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Characterization of the Domestic Horse niques R. A.
(Equus caballus)
EXPERIENTIA 32/9
Karyotype Using G- and C - B a n d i n g Tech-
BUCKLAND,JUDITH M. FLETCHER a n d ANN C. CHANDLEY 1
Medical t~esearch Council, Clinical and Population Cytogene~ics Unit, Western General Hospital, Crewe Road, Edinburgh E H 4 2 X U (Scotland), 70 March 1976. Summary. O i e m s a b a n d i n g t e c h n i q u e s h a v e b e e n used to i d e n t i f y tile c h r o m o s o m e s of t h e d o m e s t i c horse. C y t o g e n e t i c s t u d i e s in t h e d o m e s t i c h o r s e (Equus caballus, 2 n = 6 4 ) h a v e b e e n c o n d u c t e d o n o n l y a l i m i t e d scale so far. I n e a r l y studies o n t h e k a r y o t y p e s of n o r m a l horses~,3, a n d of a n i m a l s s h o w i n g sex c h r o m o s o m e m o s a i c i s m 4-~, c h r o m o s o m e p r e p a r a t i o n s s t a i n e d o n l y w i t h orcein were used. A p p l i c a t i o n of t h e G- a n d C - b a n d ing t e c h n i q u e s to h o r s e c h r o m o s o m e s h a s r e c e n t l y e n a b l e d us to i d e n t i f y a n u m b e r of c o n s t i t u t i o n a l sex c h r o m o s o m e a b n o r m a l i t i e s in infertile m a r e s 8. As i t seems likely t h a t f u r t h e r c y t o g e n e t i c s t u d i e s o n t h e horse a n d o t h e r e q u i n e species will b e c a r r i e d o u t in t h e future, we p r e s e n t here a d e t a i l e d d e s c r i p t i o n of t h e k a r y o t y p e of E. caballus b a s e d o n G- a n d C - b a n d i n g . Materials and methods. P e r i p h e r a l b l o o d was o b t a i n e d f r o m 8 m a r e s a n d a stallion. T h e s e s a m p l e s were c u l t u r e d u s i n g a m o d i f i c a t i o n of t h e whole blood m i c r o t e c h n i q u e " a n d a i r - d r i e d c h r o m o s o m e p r e p a r a t i o n s m a d e in t h e s t a n d a r d way. G i e m s a b a n d s (G-bands) were p r o d u c e d b y t h e following p r o c e d u r e . C h r o m o s o m e p r e p a r a t i o n s were i n c u b a t e d in 2 • (SSC = 0.15 M NaCI: 0.015 2~I t r i s o d i u m citrate) a t 60 ~ for 1 h, r i n s e d in deionized w a t e r , d i p p e d in a s o l u t i o n of 1 % t r y p s i n in deionized w a t e r for 5-30 sec a t r o o m t e m p e r a t u r e , rinsed, s t a i n e d in 5 % G i e m s a ( G u r r ' s R 6 6 G i e m s a in buffer, p H 6.8) for 5 - 8 min, r i n s e d a n d m o u n t e d in D P X (cf. GALLIMORE a n d RICHARDSON10). Q u i n a c r i n e b a n d s (Q-bands) were o b t a i n e d b y t h e s t a n d a r d m e t h o d e m p l o y e d in t h i s l a b o r a t o r y n, a n d Cb a n d s b y t h e b a r i u m h y d r o x i d e m e t h o d 12. Results and discussion. T h e n o r m a l diploid c h r o m o s o m e n u m b e r of E. caballus is 2 n ~ 6 4 . T h e X c h r o m o s o m e is s u b m e t a c e n t r i c a n d t h e second l a r g e s t c h r o m o s o m e in t h e c o m p l e m e n t . T h e Y c h r o m o s o m e is s i m i l a r in size to t h e s m a I l e s t a u t o s o m e s . I n a d d i t i o n t h e r e are 26 m e t a c e n t r i c or s u b m e t a c e n t r i c a u t o s o m e s a n d 36 a c r o c e n t r i c a u t o s o m e s 2. G-banding. A s t a n d a r d G - b a n d e d k a r y o t y p e of t h e dom e s t i c h o r s e h a s b e e n e s t a b l i s h e d f r o m a s t u d y of 13 wellb a n d e d m e t a p h a s e spreads, 12 of w h i c h were f r o m t h e 8 mares. T h e l e n g t h of e a c h p a i r was m e a s u r e d f r o m p h o t o g r a p h i c e n l a r g e m e n t s a n d t h e c h r o m o s o m e s a r r a n g e d , acc o r d i n g t o l e n g t h , w i t h i n 3 groups, m e t a - or s u b m e t a c e n trics, a c r o c e n t r i c s a n d sex c h r o m o s o m e s . F i g u r e s 1 a n d 2 show G - b a n d