biosystematics and conservation: a case study - annals of botany

Biosystematics and Conservation: A Case Study with Two Enigmaticand Uncommon Species of Crassula from New Zealand

P. J . DE LANGE1, P. B. HEENAN2, D. J . KEELING3, B. G. MURRAY3,*,

R. SMISSEN2 and W. R. SYKES2

1Terrestrial Conservation Unit, Department of Conservation, Private Bag 68908, Newton, Auckland, New Zealand, 2AllanHerbarium, Landcare Research, PO Box 69, Lincoln, New Zealand and 3School of Biological Sciences, The University

of Auckland, Private Bag 92019, Auckland Mail Centre, Auckland 1142, New Zealand

Received: 25 April 2007 Returned for revision: 20 June 2007 Accepted: 2 October 2007 Published electronically: 30 November 2007

† Background and Aims Crassula hunua and C. ruamahanga have been taxonomically controversial. Here their dis-tinctiveness is assessed so that their taxonomic and conservation status can be clarified.† Methods Populations of these two species were analysed using morphological, chromosomal and DNA sequencedata.† Key Results It proved impossible to differentiate between these two species using 12 key morphological characters.Populations were found to be chromosomally variable with 11 different chromosome numbers ranging from 2n ¼ 42to 2n ¼ 100. Meiotic behaviour and levels of pollen stainability were both variable. Phylogenetic analyses showedthat differences exist in both nuclear and plastid DNA sequences between individual plants, sometimes from thesame population.† Conclusions The results suggest that these plants are a species complex that has evolved through interspecifichybridization and polyploidy. Their high levels of chromosomal and DNA sequence variation present a problemfor their conservation.

Key words: Chromosome variation, Crassula, Crassula hunua, Crassula ruamahanga, Crassulaceae, conservation,phylogenetics, taxonomy, New Zealand flora.

INTRODUCTION

Remarkably little attention has been paid to the widespreadchromosome polymorphism that is often found when exten-sive studies are made of plant populations (Parker andWilby, 1989; Vaughan et al., 1997; Murray and Young,2001) in the context of plant conservation (Allendorf andLuikart, 2006). For example, recent books (Avise andHamrick, 1996; Henry, 2006) make no mention of thepossible significance of chromosome variation in plant con-servation. Intra-specific chromosome variation can rangefrom structural variants, such as inversions and transloca-tions, which occur as polymorphisms in plant populations,through to variation in the number of whole or partialgenomes that are present (polyploidy). Translocationpolymorphisms, for example, have been found in populationsin a variety of genera in different plant families[Campanulaceae (James, 1965); Onagraceae (Bloom,1974); Poaceae (Sieber and Murray, 1981)] and can, atleast in some cases, be shown to be adaptive mechanismsfor conserving genetic variation (Cleland, 1972; James,1982). Similarly, examples of euploid and, to a lesserextent, aneuploid variation have also been found in taxono-mically diverse groups (Murray, 1976; Murray and Young,2001); these polyploids often have different distribution pat-terns to the diploids and extend the range of the species(Levin, 2002). All of this chromosome variation has animpact on plant conservation, for ensuring that maximumgenetic variation is retained in a species or that reintroduction

programmes do not mix plants of different ploidy levels andthus contribute to reduced fertility via inter-ploidy hybrids(Young and Murray, 2000). This latter problem has longbeen recognized by animal conservation scientists who areoften working with captive populations that, upon cytoge-netic analysis, are found to be chromosomally differentiated(Benirschke and Kumamoto, 1991). There is also a problemof defining the conservation units when populations arefound to be chromosomally variable.

Taxonomy also has a major role to play in conservation,in providing an accurate and authentic name for the plantsof interest, providing a description so that the plant canbe identified, and establishing species relationships and dis-tinctiveness. Taxonomic uncertainty can lead to unnecess-ary conservation effort as segregates can be described asdistinct species that on further study and inspection canbe seen as part of a larger continuum (Pillon and Chase,2007). In this paper, extensive chromosome variation isdescribed in what were believed to be two threatenedspecies of Crassula, that, as a result of these investigations,are combined as a single taxon.

The taxonomic history of Crassula hunua and C. ruamahanga

Crassula hunua A.P.Druce and C. ruamahangaA.P.Druce were first described by Kirk (1899) as Tillaeapusilla Kirk and T. acutifolia Kirk. He distinguishedT. pusilla from the other New Zealand species by the‘leaves spreading, obtuse peduncles thickened upwards’and T. acutifolia by the ‘leaves acute, sepals exceeding* For correspondence. E-mail [email protected]

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the petals’. Kirk (1899) also distinguished plants from theWairoa Falls, Hunua, near Auckland as T. pusilla var.brevia Kirk, stating that these differed from the type bythe ‘peduncles usually shorter than the leaves, not thickenedupwards, carpels more obtuse’.

Cheeseman (1906, 1925) accepted both species, andadded to the descriptions and distinguishing characters pro-vided by Kirk (1899). For T. pusilla s.l., he noted that plantswere ‘intricately branched, matted. Leaves thin, obtuse orsubacute, 1/15–1/12 inches. Petals rather longer than thecalyx’, whereas T. acutifolia had ‘Leaves thin, acute or api-culate, 1/15–1/10 inches. Petals shorter than the calyx’.Cheeseman (1906, 1925) considered both species to beallied, keying them out closely together, and remarkingthat T. acutifolia ‘has precisely the habit of T. pusilla, butappears to differ in the narrower and more acute leaves,and in the calyx-lobes exceeding the petals’. He alsoadded the caution for T. acutifolia that he had ‘seen nospecimens except those in Mr Kirk’s herbarium, whichare few and incomplete’. Cheeseman (1906, 1925) did notmention Tillaea pusilla var. brevia.

Allan (1961) also accepted both species, but unlikeCheeseman (1906, 1925), in the key to Tillaea he artificiallyheightened the differences between them by comparing themwith species from section Glomeratae Haw. and he placedT. pusilla in two locations. First, he aligned it with the verydifferent T. debilis Colenso, because both possess acute tosubacute sepals, and then distinguishing T. pusilla by the‘petals acute to subacute; leaves linear to linear-lanceolate;carpels 2–4-seeded’. Secondly, he placed it close toT. helmsii Kirk and T. multicaulis Petrie on the basis of the‘calyx seg[ment]s or sepals obtuse’ species from which itdiffers by the ‘fl[ower]s minute, inconspicuous; leavesthin’. Allan (1961) placed T. acutifolia with the distinctiveT. sieberiana Schult. et Schult.f. because both had acutesepals, separating T. acutifolia by the ‘stems prostrate,ascending only at tips, greenish leaves thin’. RegardingT. pusilla var. brevia, Allan (1961) repeated Kirk’s proto-logue and typified the name but he did not venture anythingfurther about its taxonomic status.

This remained the state of knowledge for New ZealandTillaea until Toelken (1981), revised the Australianspecies of Tillaea and transferred them to Crassula. Soonafter, Druce and Given (1984) followed suit for the NewZealand species and transferred T. pusilla andT. acutifolia to Crassula L., as C. pusilla (Kirk)A.P.Druce & D.R.Given and C. acutifolia (Kirk)A.P.Druce & D.R.Given. However, these combinationswere predated by C. acutifolia Lam (1786), and C. pusillaSchonland (1913), and were therefore illegitimate.Consequently, Druce and Sykes (in Connor and Edgar,1987) effected the full and legitimate transfer of both taxato Crassula by adopting the nomina nova Crassula hunuaA.P.Druce (; Tillaea pusilla Kirk) and C. ruamahangaA.P.Druce (; Tillaea acutifolia Kirk). Neither Druce andGiven (1984) nor Druce and Sykes (in Connor and Edgar,1987) offered any further advance on the distinction ofeither species, beyond cautioning that the ‘indigenousNew Zealand species are imperfectly known and described. . . [and] are the subject of study by A. P. Druce’.

