25 25 10 8 25

VoucherA B B

2311812A 2311833A 2311808A 2311811A 2311810A 2311809A B

2311821A B B

2311827A

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Table 2. All characters measured for morphometric comparison Characters 60
65 showed no variation and were excluded from analysis No.

Morphological character

No.

Morphological character

1 2

Angle between centres of petal and dorsal sepal Area between callus ridges white/cream/ golden yellow (0/1/2) Bract colour green/green with purple tinge/purple (0/1/2) Callus length Callus width at widest point Callus with/without purple markings (0/1) Distance between dorsal sepal and labellum (midlobe) apices Distance between dorsal sepal apex and base at back (dorsal sepal curvature) Distance between labellum mid-lobe and lateral lobe at widest points Distance between petal apex and base at back (petal curvature) Distance between petal apices Dorsal sepal area of colour (%) Dorsal sepal colour diffuse/blotchy (0/1) Dorsal sepal colour intensity white
purple (scale 0
5) Dorsal sepal edges smooth/wavy/crinkled (0/1/2) Dorsal sepal length Dorsal sepal width at widest point Dorsal sepal width two mm from apex Flower and bud number (combined) Flower diameter Labellum lateral lobe edge square/rounded (0/1) Labellum lateral lobe lenth Labellum lateral lobe width Labellum mid-lobe area of colour (%) Labellum mid-lobe colour diffuse/blotchy (0/1) Labellum midlobe colour intensity white
purple (scale 0
5) Labellum midlobe curvature (scale 0
5) Labellum midlobe distance between ridge apex and highest point Labellum midlobe edges smooth/wavy/crinkled (0/1/2) Labellum midlobe length Labellum midlobe ridge height Labellum midlobe width Lateral sepal colour green
purple (scale 0
5)

34

53 54 55 56 57 58 59

Lateral sepal curled tightly longitudinally/ open slightly/very open (0/1/2) Lateral sepal length Lateral sepal width at widest point Leaf width Ovary colour green
purple (scale 0
5) Ovary length Ovary width Pedicel length Petal apex acute/obtuse (0/1) Petal claw colour green
purple (scale 0
5) Petal claw edge straight/with kinks on one side/ kinks on both sides (0/1/2) Petal claw gradually expanding into lamina/ distinct from lamina (0/1) Petal claw length Petal claw width Petal colour area (%) Petal colour diffuse/blotchy (0/1) Petal colour white
purple (scale 0
5) Petal edge smooth/wavy/crinkled (0/1/2) Petal edge straight/with kinks on one side/ kinks on both sides (0/1/2) Petal length Petal width Petal width two mm from apex Petals flat/ridge in centre (0/1) Plant height Stigma colour white/pink spots/pink (0/1/2) Stigma width

No.

Uniform characters excluded from analysis

60 61

Fragrant/not (0/1) Labellum lateral lobe colour white/purple stripes/purple (0/1/2) Leaf lamina flat/bent/curved (0/1/2) Leaf number Length of stem with purple coloration Stem colour green/purple (0/1)

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

were weighted equally and considered independent. A dissimilarity matrix was produced by the Gower metric, which is the range-standardised form of the Manhattan metric and can be used to simultaneously analyse quantitative (continuous) and qualitative (binary) data (Sneath and Sokal 1973). The dissimilarity matrix was then used to produce a dendrogram by the unweighted pair group method of averaging (UPGMA) clustering algorithm, with samples in each cluster weighted equally (Belbin 2005). Ordination was produced in three dimensions by the semi-strong-hybrid multidimensional scaling method (SSH
MDS) with 50 iterations and 20 random starts. The best solution from several iterations was determined by the lowest stress value. The first analysis included all variable morphological characters measured for all individuals. Separate analyses were