e d k a r y o t y p e s of t h e s t a l l i o n a n d m a r e respectively. T h e c e n t r o m e r i c regions, w i t h t h e e x c e p t i o n of c h r o m o s o m e p a i r 11 a n d t h e Y c h r o m o s o m e , t a k e u p no stain, a f e a t u r e w h i c h t h e h o r s e k a r y o t y p e s h a r e s w i t h t h a t of c e r t a i n o t h e r species 13. T h e b a n d i n g p a t t e r n s of m a n y c h r o m o s o m e p a i r s are d i s t i n c t i v e e n o u g h to e n a b l e t h e m to b e i d e n t i f i e d w i t h reference t o F i g u r e s 1 a n d 2, a n d a d e t a i l e d d e s c r i p t i o n of these pairs was considered unnecessary. Chromosome p a i r s w h i c h h a v e p o o r b a n d i n g p a t t e r n s , h o w e v e r , are d e s c r i b e d to aid in t h e i r i d e n t i f i c a t i o n , a n d p a i r s w h i c h have similar banding patterns have their distinguishing f e a t u r e s d e s c r i b e d collectively. a) Meta- and submetacentrics. C h r o m o s o m e 5. M e t a centric, t h e s h o r t a r m h a v i n g 2 p o s i t i v e b a n d s , 1 n e a r t h e c e n t r o m e r e a n d 1 n e a r t h e d i s t a l e n d of t h e a r m . T h e long
Fig. 1. G-banded metaphase cell and karyotype from a male horse (2n = 64). Note the negatively stained centromeric region on most of the chromosomes.
a r m s t a i n s palely, a n d 3 v a g u e p o s i t i v e b a n d s c a n b e disc e r n e d in clear p r e p a r a t i o n s . T h e s e are spaced e v e n l y along the arm, the central band being the broadest. C h r o m o s o m e s 6, 7 a n d 9. T h e s e h a v e similar c e n t r o m e r e indices a n d b a n d i n g p a t t e r n s . All h a v e a b r o a d p o s i t i v e b a n d in t h e p r o x i m a l h a l f of t h e long a r m , b u t in c h r o m o some 6 t h i s is h a l f - w a y d o w n t h e long a r m , w h e r e a s in
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c h r o m o s o m e s 7 a n d 9 it is o n l y a t h i r d of t h e w a y d o w n . T h e s e 2 p a i r s m a y be d i s t i n g u i s h e d b y t h e p r e s e n c e of a prominent positive band next to the centromeric region in t h e s h o r t a r m o f c h r o m o s o m e 7. C h r o m o s o m e s 12 a n d 13. T h e s e a r e v e r y s i m i l a r in size, b u t c h r o m o s o m e 12 is m e t a c e n t r i c w h e r e a s 13 is s u m b e t a c e n t r i c . B o t h h a v e b r o a d p o s i t i v e b a n d s o n e i t h e r side of t h e c e n t r o m e r e b u t t h o s e of c h r o m o s o m e 12 t e n d t o b e d a r k e r a n d t h e n e g a t i v e l y s t a i n i n g c e n t r o m e r i c r e g i o n of
Fig. 2. G-banded metaphase cell and karyotype from a female horse (2r~ = 64).
1 The authors wish to acknowledge the assistance of Mr. PETER ROSSDALE and his veterinary colleagues from Newmarket, Suffolk, for supplying us with the blood samples. 2 K. BENIRSCHKE, L. ]~. BROWNHILL and M. M. BEATH, J. Reprod. Fert. 4, 319 (1962). 8 J. M. TRUJILLO, C. STENIUS, L. C. CHRISTIAN and S. OHNO, Chrolnosoma 13, 243 (1962). 4 p. IK. BASRUR, H. KANAGAWA and J. P. W. GILMAN, Can. J. comp. Med. 33, 297 (1969). S. BORNSTEIN, Acta vet. scan& 8, 291 (1967). 6 R. 3OUTERS, M. VANDEPLASSCHEand A. DE MOOR, Equipe Vet. J. 4, 150 (1972). N. GLUHOVSCHI, M. BISTRICEANO, A. Suclu and M. BRATU, Br. Vet. J. 126, 522 (1970). 8 a . C. CHANDLEY, J. M. FLETCHER, P. D. ROSSDALE, C. K. PEACE, S. W. RICKETTS, R. J. MCENERV, J. P. THORNE, R. V. SHORT and W. R. ALLEN, J. Reprod. Fert. Suppl. 23, 377 (1975). 9 D. A. HUNGERFORD, Stain Technol. dO, 333 (1965). 10 p. H. GALLIMORE and C. R. RICHARDSON, Chromosoma 41, 259 (1973). 11 H. J. EVANS, K. E. BUCKTON and A. T. SUM~ER, Chromosoma 35, 310 (1971). 1s A. T. SUMNER, Expl Cell Res. 75, 304 (1972). 13 H. J. EVANS, R. A. BUCI~LANOand A. T. SUMNER, Chromosoma d2, 383 (1973).
