Cardiocrinum DNA barcodes  Feed back button

In order to shed some light on the actual identity of Cardiocrinum cathayanum (hort.) and the relationship, if any, between Cardiocrinum cordatum and Cardiocrinum cathayanum, I have had DNA barcodes produced for a number of taxa in the collection.

 The barcode process 

The system of DNA barcodes for organisms is based on the concept of identitifying one or more genes which can be considered typical of the genus and species and using the sequencing of these genes to identify the organism. [1]

For plants, the chosen genes are the matK and rbcL genes in the chloroplast.

In general, it's not considered possible to identify taxa beyond the Species level [2] although claims have been made to differentiate C. giganteum giganteum from C. giganteum yunnanense [3]. However, this process also used the trnL-F gene sequence.

The criteria for identifying a taxa are arbitary but the following definitions, based on the work done by Meier, [5] are in frequent use. [4]

The 'Distance' figures refer to calculations made from the variations between the two sequences concerned. [6]

 Reference Cardiocrinum barcode sequences 

The BOLD[7] barcode database has nine entries for Cardiocrinum matK and rbcL genes.

This covers all three species and, in fact, all varieties except Cardiocrinum cordatum v. glehnii, but unfortunately there isn't rbcL data for C. cathayanum. This gap was filled by the good offices of the Royal Botanic Garden at Kew who extracted DNA from a descendant of Kew 2007-297[8], which was then sent for barcode sequencing along with the collection samples.

This gives the list of reference sequences shown below, (Plant, gene, source)

  1. Cardiocrinum cathayanum, matK, ex Kew 2007-297
  2. Cardiocrinum cathayanum, matK, BOLD, GBVU2911-13
  3. Cardiocrinum cordatum, matK, BOLD, GBVC3452-11
  4. Cardiocrinum cordatum, matK, BOLD, GBVC3453-11
  5. Cardiocrinum giganteum, matK, BOLD, GBVU2910-13
  6. Cardiocrinum giganteum var. yunnanense, matK, BOLD, GBVT2686-13
  7. Cardiocrinum cathayanum, rbcL, ex Kew 2007-297 ‡
  8. Cardiocrinum cordatum, rbcL, BOLD, GBVC3454-11
  9. Cardiocrinum giganteum, rbcL, BOLD, GBVC3455-11
  10. Cardiocrinum giganteum var. yunnanense, rbcL, BOLD, GBVT2775-13
  11. Cardiocrinum giganteum var. yunnanense, rbcL, BOLD, GBVC3456-11

‡ as of January, 2015 the rbcL gene has only been sequenced for 538bp.

Comparisons were made using one or both of the online BOLD Plant sequences IDS[9] and the Align Sequences Nucleotide BLAST[10].

Comparing all possible combinations of the above gives the tables below.

matK genes
1      BCM
20.00     CM
30.250.16    
40.380.400.20       
50.810.560.400.64 
60.750.480.260.460.24    
            1        2        3        4        5        6                               
rbcL genes
 
7      
80.00    
90.190.46     
100.000.250.41     
110.000.230.370.00    
            7        8        9        10        11                      

The values in the boxes are the p-distance. [11].

What can be understood from these comparisons is,

 Collection Cardiocrinum barcode sequences 

The plants from the collection chosen for the first set of sequences were, [Plant, Collection code, (Initial acquisition code)],

  1. Cardiocrinum cathayanum (hort.), 55o2, (7p0)
  2. Cardiocrinum cathayanum (hort.), 95o1, (13p0)
  3. Cardiocrinum cathayanum (hort.), 110p0, (110p0)
  4. Cardiocrinum cordatum, 108o3, (4p0)
  5. Cardiocrinum cordatum, 15p0, (15p0)
  6. Cardiocrinum cordatum glehnii, 98o2, (6p0)

These were chosen to investigate the two questions referred to at the start: the nature of the plants sold to me as C. cathayanum, whose quoted identity I have reason to doubt and which I refer to as C. cathayanum (hort.), and any relationship there might be between C. cathayanum and C. cordatum.

Samples were sent to the DNA Sequencing Facility of the Natural History Museum who carried out the extraction and sequencing of the DNA of the barcode genes.[12]

As the trnL gene is mentioned as a possible species identifier[13], I also arranged to have it sequenced for the collection samples and for the material provided by Kew for C. cathayanum.

 The results for C. cathayanum (hort.) 