Subsequently, the New Zealand indigenous and natura-lized Crassula were given a full treatment by Sykes (inWebb et al., 1988). There C. hunua and C. ruamahangawere retained as distinct species, being distinguished fromeach other by differences in leaf and petal shape, lengthand width, and these more detailed and comparativedescriptions included better information about the distri-bution of the species. Since Sykes’s treatment, Moar(1993) provided a detailed description of the pollen ofC. hunua, noting that ‘similar grains occur in C. kirkii(Allan) A.P.Druce et Given, C. multicaulis (Petrie)A.P.Druce et Given, and C. ruamahanga A.P.Druce’, andWebb and Simpson (2001) described and illustrated seedmorphology of C. hunua and C. ruamahanga. Murray andde Lange (1999) and de Lange et al. (2004a) reportedthat they share the same chromosome number, 2n ¼ 42,but this was based on single accessions of each species.

Conservation status of C. hunua and C. ruamahanga

Since the early 1980s there has been the recognition thatneither C. hunua nor C. ruamahanga were common, anobservation, which when coupled with the limited numberof herbarium specimens available for either species,prompted their first conservation listing of ‘rare’ (Given,1990). Subsequent revisions of the New Zealand VascularThreatened Plant List have placed C. hunua as ‘Vulnerable’(Cameron et al., 1993), ‘Endangered’ (Cameron et al.,1995; de Lange et al., 1999), and ‘Acutely Threatened/Nationally Critical’ (de Lange et al., 2004b), whereasC. ruamahanga has been regarded as ‘rare’ (Cameronet al., 1993, 1995), ‘Naturally Uncommon/Sparse’ (deLange et al., 1999) and ‘At Risk/Sparse’ (de Lange et al.,2004b). These listings have stimulated an interest in theecology, abundance and distribution of both species and,for the highly threatened C. hunua, the need to develop sen-sible management protocols has been a high priority (Dopsonet al., 1999). As a result of this interest many new locationsfor both species have been discovered, improving infor-mation on their distribution, ecology, the nature of thethreats they face and their conservation status (de Langeet al., 1998; de Lange, 2000). However, these collectionshave also heightened problems in the circumscription anddelimitation of these two species (Sykes, 2005). In particular,a number of important diagnostic characters, such as sepaland leaf size and shape, showed a tendency to gradebetween species (de Lange, 2000). Most recently, Sykes(2005), in a revised key to the indigenous and naturalizedNew Zealand species of Crassula, keyed out these speciestogether. He commented that ‘Crassula ruamahangaand C. hunua virtually intergrade and cannot be separatedproperly if the variation over the whole of their range istaken into consideration . . . with several [collections] fromthe Chatham Islands occupying an intermediate position . . .it is therefore impossible to adhere to the present taxonomy’.

With this level of taxonomic uncertainty surroundingC. hunua and C. ruamahanga, and the high conservationstatus of C. hunua, further investigations of morphological,chromosomal and nuclear and plastid DNA sequence

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variation have been undertaken to re-evaluate thetaxonomic and conservation status of C. hunua andC. ruamahanga.

MATERIALS AND METHODS

Plant material

All herbarium specimens in AK and CHR that had beendetermined as C. hunua and C. ruamahanga, togetherwith type material from AK, WELT and WELTU, wereexamined, a total of 47 specimens (a full list of the herbar-ium voucher specimens that were used for the morphologi-cal analysis is available online as SupplementaryInformation). The live material used for chromosomal andDNA analysis is listed in Table 1. For consistency, in theResults and Discussion sections the names C. hunua andC. ruamahanga, as applied to the original collections,both herbarium and live, are used, and the names used byearlier authors are treated as synonyms.

Morphological analysis

Five vegetative and seven floral characters used pre-viously to distinguish the two species were scored fromthe herbarium specimens (Table 2). (These non-succulentCrassula spp. preserve well as herbarium specimens). Tenmeasurements were made for each character from each her-barium specimen and the mean, standard deviation andrange calculated. The vegetative characters scored werelength of the third internode from the growing point,lamina length, lamina width, leaf apex angle relative tothe midrib and apiculus. The floral characters were ped-uncle length, peduncle width, flower diameter, calyx lobelength, calyx lobe apex angle relative to the midrib, petallength and petal apex angle relative to the midrib.

Principal component analysis (PCA) for the vegetativecharacters of C. hunua and C. ruamahanga was undertaken.A correlation matrix scaled to have unit variance was usedfor the PCA because the variables were in two differentunits of measurement. The two-sample Wilcoxon rank-sum test was undertaken on each of the 12 characters totest the ability of the measurement data to discriminatebetween the specimens assigned to C. hunua andC. ruamahanga at 95 % and 99 % confidence levels [theWilcoxon rank-test was selected because exploratory dataanalysis indicated the data did not have normal(Gaussian) distributions]. Scatter plots were generated toexamine some characters. The summary statistics, scatterplots, PCA and Wilcoxon rank-test were undertaken usingS-Plus (Statistical Sciences, 1998).

Chromosome and pollen analyses

Mitotic chromosomes were observed in root tips thatwere pre-treated with a saturated solution of paradichloro-benzene for approx. 18 h at 4 8C and fixed in ethanol : aceticacid (3 : 1, v/v). Initially the roots were hydrolysed in 1 M

HCl at 60 8C for 15 min, stained by the Feulgen reactionthen squashed on a slide in FLP orcein (Jackson, 1973)

and observed under the microscope. Despite theirthread-like nature, Crassula root tips required a longerthan normal hydrolysis time and the chromosomes werestill difficult to observe. Therefore a second method wasused that involved washing the fixed root tips twice with0.01 M citrate buffer (pH 4.8) and then digesting with a1 % (w/v) pectolyase (Sigma P3026) þ 4 % cellulase(Onozuka R10) enzyme mix in citrate buffer on a slide ina humid chamber for 25 min at 38 8C. The enzyme waswashed off the root tips with citrate buffer, followed by awash with 45 % acetic acid, and then the root tips weregently macerated, heated and squashed in FLP orcein. Formeiotic analysis, immature flower buds were fixed inethanol : chloroform : acetic acid (6 : 3 : 1, v/v), and theanthers dissected from the flowers and squashed in FLPorcein. Images were captured with an Axiovision CCDcamera and processed using Photoshop. Pollen stainabilitywas measured as the number of grains in which the cyto-plasm was well stained with Cotton Blue in a sample of100 grains. The diameter of pollen grains (25 per plant)stained as above was measured using an eyepiece graticuleand a �40 objective.

DNA extraction for sequencing

Sequences of the nrDNA ITS regions and intervening 5.8S RNA coding region were generated for 20 representativesof C. ruamahanga or C. hunua. ITS sequences were also gen-erated for the other New Zealand members of Crassulasection Helophytum: two individuals of C. helmsii (onefrom Australia and one from New Zealand) and a single indi-vidual each of C. kirkii and C. multicaulis. New Zealandmembers of Crassula section Glomeratae (two individualsof C. sieberiana and single individuals of C. colligata,C. mataikona and C. manaia) were also sequenced andused as outgroups for ITS. As a further outgroup, ITSsequences were also generated from a cultivated individualof C. perfoliata (subgenus Crassula; section Crassula), thetype species of the genus.

Genomic DNA was extracted using QIAGEN Dneasyw

Plant Kits (QIAGEN GmbH, D40724 Hilden, Germany)and samples of fresh, young leaves of cultivated accessionsof the species (Table 1). The ITS region was amplified fromgenomic DNA using ITS4 and ITS5 primers (White et al.,1990) and the plastid trnL intron and trnL-F spacersequences using primers c and f (Taberlet et al., 1991).Each 50 mL reaction contained 20 mM Tris–HCl (pH8.4), 50 mM KCl, 500 nmol of each primer, 2 mM MgCl2,200 mmol of each dNTP, 1 mL of genomic DNA(40–100 ng) and 1 unit of Platinum Taq (Invitrogen, LifeTechnologies, Gaithersberg, MD, USA). The same reactionconditions were used for both regions with an initial dena-turation of 2 min at 94 8C, then 30 cycles of 94 8C for1 min, 60 8C for 30 s and 72 8C for 1 min. Amplificationswere then followed by a final extension of 72 8C for5–7 min. The PCR products were purified using a HighPure PCR Purifying Kit (Roche Molecular Biochemicals,Mannheim, Germany) and the products were sequencedat the Centre for Gene Technology, University ofAuckland. Sequencing reactions were performed using the

