Evolution of Mitochondrial Malate Dehydrogenase in Birds Since The

山階鳥研報(J.Yamashina Inst. Ornith.),14:1-15,1982

Evolution of Mitochondrial Malate Dehydrogenase in Birds

Nagahisa Kuroda*, Ryozo Kakizawa*, Hiroshi Hori**, Yutaka Osaka**, Nanako Usuda* and Seiitiro Utida*

Abstract For a comparative study of electrophoretic mobility of mitochondrial malate dehydrogenase (M-MDH), red cells or breast muscle (in some cases) from 185 species (57 families, 22 orders) of birds were subjected to starch-gel electrophoresis at pH7. The cathodal M-MDH mobility of Anas platyrhynchos was used as standard, a value of 100, and relative mobilities of other birds were estimated. Thus far tested, the M-MDH enzyme mobilities did not vary within species and genera. Even within families and orders mobilities were very conservative when compared with other enzymes. Terrestrial and aquatic bird orders following the Wetmore's system showed the standard enzyme mobility 100; i. e. Struthioniformes, Procellariiformes, Sphenisciformes, Podicipediformes, Pelecaniformes, Ciconiiformes, Phoenicopteriformes, Anseriformes, Galliformes, Gruiformes and Charadrii- formes. Among these, 4 orders (Pelecaniformes, Ciconiiformes, Galliformes and Chara- driiformes) included families with mobilities higher than 100. Exceptional orders were Tinamiformes (160) and Falconiformes (140). On the other hand, arboreal bird orders generally had mobilities exceeding 100, ranging 140-360. The values increased progres- sively toward smaller bodied orders; i. e. Columbiformes (140, 190), Cuculiformes (200), Strigiformes (200), Caprimulgiformes (200), Apodiformes (220), Coraciiformes (220, 250), Piciformes (230, 300) and Passeriformes (360). An especially high mobility was found in Psittaciformes (300-360). The variations of M-MDH mobility in birds, should have evolutionary as well as eco-physiological implications.

Introduction

Since the extensive work of Sibley and his colleagues on the electrophoretic pattern of egg white proteins (Sibley 1960, 1970, Sibley & Ahlquist 1972), avian systematic information has been derived from other protein sources and nucleic acids (Sibley et al. 1974, Sibley & Wilson 1980). Recently, isoenzyme studies have been used for ellucidat- ing both interspecific relationships (Smith & Zimmerman 1976, Barrowclough & Corbin 1978, Avise et al. 1980a, b) and intraspecific population structure (Corbin et al. 1974, 1979) in birds. Among various enzymes, malate dehydrogenase is one of the most conservative proteins. Kitto & Wilson (1966) compared the 'supernatant' form of malate dehydrogenase (S-MDH) from over 100 species of birds. Most species had S-MDH of the same relative electrophoretic mobility as that of chicken S-MDH, which was given a reference value of 100. Members of ten families of Charadriiformes showed S-MDH mobility of 55. The Apodiformes including the swifts and hummingbirds had an enzyme of mobility 63. The enzyme malate dehydrogenase exists in two forms in mammals,

Received 7 January 1982 * Yamashina Institute for Ornithology , 8-20 Nampeidai-machi, Shibuya-ku, Tokyo 150. ** Nogeyama Zoological Gardens of Yokohama , 63-10 Oimatsu-cho, Nishi-ku, Yokohama 220.

1 2 N. Kuroda, R. Kakizawa, H. Hori, Y. Osaka, N. Usuda & S. Utida birds and reptiles. One form occurs in the soluble fraction of cytoplasm and moves toward the anode during starch gel electrophoresis at pH7. The other form occurs in mitochondria and moves toward the cathode. These two forms of malate dehydrogenase in birds are distinguishable by amino acid composition, immunological properties as well as by susceptibility to substrate inhibition (Kitto & Kaplan 1966, Karig & Wilson 1971). In this study, the electrophoretic mobility of mitochondrial malate dehydrogenase was compared with mainly red cells from 185 species of birds belonging to 53 families and 22 orders. The mobility of S-MDH was also estimated for all cases.

