Introduction

Banksia is a moderately sized genus currently of 212 taxa; viz. 78 species, 9 subspecies and 11 varieties (see Collins et al. 2009), plus 114 species previously included under Dryandra (Mast and Thiele 2007). There are16 named species of Banksia in the eastern states of Australia (Collins et al. 2009). Species of Banksia are often found in sclerophyllous, heathy shrublands on nutrient poor soils and have spectacular spike-like cylindrical or flattened, head-like conflorescences that are easily recognised (Mast et al. 2005). The growth habit in Banksia ranges from small prostrate woody mats to 25 m tall trees. Only one species occurs naturally outside Australia, Banksia dentata, which extends to Papua New Guinea, Irian Jaya and the Aru Islands (George 1981; Mast et al. 2002).

According to George’s (1999) classification, the Banksia spinulosa complex has the broadest latitudinal, altitudinal and ecological amplitude of any species in the genus (Thiele and Ladiges 1996). The Banksia spinulosa complex consists of four taxa distributed from north-eastern Queensland to eastern Victoria along the coast and highlands. Banksia spinulosa var. spinulosa occupies both latitudinal extremes but is replaced along the coast between the Sunshine Coast area in south-eastern Queensland and the Hawkesbury River in central eastern New South Wales by Banksia spinulosa var. collina, which also has inland outliers west to the Carnarvon National Park area. Banksia spinulosa var. cunninghamii is mostly a montane taxon distributed mostly between the Hunter River in central eastern New South Wales and eastern Victoria, with a northern disjunction in the McPherson Range along the Queensland–New South Wales border. It is broadly sympatric with, and frequently co-occurs in mixed populations alongside Banksia spinulosa var. spinulosa between the northern Blue Mountains and the Moss Vale district. Banksia spinulosa var. neoanglica is also a montane taxon, distributed from the McPherson Range and along the eastern edge of the New England Tableland, New South Wales south to the Hanging Rock area. Banksia spinulosa var. neoanglica is parapatric with a montane variant currently attributed to Banksia spinulosa var. collina in the Daves Creek area, Lamington National Park; it is allopatric with other taxa in the complex.

Most herbaria follow George (1981, 1988, 1999) in treating this complex as one species with four varieties, viz. Banksia spinulosa var. spinulosa, Banksia spinulosa var. collina, Banksia spinulosa var. cunninghamii, and Banksia spinulosa var. neoanglica. Flora of New South Wales (NSW) Online (1999 onwards) treats the Banksia spinulosa complex as comprising two species, each with two infraspecific taxa: Banksia spinulosa var. collina, Banksia spinulosa var. spinulosa, Banksia cunninghamii subsp. cunninghamii, and Banksia cunninghamii subsp. A (= Banksia spinulosa var. neoanglica), and this paper will use this treatment as a reference point. The primary reason for recognising two species was the broad sympatry of Banksia spinulosa var. spinulosa and Banksia cunninghamii subsp. cunninghamii. There appearsto be no hybridisation between these two taxa, indicating that these two taxa are reproductively isolated from one another and are therefore different biological species (Harden 2002).

These competing taxonomic treatments have created confusion, examples of which can be found in species lists for some National Parks in New South Wales (unpublished visitor brochures), which include Banksia spinulosa var. neoanglica and Banksia cunninghamii subsp. A as separate entities. Some herbaria also concurrently use two names for the same entity (see the Atlas of Living Australia). Current circumscriptions of the taxa within the Banksia spinulosa complex are based on intuitive assessment of observed morphological variation, rather than an explicit analysis of the morphological variation. Thiele and Ladiges (1996) conducted a cladistic analysis of the whole of Banksia using 92 qualitative characters and 14 morphometric characters in an attempt to clarify interspecific relationships and to provide a phylogenetic classification. As that was a genus-wide study, limited work was conducted on or within individual species.

The aims of this study were (1) to test and set the taxonomic status and circumscription of Banksia cunninghamii subsp. A; and (2) to search for novel diagnostic characters that could be used to distinguish individual taxa within the Banksia spinulosa complex (sensu George 1988).

Materials and methods Study material

Although dried herbarium specimens were available for this study, it was considered necessary to collect fresh material to adequately investigate character homology though a detailed study of different developmental stages. Existing herbarium specimens were deficient in some developmental stages and often were not suitable for destructive sampling. We made collections from locations in New South Wales and Queensland encompassing the full geographic range of Banksia cunninghamii subsp. A. Vouchers have been lodged at NE and/or NSW (Table 1). Each site was visited twice; the first time in February to observe the development of the rachis, the second time in May to observe the flowering process. During both visits observations were made and vouchers prepared.

