New chromosome counts in New Zealand species of Plantago (Plantaginaceae)

Abstract

Polyploidy is common in the widespread genus Plantago, and might be especially important in the evolutionary history of native New Zealand species of the genus. To further understanding of native New Zealand Plantago, mitotic and meiotic chromosome counts are reported from 58 individuals representing most native species and subspecies, and one introduced species, complementing previous studies by extending the number of individuals and the geographic range of taxa counted. Previous counts were confirmed for most Plantago lanigera (2n = 2x = 12), all P. raoulii, P. spathulata subsp. spathulata, P. spathulata subsp. picta, P. triandra subsp. triandra and P. triandra subsp. masoniae (2n=8x=48), most P. unibracteata (2n=10x=60), and all P. sp. ‘Sylvester’ (2n=16x=96). Novel counts include 2n=12x=72 for four individuals identified as P. unibracteata, and 2n=48 for four individuals from Sugarloaf Pass, South Island, referred to here as P. aff. spathulata. One of the eight naturalized species, P. major, was diploid with 2n=12. These results, together with other studies, suggest that all native New Zealand Plantago have a base chromosome number of x=6 and most polyploids are allopolyploids.

Keywords:

• Plantaginaceae
• Plantago
• chromosome number
• polyploidy
• allopolyploidy
• chromosome pairing
• hybridization

Introduction

Approximately two-thirds of the>200 species of the cosmopolitan genus Plantago have had at least one chromosome count (Rahn 1996, who included Bougueria and Litorella, but see Hoggard et al. 2003). The basic chromosome number of Plantago is considered to be x=6, and the ancestor of the genus was probably diploid with 2n=12 (Rahn 1996). Roughly 75% of the studied species (representing all six subgenera) have x=6 as a base number, whereas the remaining 25% of Plantago species (from three subgenera) have x=5 (Rahn 1957, 1996). In a phylogenetic study based mostly on morphological characters, Rahn (1996) inferred three independent reductions in Plantago from x=6 to x=5, as well as one reduction to x=4.

Previous cytological studies of New Zealand Plantago (all with a base number of x=6) reveal an intriguing evolutionary history in which polyploidy has clearly played an important role. The first reported counts of New Zealand Plantago were 2n=48 for P. spathulata (Rahn 1957), and n=24/2n=48 for P. raoulii, P. spathulata, and P. triandra, and 2n=24 for ‘P. brownii’ (Rattenbury 1957). (This ‘P. brownii’ is likely to be P. novae-zelandiae and was referred to as P. aff. lanigera by Dawson 2000). There was also a dubious count of 2n=18 for P. raoulii (McCullagh 1934; Dawson 2000). In these early studies, only one or very few individuals of each species were counted, and herbarium vouchers were not cited for many individuals. [The voucher for the P. raoulii accession counted by Rattenbury (1957) is probably AK 46123, but vouchers for the other three individuals do not exist at AK (Ewen Cameron, personal communication).] Subsequently, Groves & Hair (1971) greatly increased sampling of New Zealand Plantago to include counts from a total of 40 individuals of eight described species, all vouchered at CHR. They found 2n=12 for P. lanigera (five individuals), P. triantha (two individuals) and ‘P. novae-zelandiae diploid’ (six individuals); 2n=24 for ‘P. novae-zelandiae tetraploid’ (four individuals); 2n=48 for P. picta (one individual), P. raoulii (five individuals), P. spathulata (five individuals) and P. triandra (six individuals); and 2n=60 for P. unibracteata (as P. uniflora; five individuals). In addition, they found one individual from Lake Sylvester, NW Nelson to have n=48 and 2n=96, which they called P. sp. 16x (Groves & Hair 1971) and which was later dubbed P. sp. ‘Sylvester’ by Druce (1993). More recently one individual of P. obconica was found to have 2n=12 (Dawson 1989).