Materials and Methods

Animals. All blood samples and specimens of birds were collected from April to December of 1981. Blood sampling of foreign birds was mainly performed at Nogeyama Zoological Gardens in Yokohama, and also at Tokyo Tama Zoological Park and Izu Natural History Park (Rhea americana). Some dead specimens were obtained from dealers. Blood samples and carcasses of Japanese birds were collected from the Bird Rescue Centers annexed to Nogeyama Zoo, Yokohama, Shinhama Bird Reserve, Chiba and Pheasant Breeding Station, Maki-Machi, Niigata Prefecture, and also from the Bird Banding Station, Fukushimagata, Niigata Prefecture. Blood samples of the following species were collected from sites as follows: swift (Apus affinis), at nesting site in Ito City, Shizuoka Prefecture, cormorant (Phalacrocorax carbo), from a colony in Chita Peninsula, Aichi Prefecture and grebe (Tachybaptus ruficollis), from the Aquarium of Inokashira Park Zoo in Tokyo. The species and numbers of individuals used are shown in Table 2-4. Electrophoresis. About 0.2-2ml of blood was collected by brachial venipuncture using heparinized syringes or haematocrit microtubes. Blood cells were separated from

plasma by centrifugation at 5000×g for 10min and washed twice with 0.9%NaCl. Haemolysates were obtained by addition of 1 volume of saline and by freezing and thaw-

ing three times. After centrifugation, the supernatant was stored at-30℃ until used. From fresh or frozen specimens, tissue samples were taken from heart, breast and skeletal

(leg) muscles, liver and kidney. After freezing at-30℃, the exudates from thawed tissues were used as samples. Samples of hemolysates or tissue exudates were subjected to electrophoresis in hori-

zontal gel slabs (12.5×15×0.6 or 25×15×0.6cm) of 12.5% electrostarch (Lot. No. 392) at pH7.0. The amine-citrate buffer system developed by Clayton & Tretiak (1972) and modified by Numachi (1979) was used. The electrode buffer contained 0.04M citric acid and 0.068M N-(3-aminopropyl) diethanolamine, pH7.0 and the gel buffer contained 0.002M citric acid and 0.004M N-(3-aminopropyl) diethanolamine, pH7.0. Electrophoresis was performed for about 2hr in a refrigirator at 5℃ with a constant current of 5.3-6.0mA/cm2 (about 26v/cm). A box containing ice was placed on the gel for thermal stabilization during electrophoresis. After electrophoresis, gels were sliced into 1mm thicknesses with a wire gel cutter, the second slice was stained with the nitro blue tetrazolium staining mixture specific for Evolution of M-MDH in Birds 3

malate dehydrogenase as described by Fine & Costello (1963). Stained and fixed gel slices were glycerinated and preserved as dried transparent sheets by the method of Numachi (1981). Enzyme mobility was measured from the dried sheets. For comparative study of M-MDH mobility, Anas platyrhynchos was used as the standard, assigning it a value of 100, and the mobilities of other samples were expressed as relative values. In regard to S-MDH mobility, the same value as reported by Kitto & Wilson (1966) was used in this study as standard value of 100. The value 100 of S- MDH was 2.5 times higher than that of M-MDH. Chemicals. Nitro blue tetrazolium was obtained from Dotite; sodium L-malate from Wako Chemicals; N-(3-Aminopropyl) diethanolamine from Aldrich Chemical Co. , NAD from Oriental Yeast Co., Phenazine methosulfate from Sigma Chemical Co., and electrostarch from Electrostarch Co. Taxonomic sequence. The taxonomic sequence in this paper is based on Peter's check-list, Vol. 1, second ed. (Mayr & Cottrell (eds.) 1979) for Struthioniformes to Anseriformes, and the classification of Wetmore (1960) for Galliformes to Passeriformes . The species names mainly follow the reference list of Morony et al. (1975).

Results

1. Homogeneity of red cells and tissues in M-MDH mobility The cathodal mobility in starch-gel electrophoresis of mitochondrial malate dehydrogenase (M-MDH) was compared with red cells and various tissues using the duck Anas acuta and the crow Corvus macrorhynchos. Although the mobilities differed in the two species, the main-and sub-bands of breast, heart and skeletal muscles, liver, kidney, as well as red cells showed identical values within each species (Fig . 1). The

