Morphological cladistics

      As a basis for the analysis of family-wide patterns, a cladistic study of morphological characters was undertaken.  A primary objective was to see how much structure and character support were implied by morphology, and whether these data justified the elaborate classifications for the family that have been erected in the past.
      Cladistic analysis of morphological data revealed that most previously recognized subfamilies (i.e., Apostasioideae, Cypripedioideae, Epidendroideae) are monophyletic.  Orchidoideae, when the southern hemisphere Diurideae are included and the spiranthoid groups are not, are paraphyletic however, since the spiranthoid groups have been derived from them.  The vanilloids, sometimes recognized as a distinct subfamily, are sister to the epidendroids, and have often been included in that subfamily.  The vandoids, a group that have sometimes been formally recognized, were shown to be a monophyletic group.  This group shares a cellular pollinium stalk (stipe), superposed pollinia (see below), and incumbent anther that bends early in development.  Resolution at lower levels (i.e., tribal) was poor, particularly among epidendroids.

      Inclusion of vegetative characters in the analysis made a significant difference -- the previous cladistic study of Burn-Balogh and Funk (1986) used only floral characters.  One striking example involves the placement of Tropidieae.  This tribe comprises two genera, Tropidia and Corymborkis, which have long been associated with the spiranthoids because of their floral morphology, with an extended rostellum that is equal to the anther in length.  Our analysis placed Tropidieae at the base of the epidendroids, a placement confirmed by subsequent molecular results, indicating that the spiranthoid floral morphology is convergent in Tropidieae and spiranthoids.

 
 
 
 For details see Freudenstein, J. V. and F. N. Rasmussen,  What does morphology tell us about orchid relationships?  A cladistic study.  American Journal of Botany 86: 225-248.
 

Molecular studies

      Molecular data are now being collected to test the morphological pattern and to provide a stronger base of character support on which to base our hypotheses of relationship.  The work is being done in collaboration with a number of other individuals via the Royal Botanic Gardens, Kew.  Our approach is to gather data from the three plant genomes -- chloroplast, mitochondrial, and nuclear, in order to have a broad perspective on patterns in the group.  The chloroplast genes include rbcL and matK, the mitochondrial sequence is from the nad1b-c intron, and 18S data are being gathered from the nuclear genome.  My lab is focusing on nad1b-c and matK, and we are particularly interested in the Epidendroideae, the largest subfamily.
      The mitochondrial nad1b-c Type II intron exhibits a low rate of base substitution, but a rather high rate of length change.  This combination means that it is usually possible to align the sequences with a high degree of confidence.  This situation therefore provides an excellent opportunity in which to study evolution of a mitochondrial intron.
      Early subfamily-level pattern results suggest that vanilloids are not sister to epidendroids, as suggested by morphology, but rather are sister to orchioids + epidendroids (i.e., are a branch at the basal dichotomy of the monandrous orchids).  Most other results agree with the morphological pattern and with other molecular data to date.
 

Developmental aspects

      The most important characters in orchid classification have always been stamen characters.  These include stamen number, anther orientation, pollinium number, and pollinium orientation.  Even though they have been used for nearly 200 years, little detail is known about them.  Study of the development of these features gives additional information and a new perspective on homology.
      In previous work, I have studied the development of pollinium number, which in orchids is usually 2, 4 or 8, and found that a particular number can be achieved in different ways.  At earliest stages, anther primordia have a single mass of sporogenous tissue in each anther theca, which is then subdivided into the appropriate number of pollinia.  This information allows us to make more detailed statements about homology and character states, and therefore increases the amount of information in our cladistic analyses.
      For further details see Freudenstein, J. V. and F. N. Rasmussen. 1996. Pollinium development and number in the Orchidaceae. American Journal of Botany 83: 813-824.
 

      The next stamen features to be studied developmentally are the following:

1.  The anther shown on the left is erect, with the filament at the base, and is similar in general orientation to many lilioid relatives.  The majority of orchids have the orientation shown on the right, in which the anther is bent so that the apex faces outward or can even be lowermost.  This is termed an "incumbent" anther and is clearly associated with a specialized pollination strategy.  The question here is whether all incumbent anthers really are the same.  Preliminary study suggests that different parts of the stamen can be involved, and timing of anther bending has long been known to vary, suggesting that careful study may reveal systematically useful variation.
 

 
2.  Orientation of pollinia within the anther is another important feature, particularly among the epidendroid orchids.  The pollinia may be either side-by-side (juxtaposed, left diagram) or stacked on one another (superposed, right diagram).  The juxtaposed orientation is the plesiomorphic state among orchids.  There is evidence that the superposed state may be achieved in more than one way, depending upon how the anther develops.  Again, detailed study of development is expected to give a refined definition of character states for the Epidendroideae, where additional resolution is sorely needed.