35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

62 63 64 65

then conducted to further investigate relationships between species pairs (Diuris dendrobioides and D. fragrantissima; D. punctata and D. daltonii), and their constituent populations. These analyses were conducted to investigate the taxonomic status of D. fragrantissima and D. daltonii. Within-plant variation was investigated in a fourth analysis including variable floral characters measured on all flowers of 12 plants (six D. punctata (Inverleigh) and six D. daltonii (Victoria Point Road) individuals). Only ordinations are presented from these analyses. Analyses described above were repeated with the exclusion of characters found to have non-significant Monte-Carlo attributes in ordination (MCAO; see below). The first analysis of all individuals and characters was also repeated with the removal of floral colour characters (3, 12, 13, 14, 24, 25, 26,

In situ morphometric study of Diuris punctata

33, 38, 43, 48, 49, 50 and 61), to investigate the influence of floral colour in distinguishing groups. Character evaluation The relative contribution of characters to defining groups in the ordination space was evaluated with the non-parametric Kruskal
Wallis test. Higher Kruskal
Wallis values indicate an increasing contribution of characters to group differentiation. The amount of variation explained by each character was investigated by principal component correlation (PCC), which uses multiple linear regression to display vectors corresponding to the correlation of variables with ordination axes and the direction of change of variables among individuals. Values of r2, ranging from 0 to 1, increase with the amount of variation explained by a character. The robustness of the PCC was tested by MCAO with 1000 iterations. MCAO involves the random assignment of character values to objects in the ordination space and re-runs the PCC to determine the number of iterations for which r2 exceeds the actual r2 for the character. The statistical package SPSS 14.0 for Windows (SPSS Inc. 2005) was used to obtain descriptive statistics for assessment of character variation among species and populations. Analysis of variance was conducted among species to determine significant differences in the mean values of characters that were found to be informative in separating groups in all analyses. Statistical significance is recorded at P = 0.05 for all analyses.

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floral colour characters from the dataset resulted in the loss of discrete groups in the ordination (results not shown), indicating the importance of flower colour in the above ordination. The removal of non-significant MCAO characters did not alter the overall patterns in the ordination or reduce the stress value. Floral colour characters of the four species are summarised in Table 3. On the basis of floral colour, D. fragrantissima and D. dendrobioides were largely distinguished from D. punctata and D. dendrobioides by the blotchy, rather than diffuse, pattern of colour, cream or white callus rather than yellow and paler lateral sepals, bracts and ovaries. Dorsal sepal, labellum midlobe and petal colours were generally lighter mauve in D. fragrantissima and darker purple in other species. No distinct differences in floral coloration were observed between D. punctata and D. daltonii. Descriptive statistics for informative, continuous size variables are given in Table 3. Although characters were largely overlapping, significant differences were observed between several character means for each species. These results showed that D. fragrantissima is significantly shorter, with wider leaves and ovaries and more flowers than all other species. D. dendrobioides has significantly longer ovaries, more tapered petals and dorsal sepals, and D. daltonii has significantly fewer flowers than all other species and is largely intermediate between D. punctata and D. dendrobioides in all other characters. D. punctata has a significantly wider labellum than all other species and is otherwise highly variable among individuals (data not shown).

Results Phenetic relationships among Diuris punctata, D. daltonii, D. dendrobioides and D. fragrantissima

