Journal Number 108
May 2008
ORIGINAL PAPERS
Chromosomes of NZ Native Orchids  Part 1 of 2
By Murray Dawson, Landcare Research, Lincoln
In the December 2007 issue of the New Zealand Journal of Botany, Ernst Beuzenberg and I published
our chromosome counts for most of the New Zealand native orchids [1]. These counts were made
in collaboration with our coauthor Dr Brian Molloy’s longterm taxonomic study, and are the latest
addition to the Contributions to a chromosome atlas of the New Zealand flora series initiated in
1958 by Ernst and the late Dr John Hair.
The orchid manuscript was well received by the reviewers, and Professor Rod Peakall (The
Australian National University, Canberra) had this to say:
This is an exceptionally important study being the first to document in such comprehensive detail
the chromosome numbers of the NZ orchid flora. In addition, the paper includes detailed comparisons with other relevant orchid chromosome counts for related Australian and Asian orchids.
Another valuable feature provided by the document is the extensive review of the current (sometimes confusing) state of taxonomic changes. One final important feature of this study is that it highlights future groups, both in NZ and Australia, where it appears chromosome counts will be particularly useful for assisting the resolution of outstanding taxonomic questions. In all, this contribution will not only be of great interest to Australasian orchid researchers, but to orchid researchers worldwide.
Over two articles, I will summarise some of the major findings. This first part provides introductory notes and our chromosome results, and the second part will examine the wider taxonomic implications of our findings.
Background and acknowledgements
Chromosomes are key indicators of biological diversity and provide an important means of investigating relationships between plant groups. Changes in chromosomes, either structural or numerical, can produce different plant forms, and cause reproductive isolation leading to speciation. Numerical changes include the addition of whole sets of chromosomes (polyploidy) or loss or gain of individual chromosomes (aneuploidy).
We began counting chromosomes of native orchids from about 1984, when I was a young technical trainee and Ernst Beuzenberg was teaching me his chromosome techniques at the former DSIR.
However, with Ernst retiring in 1986 and Brian Molloy in 1995, and the lack of major funding for plant chromosome studies at Landcare Research from 1999, full publication of this project was stalled for many years.
Several of our chromosome counts have been cited over the years in various taxonomic revisions (many coauthored by Brian Molloy) and by members of the NZNOG (e.g., [2, 3, 4]).
Unfortunately, many of these citations are brief and rather informal. The full documentation process involves making detailed measurements, drawings, and photomicrographs of the chromosomes from microscope slides; confirming plant identifications and nomenclature (names used); checking locality information; and depositing voucher specimens in the Allan Herbarium at Lincoln. Because this process had not been completed I had to omit our unpublished orchid counts from an otherwise comprehensive index of chromosome numbers of the New Zealand flora [5].
Fortunately, in 2004 we obtained a TFBIS (Terrestrial and Freshwater Biodiversity Information System) contract to publish this outstanding work. Without this support, it would have been impossible to complete the project. Our sincere thanks go to the TFBIS Programme and our application supporters, Ian St George and Peter de Lange.
Orchid experts Mark Clements and David Jones (Centre for Plant Biodiversity Research, Canberra) have also supported us over the years. Many contributors from both sides of the Tasman have provided plant material, and we also thank the private landholders and the Department of Conservation for permission to collect live plants for our chromosome counts.
Previous chromosome counts
Prior to our 2007 paper [1], relatively few chromosome numbers of the Australasian orchids had been published. Chromosomes of 35 Australian species were counted by Peakall and James [6], but even including our counts of Australian material, there are still less than 15% of the genera and only about
6% of the described taxa from that country counted.
For New Zealand, the first chromosome count of an orchid was made in 1942 by John Hair [7], who counted Thelymitra longifolia. Forty years later, Jones et al. [8] counted Winika cunninghamii (then as Dendrobium cunninghamii). More recently, Brian Murray and Peter de Lange have also published several orchid chromosome numbers [9, 10, 11].
