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■ Haworthia Study No. 23 (2010. 8) p.6~13 「DNA vs Morphology By Dr. Hayashi」 

DNA vs Morphology   By Dr. Hayashi

 Treutlein & al. (Taxon 52: 193, 2003) reported DNA analysis by matK, rbcL and ISSR (Fig.1). They indicated that the hard leaf Haworthia group is closer to Gasteria and Astroloba than to the soft leaf Haworthia and suggested that the hard leaf Haworthia would be better separated from the genus Haworthia.

 There are many severe defects, however, in their study:-

 1. They got most material from botanical gardens in Germany, which are old collections without locality data. Fig. 3 is a photo of their “H. geraldii” sent by the authors to me. It is definitely not H. geraldii, but a form of H. attenuata. I informed them that it was H. attenuata and asked them to send photos of other materials, but no reply was received. It is obvious that they used their material without any identification of the names.

 2. Aloe aristata and Haworthia aristata have the same DNA accession (registration) number in their material table. This may be a simple mistake, but rather indicates rough checking by the Taxon editors.

 3. Sequence alignment by the authors is very rough, and they overlooked many deletions in their registered sequences.

 4. They emphasized a high bootstrap ratio in their study, indicating stability of their result. Table 1 details sequence data arranged from their data registered (after re-aliment for deletions), showing common variations among more than 3 materials.

 Base pair number supporting a group is counted in Table 1 and added to Fig. 1 by bold red numbers. For example, group ① (Woody Aloe) is supported by 7 base pairs (unique changes only) of matK (No. 5~10 in Table 1) and a rbcL bp (No. 75), and 6 base pairs (matK 1~4 & rbcL 73, 74) are common with soft leaf Haworthia (Group ③).

 For the group (Gasteria + hard leaf Haworthia +Astroloba), only 3 base pairs (matK 47, 48, rbcL 82) support the alliance of this group. As there are no competitive sequences against these 3 pairs, the group will be re-calculated even when only one of these 3 is taken in a bootstrap sampling.

  Bootstrap ratio does not guarantee the validity of the group, especially for the group supported by a few base pairs. It is rather a mathematic deceit in such cases. Table 1 may reveal the real validity of the group better than the bootstrap ratio. DNA researchers must present this kind of table.

 Fig. 4 is a cladogram by Ramdhanii & al. (Alsterworthia Int. 9: 13, 2009) using ITS1 and trnL-F. In this figure hard leaf Haworthia species are also put close to Gasteria and Astroloba, but far from soft leaf Haworthia. Some other DNA studies (mostly by chloroplast DNA) report similar results.

 The results of these DNA studies, however, disagree with traditional taxonomic grouping in Aloaceae, using genetic closeness presumed through hybridization and especially floral structure. It is well known that hard leaf Haworthia hybridizes with Astroloba or Gasteria, but it hybridizes far easier with soft leaf Haworthia.

 In morphology, all the Haworthia groups, Astroloba and Chortolirion share many unique distinctions, especially in their floral structure shown in Table 2 (cf. Fig. 5). Mark ◎ indicates unique (not found in any other groups in Aloaceae) distinctions of the group without any exceptions. Mark 〇 indicates distinctions without exception but not necessary unique. Mark △ shows unique distinctions with exceptions.

 There are many common distinctions among both (soft and hard leaf) Haworthia groups, Astroloba and Chortolirion as shown in pink in Table 2. They share 9 unique floral distinctions without any exceptions (Character No. 1~9), and 6 non-unique floral distinctions without exceptions (Char. No. 11~16). These floral distinctions strongly support alliance among these groups.

 It is also shown in Table 2 that 4 floral distinctions (Character 1, 3, 5, 7) clearly separate 3 major groups of Aloaceae (Aloe, Gasteria and Haworthia). It also indicates that Aloe shares several floral distinctions with the Gasteroid (Gasteria and Poellnitzia) and Haworthia is rather isolated from others. These accord well with the genetic closeness estimated by hybridization.

 On the other hand, there are some characters which support the alliance among hard leaf Haworthia, Astroloba and Gasteria as shown in blue in Table 2. This alliance agrees well with the result by DNA analysis to date. There are, however, only 2 unique distinctions (Char. 28, 29) in these characters and they are all leaf tissue characters.

 Some people may say that DNA data is more important and valid than morphological. It is believed that DNA regulates morphological characters and, hence, it is more reliable than the morphological data, but we should note the problem of DNA data on the following 4 points.

 1. Most DNA sequences studied in Aloaceae to date are of chloroplast DNA. No genes in nuclear DNA have been studied. Of course it is possible to trace phylogenesis by meaningless (junk) sequences. In such a case, however, there is no theoretical priority in DNA data over morphological one.

 2. It is easily presumed that chloroplast DNA may relate to leaf nature. Both Treutlein & al. (2003) and Ramdhanii & al. (2009) mostly depend on the chloroplast DNA data. It is rather natural, therefore, that their results support the alliance (grouping) by vegetative characters in Table 2 (shown in blue). But it is well known that the grouping by vegetative character has lower validity than those by floral character.

 3. Genome size in Monocotyledons is reported as Rice = 430 million (4.3 x 108) base pair, Sweet corn = 5 billion (5 x 109) bp, Wheat = 17 billion bp and Fritillaria (Liliaceae) = 120 billion bp. Though genome size is not known in Aloaceae, if it is 5 billion bp and the average size of a DNA region is 5000bp, there may be a million DNA regions in Aloe or Haworthia. Only 5 DNA regions in Aloaceae have been studied to date, 3 of which are of chloroplast. The others are meaningless regions of nuclear DNA. It is too immature to say something firm based on such studies by a few and nonsense DNA regions.

 4. The importance of data or character in taxonomy or cladistical analysis is not decided by itself, even if it is a gene or exon with important function. It is only evaluated by comparison with other data or characters.

 For example, the number of cotyledons is one of the most important characters to separate dicotyledon and monocotyledon. But it is a vegetative character and there is no theoretical reason why the number of cotyledons is so important in taxonomy. It indicates a large grouping/division in Angiospermae together with many other characters, such as the basic number of perianth, type and location of vascular bundle, type of leaf veins, existence/lack of cambium etc. A character supporting a large grouping together with many other characters is considered important.

 On the other hand, the number of stamens, a character used by Linnaeus, is not used today, though it is a floral character. This character disagrees with the grouping supported by many other characters. It is considered not important.
It is the same with DNA data. If results by DNA data disagree with a large grouping by morphological data, the researcher must collect other data (DNA/ morphology) to support his result. In many cases, if the grouping by DNA data is correct, the researcher also found many morphological characters supporting his result.

 In DNA analysis, we can get much clear data, and many taxonomic systems have been rearranged by them. As traditional major groups in the Aloaceae have firm floral distinctions, the researcher, who tries to change this grouping, should present a firm base in other floral characters as well as DNA data.