Polyploidy in Plantago is also common on a global scale. Of the species of Plantago with chromosome counts, approximately two-thirds are polyploids. Nevertheless, polyploidy has been dismissed by some as playing only a minor role in shaping diversification for most of the genus (Briggs 1973; Rahn 1996), with the important exception of sect. Oliganthos from the large subgenus Plantago (Rahn 1984). By contrast, Ishikawa et al. (2009) hypothesized that the entire subgenus Plantago has had a history of allopolyploidy both among and within sections. Indeed, of the 24 species of Plantago that are known to be hexaploid (2n=6x=36) or higher, 20 are from the subgenus Plantago, including nine from sect. Oliganthos and four from sect. Mesembrynia. For the most part, these two sections comprise species from the southern hemisphere, including all but one New Zealand native species (P. aucklandica, sect. Plantago) (Rahn 1996). When chromosome number was mapped onto a recent molecular phylogeny of Australasian species of these sections, it was shown that polyploid series have evolved at least four times in the Australasian group (Tay et al. 2010a). Tay et al. (2010a) also concluded that sections as currently circumscribed are not all monophyletic. We believe that detailed cytological studies will play a key role in resolving the historically difficult taxonomy of the New Zealand species of Plantago, as was the case with their southern South American counterparts (Rahn 1984). With this aim in mind, we publish here the chromosome counts for accessions of most native species of New Zealand Plantago and one naturalized species.

Materials and methods

The individual plants used in this study were collected in the field, and the identifications and places of collection are listed in Table 1. When several individuals of a particular species of Plantago were collected at the same locality, they were given the same collector's number with a different letter (e.g. 250X, 250Y). One of these individuals was pressed to make a voucher for the population and these have been deposited either in WELT or WELTU (Table 1). The other plants have been kept alive and transplanted to greenhouses for this study and for further molecular cytogenetic research. A photograph of each plant included in this study was also taken, and attached to the voucher specimen together with a record of its chromosome number. When our molecular cytogenetic studies are completed the entire plant will be vouchered in WELT.

Table 1  The species, chromosome numbers, collection details and herbarium vouchers of the Plantago accessions used in this study.