Defining species boundaries with morphometric data

Two major groups were clearly evident at the 40% dissimilarity level (Fig. 2) in a UPGMA dendrogram produced from the morphometric dataset. Group 1 contained all D. punctata and D. daltonii individuals, and Group 2 contained all D. fragrantissima and D. dendrobioides individuals. D. daltonii individuals grouped within D. punctata, forming only one subcluster with some individuals of D. punctata and indicating that the species is unlikely to warrant recognition at this level. D. fragrantissima and D. dendrobioides formed two distinct clusters within Group 2. In situ D. fragrantissima individuals clustered closely but did not form a group separate from ex situ individuals. The same major clusters were evident in the ordination (Fig. 3), with two discrete groups forming. One contained all D. punctata and D. daltonii individuals and the other all D. fragrantissima and D. dendrobioides. D. fragrantissima and D. dendrobioides clustered closely with slight overlap. D. punctata and D. daltonii overlapped, although there was some segregation in the third dimension. Characters found to be most informative in defining groups, i.e. those with the highest Kruskal
Wallis values, are displayed as vectors in the ordination space. Characters most informative in separating groups were 3 (bract colour, KW = 159.47, r2 = 0.639), 12 (dorsal sepal colour area, KW = 162.81, r2 = 0.694), 24 (labellum mid-lobe colour area, KW = 164.06, r2 = 0.619), 49 (petal colour diffuse/blotchy, KW = 153.16, r2 = 0.635) and 54 (petal width, KW = 162.71, r2 = 0.619). The removal of

The three-dimensional ordination of D. fragrantissima and D. dendrobioides individuals is shown in Fig. 4. The two species clustered separately, but with no clear disjunction between groups. In situ D. fragrantissima individuals generally clustered closer to D. dendrobioides individuals. The two populations of D. dendrobioides were indistinguishable in the ordination. The most-informative characters separating the two species were 14 (dorsal sepal colour 0
5, KW = 59.24, r2 = 0.660), 18 (dorsal sepal width two mm from apex, KW = 56.86, r2 = 0.678), 26 (labellum midlobe colour 0
5, KW = 59.94, r2 = 0.710), 55 (petal width 2 mm from apex, KW = 66.18, r2 = 0.749) and 57 (plant height, KW = 63.31, r2 = 0.649). Vectors depicting directions of change indicated that D. fragrantissima has more rounded petal and dorsal sepal apices, shorter height and paler flower colour, particularly in ex situ individuals. The three-dimensional ordination of D. punctata and D. daltonii individuals is shown in Fig. 5. The two species overlapped in the ordination space, confirming the lack of distinction between them. No clear population subgroups were evident; however, individuals appeared to group more closely on the basis of geographic proximity. Vectors indicated greater labellum and petal size and more rounded petal apices in D. punctata, particularly in northern populations (character 30: KW = 98.45, r2 = 0.669; 32: KW = 82.96, r2 = 0.444; 42: KW = 75.97, r2 = 0.567; 53: KW = 88.67, r2 = 0.683 and 54: KW = 85.59, r2 = 0.455).

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1

Diuris punctata and D. daltonii

2a D. dendrobioides

2

2b D. fragrantissima (in situ and ex situ

(

0.75

0.57

0.40 0.22 Dissimilarity

0.05

Fig. 2. UPGMA dendrogram of species of Diuris punctata group in Victoria. Group 1 comprises all D. punctata and D. daltonii individuals and Group 2 comprises all D. dendrobioides (Subgroup 2a) and D. fragrantissima (Subgroup 2b) individuals. Placement of D. daltonii (solid lines) within D. punctata and D. fragrantissima in situ (dotted lines) among ex situ individuals are shown. Not all populations are identified because of lack of observed clustering.

The high stress value (0.2089), however, indicated variability of the data and lack of true groups. Influence of within-plant floral variation on phenetic relationships among species Diuris punctata (Inverleigh) and D. daltonii (Victoria Point Road) clustered separately in the three-dimensional ordination

of floral-character measurements (Fig. 6). Flowers of individual plants were not identical (did not cluster together); therefore, variation resultant of flowering stage should not affect overall results. The most informative characters (i.e. highest Kruskal
Wallis values) in separating flowers on the same plant were 10 (petal curvature, KW = 23.94), 11 (distance between petal apices, KW = 23.96), 20 (flower diameter, KW = 23.93) and 46 (petal claw length, KW = 23.92).

In situ morphometric study of Diuris punctata

1.197

Discussion

3

Phenetic relationships among Diuris punctata, D. daltonii, D. dendrobioides and D. fragrantissima

24 12

–1.406 1.323

Dimension 1

–1.292

Dimension 3

1.124

54

3

–1.496

49

–1.292

295

(Table 3). In both populations, the majority of individuals had no more than two flowers, so frequency distributions of withinplant floral characters are not shown.