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TABLE 1. The chromosome number, pollen stainability, origin, herbarium voucher number and GenBank accession numbersof the Crassula plants used in this study

n 2nPollen

(%) Origin Voucher

GenBank numbers

ITS cpDNA

C. hunuaII þ I 78 60 North I., North Auckland, Clevedon Bridge, Wairoa River AK 294547 EF436533 –– 42 – North I., South Auckland, Hunua, Wairoa Falls AK 288129* – –II þ I 98 96 North I., South Auckland, Hunua, Wairoa Falls, AK 298407 EF436528 –47II (94) 97 #1 North I., South Auckland, Hunua, Wairoa Falls, AK 298402 – –49II (98) 93 #2 North I., South Auckland, Hunua, Wairoa Falls, AK 294737 EF436529 EF43651347II (94) 96 #3 North I., South Auckland, Hunua, Wairoa Falls, AK 298404 – –47II (94) 95 #4 North I., South Auckland, Hunua, Wairoa Falls, AK 298405 – –II þ I (94) 98 #5 North I., South Auckland, Hunua, Wairoa Falls, AK 298406 – –49II (98) 97 #6 North I., South Auckland, Hunua, Wairoa Falls, AK 294739 – –49II (98) 91 #7 North I., South Auckland, Hunua, Wairoa Falls, AK 294738 – –47II 94 2 North I., South Auckland, Auckland City, Mt Albert

Fowlds Park (naturalized in bowling green turf)AK 294551 EF436537 EF436510

II þ I 70 58 Chatham I., Te Whanga Lagoon AK 291565 EF436541 EF43652047II (94) 88 Chatham I., Lake Huro, Mangape Stream AK 298410 EF436532 –

C. ruamahanga47II (94) 99 North I., South Auckland, Kawhia Harbour, Rakaunui

Scenic ReserveAK289902 EF436535 EF436517

– 42 – North I., Wellington, Lake Wiritoa, Scoutlands AK234448 – –45II 90 88 North I., Wellington, Lake Wiritoa, Scoutlands AK 294740 EF436536 EF43652332II (64) 85 North I., Hawke’s Bay, Mangarouhi Stream AK286171 AY787411 EF43650634II (68) 83 North I., Wellington, Carter’s Bush AK 289899 EF436542 EF43651448II (96) 93 North I., Wellington, Wainuioru River, Admiral Farm

StationAK 289901 EF436543 EF436516

II þ I (70) 34 North I., Wellington, Lake Wairarapa Domain AK 289134 EF436534 EF436512– 42 – North I., Wellington, Lake Wairarapa, near Hinaburn AK 294741 – –50II (100) 98 South I., Westland, Barrytown Flats, Maher’s Swamp AK 289900 EF436545 EF436515II þ I (70) 2 South I., Westland, Lake Kaniere AK 294552 EF436525 EF436524II þ I c.84 85 South I., Southland, Invercargill, Andersons Park AK 287202 EF436538 EF436511II þ I (78) 14 Stewart I., Rakeahua Valley Mouth AK 290559 EF436526 EF436518II þ I (78) 29 Stewart I., Rakeahua Valley AK 290558 EF436527 EF436519

C. colligata ssp.colligata

– – – Three Kings Is., West I. AK 298030 EF436549 –C. helmsii

II þ I (42) – Australia, Victoria, unspecified location AK 289904 EF436531 EF436522– 14 – South I., West Coast, Westport, Cape Foulwind AK 256182* AY787405 EF436504

C. kirkii– 84 – North I., Wellington, Wainuiomata River Mouth AK 284526* – –II þ I 78 – North I., Cape Turakirae Scientific Reserve AK 286752 EF436539 EF436505

C. manaia– – – North I., Taranaki, Stent Road, near Warea AK 288422 EF436548 –

C. mataikona– – – North I., Wellington, Ohairu Bay AK 285422 – –– – – North I., Taranaki, Stent Road, near Warea AK 288421 – –

C. moschata– – – North I., Wellington, Moa Point (cultivated) AK 288299 EF436546 –

C. multicaulis28II 56 – South Island, Otago, Rock & Pillar Range, above Taieri

RiverAK 290642 EF436544 EF436521

C. peduncularis– – – North I., Tararua, Cape Turakirae Scientific Reserve AK 286751 AY787409 EF436508

C. perfoliata– – – North I., North Auckland, Sunnynook AK 285781 AY787411 –

C. sieberiana– – – North I., Hawkes Bay, Napier, Ahuriri Lagoon AK 296072 – EF436507– – – North I., South Auckland, Cornwallis, Puponga Point AK 285560 EF436547 –

C. sinclairii15II 30 – South I., Taramoa, Oreti Beach, Long White Lagoon AK 297928

Lake Wairarapa AK288396 EF436540 EF436509

Values in the 2n column in brackets have been calculated from the n values to aid comparisons.II, bivalents; I, univalents; – , measurements not made.* Chromosome numbers taken from Murray and de Lange (1999) and de Lange et al. (2002).


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same primers as the PCR amplifications and the 3.1 ABIPrismTM BIG DYE Terminator Sequencing Kit.Sequencing products were then run on an ABI PrismTM

3100 DNA Sequencer.Forward and reverse sequences were aligned and edited

using the Contig Expressw module of Vector NTI Advance(InformaxTM, InvitrogenTM Life Science Software,Frederick, MD, USA) and the edited DNA sequences werecompared using ClustalX (Thompson et al., 1997).

Parsimony analysis of ITS sequences was undertakenusing PAUP*4.0b10 (Swofford, 2003). Branch and boundsearches were employed, guaranteeing that all shortesttrees were found. DELTRAN character optimization

was used. Bootstrap values were generated from 1000replicate branch and bound searches. The NeighborNet algorithm as implemented in Splitstree 4.0 (Husonand Bryant, 2006) was used to analyse ITS sequenceswith the inclusion of mixed sequences with multiple basesignals coded using ambiguity codes. In calculatinggenetic distances between pairs of sequences, SplitsTree4.0 treats a difference between a single base signal and amixed signal coded using ambiguity codes as half of achange from one base to another. Plastid trnL intron andtrnL-F spacer sequences were analysed using mediannetwork analysis as implemented in Spectronet 1.1(Huber et al., 2002).

TABLE 2. Characters used by various authors to distinguish C. hunua and C. ruamahanga

Character Taxon Kirk (1899)Cheeseman

(1925) Allan (1961)Webb et al.

(1988)

Present study

Mean+ s.d. Range

Internode (mm) C. hunua Usually not .5 2.5+0.9 1.5–3.6C. ruamahanga Up to 10 4.2+2.1 1.9–9.8

Leaf length* (mmunless indicated)

C. hunua 1/16–1/14 inch 1/15–1/10 inch 1–2 0.7–2.8 1.7+0.4 0.8–2.1

C. ruamahanga 1/16 inch 1/15–1/12 inch 1.5–2.5 1.3–5.0 2.0+0.6 1.1–2.9Leaf width (mm) C. hunua 0.3–0.6 0.5+0.1 0.3–0.7

C. ruamahanga 0.4–1.0 0.5+0.1 0.3–0.8Leaf tip angle(degrees)

C. hunua Obtuse Obtuse orsubacute; acute

Obtuse tosubacute

Acute 31.3+17.3 8.4–58.4

C. ruamahanga Acute or apiculate Acute or apiculate Acute to apiculate(sometimeslong-apiculate)

Sharply acute 19.3+7.5 5.6–31.5

Apiculus(degrees)

C. hunua – 0.04+0.11 0.0–0.1

C. ruamahanga Shortlyacuminate or

apiculate

0.08+0.09 0.0–0.3

Peduncle length(mm)

C. hunua Usually longerthan leaves

Longer or shorterthan leaves

Short . Calyx,0.5–1.3

0.9+0.4 0.2–1.9

C. ruamahanga Sessile or onpeduncles shorterthan leaves

Shorter thanleaves

Sessile orsubsessile

. Calyx,0.5–1.0

1.0+0.6 0.1–5.4

Peduncle width(mm)