Observations and microscopy

Micromorphology was examined using Leica MZ8 and MZ9 stereomicroscopes fitted with eyepiece graticules. Images were taken using a Wild M400 photomacroscope fitted with a Nikon DS-5M-L1 Digital Sight Camera System. Exploratory scanning electron microscopy of styles was undertaken using air and silca gel-dried samples mounted on double-sided carbon tabs on aluminium stubs. Specimens were coated with gold in a Neocoater sputter coater and examined at 15 kV using a Neoscope JCM-5000 bench-top SEM.

Phenetic analysis Selection of characters

The character list was primarily constructed to include leaf, floral, stem and fruit morphology. Assessment of descriptions of the taxa in the Banksia spinulosa complex (George 1981, 1988; Thiele and Ladiges 1996; Harden 2002) led to the selection of characters for the inclusion in the phenetic analysis. Additional characters were considered based on observed differences in the field (Table 2). Wherever possible, quantitative characters were used to reduce subjectivity and to avoid artefacts resulting from the conversion of continuous variables into categorical ones. Qualitative character states were scored as either 1 or 2. Quantitative characters for each OTU were the mean of up to 10 measurements where possible.

Colours, however, were treated as multistate characters to maximise accuracy and repeatability, which allowed for some natural variation, thus avoiding spurious over-precision (see below). Royal Horticultural Society (RHS) colours were used to compare adaxial and abaxial leaf surfaces prior to, and after drying, as well as styles before and after anthesis. Each RHS colour was allocated a number from 1–26 (Table 3).

All leaf measurements were taken from leaves in the middle of a branchlet, selected from the whorl of branchlets subtending a resting terminal bud or conflorescence; leaves were measured after drying. Conflorescence characters such as number of floral pairs were counted live on the plant. Infructescences were measured vertically with a steel ruler and the circumference was measured with a sewing tape measure.

Infructescences were placed on a gas burner for 1–3 min then left on brown paper for two days in a dry place. Seeds were extracted using a pair of forceps and measured under a stereomicroscope using a calibrated eyepiece graticule.

Dataset

A dataset (Appendix 1) was maintained in Microsoft Excel and exported to PATN v. 3 for Windows (Belbin and Collins 2006). The characters were range-standardised and a distance matrix calculated using the Gower distance metric (Wills et al. 2000). Three-dimensional ordination plots were generated from the distance matrix using semi-strong hybrid multidimensional scaling (SSH MDS) with 100 random starts and 200 iterations to minimise stress. Flexible UPGMA (Beta-value = -0.1) phenograms, 3D ordination scatter plots, and correlation of characters with ordination pattern (PCC) were produced directly within PATN. The criteria for circumscribing distinct taxa were: (1) the OTUs representing the putative taxa formed discrete groups that did not overlap those of any other groups of OTUs in both cluster and ordination analysis and (2) the OTUs within these groups showed an amount of morphological heterogeneity similar to that of the other putative species in the Banksia spinulosa complex included in the analysis (Plunkett et al. 2009). In total 23 characters were used, 11 morphometric, five binary, seven multistate qualitatively coded morphological characters (Table 2).

Diagnostic qualitative morphological characters Conflorescences

The conflorescences of all taxa in the Banksia spinulosa species complex consist of an elongate woody rachis that has three types of bracts. Below the base of the rachis on the short peduncle are the involucral bracts. The second type of bract is the common bract each of which subtends a flower pair on the conflorescence axis. The third type of bract, a smaller floral bract, subtends each flower in a pair (Johnson and Briggs 1975; George 1981; Thiele and Ladiges 1996). In the early stage of conflorescence development, flower pairs start to develop along the rachis basipetally. The flowers emerge from each side of the floral bracts and above and below each large common bract. Bracts and flower pairs are arranged in vertical columns on the rachis. This pattern is visually enhanced with the development of styles. The vertical striping pattern remains until the perianth and the styles have senesced or fallen from the rachis (George 1981; Thiele and Ladiges 1996; Collins et al. 2009).

Structure of the perianth (floral pairs)

The perianth segments or tepals in Banksia each consist of a limb and a claw (Thiele and Ladiges 1996). In Banksia andmost other Proteaceaethe perianth is made up of four tepals (Wrigley and Fagg 1989; Weston 2006).