Fig. 1. Electrophoretic patterns of M- and S-MDH in various tissues from Corvus macrorhynchos and Anas acuta. 4 N. Kuroda, R. Kakizawa, H. Hori, Y. Osaka, N. Usuda & S. Utida

mobilities of sub-bands were always slower than those of the main bands, which were the recorded values of enzyme mobilities. Since the activity of red cell M-MDH was lower than the tissue enzyme, the density of red cell sub-bands was usually weak, and often only the main band appeared. The same mobilities of red cell and breast muscle enzymes were also observed in the shearwater Calonectris leucomelas, the roller Eury- stomus orientalis, and several species in Ardeidae, Anatidae, Phasianidae, Columbidae, Psittacidae and Passeriformes. In this study, red cells were used as the main material, but in some cases breast muscle was used when fresh blood samples were not available. 2. Intraspecific homogeneity in M-MDH mobility To test the intraspecific variation in mobility of M-MDH, breast muscle exudates from 100 starlings (Sturnus cineraceus) were subjected to electrophoresis. No variation was detected in the mobilities and relative intensities of the sub-bands. Similarly, no intraspecific or interspecific variations were found in red cell MDH when 125 individuals from 26 species of Anatidae were compared, including 24 Aix galericulata, 10 Dendrocygna iavanica, 9 Dendrocvgna bicolor and 7 Anas poecilorhyncha (Fig. 2). 3. Birds with relative M-MDH mobility 100 (Table 1 and 2, Fig. 3) As stated above, 26 species of Anatidae (Table 2) showed identical mobilities, namely 100. One species, Chauna chavaria (Anhimidae) also indicated the same mobility as that of Anatidae. Therefore, all members in Anseriformes seem to have M-MDH of uniform mobility 100. Although the number of species tested was few, the following 6 orders also had M-MDH mobilities of 100; i. e. Struthioniformes, Procellariiformes, Sphenisciformes, Podicipediformes, Phoenicopteriformes and Gruiformes. The species sampled are shown in Table 2. In Galliformes, 17 species of Phasianidae and 1 species of Megapodiidae had M- MDH mobilities of 100, but higher mobilities of 140 were found in 3 species of Cracidae. Therefore, differences in mobilities exist between families in the order Galliformes. Similarly, Phalacrocoracidae in Pelecaniformes, Threskiornithidae in Ciconiiformes

Fig. 2. Electrophoretic patterns of M- and S-MDH in vairous species of Anatidae together with Milvus migrans and Corvus corone. Evolution of M-MDH in Birds 5

Fig. 3. Electrophoretic patterns of M- and S-MDH in various species of birds.

and Charadriidae and Laridae in Charadriiformes showed an enzyme mobility of 100 in all species examined (Table 2). In these orders, other families tested had enzyme mobilities higher than 100. An exceptional case was indicated in Scolopacidae in which 7 species had M-MDH mobility 250, but only Numenius phaeopus showed a mobility 100. Avian species belonging to the above eleven orders (Table 1) are terrestrial or aquatic. A turaco, Musophaga rossae (Musophagidae, Cuculiformes) showed an enzyme mobility of 100. This was rather an exceptional case, since the arboreal birds usually had M-MDH mobilities higher than 100 as shown in the following section. 4. Birds with relative M-MDH mobility from 100 to 300 (Table 1 and 3, Fig. 3) From the above results it was expected that Tinamiformes would have the enzyme mobility of 100. The relative value exhibited was, however, 160 in 5 individuals of Eudromia elegans (Table 3). Another high mobility of 140 was found in 6 species of Accipitridae in Falconiformes. Although Falconiformes are now placed next to Ci- coniiformes systematically (Table 1), they are for the most part arboreal. In the orders which included the families with enzyme mobility 100, the following 6 families showed mobilities higher than 100. These values were 130 in Pelecanidae (Pelecaniformes), 130 in Ciconiidae and 150 in Ardeidae (Ciconiiformes), 140 in Cracidae (Galliformes), and 250 in Scolopacidae (with exception of Numenius) and 190 in Alcidae (Charadriiformes). 6 N. Kuroda, R. Kakizawa, H. Hori, Y. Osaka, N. Usuda & S. Utida

Table 1. Relative mobility of M-MDH in avian orders and families. The order sequence from Struthioniformes to Anseriformes follows Peters checklist Vol. 1 (1979) and from Galliformes to Passeriformes Wetmore's systematic list (1960). Evolution of M-MDH in Birds 7

Table 2. Birds with M-MDH of relative mobility 100.

The numbers in parentheses indicate the number of individuals examined. 8 N. Kuroda, R. Kakizawa, H. Hori, Y. Osaka, N. Usuda & S. Utida

Table 3. Birds with M-MDH of relative mobility from 130 to 300.

The numbers in parentheses indicate the number of individuals examined.