C. hunua Thickened 0.3+0.1 0.1–0.7

C. ruamahanga 0.2+0.1 0.1–0.6Flower diameter(mm unlessindicated)

C. hunua Minute 1/15 inch +1.75 2.2–3.0 1.6+0.3 1.0–2.8

C. ruamahanga Minute 1/20–1/15 inch +1.5 1.8–2.5 1.6+0.4 0.9–2.7Calyx length(mm)

C. hunua , Petals 0.5–0.7 0.7+0.2 0.3–1.1

C. ruamahanga . Petals 0.8–1.0 1.1+0.3 0.5–1.7Calyx lobe apexangle (degrees)

C. hunua – Acute Obtuse Obtuse orapiculate

35.7+12.3 20.0–60.0

C. ruamahanga Acuminate Acuminate Acuminate Acute 22.2+9.6 5.0–50.0Petal length (mm) C. hunua Longer than

sepals. Calyx . Calyx lobes 1.0–1.4 1.0+0.2 0.6–1.5

C. ruamahanga Shorter thansepals

, Calyx , Calyx lobes 1.0–1.3 1.2+0.3 0.5–1.7

Petal apex angle(degrees)

C. hunua - Acute or subacute Acute to subacute Subacute 31.0+12.3 10.0–60.0

C. ruamahanga acute - Acute to subacute Acute orsharply acute

28.6+18.9 8.0–64.0

Character mean, standard deviation and range are presented from the present study (vegetative character sample sizes: C. ruamahanga n ¼ 30;C. hunua n ¼ 17; floral character sample sizes: C. ruamahanga n ¼ 17; C. hunua n ¼ 8).

* Leaf length differs in Cheeseman (1906, 1925) between the keys and descriptions for T. acutifolia and T. pusilla. The measurements presentedhere are those given by Cheeseman (1906, 1925) in the description.


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RESULTS

Morphology

For the vegetative characters, ten measurements wereobtained from each of the 47 available herbarium speci-mens, but only 25 of these specimens could be scored forfloral characters. In addition, many specimens had only asmall number of flowers so all ten measurements of allcharacters were obtained for only five of the 25 specimens.Therefore, for the vegetative data, a PCA and summarystatistics were undertaken, but for the incompletelysampled floral data only summary statistics were used.

Only three of the five vegetative characters showed stat-istically significant mean differences (internode length,lamina length, leaf apex angle), but in each case therange of values either overlapped or one was a subset ofthe other (Table 2 and Fig. 1). Similarly only two of theseven floral characters had significantly different meanvalues, but again they showed either an overlapping range(calyx lobe length) or the range of one was a subset ofthe other (calyx lobe angle; Table 2). The means for theother characters were not significantly different (Table 2).

As mean internode length and leaf length are the onlytwo vegetative characters that were significantly differenta scatter plot was used to assess their combined effect; allC. hunua samples grouped on the lower left of the graphwhereas the C. ruamahanga samples were scattered acrossthe graph (Fig. 2A). Similarly, as mean calyx length andcalyx lobe apex angle are the only two floral charactersthat are significantly different they were also combined ina scatter plot (Fig. 2B). Again the samples of C. hunuagrouped on the lower half of the graph and theC. ruamahanga ones were scattered across the graph.Thus, although mean internode length, leaf length and

calyx length and calyx lobe apex angle are significantlydifferent for C. hunua and C. ruamahanga, each charactershows considerable overlap and they do not allow the deli-mitation of discrete groups corresponding to C. hunua andC. ruamahanga.

The principal component analysis of the five vegetativecharacters did not distinguish discrete groups that corre-spond to C. hunua or C. ruamahanga (Fig. 2C). The firstand second co-ordinates explained 36 % and 20 % of thetotal variation, respectively. Two additional PCA analyseswere also undertaken to examine patterns of variation, butthe results of these analyses are not presented. For thefirst one, to assess whether there was a geographic com-ponent to the morphological variation, the specimenswere coded for latitude by dividing New Zealand into 13bands of equal degrees of latitude from north to south.For the second, specimens from the restricted geographicarea around Wairoa Falls (ten specimens collected anddetermined as C. hunua) and Wairarapa (seven specimensdetermined as C. ruamahanga) were coded separatelyfrom all other specimens. In neither analysis was thereany geographic pattern, and specimens from differentlatitudes and geographic locations were intermixed.Examples of plants from the geographical range of thespecies are shown in Fig. 3.

Chromosome number, pollen size and stainability

Crassula chromosomes are small, and mitotic spreadswere particularly difficult to obtain as the fine roots con-tained few dividing cells due to the small size of their meri-stems. In addition, the roots were particularly hard andneeded a long hydrolysis time with the Feulgen methodthat then tended to degrade the chromosomes. The enzyme-

FI G. 1. Comparative measurements of (A) internode length; (B) leaf length; (C) leaf apex angle; (D) apiculus length; (E) calyx length; (F) calyx lobeapex angle in the selected specimens of Crassula hunua (h) and C. ruamahanga (r).


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based method yielded much better chromosome spreads(compare A and B in Fig. 4) though the small number ofdividing cells in a root tip meristem remained a problem.Consequently, a switch was made to observe meiosis;these observations were considerably more successful,

although there were again only a small number of cells atthe appropriate stage in the small anthers, and only five toten cells were analysed for each plant. Differentiating biva-lents from univalents was difficult as the chromosomes areso small and numerous.

Plants of C. hunua and C. ruamahanga from 16 localitiesspanning much of the range of the species were examinedand 11 different chromosome numbers were found (Fig. 4and Table 1). These ranged from 2n ¼ 42 in plants fromLake Wairarapa to 2n ¼ 100 in a plant from Maher’sSwamp in Westland. Plants collected from the same localitycan have different chromosome numbers as seen in the largestsample of plants in the present study, from the type localityfor C. hunua at Wairoa Falls, Hunua. In most plants encom-passing a wide range of chromosome numbers, meiosisappeared to be regular and bivalents could be seen clearlyaligned on the metaphase plate (Fig. 4C, E, G). In otherplants, however, univalents were clearly identified aschromosomes that were not aligned on the metaphase plateand were smaller than the bivalents (Fig. 4D, F, H). Onlybivalents, in the majority of cases ring bivalents with twochiasmata, and univalents were observed in the presentsample of plants. No multivalents were found.

Chromosome numbers were also determined for threeother species in section Helophytum (Eckl. & Zeyh.)Toelken for which they were previously unknown(Table 1). Each had a different chromosome number,C. sinclairii had 2n ¼ 30, C. multicaulis had 2n ¼ 56 andC. helmsii had 2n ¼ 14 in a New Zealand plant and 2n ¼42 in one from Australia. Univalents were observed atmeiotic metaphase I in the Australian C. helmsii, but inthe other species meiosis was regular.

The pollen stainability of the C. hunua andC. ruamahanga plants was also variable, ranging from 2 %to 98 %, and low stainability was not always correlatedwith the regularity of chromosome pairing at meiotic meta-phase I. For example, the Bowling Green plant of C. hunuashowed regular bivalent formation but only 2 % pollenstainability, whereas the plant of C. ruamahanga fromLake Wiritoa, which also showed regular bivalent for-mation, had 88 % pollen stainability. Pollen diameterswere measured in the plants with relatively high stainability(.80 %) and these showed a clear correlation with chromo-some number (r ¼ 0.808).

DNA sequences

A heuristic search of the ITS sequence matrix recovered25 664 trees of length 259, a strict consensus tree generatedfrom these is shown on the left-hand side of Fig. 5. One ofthe shortest trees is also shown as a phylogram (Fig. 6).Many of the C. ruamahanga and C. hunua ITS sequencesdisplayed multiple signals at several sites indicating theamplification of multiple sequence types. Where these mul-tiple signals coincide with potentially parsimony informa-tive sites in the sequence alignment these sequences wereremoved from the analysis and a branch and bound searchconducted. This found three shortest trees of length 259(CI ¼ 0.853, RI ¼ 0.953, excluding uninformative charac-ters). The strict consensus of these three trees is shown on

FI G. 2. Scatter plots of leaf length against internode length (A), calyxlobe apex angle against calyx length (B) and principle component analysis(C) of the morphological variation in Crassula hunua (h) and

C. ruamahanga (r).


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FI G. 3. Examples of cultivated accessions of Crassula hunua and C. ruamahanga (plants cultivated under uniform conditions for 2 years) arrangedgeographically north to south: (A) C. hunua, Clevedon Bridge; (B) C. hunua, Fowlds Park; (C) C. hunua, Wairoa Falls (type locality for C. hunua);(D) C. ruamahanga, Kawhia Harbour, Rakaunui Scenic Reserve; (E) C. ruamahanga, Lake Wiritoa; (F) C. ruamahanga, Carter’s Bush ScenicReserve; (G) C. ruamahanga, Maher’s Swamp; (H) C. ruamahanga, Lake Kaniere; (I) C. ruamahanga, Anderson’s Park, Invercargill;

(J) C. ruamahanga, Rakeahua Valley.