49

54

Dimension 2

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24 12

Dimension 1

1.323

Fig. 3. Three-dimensional SSH (MDS) ordination of species of Diuris punctata group in Victoria. Key: D. punctata (&), D. daltonii ( ), D. dendrobioides ( ), D. fragrantissima: in situ (˛) and ex situ (¤). Individual populations are not shown because of the lack of observed clustering. Vectors depict direction of change in characters with highest Kruskal
Wallis values: 37 (labellum midlobe colour area, KW = 164.06, r2 = 0.619), 24 (dorsal sepal colour area, KW = 162.81, r2 = 0.694), 48 (petal width, KW = 162.71, r2 = 0.619), 65 (bract colour, KW = 159.47, r2 = 0.639), and 62 (petal colour diffuse/blotchy, KW = 153.16, r2 = 0.635). Stress = 0.1624.

The directions of character change among the basal and apical flowers of individuals, depicted as vectors in the ordination space, showed expected increase in floral size with age. Flower width was recorded for the distance between the outer edges of the petals; therefore, these results indicated that petals open further laterally as they age. Character 9 (distance between mid-lobe and lateral lobe at widest point, KW = 23.94) was most informative in separating the two species. The direction of change shown by the vector in the ordination space (Fig. 6) indicated that petal claws are longer in D. daltonii (Victoria Point Road) than in D. punctata (Inverleigh), contradicting the greater overall mean of this character in D. punctata

Results support the recognition of three species: D. punctata, D. fragrantissima and D. dendrobioides. D. fragrantissima and D. dendrobioides formed two close, but separate clusters, which were clearly distinct from D. punctata (including D. daltonii) (Figs 2, 3). Although few discrete diagnostic features were recorded among all four species, the consistent separation of D. fragrantissima, D. dendrobioides and D. punctata indicates phenetic distinction between these species. However, D. daltonii nested within D. punctata in both the dendrogram and the ordination of the four species. Further analysis including only D. punctata and D. daltonii also revealed no clear distinction between the two species. D. daltonii was recently elevated to the species level from a variety of D. punctata (Jones and Clements 2004); however, even the original recognition of this taxon as a distinct variety of D. punctata was questioned because of the extensive morphological variation within and between populations of D. punctata (Dockrill 1964). Furthermore, Clements (1989) stated that the variety was doubtful and was collected close to the western edge of the distribution of D. punctata. Our results indicated that D. daltonii does not warrant recognition at the species level and supported the findings of an earlier molecular study based on AFLP data (Smith et al. 2007a). The use of morphometric data in defining species boundaries within the Diuris punctata species complex Phenetic analysis of morphological data produced a dendrogram with general topology similar to the dendrogram derived from the results of a previous molecular study of the D. punctata species complex which used AFLP data (Smith et al. 2007a). This confirmed the clustering of D. daltonii within D. punctata and supported the current recognition of D. fragrantissima at the species level and therefore its recognition as a unique entity for conservation. Congruence between morphological and molecular datasets validated the use of morphological studies in defining species boundaries and hence conservation units, provided that sample sizes and measured characters are sufficient. For example, morphological analysis confirmed the molecular distinction between four of five varieties of Calopogon tuberosus and the unwarranted recognition of var. latifolius (Goldman et al. 2004a, 2004b). Molecular data revealed some clustering of populations that was not evident in the dendrogram based on morphological data. This included separation of the two D. dendrobioides populations, despite their geographical proximity relative to populations of D. punctata, which form a single cluster. This pattern may occur because genetic divergence among populations is unexpressed phenotypically, because of greater intrapopulation variation in morphological characters, because of insufficient recorded morphological characters to fully reveal morphological differences among populations or because of a combination of