From Columbiformes to Piciformes, the arboreal birds, the mobilities spanned from 140 to 300, with the trend becoming gradually higher as the orders progress toward the Passeriformes (Table 1). Specifically the values were 140 or 190 in Colum- biformes, 200 in Cuculiformes, Strigiformes and Caprimulgiformes, 220 in Apodi- formes, and 220 to 300 in Coraciiformes and Piciformes. Among Strigiformes, an exceptional mobility of 130 was observed in Otus bakkamoena. Evolution of M-MDH in Birds 9

5. Birds with relative M-MDH mobility more than 300 (Table 1 and 4, Fig. 3) Although Psittaciformes have been placed next to Columbiformes taxonomically (Table 1), they nevertheless showed a high mobilities of 300 to 360. Two species of Loriidae and 3 species of Cacatua (Cacatuidae) showed mobilities of 330. Only Nymphicus hollandicus had a mobility 360. In the new world taxa of Psittacidae, the

Table 4. Birds with M-MDH of relative mobility from 300 to 360.

The numbers in parentheses indicate the number of individuals examined. 10 N. Kuroda, R. Kakizawa, H. Hori, Y. Osaka, N. Usuda & S. Utida mobilities were 300 in 3 species of Ara and 330 in 2 species of Amazona, whereas, 6 species of Pacific and Afro-Asian taxa (Psittacidae) indicated a mobility 360, with the exception of 320 in Agapornis personata. In Passeriformes, all the species sampled had the highest M-MDH mobility values of 360, including 2 species of sub-oscines, Rupicola peru- viana (Cotingidae) and Pitta sordida (Pittidae) as well as 37 species of oscines belonging to 12 families: Hirundinidae, Laniidae, Muscicapidae, Zosteropidae, Emberizidae, Icteridae, Fringillidae, Estrildidae, Ploceidae, Sturnidae, Oriolidae and Corvidae (Table 4). 6. Mobility variation of S-MDH The anodal mobilities of supernatant malate dehydrogenase (S-MDH) were found to be in good agreement with the results of Kitto & Wilson (1966). A basal mobility of 100 was found in all the species of 18 orders tested; i.e. Struthioniformes, Tinami- formes, Procellariiformes, Sphenisciformes, Podicipediformes, Pelecaniformes, Ciconi- iformes, Phoenicopteriformes, Falconiformes, Anseriformes, Galliformes, Gruiformes, Columbiformes, Psittaciformes, Strigiformes, Coraciiformes, Piciformes and Passeri- formes. The species which showed S-MDH mobilities other than 100 were: 2 species of Cuculidae (130), 17 species of 4 families in Charadriiformes (60), one species (Apus affinis) of Apodidae (70), and one species (Caprimulgus indicus) of Caprimulgidae (27). An exceptional mobility of 135 was found in Carpodacus erythrinus, Fringillidae, Passeri- formes.

Discussions

In the course of our study, we found the enzyme malate dehydrogenase (MDH) to be generally more conservative in nature than other enzymes, such as lactate dehydrogenase, 6-phosphogluconate dehydrogenase and glucosephosphate isomerase (Utida, Kakizawa and Kuroda, unpublished results). This conservative nature of MDH has already been pointed out by Kitto & Wilson (1966) in their comparative study of avian supernatant MDH (S-MDH), and they mentioned that the mitochondrial MDH (M- MDH) is less conservative than S-MDH. Our data confirm this point and that the electrophoretic mobility of M-MDH showed inter-taxa variation as well as intra-taxa sta- bility at order, family and even generic levels. The currently used taxonomic sequence of avian orders follows Wetmore's system (Wetmore 1960) beginning with phylogenetically old (from fossil evidence) terrestrial and aquatic bird orders and ending with arboreal and possibly more evolved groups. The relative M-MDH mobility 100 is likely to be the basic value, since this mobility was found widely in terrestrial and aquatic bird orders. As shown in Fig. 4, eleven orders of such birds have the mobility of 100, although 4 orders include families with mobilities higher than 100. On the other hand, the arboreal bird orders have the values of M-MDH mobilities exceeding 100, ranging from 140-360. Moreover, these values increase as the orders progress from Columbiformes to Passeriformes. The highest and stable mobility of 360 was found only in Passeriformes. A higher mobility of M- MDH in pitohoui (Pitohui kirhocephalus, Passeriformes) than of chicken enzyme has been reported by Karig & Wilson (1970). Evolution of M-MDH in Birds 11

Fig. 4. Tentative evolutionary diagram of avian families based on M-MDH mobilities. Numbers from 100 to 360 indicate M-MDH mobility and numbers in parenthesis indicate the order sequence shown in Table 1 (As modified from Sibley's diagram, 1960). 12 N. Kuroda, R. Kakizawa, H. Hori, Y. Osaka, N. Usuda & S. Utida