Structure of the style

The conflorescences in the Banksia spinulosa complex have the appearance of being a particular colour, i.e. black, red, yellow orange, or purple. It is the styles that are most boldly coloured with red, black, green, yellow or purple pigment, not the limb and claw (George 1981; Collins et al. 2009). All styles in the Banksia spinulosa species complex are hooked and extend up to 3 mm past the limb and claw just prior to anthesis. The distal part of the style is modified as a pollen presenter and the stigmatic cavity is located at the apex of the style. The style is released from the limb upon anthesis (Thiele and Ladiges 1996; Weston 2006). All styles in the Banksia spinulosa complex have similar surfaces. Scanning electron microscopy was performed on the style surfaces and no distinguishing features were found.

Leaf morphology

All taxa within the Banksia spinulosa complex have leaves that are scleromorphic in texture, discolourous, and linear in shape. The indumentum on the abaxial leaf surface is felted and the midvein is raised on the abaxial surface of all leaves in all taxa within the complex. Continuous variation was found in the colour of adaxial and abaxial leaf surfaces both within and between populations of all taxa within the Banksia spinulosa complex.

Lignotubers

The term lignotuber refers to a woody swelling which may take the form of an extensive subterranean lignotuber, basal lignotuber, or an above ground lignotuber (Mibus and Sedgley 2000). The development of a lignotuber is considered to have evolved repeatedly in different lineages in response to increased fire frequency (Whelan and York 1998).

Results and discussion Phenetic analysis

Ordination (Figure 1) and clustering (Figure 2) of the data matrix found five distinct groups of OTUs in the Banksia spinulosa complex: corresponding to a priori names Banksia spinulosa var. collina sens. lat., Banksia spinulosa var. collina × Banksia spinulosa var. spinulosa from near the New South Wales locations of Morisset, Bouddi and Calga, Banksia spinulosa var. spinulosa, Banksia cunninghamii subsp. cunninghamii, B. cunninghamii subsp. A. The phenogram displays the same five groups of OTUs (Figure 2). Even when we reran the analyses excluding all the binary characters (Characters 11, 20–23; ordination and phenogram not presented), the same five groups of OTUs were obtained, which, along with the very low stress value (Figure 1) indicate that the results are robust. Twelve of the 23 characters, including quantitative, binary and multistate characters had correlated more than 70% with the ordination (Table 4) indicating sound choice of characters, a broad base of evidence for the patterns obtained, and confidence in the results obtained.

The cluster of OTUs of Banksia spinulosa from Morisset, Bouddi and Calga (Table 1) is characterised by red styles, at Morisset and Bouddi and black styles at Calga, multi-stemmed habit and occurs between the Hawkesbury River and Hunter Valley. Herbarium specimens from these locations have been determined by A.S. George and other botanists as “Banksia spinulosa var. collina × Banksia spinulosa var. spinulosa”. George (1981) considered this group of OTUs to be an intergrade between Banksia spinulosa var. spinulosa and Banksia spinulosa var. collina. These populations do not fall in a position intermediate between Banksia spinulosa var. collina and Banksia spinulosa var. spinulosa in the ordination diagram, nor do they segregate into three clusters representing parental species and hybrids. There is thus no clear phenetic evidence of either an intergrade or a mixture of hybrids and parental species between the Hawkesbury River and Hunter Valley. The taxonomic status of these populations and their relationships to others remains unclear. This cluster of OTUs could represent a distinct species, but we will investigate this question and the broader relationship between Banksia spinulosa var. collina and Banksia spinulosa var. spinulosa further before making any formal taxonomic changes to these taxa.

Slight outliers in the Banksia spinulosa var. collina cluster represent some discontinuous morphological variation, which we also plan to investigate.

Taxonomic inference

Given the consistent clear cut groups in the ordination and cluster analysis across a broad geographic and morphological range of OTUs (Table 1), we propose the following taxonomic arrangement, which we use hereafter in this paper: recognising Banksia cunninghamii subsp. cunninghamii as Banksia cunninghamii sensu stricto; recognising Banksia spinulosa var. collina as Banksia collina sensu lato; recognising Banksia spinulosa var. spinulosa as Banksia spinulosa sensu stricto; formalising Banksia cunninghamii subsp. A at species rank under the name Banksia neoanglica . Although the OTUs of Banksia spinulosa from the Morisset and Bouddi populations could be considered to constitute a distinct species on the evidence we present here, we refrain from recognising these populations as a distinct taxon until we have more thoroughly tested the hypothesis that they are part of an extensive hybrid swarm and searched for any additional populations that might provide evidence for integradation between Banksia collina and Banksia spinulosa.