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Table 3. Mean and standard deviation (in parentheses) of selected variables for the four Diuris punctata group species in Victoria Significant (P < 0.05) differences between means are indicated by different letters Character

D. fragrantissima

D. dendrobioides

D. daltonii

D. punctata

Plant height (mm) Leaf width (mm) Flower no. Ovary length (mm) Ovary width (mm) Lateral sepal length (mm) Petal width 2 mm from apex (mm) Petal claw length (mm) Dorsal sepal width 2 mm from apex (mm) Labellum midlobe width (mm) Distance between petal apices (mm) Colour ovary, bract and lateral sepals Colour petals and dorsal sepal

195.8 (62.5) 8.3 (2.7) 7.2 (2.4) 8.2 (1.2)a 4.3 (0.8) 56.0 (14.2)a 4.9 (1.0)ab

365.5 (73.2)(A) 6.4 (1.8) 5.0 (1.7) 9.4 (1.6) 3.6 (0.5) 54.9 (10.1)ab 2.9 (0.6)

304.0 (66.8) 5.0 (1.2) 2.2 (0.7) 8.6 (1.4)a 3.2 (0.4)a 50.3 (9.1)a 4.6 (0.7)a

349.5 (103.6)a 5.6 (1.6) 2.9 (1.1) 8.5 (1.4)a 3.3 (0.5)a 57.8 (12.2)a 5.0 (1.0)b

6.7 (1.6) 5.5 (1.2)a

7.9 (1.1)a 3.2 (0.9)

7.7 (1.0)a 5.2 (1.0)a

8.1 (1.3)a 5.5 (1.1)a

10.3 (1.7)a

9.9 (1.1)a

10.2 (1.0)a

12.2 (2.1)

19.3 (7.4)a

19.0 (5.5)a

24.9 (7.1)b

26.0 (9.2)b

Green
diffuse purple

Green
diffuse purple

Dark purple

Dark purple

White with blotchy or diffuse pale mauve White
cream

White with blotchy purple

Mauve to purple, colour diffuse

Mauve to purple, colour diffuse

White
cream

Generally golden yellow

Generally golden yellow

Colour callus

these. Further insight may be gained by investigation of the ecology and phenology of studied populations. HarastovaSobotkova et al. (2005) showed that molecular (RAPD) data distinguished between two weakly morphologically differentiated subspecies of Neotinea ustulata with differing flowering phenologies. Examples such as these support the use of a combination of morphological and molecular techniques, particularly in resolving complex taxonomic relationships, as recommended by several authors (e.g. Schuiteman and de Vogel 2003; Bateman and Hollingsworth 2004; Shipunov et al. 2005). Potentially confounding the interpretation of both previously published molecular data (Smith et al. 2005, 2007a) and morphological data presented here, are recent, severe habitat losses and demographic decline, particularly in D. punctata and D. fragrantissima (DSE 2004; Murphy et al. 2006). It is feasible that genetically or morphologically discreet taxa identified here represent artificially restricted samples from a disrupted cline, rather than naturally divergent entities present before fragmentation. The extensive decline of D. fragrantissima has resulted in its high conservation priority relative to other species of the D. punctata complex. However, the risk of losing variation within the group as a whole highlights the importance of identifying extant taxa and providing conservation management targeted to maintain current levels of diversity in the D. punctata complex, irrespective of any future taxonomic changes. The utility of morphological characters in defining species boundaries and field identification is discussed below. Floral colour, shape and size are regularly considered important distinguishing features within the D. punctata species complex (e.g. Dockrill 1964; Clements 1989; Bishop 2000; Jones and Clements 2004). In the present study, floral characters largely explained distinctions between groups, with floral-colour characters most informative in separating