Species	Chromosome number		Collection details	Herbarium voucher
	n	2n
Plantago lanigera		12	HMM267X, SI, Otago, Rock and Pillar Range	WELTU 20126
		12	HMM273/2X, SI, Otago, Rock and Pillar Range	WELTU 20130
		12	HMM274Y, SI, Otago, Rock and Pillar Range	WELTU 20127
		12	MLT036, SI, Otago, The Remarkables	WELTU 20140
		12	MLT040, SI, Otago, The Remarkables	WELTU 20128
		12	MLT041, SI, Otago, The Remarkables	WELTU 20129
	6II	12	PGJ2611X, SI, Otago, The Remarkables	WELTU 20142
		12	PGJ2611Y, SI, Otago, The Remarkables	WELTU 20142
Plantago raoulii		48	BVS s.n. B, SI, Bluff Hill	WELT SP086802
		48	AD s.n. cult	Cult. only; no voucher
		48	‘HMM273/2Y’ ^a	Cult. only; no voucher^a
		48	‘ HMM274X’ ^a	Cult. only; no voucher^a
		48	HMM277B, NI, East Cape, Ruatoria	WELT SP086773
		48	HMM280D, NI, Bay of Plenty, Waiorere	WELT SP086776
		48	HMM281E, NI, Bay of Plenty, Puketapu	WELT SP086777
		48	MLT016, NI, Wellington, Owhiro Bay	WELTU 20151
		48	MLT017X, NI, Wellington, Owhiro Bay	WELTU 20157
		48	MLT017Y, NI, Wellington, Owhiro Bay	WELTU 20157
		48	MLT020, NI, Maungatawharau, SW of Waiouru	Cult. only; no voucher
		48	MLT054, NI, Taranaki, Manaia	WULTU 20156
Plantago spathulata subsp.
spathulata		48	JB s.n., SI, Otago, Old Man Range	WELT SP087211
		48	JMP09-119c, SI, Marlborough, Flaxbourne River	WELT SP087209
		48	PGJ2568, NI, Wellington, Cape Palliser	WELTU 20119
	24II		PGJ2629X, SI, Marlborough, Marfells Beach	WELTU 20117
P. spathulata subsp. picta		48	GA s.n. E, NI, East Cape, Tolaga Bay	WELT SP086772
		48	GA s.n. J, NI, East Cape, Tolaga Bay	WELT SP086772
		48	GA s.n. K, NI, East Cape, Tolaga Bay	WELT SP086772
		48	HMM278C, NI, East Cape, E Cape	WELT SP086774
		48	HMM278E, NI, East Cape, E Cape	WELT SP086774
Plantago aff. spathulata?		48	MLT049W, SI, Otago, Sugarloaf Pass (MJT s.n.)	WELTU 20133
		48	MLT049X, SI, Otago, Sugarloaf Pass (MJT s.n.)	WELTU 20133
	24II		MLT049Y, SI, Otago, Sugarloaf Pass (MJT s.n.)	WELTU 20133
	24II		MLT049Z, SI, Otago, Sugarloaf Pass (MJT s.n.)	WELTU 20133
Plantago triandra subsp. triandra		48	MLT021X, SI, Nelson, Lake Sylvester	WELTU 20163
		48	MLT025, SI, Nelson, St Arnaud	WELTU 20162
		48	MLT029X, SI, West Coast, Harihari	WELTU 20164
P. triandra subsp. Masoniae	48		BVS s.n. A, SI, Southland, Bluff Hill	WELT SP086801
		48	HMM283X, SI, Nelson, Wharariki Beach	WELT SP087213
		48	MLT048, SI, Nelson, Paturau coast	WELTU 20168
		48	MLT055X, NI, Taranaki, Manaia	WELTU 20167
Plantago unibracteata (decaploid)		60	HMM273/1X, SI, Otago, Rock and Pillar Range	WELTU 20170
		60	MLT026Y, SI, Nelson, Rainbow Ski Field	WELTU 20174
		60	PGJ2603, SI, Otago, Cardrona Ski Field	WELTU 20171
P. unibracteata (dodecaploid)		72	MLT024W, SI, Nelson, Lake Sylvester	WELTU 20175
		72	MLT024Y, SI, Nelson, Lake Sylvester	WELTU 20175
		72	MLT026X, SI, Nelson, Rainbow Ski Field	WELTU 20174
		72	BVS s.n. A, SI, Nelson, Tableland near Balloon Hut	WELTU 20215
		72	MLT028X, SI, Wilberg Range	WELTU 20172
		72	PGJ2599Y, SI, Wilberg Range	WELTU 20160
Plantago sp. ‘Sylvester’		96	BVS s.n. B, SI, Nelson, Tableland near Balloon Hut	WELTU 20215
		96	BVS s.n. C, SI, Nelson, Tableland near Balloon Hut	WELTU 20215
		96	DG10142A, SI, Nelson, Boulder Lake	WELTU 20216
		96	MLT022A, SI, Nelson, Little Lake Sylvester	WELTU 20150
		96	MLT022B, SI, Nelson, Little Lake Sylvester	WELTU 20150
		96	MLT023A, SI, Nelson, Little Lake Sylvester	WELTU 20149
		96	MLT023B, SI, Nelson, Little Lake Sylvester	WELTU 20149
Plantago major		12	PGJ2566A, NI, Wellington, Pukerua Bay	WELTU 20178
		12	PGJ2566B, NI, Wellington, Pukerua Bay	WELTU 20178
Note: The following abbreviations are used for collectors and localities: AD, Arnold Dench; BVS, Barry Sneddon; DG, David Glenny; GA, Graeme Atkins; HMM, Heidi Meudt; JB, John Barkla; JMP, Jessie Prebble; MJT, Michael J. Thorsen; MLT, Mei Lin Tay; PGJ, Phil Garnock-Jones; NI, North Island; SI, South Island. Accessions with the same collection number but a different letter represent different individuals collected at the same locality and thought to be of the same population at the time of collection. ^aThe two octoploid individuals labelled in the greenhouse as ‘HMM273/2Y’ and ‘HMM274X’ were putatively collected from the Rock and Pillar Range alongside diploid P. lanigera (HMM273/2X, HMM274Y) as well as decaploid P. unibracteata (HMM273/1X). However, inspection of these two octoploid individuals currently growing in the greenhouse shows that they do not match the vouchers nor the photos taken previously (Februsry 2007) in the greenhouse of HMM273 and HMM274. Based on seed and vegetative characters, these two octoploid individuals are clearly P. raoulii, a species which is not known from Rock and Pillar Range, nor (more generally) from high-elevation habitats. Therefore, it is highly likely that these two octoploid individuals established themselves in the pots labelled ‘HMM273/2Y’ and ‘HMM274X’ from seed of other P. raoulii individuals growing in the greenhouse. Vouchers of these two plants will be made and deposited at WELT when cytological studies are finished.