Morphological analysis Growth forms within the Banksia spinulosa species complex

Banksia cunninghamii sensu stricto is a single-stemmed tree to 7 m tall, and is non-lignotuberous. Banksia spinulosa sensu stricto forms a multi-stemmed, rounded shrub to 3 m high. The lignotuber is subterranean. Banksia collina sensu lato is a multi-stemmed upright shrub to 3 m tall, with a subterranean lignotuber (Harden 2002; George 1981).

Banksia neoanglica has a variety of growth forms ranging from small rounded multi-stemmed shrubs to single-stemmed trees. The growth forms of Banksia neoanglica appear to be related to the degree of exposure of plants to fire. At sites where there have been no fires for more than 15 years, such as at Binna Burra, Lamington National Park, Queensland and some parts of Gibraltar Range National Park, New South Wales (Pers. Comm. Justin Kreis Ranger Glen Innes National Park), Banksia neoanglica is a single-stemmed tree and exhibits all the traits of an obligate seeder such as a greater infructescence load and spontaneous opening of the follicles. In the tree form, Banksia neoanglica has a slight swelling at the base of the trunk just below the soil or there are epicormic buds which often develop into branches, well above ground level, similar to those of some eucalypts (Burrows 2008). The multi-stemmed form has a substantial subterranean lignotuber and requires fire to open follicles and has a greatly reduced infructescence load.

Individual adult morphological features

Styles: The structure of the conflorescence, including perianth and styles is similar for all taxa in the Banksia spinulosa complex. Size, shape and colour of the individual parts of the conflorescence, however, differ considerably across the species. Style colour in the Banksia spinulosa complex varies depending on the proportions of chlorophyll (green), carotenoid (yellow to orange), anthoxanthin (yellow) and anthocyanin (red to purple) pigments that develop in them (Grotewold 2006). The style colour in Banksia neoanglica, Banksia spinulosa sensu stricto and Banksia cunninghamii sensu stricto usually grades from red to maroon to purple during conflorescence development, then the style becomes discolorous at anthesis, with the apex becoming dark purple to black. This is a consistent character within and between populations of three species in the Banksia spinulosa complex. The exception is Banksia collina sensu lato which has concolourous green styles both before and after anthesis. We found no black-styled Banksia collina sensu lato within the geographical range of this project.

The style apex in B. cunninghamii sensu stricto seems to have substantially more anthocyanin pigment than either Banksia spinulosa sensu stricto or Banksia neoanglica. In Banksia cunninghamii sensu stricto we observed that the style length is usually longer than either Banksia spinulosa sensu stricto or Banksia neoanglica and is a similar length to Banksia collina sensu lato. The black pigmentation of the styles of Banksia cunninghamii sensu stricto starts to develop one third of the way along the style above the ovary. In Banksia spinulosa sensu stricto and Banksia neoanglica the dark pigmentation in the style develops one half to two thirds of its length above the ovary. In all populations in the Banksia spinulosa complex with the exception of Banksia collina sensu lato we observed what appeared to be yellow-styled conflorescences. Upon closer inspection they are green styled and appear to have less chlorophyll in both the styles and leaves than is found in Banksia collina sensu lato which is also green-styled. Green styled variants are found in less than 2% of any one population except in Banksia collina. Polymorphism is a common trait in Proteaceae where, for example, 40% of all species of Protea exhibit variation in the bract, style and perianth colour (Carlson et al. 2010). It is often unclear whether these variants are transient mutant individuals or this feature is a persistent polymorphism (Carlson et al. 2010). In the case of the Banksia spinulosa complex, however, the variants comprise less than 2% of a population and were not found in every population; therefore it is unlikely to be persistent polymorphism.

Perianth colour: The colours of the perianth in the Banksia spinulosa complex vary according to their developmental stage and their exposure to sunlight. The perianth colours can vary within and between populations in all four of the species in the Banksia spinulosa complex. The factor that seems to have the most influence on the perianth colour in the early stages of development is exposure to sun, often mediated by the position of an conflorescence on the outside or inside branches of the plant or by shading from other plants. In Banksia spinulosa sensu stricto, Banksia collina and Banksia neoanglica, the conflorescences that are exposed to full sun tend to have orange or yellow perianths. Those that are exposed to a limited amount of sun tend to be green. The perianth colour of Banksia cunninghamii sensu stricto is diagnostic for the species. At maturity the perianth always has a distinct pink hue and this colouring continues through to anthesis. The pink hue does not vary between and within populations of Banksia cunninghamii sensu stricto, nor does exposure to full sun orfull shade effect the colour of the perianth at maturity.