Chromosomes of New Zealand orchids previously counted by others are:
Anzybas carsei (2n = 36) [9]
Cryptostylis subulata (2n = c. 60) [11]
Microtis parviflora (2n = 44) P. J. de Lange in [1]
Myrmechila trapeziformis (2n = c. 40) [10]
Pterostylis cernua (2n = 44) [10]
Simpliglottis valida (2n = 40) [11]
Thelymitra longifolia (2n = 26) [7]
Winika cunninghamii (2n = 38) [8]
Our chromosome counts
We counted the chromosomes of many orchid species and genera from Australasia for the first time. For
New Zealand, we made 190 counts of 80 species and about 25 undescribed taxa, tagname entities, and hybrids.
The following list shows the range and diversity of chromosome numbers that we obtained for New Zealand
(for more details and our counts of Australian material, please refer to our 2007 paper [1]):
Acianthus sinclairii (2n = 40)
Adelopetalum tuberculatum (2n = 38)
Adenochilus gracilis (2n = 38)
Anzybas carsei & A. rotundifolius (2n = 36)
Aporostylis bifolia (2n = 40)
Calochilus aff. herbaceus (2n = 22);
C. paludosus & C. robertsonii (2n = 24)
Corunastylis nuda & C. pumila (2n = 44)
Corybas cheesemanii (2n = 54+2)
Cryptostylis subulata (2n = 64)
Cyrtostylis oblonga (2n = 44(+2));
C. rotundifolia (2n = 44+2)
Danhatchia australis (2n = 22)
Diplodium alobulum, D. brumalis, D. trullifolium (2n = 50)
Drymoanthus adversus (2n = 76);
D. flavus (2n = 38)
Earina aestivalis (2n = 40, 41);
E. autumnalis (2n = 40);
E. mucronata (2n = 40(+0  2))
Gastrodia cunninghamii & G. minor (2n = 40);
G. aff. sesamoides & G. “long column” (2n = 38  40)
Hymenochilus tanypodus (2n = 54); H. tristis (2n = 52)
Ichthyostomum pygmaeum (2n = 38)
Linguella puberula (2n = (48), 50)
Microtis oligantha & M. parviflora (2n = 44);
M. unifolia (2n = 88)
Molloybas cryptanthus (2n = 34)
Nematoceras acuminatum, N. iridescens, N. macranthum, N. orbiculatum, N. papa (2n = 36);
N. aff. trilobum
(2n = 36 & 2n = 72)
Orthoceras novaezeelandiae (2n = 42, 44)
Petalochilus aff. carneus (2n = 40);
P. chlorostylus (2n = 39, 40, 41);
P. minor (2n = 39, 40)
Plumatichilos tasmanicum (2n = 50  54)
Prasophyllum colensoi & P. hectorii (2n = 42)
Pterostylis agathicola, P. areolata, P. aff. areolata, P. auriculata, P. australis, P. banksii, P. cardiostigma, P. graminea agg., P. humilis, P. irsoniana, P. micromega, P. montana sens. str., P. paludosa, P. patens, P. porrecta, P. venosa (2n = 44);
P. aff. montana agg. (2n = (43), 44);
P. foliata & P. silvicultrix (2n = 44  46);
P. oliveri (2n = 46)
Simpliglottis cornuta & S. valida (2n = 40)
Singularybas oblongus (2n = 34)
Spiranthes novaezelandiae & S. “Motutangi” (2n = 30)
Stegostyla lyallii (2n = 47, 48)
Thelymitra longifolia, T. aff. longifolia agg., T. malvina, T. aff. pauciflora, T. sanscilia (2n = 26)
Thelymitra aff. ixioides (2n = 28)
Thelymitra aemula, T. cyanea, T. formosa (2n = 40)
Thelymitra ×dentata (T. longifolia × T. pulchella) (2n = (45), 46). Natural hybrid
Thelymitra longifolia × T. pulchella (2n = 45, 46). Artificial hybrid
(Thelymitra longifolia × T. pulchella) × T. pulchella (2n = 52). Artificial hybrid
Thelymitra nervosa (2n = 54)
Thelymitra “Ahipara” & T. “darkie” (2n = 60)
Thelymitra carnea (2n = 62)
Thelymitra hatchii & T. pulchella (2n = 66)
Thelymitra tholiformis (2n = 65, c. 66)
Thelymitra “rough leaf” (2n = 84)
Thelymitra aff. “rough leaf” (2n = c. 84)
Townsonia deflexa (2n = 28)
Waireia stenopetala (2n = 40)
Winika cunninghamii (2n = 40)
The term "2n" is used to indicate that vegetative (nonsexual) tissue was counted. For the orchids this includes root tips, rootstem tubers, and pseudobulbs.