Mitotic chromosomes were observed in root tips that were pretreated with a saturated solution of paradichlorobenzene for ~ 18 h at 4 °C and fixed in ethanol/acetic acid (3:1, v/v). The roots were hydrolysed in 1 M HCl at 60 °C for 6–8 min, transferred to 45% acetic acid and then macerated on a slide in a drop of FLP orcein (Jackson 1973), heated over a spirit flame, flattened under a coverslip and observed under the microscope. Meiotic chromosomes were obtained from immature anthers that were fixed in ethanol/chloroform/acetic acid (6:3:1) and stained with FLP orcein as above.

Results and discussion

Chromosome numbers were determined for 58 individuals representing most species and subspecies of native New Zealand Plantago and one introduced species (Table 1). Our sampling of these species complements that of Groves & Hair (1971), which means the chromosome counts now more fully represent their geographical ranges on the North and South Islands. Our results confirm previous counts for most of the species, but we have found potentially new numbers for two species, including dodecaploid (2n=12x=72) for P. unibracteata, which is the first time this chromosome number has been recorded from New Zealand material.

All the plants of P. raoulii, P. spathulata subsp. spathulata, P. spathulata subsp. picta, P. triandra subsp. triandra and P. triandra subsp. masoniae were octoploid (2n=8x=48), as in previous studies.

Confirming previous reports, most plants of P. lanigera sampled were diploid (2n=2x=12). Octoploid (2n=8x=48) plants of individuals identified in the field as P. lanigera by the collector (Michael J. Thorsen, Department of Conservation) were counted from four individuals from Sugarloaf Pass (Fig. 1A). The leaf morphology of these plants indeed approaches P. lanigera, and yet we are not completely satisfied with this identification. Also, because these individuals cluster closely with P. spathulata and P. raoulii, and not P. lanigera, using ITS sequences (Tay et al. 2010b) and AFLP markers (Heidi M Meudt in press), we refer to them here as ‘P. aff. spathulata?’ pending further morphlogical study. No tetraploid individuals were found in our sampling of P. lanigera plants. Tetraploids (2n=4x=24) have been previously reported for P. novae-zelandiae (sometimes considered to be a synonym of P. lanigera, and also called P. aff. lanigera by Dawson 2000) by Groves & Hair (1971) from both Canterbury and Fiordland and by Rattenbury (1957) from Otago. Plantago lanigera/P. novae-zelandiae thus appears to be variable chromosomally and more intensive sampling is needed to determine the geographical extent of its chromosome races.

Fig. 1  Somatic chromosomes of Plantago species. (A) Plantago aff. spathulata? (MLT049W; WELTU 20133) 2n=48; B, Plantago unibracteata (MLT 024W; WELTU 20175) 2n=72; C, Plantago sp. ‘Sylvester’ (BVS s.n. C; WELTU 20215) 2n=96. Scale bar=10 µm.