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FI G. 4. (A, B) Mitotic and (C–H) meiotic chromosomes of Crassula hunua and C. ruamahanga: (A) C. hunua, Clevedon Bridge 2n ¼ 78; (B) C. hunua,Wairoa Falls, 2n ¼ 98; (C) C. ruamahanga, Mangarouhi Stream, 2n ¼ 64, all II; (D) C. hunua, Te Whanga Lagoon, 2n ¼ 70, II þ I; (E) C. ruamahanga,Carter’s Bush, 2n ¼ 68, all II; (F) C. ruamahanga, Stewart I., 2n ¼ 78, all II; (G) C. hunua #2, Wairoa Falls, 2n ¼ 98, all II; (H) C. hunua #5, Wairoa

Falls, 2n ¼ 94, II þ I. Scale bars ¼ 10 mm.


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the right-hand side of Fig. 5. A further 12 sequences wereidentical. All but one of these was removed prior to parsi-mony analysis, but they were added back to the redrawntree (Fig. 5). Also shown in Fig. 5 are the chromosomenumbers for each individual, where these are known.

In all trees the C. ruamahanga and C. hunua ITSsequences appear as part of a strongly supported cladethat also includes the C. helmsii, C. multicaulis,C. sinclairii and C. kirkii sequences (for simplicity referredto as the C. hunua clade). Crassula moschata is the sister tothis clade, and the sequences of C. perfoliata (sectionCrassula), C. peduncularis (section Helophytum) andthose of the section Glomeratae samples are much moredistantly related (Fig. 6).

The sequence variation within the C. hunua clade is com-plicated, and the presence of multiple sequences withinindividuals makes these data unsuitable for analysis undermost phylogenetic analysis programs. However, theprogram Splitstree 4 is capable of recovering genetic dis-tance information from direct sequences by including ambi-guity codes to represent mixed signals. Interpretation of thegraphs produced by SplitsTree 4, as all representations ofphylogenetic relationships, must be cautious, but theNeighbor Net graph shown (Fig. 7) provides a visual

representation of the patterns of similarity in the sequences.ITS sequence variation is also shown in Table 3 (variablecharacters only).

Plastid trnL intron and trnL-F spacer sequences weregenerated for a subset of 12 of the C. hunua andC. ruamahanga samples, the two C. helmsii provenances,one of the two C. kirkii samples and the C. multicaulis,C. peduncularis, C. sieberiana and C. sinclairii individuals.Further sequences were not generated because it was clearno simple pattern was emerging. Within the New Zealandmembers of the C. hunua clade, six distinct plastid haplo-types were identified. Relationships among these haplo-types were assessed by median network analysis using theSpectronet 1.1 program and are shown in the mediannetwork (Fig. 8). Haplotype A was found in C. sinclairiiand C. hunua Te Whanga Lagoon. Haplotype B wasfound in C. hunua Wairoa Falls and C. multicaulis andC. helmsii Australia. Haplotypes C and D were found invarious geographically diverse C. ruamahanga andC. hunua plants and haplotype D was also found inC. kirkii. Haplotype E was only found in C. ruamahangafrom Rakaunui Inlet and haplotype F was only found inC. helmsii from Westport. In general there was little corre-spondence between plastid and ITS sequence groupings.

FI G. 5. Strict consensus of the most parsimonious trees for the full ITS sequence set (left) and reduced set of sequences with those showing additivesignal at potentially parsimony informative sites removed (right). Numbers above branches are bootstrap values greater than 50 % derived from 1000

replicates.


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DISCUSSION

Morphology

The present analyses of morphological characters that havetraditionally been used to distinguish C. hunua andC. ruamahanga do not support the continued recognitionof two species. Although the means of several characters

were significantly different, the range of variation for thecharacters showed that there is either no differencebetween species or the variation of one species is essen-tially a subset of the variation of the other. Furthermore,as shown by the PCA of vegetative characters (Fig. 2C)and the scatter plots (Fig. 2A and B), combining charactersdoes not support the recognition of discrete groups. These

FI G. 6. Phylogram for one selected most parsimonious tree for the full ITS sequence set.


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combined analyses are particularly informative, since theyeach involved characters for which means are statisticallysignificantly different.

The failure to retrieve any discrete groups in the analysesof morphological characters presented here infers that thenatural variation of the C. hunua/C. ruamahanga complexis mosaic with no obvious taxonomic or geographicpattern. Therefore, on the basis of their morphology weconsider that it is not possible to distinguish two species.

Chromosome and pollen

Crassula hunua and C. ruamahanga present a uniqueexample of chromosome diversity in the New Zealandendemic flora. There is a greater than 2-fold variation inchromosome number in the plants that were sampled withno obvious variation around a single basic number.de Lange et al. (2004a) suggested that the basic numberfor the genus was x ¼ 7, as at that time all the NewZealand species that had been determined containedmultiples of seven chromosomes. In a phylogenetic studybased on matK sequence data, Mort et al. (2001) proposedthat x ¼ 8 is ancestral in Crassula and Crassulaceae buttheir study did not include species from section Tillaea.Regardless of whether the basic number is x ¼ 7 or 8,there appears to be both euploid and aneuploid variationin the C. hunua/C. ruamahanga complex. This raises

questions about the meiotic pairing behaviour of theplants, as no multiple chromosome configurations wereobserved. On the basis of their meiotic pairing behaviour,the plants fall into two groups, one in which there isregular bivalent formation, despite a range of chromosomenumbers, and a second in which there is an appreciable fre-quency of univalent formation. High polyploids and aneu-ploids would be expected to show multivalent formationand the lack of multivalents is not a consequence of lowchiasma frequencies; the majority of bivalents have twochiasmata, so autopolyploids would be expected to formmultivalents (Jackson and Casey, 1982). Bivalent promot-ing mechanisms are also common in polyploid plants(Sears, 1976; Jackson, 1982; Jauhar and Joppa, 1996) andmay be operational in C. hunua/ruamahanga, but clearlymore work is needed to fully elucidate the nature of thechromosome variation that has been discovered. The for-mation of univalents at meiosis can also be indicative ofhybridity as the genomes of the different parental speciesmay fail to pair with each other. The phylogenetic analyses,discussed below, are suggestive of hybridization betweenspecies of section Helophytum, so a further possibility isthat some of the plants show allopolyploid meioticbehaviour.

Crassula helmsii, C. kirkii, C. hunua and C. ruamahangaall show intraspecific chromosome variation, a relativelyrare phenomenon in the New Zealand native flora. Murray

FI G. 7. Neighbor Net graph for ITS sequences of Crassula hunua clade (as defined in text).