D. fragrantissima and D. dendrobioides from D. punctata and D. daltonii. The removal of flower-colour variables from our dataset resulted in the loss of groups in the resultant ordination, thus highlighting the importance of flower colour as a defining feature of these groups and the usefulness of this character for distinguishing species in the field. Examples of the floral morphology observed in the present study are shown in Smith et al. (2007b). Vegetative characters were mostly uninformative, although plant height distinguished the shorter D. fragrantissima from all other species. Even though vegetative characters have been excluded in some earlier revisions of taxa within Orchidaceae (e.g. Burns-Balogh and Funk 1986), recent studies have shown their value in resolving relationships at various taxonomic levels (e.g. within Orchidaceae, Freudenstein and Rasmussen 1999; tribe Vandeae, Carlsward et al. 2006; putative hybrids of Liparis kumokiri and L. makinoana, Yoon Chung et al. 2005). The exclusion of vegetative characters has resulted in the misplacement of the genus Tripodia among the spiranthoids (Freudenstein and Rasmussen 1999). Further, in their morphological analysis of Orchidaceae; Freudenstein and Rasmussen (1999) showed that incorporation of vegetative characters provided a test of homoplasy in floral characters, which are likely to be under greater selective pressures as a result of specialised insect-pollination mechanisms. Quantitative characters were largely indiscrete and overlapping, limiting their use in field identification of the studied Diuris species and highlighting the importance of incorporating a variety of characters into morphological studies rather than the subjective selection of a few ‘key’ characters. Analysis of variance revealed significant differences among the character means for each species, showing overall trends such as shorter height and greater flower number in D. fragrantissima, and

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1.180

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32 54 30 42

18 55

53

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26 –1.357

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–1.068 –1.412

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26 14

42 30 53 32 54

55 18

–1.171 –1.573

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Fig. 4. Three-dimensional SSH (MDS) ordination of Diuris fragrantissima and D. dendrobioides in Victoria. Key: D. fragrantissima: in situ (˛) and ex situ; Melbourne Zoo (¤), Royal Botanic Gardens Melbourne ( ), D. dendrobioides: Bonegilla ( ), and Boorhaman (). Vectors depict direction of change in characters with highest Kruskal
Wallis values: 49 (petal width 2 mm from apex, KW = 66.18, r2 = 0.749), 1 (plant height, KW = 63.31, r2 = 0.649), 35 (labellum midlobe colour 0
5, KW = 59.94, r2 = 0.710), 22 (dorsal sepal colour 0
5, KW = 59.24, r2 = 0.660) and 20 (dorsal sepal width 2 mm from apex, KW = 56.86), r2 = 0.678). Stress = 0.1787.

longer ovaries and more tapered petals and dorsal sepals in D. dendrobioides. D. daltonii had fewer flowers than all other species, although a difference in means between D. daltonii and D. punctata of
0.67 (i.e. less than one flower) indicated that this would not be a useful distinguishing character in the field. Other than having a significantly wider labellum than all other species, D. punctata showed a lot of inter-population variation. Vectors depicting directions of change in the ordination space (Fig. 4) indicated that D. fragrantissima has more rounded petal

Dimension 1 1.543

Fig. 5. Three-dimensional SSH (MDS) ordination of Diuris punctata and D. daltonii in Victoria. Key: D. punctata: Glenrowan (&), Bonegilla (&), Mt Eliza ( ), Mornington ( ), Inverleigh ( ), D. daltonii: McGiverns Road ( ), McCutcheons Road ( ), Victoria Point Road ( ). Vectors depict direction of change in characters with the highest Kruskal
Wallis values: 29 (labellum midlobe length, KW = 98.45, r2 = 0.669), 47 (petal length, KW = 88.67, r2 = 0.683), 48 (petal width, KW = 85.59, r2 = 0.455), 30 (labellum midlobe width, KW = 82.96, r2 = 0.444) and 64 (petal apices obtuse/acute, KW = 75.97, r2 = 0.567). Stress = 0.2089.

apices, shorter height and paler flower colour than D. dendrobioides, particularly in ex situ individuals. The close clustering of D. fragrantissima and D. dendrobioides may be a reflection of similar characters that were not shared with D. punctata, including cream, rather than yellow, callus colour, and blotchy rather than diffuse floral colour. D. dendrobioides is described as having longer lateral sepals than D. punctata and D. fragrantissima (Bishop 2000); however, in the present study lateral sepal length was not informative in distinguishing these species, with similar means and variances measured for this character in all populations (Table 3). Bract colour was a good distinguishing character for sympatric D. punctata and D. dendrobioides at Bonegilla.