It is quite an interesting phenomenon that parallel trends exist between the increase in electrophoretic mobility of M-MDH and the progress of avian orders toward Passeri- formes. However, terrestrial and aquatic birds are generally larger in size and slower activity, while arboreal birds are generally smaller in size and more active as their lives become more arboreal and their metabolic rates increase. Since M-MDH acts via NADH of the respiratory chain and differences in mobility may be related to amino acid sequences, its properties can be expected to correlate with metabolic rate. It is not obvious why electrophoretic mobility would be correlated with metabolism, but the net charge on the molecule of M-MDH could relate to the action of malate-aspartate shuttle . For clarification of exceptional cases to the general trends more data are necessary, but we shall here describe some results of interest. Tinamidae, though generally con- sidered to be related to Struthioniformes, showed the M-MDH mobility 160. Tinamous , though terrestrial, is quite active in territorial defense and courtship. They have large pectoral muscles and fly fast, but short distances perhaps limitted by small heart size. Therefore, they expend much energy in intermittently daily activities and necessarily maintain a high metabolic rate. In Pelecaniformes, Pelecanidae showed 130, Phalacro- coracidae 100 and in Ciconiiformes, Threskiornithidae showed 100, while Ciconiidae showed 130 and Ardeidae 150. Among Charadriiformes, Charadriidae and Laridae showed 100, Scolopacidae 250 and Alcidae 190. These variations would indicate an evolutionary excursion of groups (to higher M-MDH mobility) from their basic or original M-MDH mobility 100. Within a family, variations were found among genera in Scolo- pacidae, Columbidae, and Strigidae. Such a heterogeneity within relatively lower taxonomic levels might involve some eco-physiological adaptive mechanisms. The high M-MDH mobility 140 of Cracidae among Galliformes in which Phasiani- dae and Megapodiidae show 100, suggests an evolutionary peculiarity of the Cracidae , which may support the work of Jolles et al. (1976) on the difference of amino acids sequence in chachalaca egg-white lysozyme as compared with Phasianidae and Anatidae, although Sibley's (1970) egg-white pattern was close to the Phasianidae. It is also to be noted that Musophagidae had the mobility 100 as against 200 of Cuculidae . This might suggest its possible remote terrestrial origin. Finally, our most notable finding was the high M-MDH mobility in Psittaciformes , 300-360, reaching the highest level 360 for Passeriformes. The high heterogeneity of mobility in Psittaciformes would still reflect an unstable evolutionary condition as compared with very stable mobility in the Passeriformes. The Psittaciformes and Passeriformes are the only two bird orders in which some species are capable of "talking" or imitating human or other sounds . This is not only functionally based on their multi-syringeal muscles, which are 3 or more (passerines) pairs, all the other orders having 1 or 2 pairs, but also supported by high brain development, especially of its Wulst (hyperstriatum layer). This part of avian brain shows a variety of developmental levels and positions, and it is highly developed both in Psittaci- formes and Passeriformes, although it is located in the rostral portion of the hemisphere in the passerines (raven) and in the mediocaudal portion in the parrots (lovebird) (Godman & Schein (eds.) 1974). According to the reversal experiments (switching of position or color) of Gossette et al. (1966), the superior order in performance was (from Evolution of M-MDH in Birds 13 best to worse) from magpies (Passeriformes), parrots (Psittaciformes) to quail and chickens (Galliformes), and the "magpie and parrots had very similar curves and so did quail and chickens", "but with a sharp break" between magpie-parrot and quail-chicken curves. Furthermore, Portman & Stingelin's (1961) "telencephalization index" (relative forebrain size for brain stem), showed the highest index values in Psittaciformes and Passeriformes, the Galliformes being among the lowest. It was therefore confirmed that Psittaciformes are only next to Passeriformes in brain development as well as in the M-MDH mobility. However, this may not prove, without more evidence, direct evolutionary relationships or taxonomic affinities between the two orders. Slibey (1970) discussed historical works on anatomical and other aspects of Psittaciformes in relation with Columbiformes and Cuculiformes, and his egg-white protein study showed the re- semblance with Columbiformes. Psittaciformes, therefore, may have experienced a marked convergent evolution, approaching Passeriformes level, from Columbo-Psittaci- Cuculi order-group. Summing up the above, the necessity exists for further investigations with more species and families in order to substantiate the now tentative evolutionary dendrogram. According to Prof. C. G. Sibley (personal communication), a complete reconstruction of avian phylogeny with DNA-DNA hybridization technique is in progress. The method of microcomplement fixation is also contributing to our new understanding of avian phyletic evolution (Prager & Wilson 1980).