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TABLE 3. Variable nucleotide positions in the ITS sequences of Crassula hunua and C. ruamahanga

nrDNA ITS1 and ITS2 base pair position

Specimen sequenced 152 170 203 215 224 236 284 523 534 584 602 685 725 728 732 745

Crassula ruamahanga A G C/T A T C C T A T C C/T T A T TNorth Island, Southern Wairarapa, Lake Wairarapa,Lakeside Reserve (AK 284533)C. ruamahanga A G/T C/T A T C C T A C/T C/T C/T T A T TSouth Island, Barrytown Flats, Mahers Swamp (AK289900)C. ruamahanga A G T A T C C T A C T T T T T TSouth Island, Invercargill, Anderson’s Park (Thomson’sBush) (AK 287202)C. hunua A T T A T C C T A C T T T T T TChatham Island, Te Whaanga Lagoon (AK 291565)C. ruamahanga A T T A T C C T A C C T T T T TStewart Island, Rakeahua Valley(AK 290558)C. ruamahanga A G/T T A T C C T A C/T C/T T T T T TNorth Island, Southern Wairarapa, Carter’s Bush ScenicReserve (AK 289899)C. ruamahanga A G/T T A T C C T A C/T C/T T T T T TNorth Island, Eastern Wairarapa, Admiral Road FarmStation (AK 289901)C. ruamahanga A G/T T A T C C T A C/T C/T T T T T TStewart Island, Rakeahua Valley Mouth (AK 290559)C. ruamahanga G A T T T T T T T T C T C A – TNorth Island, Northern Wairarapa. Mangarouhi Stream(AK 286171)C. hunua G A T T T T T – T T C T C A – TNorth Island, Hunua, Wairoa Falls (AK 288129)C. hunua G A T C/T C/T T T T T T C T C A – CNorth Island, Auckland City, Mt Albert, Rocky NookBowling Club (AK 294551)C. ruamahanga G A T T T T T T T T C T C A – TNorth Island, Kawhia Harbour, Rakaunui Scenic Reserve(AK 289902)

FI G. 8. Median network of plastid trnL intron and trnL-F intergenic spacer sequences for the Crassula hunua clade as defined in the text. Haplotypes arelabelled A–F.


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et al. (1989) calculated that approx. 3 % of species that hadbeen examined chromosomally showed evidence of intras-pecific chromosome variation, and a recalculation, usingthe comprehensive list produced by Dawson (2000), inwhich chromosome numbers are reported for .80 % ofspecies, gives a similar percentage. However, this may bean underestimate as in many cases only single plants of aspecies have been examined and it is likely that more exten-sive sampling will reveal intraspecific variation in othertaxa. Most of the variation that has been found relates topolyploid races and these have been well documented inseveral genera including Veronica [as Hebe] (Murrayet al., 1989; Dawson and Beuzenberg, 2000), Pratia(Murray et al., 2004) and several genera of Poaceae(Murray et al., 2005). Aneuploid variation, as found inC. hunua/ruamahanga, is virtually unknown in the NewZealand angiosperm flora, but well-documented examplesare reported from elsewhere and include several groups ofCrassulaceae and other families (Lewis and Oliver, 1971;Uhl and Moran, 1999; Murray and Young, 2001).

DNA sequences

Two major groups of ITS sequences can be defined forthe purpose of this discussion. One includes onlyC. ruamahanga and C. hunua sequences (group I). Onlytwo of the sequences in group I displayed any additivity intheir sequences, and each of these had only one mixed baseeach, both at positions invariant in the rest of the samples.Group II includes sequences of individuals identified as C.hunua and C. ruamahanga and the sequences of C. kirkii,C. helmsii, C. multicaulis and C. sinclairii. All of theplants identified as C. hunua or C. ruamahanga with groupII ITS sequences displayed a considerable degree of mixedsequence with the exception of the C. ruamahanga samplefrom Te Whanga Lagoon, Chatham Island which displayedan ITS sequence identical to that displayed by C. sinclairiiexcept for one mixed base in the C. sinclairii sequence(Table 3). The Te Whanga Lagoon C. ruamahanga andC. sinclairii samples also shared the distinctive A plastidhaplotype not found in any other plants, providing corrobora-tion of a close relationship between the plants. Some mixedsequences are attributable to combinations of othersequences represented in the graph (Fig. 7). For example,the C. ruamahanga Maher’s Swamp sequence can beobtained by combining the sequences of the C. helmsii orC. ruamahanga Lake Kaniere samples with those of theC. ruamahanga Rakeahua Valley Mouth, Carter’s Bush, TeWhanga Lagoon or Admiral Farm samples. Therefore, it ispossible that at least some of the plants displaying multiplesequences are recent hybrids or have a hybrid ancestry. Nomixing between group I and II sequences was detected.

Given the high ploidy of many of the plants sampled in thisstudy, it is possible that direct sequencing of PCR productshas not provided a true reflection of the full diversity ofITS sequences present in individuals. Those individualsthat displayed a single sequence may also have multipleITS sequence types not detected in this study as a result ofimbalanced copy number between homoeologous nrDNArepeats (possibly as a result of concerted evolution favouring

one homoeologue) or PCR bias. Failure of direct sequencingto recover all the ITS sequence types present in a genome hasbeen documented (Rauscher et al., 2002), as have differencesin copy number of different homoeologous nrDNA repeats(Rauscher et al., 2004). In general, where hybridizationand especially allopolyploidy are important factors in theevolution of a group, ITS sequence evolution can be idiosyn-cratic (Alvarez and Wendel, 2003).

Together with the chromosome and pollen stainabilitydata, these complex sequence patterns are most readilyexplained by hypothesizing a high frequency of hybridiz-ation between biological species and a mixture of allo-and possibly autopolyploidy along with the persistence ofhybrid individuals in nature through vegetative reproduc-tion. Despite the lack of sequences displaying an additivecombination of group I and group II sequence types, onepossibility is that the group I ITS sequences represent theC. ruamahanga/hunua ancestral sequence and the individ-uals identified as C. ruamahanga or C. hunua that displaygroup II sequences have hybrid origins with some of theirancestors including one or more of the other Crassulaspecies of section Helophytum. Another possibility is thattwo cryptic lineages are represented in C. ruamahangaand C. hunua, one of which (group II) has undergonemore hybridization with other species of sectionHelophytum than the other, but both of which have givenrise to a polyploid series. Other more complicated expla-nations are also plausible, and it seems likely that at leastsome of the individuals displaying group I ITS sequencesalso have hybrid origins, given that group I includes indi-viduals that show univalent formation and/or poor pollenstainability. In particular, the possession of a plastid C hap-lotype, not otherwise sampled from plants with group I ITSsequences, may indicate a hybrid origin of theC. ruamahanga Anderson Park, Invercargill sample.Other processes, such as the independent sorting of ances-tral polymorphisms at different genetic loci may also havecontributed to the phylogenetic incongruence observedbetween nuclear and plastid markers in this study and thediscrepancies between relationships indicated by analysisof either sequence data set with morphologically definedtaxa [for general discussion of these see Avise (2000);and for examples see Smissen et al. (2004), Fehrer et al.(2007) and references therein].

Unravelling the details of this hypothetical reticulateevolution in the C. hunua clade will require extensive popu-lation level sampling to address issues such as the level ofsexual reproduction in different populations, the fertility ofdifferent hybrid combinations and phylogenetic relation-ships. AFLP or other multilocus DNA fingerprinting tech-niques may be informative in addressing these questions,and may potentially provide an assessment of nuclear DNAphylogenetic relationships independent of ITS. Clearly inCrassula it is not logical to rely too heavily on sequencesfrom a single region of the genome. Cloning of ITS PCR prod-ucts to explore the diversity of sequences present in thosespecimens displaying mixed signals in direct sequencesmight allow the identification of a larger number of variantswithin individuals, and allow for more robust phylogeneticanalysis of these. The use of sequence-specific primers


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would allow a further test of whether PCR-bias or copynumber differences are resulting in a distorted view ofITS sequence diversity within individuals. While theseexperiments might give a better picture of the complexitiesof ITS sequence evolution in the group, given their cytoge-netic diversity, it is doubtful that these data would be ofdirect relevance to the taxonomic and conservation issuesof Crassula in New Zealand. Despite the sampling andtechnical limitations of the DNA sequence data presentedhere, it is clear that issues of population and breedingbiology within these Crassula taxa need to be addressedbefore DNA sequence variation can be meaningfully incor-porated into historical accounts of evolution within theC. ruamahanga clade. However, the DNA sequence data,particularly the incongruence between nuclear and plastidmarkers do suggest a complex phylogenetic history inaccord with the cytological data presented here. The otherfour Crassula species, C. helmsii, C. kirkii, C. multicaulisand C. sinclairii, which also group in the C. ruamahangaclade, show clear morphological distinctions fromC. ruamahanga and are therefore not included in the recir-cumscribed C. ruamahunga.