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20 11

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9

10

*

*

46 *

*

* * *

* * * * *

Dimension 3

Dimension 1

2011 10

9

*

*

46

*

*

* * **

* *

* * Dimension 1

Fig. 6. Three-dimensional SSH (MDS) ordination showing floral variation within individual plants of Diuris daltonii (Victoria Point Road: ) and D. punctata (Inverleigh: ). Apical flowers are shown (*). Vectors depict direction of change in characters with the highest Kruskal
Wallis values: 56 (distance between petal apices, KW = 23.96), 50 (petal curvature, KW = 23.94), 46 (distance between labellum midlobe and lateral lobe at widest points, KW = 23.94), 8 (flower diameter, KW = 23.93) and 54 (petal claw length, KW = 23.92). Stress = 0.1395.

Characters used by Clements (1989) to distinguish D. fragrantissima from D. punctata were confirmed in the present study, with the exception of fragrance, which was found in all populations of all species examined, although fragrance intensity varied within both populations and individual plants, and was weather-dependent (data not shown). Slight differences in biologically active floral-odour components have been shown to be sufficient to attract different pollinator species in Chiloglottis (Mant et al. 2002;

Schiestl et al. 2003) and Ophrys (Schiestl and Ayasse 2002) and may indicate a shift towards reproductive isolation and speciation. Analysis of scent in the D. punctata species complex may provide a new suite of characters for species resolution on the basis of biological interactions. Diuris punctata and D. daltonii did not cluster separately in ordination resulting from analysis of all measured individuals; however, populations clustered according to geographic proximity, indicating clinal variation rather than distinct speciation events. Mean plant height, lateral sepal length and labellum width were found to be significantly smaller in D. daltonii than D. punctata; however, these differences were not sufficient to separate the species in ordination (Figs 3 and 5). In the analysis of floral variation in one population of each species, the disjunction between the populations was clear and largely a result of variation in Character 9 (distance between midlobe and lateral lobe at the widest points). The overall species mean for this character was found to be significantly greater in D. daltonii than D. punctata, supporting Jones and Clements (2004) who described D. daltonii as distinct from D. punctata because of a longer ‘neck’ on the labellum midlobe. This character was not, however, found to be informative in separating groups in an ordination of the two species, having a relatively low Kruskal
Wallis value (28.24). The present study revealed some discrepancies with the description of D. daltonii (Jones and Clements 2004) and, therefore, its elevation to rank of species. For example, petal claws were not found to be longer, nor were flowers found to be darker in colour in D. daltonii than in D. punctata. While flower parts were generally smaller in D. daltonii than D. punctata (data not shown), mean differences of less than 2 mm in all measurements indicated that this would not be a useful field character. A molecular phylogeny of the genus may shed further light on the taxonomic placement of this species. Physical and biotic factors are known to influence plant phenotype (Kores et al. 1993; Edelkraut and Güsewell 2006; Wang 2007) and can result in morphological separation which is a reflection of habitat differences rather than taxonomic distinction on the basis of underlying genetic differentiation. Goldman et al. (2004b) recognised the potential influence of ecophenotypical variation in their morphometric circumscription of species and infraspecific taxa in Calopogon and grew plants in identical conditions for 24 months before measurement to reduce the impact of environmental variables. In the present study, D. fragrantissima plants cultivated ex situ where water and nutrients were not limiting were more robust than in situ plants, and produced larger flowers in greater numbers. Although ex situ D. fragrantissima plants were sourced outside (within 10 km) the existing remnant site, their genetic similarity to in situ D. fragrantissima individuals was shown in a previous study (Smith et al. 2007a), with all individuals clustering together. The influence of within-plant floral variation on phenetic relationships among species A potential source of error in morphological studies is measurement of flowers at different flowering stages but