Including our contribution and those from previous workers, about 75% of the species representing all the native orchid genera in New Zealand now have chromosome counts.
Polyploids
Polyploid plants have more than two of the basic (haploid) sets of chromosomes in the nucleus of the cells.
The prefixes tri, tetra, penta, octa, etc. are used to denote the level of ploidy. We found tetraploidy (plants with four times the haploid number) in three genera of New Zealand orchids.
Drymoanthus flavus is diploid (2n = 38) but D. adversus is tetraploid (2n = 76); similarly, Microtis oligantha and
M. parviflora are diploid species (2n = 44) whereas M. unifolia is tetraploid (2n = 88).
Nematoceras is interesting. All New Zealand species are diploid except for within the N. trilobum aggregate where both diploid (2n = 36) and tetraploid (2n = 72) taxa occur. We found predominantly diploids in the North Island, and only tetraploids in the South Island and Chatham Island.
Further chromosome counts are needed to see if this pattern holds up, and to help resolve the 25
or so taxa said to occur in this species complex.
Allopolyploids and chromosome evolution in Thelymitra
Including all Australasian material, there is a remarkably wide range of chromosome counts in Thelymitra, 2n = 26, 28, 32, 36, 40, 45, 46, 52, 54, 56, 57, 58, 60, 62, 65, 66, 70, 84, and 93.
These numbers do not form a simple polyploid series, and in 1998 we explained some of them by natural hybridism, allopolyploidy, and speciation [12]. In our most recent paper [1] we presented additional chromosome counts and interpretation on how these numbers are related.
Allopolyploidy was confirmed only in Thelymitra. Allopolyploids (also known as amphidiploids) are polyploids with chromosomes derived from different species. For example, if two progenitor species, represented by T. longifolia (2n = 26) and T. aff. ixioides (2n = 28), hybridised naturally, they would produce F1 hybrids with the intermediate chromosome number of 2n = 27.
These plants could represent a transition phase as they may not be fertile, because the chromosome complement is unbalanced and the chromosomes cannot pair evenly at meiosis (the sexual cell division cycle). This chromosome set of 2n = 27 would need to double to pair evenly and hence produce fully fertile and reproductively isolated derivatives, in this case represented by the allopolyploid T. nervosa (previously known as T. decora) with 2n = 54. And so on for other examples:
T. carnea, 2n = 62, an Australian and New Zealand allopolyploid possibly between T. pauciflora
(2n = 26) and
T. flexuosa (2n = 36);
T. rubra, 2n = 62, an Australian allopolyploid with similar origins to T. carnea;
T. tholiformis, 2n = 65 & c. 66, a New Zealand allopolyploid between T. aemula (2n = 40) and T. aff. pauciflora
(2n = 26);
T. hatchii, 2n = 66, a New Zealand allopolyploid between T. longifolia (2n = 26) and T. formosa
(2n = 40);
T. pulchella, 2n = 66, a New Zealand allopolyploid between T. longifolia (2n = 26) and T. cyanea
(2n = 40).
We obtained other interesting numbers for some of the undescribed entities. Two similar taxa from Northland, Thelymitra “Ahipara” and T. “darkie”, both share 2n = 60.
A higher chromosome number of 2n = 84 was found in both Thelymitra “rough leaf” from North Auckland and a similar undescribed Thelymitra from Shag Point, Otago. This same number was also found in some Tasmanian Thelymitra, including T. viridis. Closer comparisons are needed between some of these undescribed New Zealand entities and Australian taxa.
2n = 26 and 2n = 28 are the lowest chromosome numbers found in Thelymitra and are considered functional diploid species. If the “original” number is 2n = 28, then it is likely that the lower number,
2n = 26, is derived from the loss of one chromosome pair through aneuploidy.
There are a few species with chromosome counts (2n = 36, 2n = 40) that are intermediate between the low diploids (2n = 26 & 2n = 28) and the high allopolyploids (starting from 2n = 52). These are fertile species likely to have hybrid origins between the diploids and allopolyploids.