Taxonomy

As a result of the morphological, chromosomal and DNAsequence data presented here, and in the context of the pre-ceding discussion, it is not possible to retain the two speciesC. hunua and C. ruamahanga. We consider these to be con-specific and therefore accept only one species in thiscomplex and reduce the other to synonymy. However,C. ruamahanga as recircumscribed probably includes anumber of ‘biological species’ and hybrids between them,but accepting a widespread and variable species is theonly practical and realistic taxonomic option. As bothC. hunua A.P.Druce and C. ruamahanga A.P.Druce werevalidly and effectively published simultaneously by thesame author, under Article 11.5, Note 2, of theInternational Code of Botanical Nomenclature (McNeillet al., 2006) either name has equal priority, and so achoice can be effected by adopting one of these names,or its final epithet in the required combination, and simul-taneously rejecting or relegating to synonymy the other.Accordingly, Crassula ruamahanga A.P.Druce wasselected as the preferred name because the subacute,acute to sharply acute leaves normally associated withthis name is the more usual vegetative state found in popu-lations of this species throughout New Zealand. Plants withthe alternative condition, obtuse leaf apices are virtuallyrestricted to the Wairoa Falls, Hunua. It should be notedthat this latter condition, while frequent at the HunuaFalls, has proved unstable over time in cultivation, withplants from that site reverting to the acute to sharplyacute leaves state ‘typical’ of C. ruamahanga.

Crassula ruamahanga A.P.Druce, N. Z. J. Bot. 25, 128(1987) emend. de Lange et Heenan ; Tillaea acutifoliaKirk, Stud. Fl. N. Z. 143 (1899) ; Crassula acutifolia(Kirk) A.P.Druce et Given, N. Z. J. Bot. 22, 583 (1985)non. C. acutifolia Lam., Encyc. II, 175 (1786)

Type collections. ‘NORTH Island: Hurunuiorangi (flowersnot seen). SOUTH Island: Winton Forest, Southland,T. K[irk], Dec.’

Lectotype (vide Allan, Fl. New Zealand 1 : 199. 1961).Hurunuiorangi, May 17, 1877, T. Kirk. WELT SP050125a

¼Crassula hunua A.P.Druce, N. Z. J. Bot. 25, 128 (1987)

;C. pusilla (Kirk) A.P.Druce et D.R.Given, N. Z. J. Bot.22, 583 (1985) non C. pusilla Schonland Rec. AlbanyMus. II, 451 (1913)

;Tillaea pusilla Kirk, Stud. Fl. N.Z. 143 (1899)

Type collections. ‘NORTH Island: banks of streams, &c.Kawakawa, Bay of Islands, T. K[irk]. AucklandCheeseman! Dec.’

Lectotype (vide Allan 1961 : 198). ‘WELT SP050132!,Tillaea pusilla T. Kirk, Stud. Fl. N.Z., Auckland,Mr Cheeseman.’

Notes. It has not been possible to locate the Kawakawa Riverspecimens that Kirk (1899) cited in his protologue, and thatCheeseman (1906, p. 142) evidently saw. Kirk (1899) statesclearly that he used material gathered by Cheeseman fromAuckland in December, yet the label details on WELTSP050132 suggests he was unsure if Cheeseman had col-lected that specimen. Cheeseman (1906, p. 142) makes itclear he did, but not from Auckland, which he does notmention at all, stating rather that he collected it from theWairoa Falls, near Hunua. Whatever the exact details, it is‘Auckland’ we must assume in a broad geographic senseand not the more specific Wairoa Falls, Hunua which muststand as the type locality for Tillaea pusilla. ¼ Tillaeapusilla var. brevis Kirk, Stud. Fl. N.Z. 143 (1899) (‘brevia’)

Type collection. ‘NORTH Island: Wairoa Falls, Hunua,T. K[irk].’

Lectotype (vide Allan 1961). WELT SP050134!, Tillaeapusilla var. brevia T. Kirk, Stud. Fl. N.Z., Wairoa Falls,Hunua, T. Kirk, Dec 1868.

Notes. Allan (1961, p. 198) typified this taxon in the follow-ing manner ‘Type: specimens in W[ELT] labelled as var.brevia Wairoa Falls, Hunua, Dec 1868’. At WELT there isnow only one gathering, WELT SP050134 (L. Perrie,WELT herbarium, Te Papa Tongarewa – The Museum ofNew Zealand, New Zealand, ‘pers. comm.’), and this muststand as the lectotype. We prefer to regard it as lectotypebecause we cannot be sure that there are no other specimensof this gathering in herbaria known to hold Kirk material, andAllan’s wording suggests there might have been. WELTSP050135, though matching Kirk’s protologue as to location,name and authors hand was collected in March 1867.Although Kirk (1899) does not provide a date for his typecollections in his protologue, by Allan’s full and direct refer-ence to a specimen including collection date as lectotype,WELT SP050135 must now stand as a paralectotype.

Notes. Because C. ruamahanga now includes both C. hunuaand C. ruamahanga and our circumscription differs substan-tially from that of the taxon originally described by Kirk


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(1899), and, in addition, characters either not seen or una-vailable to Kirk have been included, formal recognition ofthis is required (see Article 47, Recommendation 47A.1. ofthe ICBN; McNeill et al., 2006).

Description. Diminutive, perennial herb forming diffuse toextensive, compact to loose, yellow-green, green to darkgreen, moss-like mats; stems much branched, prostrate,rooting at nodes, scarcely ascending at tips: internodes1.5–9.8 mm, terete, 0.3–0.6 mm diam. Leaves connate atbase, 0.7–2.9 � 0.3–1 mm, 0.3–0.5 mm thick, linear-oblong, linear-lanceolate, lanceolate or elliptic-lanceolate,flattened to slightly concave above, convex beneath, apexsometimes obtuse, usually subacute, acute to sharplyacute; shortly acuminate or with an apiculus up to0.3 mm long, caducous. Flowers solitary in leaf axils, star-like, sweetly fragrant, 4-merous, peduncle 0.2–5.4 mmlong, 0.7–0.7 mm wide, not or scarcely elongating at fruit-ing. Calyx lobes 0.5–1.7 � 0.3–0.5 mm, triangular totriangular-ovate, obtuse, acute,+ acuminate, usually withan apiculus up to 0.1 mm long, caducous. Corolla 0.9–3.2 mm diam.; petals 0.5–1.7 � 0.3–0.6 mm, narrowlyelliptic-ovate, ovate to triangular-ovate, white or pink-flushed, subacute, acute to sharply acute, � calyx.Stamens 4, filaments 0.9–1.2 mm, white, terete; anthers0.4–0.6 mm, rose-pink; pollen (vide Moar, 1993) paleyellow, usually anisopolar, tricolporate, sometimes tetracol-porate; ectoaperture broad, long, generally branching, andoften fusing to form a polar cap at one, or both, poles;polar axis 13–14 mm, equatorial axis 12–15 mm. Scales4, 0.3–0.5 mm long, narrowly to broadly cuneate.Follicles 4, 0.9–1.2 � 0.6–1 mm, ovoid to ellipsoid,turgid, recurved, smooth. Style minute, approx. 0.01 mm,recurved. Seeds (vide Webb and Simpson, 2001) 2–4 perfollicle, 0.4 � 0.25 mm, narrowly elliptic, elliptic-oblong,to oblong, more or less terete to almost square in section,apex and base truncate, base sometimes with a slight pro-jection. Testa dull, dark henna, dark purple-brown,orange-brown or dark red-brown, almost smooth or withan indistinct regulate-colliculate pattern. Fl. (Aug-)Jan(–Apr). Fr. (Sep–)Jan(–May). Chromosome number 2n ¼42 to 2n ¼ 100 (vide Murray and de Lange, 1999; deLange et al., 2004a; this paper).