Acknowledgements

The authors wish to express hearty thanks to Dr. Ken-ichi Numachi, Otsuchi Marine Research Center, Ocean Research Institute, University of Tokyo, Professor Shigeru Oba and Dr. Tadashi Aotsuka, Department of Biology, Tokyo Metropolitan University and Dr. Kazuo Moriwaki and Dr. Mitsuru Sakaizumi, Department of Cyto- genetics, National Institute of Genetics, Mishima, for kindly providing us with their electrophoresis technique. Thanks are also due to many people for collecting blood samples and bird specimens; i. e. Professor Norio Kondo, Tokyo University of Agriculture, Mr. Yoshitake and Mrs. Sumiko Hasuo, Gyotoku Bird Observatory, Shinhama Bird Reserve, Chiba Pref., Mr. Tatsuo Kazama, Nature Protection Section, Life Environment Division, Niigata Pref., Mr. Masaru Saito, Tokyo Tama Zoological Park, Mr. Hiroshi Sugiura, the Aquarium of Inokashira Park Zoo, Mr. Tetsuya Ishihara, Ishihara Animals and Birds Co., Mr. Masashi Yoshii and members of Bird Banding Team, Mr. Akio Sasa- gawa and Mr. Naoya Masuda in our Institute. We are grateful to Director Yoshimaro Yamashina who gave us kind support for our present study. Finally, we wish to express our special gratitude to Prof. C. B. Sibley, Peabody Museum of Natural History, Yale University, for his instructive comments on our manuscript and information on recent avian biochemical taxonomy. We also thank Dr. Ebert A. Ashby, Head of Tokyo Regional Office, National Science Foundation, for reading the manuscript and for valuable discussions. 14 N. Kuroda, R. Kakizawa, H. Hori, Y. Osaka, N. Usuda & S. Utida

References

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鳥類におけるミトコンドリア内リンゴ酸脱水素酵素(M-MDH)の進化

22目57科185種の鳥類の血球(一部胸筋)を試料として,澱粉ゲル竜気泳動法(pH 7)により,ミ

トコンドリア内リンゴ酸脱水素酵素(M-MDH)の陰極側への移動度を測定した。移動度の表現は,マガモ血球のM-MDH移動度を100と定めたときのこれに対する相対値である。鳥類M-MDHの移動度は

各種組織間に差がなく,またこれまで種内,属内での変異は見られなかった。さらに科内,目内での変異

も比較的少なく,他の酵素(アイソザイム)にくらべて極めて均一性の高い酵素である。

ダチョウ目,ミズナギドリ目,ペンギン目,カイツブリ目,ペリカソ目(ウ科),コウノトリ目(トキ

科),フラミンゴ目,ガンカモ目,キジ日(ツカツクリ科・キジ科),ツル目,チドリ目(チドリ科・カモメ科)に属する鳥類は何れも移動度100を示した。これらの目は比較的に原始的とされる地上・水生鳥類

の大部分を含んでいる。しかし,ペリカン目のペリカン科(130),コウノトリ目のコウノトリ科(130)・

サギ科(150),キジ目のホウカンチョウ科(140),チドリ目のシギ科(250)・ウミスズメ科(190)では

100以上の移動度が見られた。また,地上性のシギダチョウ目は例外的に160の,コウノトリ目に比較的

近いとされるワシタカ目(ワシタカ科)は140の値を示した。

一方,いわゆる樹上性の鳥類では140から360までの移動値が得られ,ハト目からスズメ目へと次第に高い値を示す傾向が見られた。すなわちハト目(140,190),ホトトギス目(200),フクロウ目(200),

ヨタカ目(200),アマツバメ目(220),ブッポウソウ目(220,250),キツツキ目(230,300),スズメ目

(360)である。ハト目に近いとされるオウム目では,300から360のスズメ目に近い値が得られた。

以上の結果から,電気泳動法によるM-MDHの移動度は,科・目を含む高いレベルでの進化を反映しているように思われる。

黒田長久,柿澤亮三,臼田奈々子,内田清一郎:山階鳥類研究所.〒150東京都渋谷区南平台町8-20. 堀浩,大阪豊:横浜市野毛山動物園.〒220横浜市西区老松町63-10.