In situ morphometric study of Diuris punctata

treating the results as comparable. In a study of clinal variation within inflorescences of European orchids within the subtribe Orchidinae (Pridgeon et al. 2001), Bateman and Rudall (2006) described the importance of measuring flowers from a consistent location within inflorescences and considering effects of the cline on floral morphology. An assessment of within-plant variation in the present study showed that inter-population variation was greater than variation within populations or individuals. By restricting measurement to basal flowers and using ratios to standardise size variables, flowering stage did not adversely affect the resolution of groups. Shrinkage of excised plant structures was also shown to be a potential source of measurement error (Bateman and Rudall 2006), which was overcome in the present study by conducting all measurements in situ on undamaged, open flowers. Changes in floral morphology, including colour and odour, can be important in creating reproductive-isolation barriers through selective pressure from different pollinators (Cozzolino and Widmer 2005) and thereby driving speciation. Pollinators are known to discriminate among variable floral traits, such as gynostemium length (Ushimaru and Nakata 2001), petal colour (Stanton et al. 1989), odour (Galen 1989; Schiestl and Ayasse 2002), flower number (Ehlers et al. 2002) and flower height (Peakall and Handel 1993). Where changes to floral morphology lead to an increase in pollinator specificity, for example in sexually deceptive orchids, the differences can be maintained because of reduced gene flow among variants. In contrast, where floral changes result in lower pollinator specificity, for example in cases of floral mimicry, gene flow between sympatric taxa may actually increase (Cozzolino and Widmer 2005). Potentially, various pollination strategies found in Diuris may act to maintain a broad gene pool and consequent high levels of similarity among some taxa. D. maculata and D. aurea, both yellow/brown-flowered species, have been shown to employ food-deceptive pollination strategies (Beardsell et al. 1986; Indsto et al. 2006, 2007), whereas D. alba, a non-Victorian member of the D. punctata group, provides a small nectar reward (Indsto et al. 2007). Further investigations into the pollination biology of the Victorian D. punctata group may reveal potential selective pressures driven by pollinators. This may have implications for habitat management, including the maintenance of sympatric plants such as legumes mimicked by Diuris species (Beardsell et al. 1986). Conclusions Morphological data were informative in resolving relationships among D. punctata, D. daltonii, D. dendrobioides and D. fragrantissima in Victoria. The phenetic distinction between D. punctata, D. fragrantissima and D. dendrobioides was evident. However, D. daltonii is recognised at varietal rather than species rank. Congruence with a previous molecular study (Smith et al. 2007a) validated the use of morphological data in defining species boundaries. Although species were largely distinguished by floral characters, floral variation within plants was insufficient to influence the overall morphological patterns observed among the four Diuris taxa. Conducting the present study in situ enabled the incorporation of additional characters and larger sample sizes than are possible

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when measurements are restricted to herbarium specimens and minimal destructive sampling enforced by restrictions on the collection of threatened species. Acknowledgements We thank Colin Knight (Melbourne Zoo), Glen Johnson and Andrew Pritchard (Department of Sustainability and Environment), Dr Fiona Coates (Arthur Rylah Institute for Environmental Research), Dave and Lyn Munro, Neil and Judy Anderton, Russell Mawson, Eileen Collins and Anusha Babbar for assistance in the field. We also thank Professor Pauline Ladiges (University of Melbourne) and two anonymous reviewers for comments on an earlier draft and Professor Mark McDonnell (University of Melbourne) for assistance with manuscript preparation.

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Manuscript received 11 March 2008, accepted 24 July 2008

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