Representative specimens. NORTH ISLAND: NORTHAUCKLAND: Northern Wairoa River, T. F. Cheesemans.n., n. d., AK 4554. SOUTH AUCKLAND: Wairoa Falls,T. Kirk 153, n.d., AK 11433; Falls of the southern Wairoa,T. F. Cheeseman s.n., Apr 1896, AK 4555; Wairoa RiverGorge, R. O. Gardner 4344, 3 Oct 1984, AK 168426;Waihou (Thames) River, at Hikutaia Wharf, R. Mason7304, 30 Nov 1959, CHR 113044; Rakaunui ScenicReserve, Rakaukeke Inlet, Tawairoa Stream, P. J. de Lange4312, 3 Apr 2000, AK 252131. WELLINGTON:Wanganui, Lake Wiritoa, Scoutlands, C. C. Ogle 3451, 31Jan 1999, CHR 518730; Northern Wairarapa, MangarouhiStream, P. J. de Lange 5994, 14 Apr 2004, AK 286171;Wairarapa, Carter’s Bush, L. B. Moore s.n., 29 Nov 1958,CHR 124456; Ruamahanga River, SE of Carterton,A. P. Druce s.n., Oct 1973, CHR 208995; Lake Wairarapa,Lakeside Reserve (NE end), P. Enright s.n., 16 Nov 2003,

AK 284533; Wellington Harbour, Mokopuna Island,P. J. de Lange 1580 & G. M. Crowcroft, 4 Sept 1992, AK212059. SOUTH ISLAND: NELSON: Westport Domain,Buller River, R. Mason s.n. & N. J. Moar 1739, 25 Jan1953, CHR 81569. WESTLAND: Barrytown Flats, MaherSwamp, P. J. de Lange 1002, 1 Sept 1991, CHR 473589.CANTERBURY: Ashburton River, H. H. Allan s.n., n.d.,CHR 11950; South Canterbury, near Pareora River,R. Mason s.n., 9 May 1945, CHR 51479. OTAGO: Otago,Mihiwaka Hill, P. N. Johnson s.n., 3 May 1981, CHR363947; SOUTHLAND: Winton Forest, T. Kirk s.n., n.d.,AK 4557; Invercargill, Andersons Park (Thomson’s Bush),B. D. Rance s.n., 25 Jan 2004, AK 287202; Waitutu Forest,east of Lake Poteriteri, C. C. Ogle 1068, 21 Jan 1984, CHR417095; Waitutu Forest, west of Waitutu Hut, aboveWaitutu River, B. D. Rance s.n., 5 Feb 2007, AK 298466;Lake Manapouri at mouth of Spey River, M. J. A. Simpsons.n., 16 Feb 1959, CHR 111815; Lake Manapouri, SupplyBay, B. D. Rance s.n., 17 Jan 2007, AK 298442.STEWART (RAKIURA) ISLAND: Mouth of theRakeahua Valley (ex cultivated), P. J. de Lange 6492, 29Apr 2005, AK 290559; Rakeahua Valley (ex cultivated),P. J. de Lange 6491, 29 Apr 2005, AK 290558.CHATHAM ISLANDS: Rekohu (Chatham Island), LakeHuro, Mangape Stream outlet, P. J. de Lange CH223 &G. M. Crowcroft, 29 Mar 1996, AK 229937; Pitt Island,G. Walls s.n., 16 May 1999, CHR 535256.

For a revised key to New Zealand Crassula SectionHelophytum, see Table 4.

Conservation implications

With the recircumscription of C. ruamahanga to includeplants previously known as C. hunua, a reassessment of theconservation status of C. ruamahanga is warranted.Previously, C. hunua sensu A.P.Druce had been assessedusing the New Zealand Threat Classification System(Molloy et al., 2002) as ‘Acutely Threatened/NationallyCritical’ (de Lange et al., 2004b), because the specieswas believed to be restricted to two sub-populations(Wairoa Falls and the adjacent Gorge, and the ChathamIslands), which collectively occupy an area of �1 ha.

With the reduction of C. hunua into the synonymy ofC. ruamahanga, the recircumscribed C. ruamahanga isnow more widespread than before with extant populationsranging from the Wairoa Falls, Hunua, in the NorthIsland, through most of the South Island and Stewart andChatham Islands. Within this range, the species has a natu-rally sparse distribution but at some localities (e.g. in thesouthern Wairarapa and Southland) it may be locallycommon. The range of habitats occupied is varied withthe species being most commonly recorded from ephem-eral, often muddy or silty pools within lowland alluvialforest and from lake margin turf communities. Thespecies may also be an urban weed, and it has been foundin bowling green turf and in damp shaded sites inAuckland, Wellington, Christchurch and Dunedin. Evenwith the combination of the two taxa some range contrac-tion is known, as C. hunua sensu A.P.Druce has not beenrecorded north of the Wairoa Falls, Hunua since 1906,


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when Cheeseman last reported it from the Wairoa Rivernear Dargaville. In contrast to its apparent extinction fromthe Northland Peninsula, there have been discoveries ofnew populations, often from quite unexpected locations inthe northern Wairarapa, Wanganui and Waikato regions(de Lange et al., 1998; de Lange, 2000). These discoverieshave increased the number of known populations and it ispossible that C. ruamahanga could be rediscovered inNorthland. Therefore, it is concluded that the recircum-scribed species is most appropriately assessed using theNew Zealand Threat Classification System as ‘At Risk/Sparse’, a conservation rating which most accuratelyreflects the current level of knowledge about the ecology,distribution, range of habitat preferences and the nature ofthreats it faces.

The chromosomal and DNA sequence variation that hasbeen uncovered in these studies pose questions concerningthe conservation of C. ruamahanga. In addition, they high-light the need for further critical study at the populationlevel using a range of different molecular markers. Thisstudy has also shown that the range of molecular markers(plastid DNA and nrDNA) used for taxonomic research,when undertaken on multiple samples, spanning a largegeographic range, are not necessarily reliable in thisgroup. For all these reasons it is important to conservethese plants at the maximum number of sites if the fullrange of variation is to be maintained.

SUPPLEMENTARY INFORMATION

Supplementary information is available online at www.aob.oxfordjournals.org and lists herbarium specimens used formorphological analysis of Crassula hunua andC. ruamahanga.

ACKNOWLEDGEMENTS

We acknowledge the considerable collecting efforts, adviceand comments received from the following former or

current Department of Conservation Staff, M. Thorsen,G. Foster, R. Stanley, P. Knightbridge, B. Rance,A. Baird, B. Gibb and I. Keenan. C. Ogle of Wanganuiand P. Enright, of Featherston, Wairarapa, helped procurelive material from their respective regions. We also thankR. K. Brummitt (Royal Botanic Gardens, Kew),H. R. Toelken (AD, Adelaide, South Australia) andC. Ogle for comments and advice and L. Perrie andB. V. Sneddon in locating type material. The late F. Pitt(WELT) helped resolve typification issues by clarifyingaspects of the acquisition and curation of the Kirk herbar-ium, now housed at WELT, Te Papa, Museum of NewZealand, Wellington. Funds for this study were providedby the New Zealand Department of Conservation,Conservation Management Units Investigation 3838 andthe Foundation for Research, Science and Technology.

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TABLE 4. Revised key to New Zealand Crassula Section Helophytum

1. Flowers often in small clusters in the leaf axils, sometimes solitary, inconspicuous; petals closely appressed to carpels orwith spreading tips, usually , calyx lobes, sometimes equal to calyx lobes, or nearly so Section GlomerataeFlowers always solitary in the leaf axils, conspicuous; petals spreading and corolla star-like, usually significant , calyxlobes, rarely nearly equal 2. Section Helophytum

2. Leaves often .7 � 2 mm; flowers 4–7 mm diam.; plants terrestrial C. moschataLeaves ,7 � 1.6 mm; flowers , 4 mm diam.; plants aquatic to semi-aquatic, if terrestrial then leaves always smaller 3

3. Plants strict summer annuals. Pedicels elongating to 13 mm long at fruiting; seeds .10 per follicle C. peduncularisPlants perennial. Pedicels �7 mm long, scarcely elongating at fruiting; seeds �5 per follicle 4

4. Leaves+ triangular-lanceolate, clustered and imbricate at stem nodes, acuminate, petals distinctly rounded C. multicaulisLeaves linear to elliptic, or if+ lanceolate then not triangular lanceolate, usually opposite, occasionally clustered at stemnodes; petals obtuse, subacute to acute 5

5. Petals 0.9–1.5+ calyx lobes 6Petals 2–3 � calyx lobes 7

6. Leaves (0.7–)2(–2.8) � (0.3–)0.6(–1) mm, not noticeably succulent; calyx lobes (0.5–)0.8(–1.6) mm long; petals equalto or 2 � width C. ruamahangaLeaves (2.5–)8(–10) � 0.7–1.6 mm, obviously thick and succulent; calyx lobes 1–1.5 mm long, petals with length 1.7–1.8 � width C. helmsii

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