Most plants of P. unibracteata that we sampled were decaploid (2n=10x=60), but four Nelson individuals were found to be dodecaploid (2n=12x=72), including two individuals identified as P. unibracteata from Lake Sylvester, as well as one individual from Tableland near Balloon Hut (Fig. 1B). This third individual was originally identified as P. sp ‘Sylvester’ by the collector, but we identified it as P. unibracteata based on morphology, and AFLP markers clearly place it with other P. unibracteata and not with P. sp. ‘Sylvester’ (Heidi M Meudt in press). No New Zealand species were known previously to have 2n=72, which has been found in only four other Plantago species worldwide (three South American species from sect. Oliganthos, and the European P. altissima in subg. Albicans; Rahn 1957, 1984). Whether these New Zealand individuals represent another chromosomal race of P. unibracteata, or a new alloploid species, involving an octoploid (2n=8x=48) and the 16-ploid (2n=16x=96) or perhaps more likely P. unibracteata (2n=10x=60) and a diploid species (2n=2x=12), requires further investigation.

Plants of the putative undescribed species P. sp. ‘Sylvester’ (Druce 1993) were verified to have 2n=16x=96 (Fig. 1C). Groves & Hair (1971) counted one individual from Lake Sylvester, and here we report counts of seven additional individuals from three other localities, i.e. Boulder Lake, Tableland near Balloon Hut and Little Lake Sylvester. These data confirm the presence of this entity from several places in north-west Nelson. Only one other species of Plantago is known to share the 2n=96 count, P. correae (sect. Oliganthos) from southern South America (Rahn 1984).

Finally, we report 2n=12 for a naturalized plant from which Tay et al. (2010a) previously obtained an ITS DNA sequence (as ‘P. sp.’; Genbank number FJ024620). This sequence is identical to another Genbank sequence (AY101862) from an individual identified as P. asiatica. The sequence is also 99% similar (1–3 nucleotide differences) to other Genbank sequences of individuals identified as P. asiatica (AJ548977), P. major (AY101861, FJ024619) and P. hostifolia (AB281166). Our count of 2n=12 matches all previous chromosome counts of P. major, but not those of P. asiatica (2n=24, Rahn 1957, 1996). The plants we counted fall within the published morphological range of P. major but differ from many New Zealand collections in having narrower, five-veined, cuneate leaves and shorter flowering portions to their spikes. For these reasons, we have identified the plants we examined as P. major rather than P. asiatica. It is possible that these represent a separate introduction of this widespread and variable species.

Meiosis was observed in three octoploid plants (P. aff. spathulata?, MLT 049Y and MLT 049Z; P. spathulata subsp. spathulata, PGJ 2629X) and in all cases chromosomes formed 24 bivalents at metaphase I. Bivalent formation was also reported by Groves & Hair (1971) for octoploid P. spathulata and 16-ploid P. sp. ‘Sylvester’, and together with the results from Ishikawa et al. (2009) this meiotic chromosome pairing behaviour would suggest that many, if not all, of the New Zealand polyploid species of Plantago are allopolyploids. Indeed, the identity and nature of all New Zealand Plantago, but especially P. sp. ‘Sylvester’ and the higher ploidy individuals of P. lanigera and P. unibracteata mentioned above, are being explored further with DNA sequencing (Tay et al. 2010b), molecular cytogenetics (C Wong & BG Murray unpublished observations), morphometrics (HM Meudt in press), and AFLP DNA fingerprinting (HM Meudt unpublished observations.

Acknowledgements

We thank Lesley Milicich for assistance with maintaining live plants in the Victoria University greenhouses and Dirk Albach for taxonomic advice. We also thank the Department of Conservation (especially Graeme Atkins, John Barkla, and Mike Thorsen), Arnold Dench, David Glenny, Pete Lockhart, Bill and Nancy Malcolm, Jessie Prebble and Barry Sneddon for assistance with field collections. This research was funded in part by the New Zealand Foundation for Research Science and Technology through the Defining New Zealand's Land Biota OBI.