Naturally occurring F1 hybrids with the odd number of 2n = 45 were found for Thelymitra ×dentata of New Zealand and T. ×irregularis of Australia. These numbers are intermediate with their suspected parent species, but the chromosome complements are unbalanced and as a consequence the plants are sterile.
Aneuploids
As previously mentioned, aneuploidy is a variation in chromosome number involving the loss (or gain) of one or two chromosomes. Aneuploidy appears to be extensive for the Australasian orchids, and occurs at several levels:
Within the same species or apparently even within the cells of the same plant: Earina mucronata,
2n = 40, 41, 42; Petalochilus chlorostylus, 2n = 39, 40, 41; Pterostylis aff. montana agg., 2n = 43, 44;
Stegostyla lyallii, 2n = 47, 48.
Between species in the same genus. I have already mentioned the example of an aneuploid reduction within Thelymitra of 2n = 28 to 2n = 26, but there is also aneuploidy within the related genus Calochilus from 2n = 24 to 2n = 22. Other examples are: Adelopetalum, from 2n = 38 to 2n = 36; Hymenochilus, from 2n = 54 to 2n = 52 (to 2n = 48?); Prasophyllum, from 2n = 44 to 2n = 42.
Between subgenera: Pterostylis subg. Pterostylis, with 2n = 42, derived 2n = 44 found in the other two subgenera.
Between related genera: Calochilus, 2n = 24, possibly derived from 2n = 26 in Thelymitra; Molloybas and Singularybas, 2n = 34, from 2n = 36 found elsewhere in the Corybas alliance (Anzybas and Nematoceras).
As I will explain in Part 2, chromosome characters have proven remarkably informative for the Australasian orchids, not only at the species level, but in some cases up to the subtribal level.
However, it must be stressed that chromosome information on its own should be treated cautiously
at higher taxonomic levels and that orchid classification is certainly not straightforward.
We have not solved all of the problems and orchidologists will still have to make their own decisions based on our additional chromosome evidence. I should also mention that the plant names used in
the original paper [1] and in these two articles follow Dr Brian Molloy’s taxonomic concepts.
References
01. Dawson MI, Molloy BPJ, Beuzenberg, EJ 2007. Contributions to a chromosome atlas of the New Zealand flora 
39. Orchidaceae. New Zealand Journal of Botany 45: 611  684.
02. Campbell M 1996. Australasian hybrids. New Zealand Native Orchid Group Journal 60: 13  15.
03. Hatch ED 2000. Chromosome counts in the New Zealand orchids. NZ Native Orchid Group Journal 76: 10.
04. Scanlen E 2005. Caladenia surprises. New Zealand Native Orchid Journal 94: 29  36.
05. Dawson MI 2000. Index of chromosome numbers of indigenous New Zealand spermatophytes.
New Zealand Journal of Botany 38: 47  150.
06. Peakall R, James SH 1989. Chromosome numbers of some Australian terrestrial orchids. Lindleyana 4: 85  88.
07. Hair JB 1942. The chromosome complements of some New Zealand plants. 1. Transactions of the Royal Society
of New Zealand 71: 271  276.
08. Jones K, Lim KY, Cribb PJ 1982. The chromosomes of orchids VII. Dendrobium. Kew Bulletin 37: 221  227.
09. Murray BG, de Lange PJ 1999. Contributions to a chromosome atlas of the New Zealand flora  35.
Miscellaneous families. New Zealand Journal of Botany 37: 511  521.
10. de Lange PJ, Murray BG 2002. Contributions to a chromosome atlas of the New Zealand flora  37.
Miscellaneous families. New Zealand Journal of Botany 40: 1  23.
11. de Lange PJ, Murray BG, Datson PM 2004. Contributions to a chromosome atlas of the New Zealand flora 
38. Counts for 50 families. New Zealand Journal of Botany 42: 873  904.
12. Molloy BPJ, Dawson MI 1998. Speciation in Thelymitra (Orchidaceae) by natural hybridism and amphidiploidy.
In: Ecosystems, entomology and plants: proceedings of a symposium held at Lincoln University to mark the
retirement of Bryony Macmillan, John Dugdale, Peter Wardle and Brian Molloy. 1 September 1995.
The Royal Society of New Zealand Miscellaneous Series 48: 103  113.