Common bracts: Common bracts have been mentioned in previous studies (Johnson and Briggs 1975; Thiele and Ladiges 1996; George 1981) but the bract surfaces had not been mentioned before this study or used to draw taxonomic conclusions. Close examination, especially at early stages of development, of the abaxial surface of the common (or flower pair) bracts found them to have differences in shape, texture, colour, and surface (Figure 3A–D) which covary in line with the entities recognised here (Figures 1–2) within the Banksia spinulosa complex. We will characterise these differences for use in future expanded phenetic analysis and description of taxa in Banksia. Floral bracts were not examined in detail in this study.

Involucral bracts: Involucral bracts appear to be taxonomically informative at the species level in the study group. The involucral bracts of Banksia cunninghamii sensu stricto are caudate with an abaxial ‘spine’ (Figure 4A). The involucral bracts in Banksia spinulosa sensu stricto (Figure 4B) are longer and more scleromorphic, with little or no hair and no external spine. In Banksia neoanglica (Figure 4C) these bracts are more hirsute without an external spine and in Banksia collina sensu lato (Figure 4D) the involucral bracts are shorter, have no external spine and limited hair. There are differences in the distal and proximal portions of the involucral bracts (Figure 4A–D) in each species that warrant further examination.

Taxonomic conclusions

The diversity of species concepts in the biological literature is an asset, not a liability when considering the Banksia spinulosa complex and are an integral part of biological theory. We have taken into account the co-varying morphological discontinuities, the phenetic species concept, geographical and ecological isolation and the biological species concept of reproductive isolation. The use of differing concepts has been useful in suggesting multiple lines of evidence for testing taxonomic boundaries in the Banksia spinulosa complex (cf. de Queiroz 2007). Clear taxonomic groups were obtained based on the results of the morphometric analyses and corroborated by new characters (cf. Thiele and Ladiges 1996) such as the abaxial surface of the common bract (Figure 3), the number of floral pairs around the circumference of the conflorescence and obvious differences in the involucral bracts (Figure 4A–D). Additionally, given the ecological isolation, reproductive isolation and morphometric differentiation of at least three of the taxa in the Banksia spinulosa complex, there is a compelling case to recognise Banksia spinulosa sensu stricto, Banksia cunninghamii sensu stricto and Banksia neoanglica as separate species (Table 5). Banksia collina sensu lato is considered heterogeneous and in need of further study, but is not readily confused with Banksia neoanglica. Similarly Banksia spinulosa from the Morisset, Bouddi and Calga requires further study but is distinct from Banksia neoanglica.

The geographic distribution of Banksia neoanglica falls within the biogeographic region known as the “Macpherson–Macleay Overlap” of Burbidge (1960), which is a biogeographically distinctive and rich area (Crisp et al. 1999) with many species of plants and invertebrates endemic to the area.

Banksia cunninghamii sensu stricto and Banksia neoanglica have often been misidentified because Banksia cunninghamii sensu stricto, on occasions, has a brown indumentum; Banksia neoanglica sometimesalso exhibitsbrowning on the abaxial leaf surface. This character has been used in the past as an aid to distinguishing Banksia cunninghamii sensu stricto and the two other ‘varieties’ recognised at that time (George 1981; Harden 2002). Indeed, this attribute occurs in both Banksia neoanglica and Banksia cunninghamii sensu stricto. Drying of the specimens in both of these species can cause browning on the abaxial leaf surface. The browning of the abaxial leaf surface should not be used as taxonomic marker or an identification tool.

Future directions

Disjunct populations in central and northern Queensland currently assigned to Banksia spinulosa var. spinulosa warrant inclusion in a more broadly framed analysis, as do the northern and southern populations of Banksia collina sensu lato and Victorian populations of Banksia cunninghamii sensu stricto. There are also other populations of Banksia that clearly belong with the Banksia spinulosa group but are as yet unstudied. Further work is needed to enable suitable placement of these populations. Analysis using molecular data, together with expanded use of the novel characters presented here, would likely resolve these long-outstanding taxonomic issues.

Taxonomic treatment