The tables below show the results of the comparisons with the samples themselves and with the reference samples.

matK genes

55o2    
95o10.00   
110p00.28 0.28 
    55o2      95o1    110p0  
55o20.85 0.960.570.710.000.28
95o10.85 0.950.560.710.000.28
110p00.73 0.810.490.610.270.00
Ref.    1        2        3        4        5        6    

rbcL genes

55o2    
95o10.00   
110p00.18 0.18 
    55o2      95o1    110p0  
55o20.19 0.180.390.000.19
95o10.19 0.180.390.000.19
110p00.00 0.000.190.000.00
Ref.     7           8          9         10        11    

trnL genes

55o20.74     
95o10.74 0.00   
110p00.62 0.120.12  
 Kew ref.   55o2      95o1   110p0 

 Discussion 

In view of the fact that all differences fall within the BCM range, no certain identification can be made on the basis of these DNA Barcodes and morphology remains the best indicator of taxonomy.

However, there are variations greater than 0.2 and these can be used to suggest the following,

C. cathayanum comparison graph    Data

I shall continue to label then as C. cathayanum (hort.), but do not intend to change my current taxonometric key which identifies them as C. giganteum yunnanense.

 C. cathayanum & C. cordatum 

My uncertainty about C. cathayanum is caused by the fact that the only difference I have found in the descriptions of cathayanum and cordatum is in the way the leaves are distributed along the flowering stem.

cathayanum: "Leaves absent in proximal part of stem except basal ones, crowded in middle part, laxly spirally alternate in distal part" [14]
cathayanum image
cordatum image
cordatum: "leaves loosely whorled on lower half of the stem" [15]

The problem with these descriptions is that C. cordatum can have the distribution described for cathayanum as shown in the pictures below.

Cordatum image
Cordatum glehnii image

Therefore, the possibility exists that C. cathayanum & C. cordatum are not separate species but varieties of one species and the DNA barcodes were produced to investigate this possibility.

The tables below show the results of the comparisons with the samples themselves and with reference samples of C. cathayanum and C. cordatum.

matK genes

15p0    
98o2*   
108o30.00 * 
    15p0      98o2    108o3  
* No matK sequence was obtained for 98o2
15p00.24 0.270.000.12
98o2* ***
108o30.24 0.270.000.12
Ref.     1          2          3          4     

rbcL genes

15p0    
98o20.00   
108o30.00 0.00 
    15p0      98o2    108o3  
15p00.00 0.00
98o20.00 0.00
108o20.00 0.00
  Ref. 7 
(cathay.)
Ref. 8
  (cord.)  

trnL genes

15p01.11    
98o21.11 0.00   
108o31.11 0.000.00  
 Kew ref.   15p0      98o2   108o3 

 Discussion 

Little can be deduced in the absence of a matK sequence for 98o2.

C. cathayanum comparison graph    Data

The results tend to support the conjecture that C. cathayanum and cordatum are closely related and I shall try to get barcodes from other strains of C. cordatum in future years.

In the meantime I've altered my taxonometric key as I no longer believe that the leaf distribution can be used to distinguish C. cathayanum from cordatum. For the time being, I use the unsatisfactory, hopefully temporary criterion of 'collected location: China' to distinguish them and would be very grateful to anyone who can offer a better!

To do list,

[1] The Consortium for the Barcode of Life. What is DNA barcoding?
[2] Choosing and using a Plant DNA Barcode.
[3] [13] Evaluation of seven DNA barcodes for differentiating closely related medicinal Gentiana species and their adulterants, p.2 Chin Med. 2013; 8: 16, Li M, quoting, [11], Ling KH, Lam H, Shaw PC, Cheng L, Techen N, Khan LA, Chang YS, But PP. Cardiocrinum seeds as a replacement for Aristolochia fruits in treating cough. J Ethnopharmacol. 2010;8:429432. doi: 10.1016/j.jep.2010.04.040.
[4] Turning DNA barcodes into an alternative tool for identification.
[5] DNA Barcoding and Taxonomy in Diptera: A Tale of High Intraspecific Variability and Low Identification Success Syst. Biol. 55(5)715-728,2006
[6] Distance methods
[7] Barcode of Life Data, (BOLD), Systems
[8] I'm grateful for the help provided by Sven Landrein, (Horticultural Botanist), & László Csiba, (DNA Bank Manager), The Royal Botanic Garden, Kew.
[9] BOLD IDS
[10] Nucleotide BLAST
[11] Distance methods, p2
[12] I'm grateful for the help provided by Julia Llewellyn-Hughes, DNA Sequencing Facility Manager, and her staff at The Natural History Museum, London.
[14] "Lilium cathayanum E. H. Wilson, Lilies East Asia, 99. 1925", Flora of China, 24: 135. 2000.
[15] Flora of Japan, (in English), 1953, p.297