Fig. 3. Horse metaphase chromosomes and karyotype, stained with the C-band technique. All but 2 pairs contain eentromeric heterochromatin. Note also that pair 13 is heteromorphie for its heterochromatin content (arrow).
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Fig. 4. Male horse cell which was Q-banded prior to C-banding to identify the pairs which Iack centromeric heterochromatin. Tile figure shows these to be pairs 8 avd 11. Note that the homologues of pair 13 (double arrows) have the same heterochromatm content in this individual.
t h i s c h r o m o s o m e is larger. T h e s e c h r o m o s o m e pairs m a y be u n e q u i v o c a l l y d i s t i n g u i s h e d f r o m one a n o t h e r b y Cb a n d i n g , as c h r o m o s o m e 12 h a s a n i n t e r s t i t i a l C - b a n d o n its long a r m (see below a n d F i g u r e s 3 a n d 4). b) Acrocentric chromosomes. C h r o m o s o m e s 14 a n d 15. C h r o m o s o m e 14 h a s a n e g a t i v e b a n d h a l f w a y d o w n t h e a r m , a n d d i s t a l to t h i s it s t a i n s darkly. 3 p o s i t i v e b a n d s m a y u s u a l l y be resolved in t h i s region. P r o x i m a l t o t h e negative band the chromosome stains palely with 3 dark, n a r r o w b a n d s . C h r o m o s o m e 15 also h a s a series of n a r r o w p o s i t i v e b a n d s d o w n its l e n g t h , b u t t h e s e o n l y n u m b e r 5, a n d are e v e n l y s p a c e d on a u n i f o r m pale s t a i n i n g b a c k ground. C h r o m o s o m e 17. T h e r e is a n e g a t i v e b a n d a t h i r d of t h e w a y d o w n t h e arm. 2 n a r r o w p o s i t i v e b a n d s m a y b e resolved p r o x i m a l l y to this, a n d 2 b r o a d p o s i t i v e b a n d s distally. C h r o m o s o m e 18. T h e r e is 1 n a r r o w p o s i t i v e b a n d i m m e d i a t e l y below t h e c e n t r o m e r i c region a n d 2 b r o a d e r ones in t h e m i d d l e of t h e c h r o m o s o m e . D i s t a l to t h i s is a n e g a t i v e b a n d , w h i c h in t u r n is b o u n d e d b y a b r o a d positive b a n d . C h r o m o s o m e s 19 a n d 20. B o t h h a v e p o s i t i v e b a n d s a t h i r d of t h e w a y d o w n t h e i r arms, w i t h b r o a d n e g a t i v e b a n d s d i s t a l to these, t h e tips of t h e c h r o m o s o m e s b e i n g dark. H o w e v e r , t h e s e f e a t u r e s are all m o r e p r o n o u n c e d i n c h r o m o s o m e 20 a n d i t is n o t i c e a b l y smaller. C h r o m o s o m e 23. T h i s c h r o m o s o m e h a s a poor b a n d i n g p a t t e r n . T h e d i s t a l h a l f of t h e l o n g a r m s t a i n s d a r k l y as does a n a r r o w region r o u n d t h e c e n t r o m e r e . C h r o m o s o m e s 25 a n d 27. T h e s e b o t h s t a i n d a r k proxim a l l y a n d pale distally. T h e d a r k region m a y be resolved i n t o 2 b a n d s w h i c h are b o t h t h e s a m e size in c h r o m o s o m e 27. T h e m o r e p r o x i m a l b a n d in c h r o m o s o m e 25 is t h e s a m e size as these, b u t t h e m o r e d i s t a l is smaller.
C h r o m o s o m e 26. This c h r o m o s o m e s t a i n s a l m o s t uniformly, t h e r e b e i n g suggestions of p o s i t i v e b a n d s in t h e m i d d l e ot t h e a r m a n d a t t h e d i s t a l t i p of t h e c h r o m o somes. C h r o m o s o m e s 28-31. C h r o m o s o m e 28 h a s 2 p o s i t i v e b a n d s , while c h r o m o s o m e 29 h a s o n l y one, w h i c h is proxim a l to t h e c e n t r o m e r e . C h r o m o s o m e 30 h a s 1 d i s t i n c t p o s i t i v e b a n d a t its distal tip, a n d c h r o m o s o m e 31 s t a i n s palely, h a v i n g a n a r r o w p o s i t i v e b a n d j u s t below t h e c e n t r o m e r i c region. c) Sex chromosomes. T h e s h o r t a r m of t h e s u b m e t a c e n t r i c X c h r o m o s o m e h a s 2 n a r r o w p o s i t i v e b a n d s , one a t t h e c e n t r o m e r i c region a n d t h e o t h e r h a l f w a y a l o n g its length. T h e long a r m h a s a b r o a d i n t e n s e l y s t a i n e d b a n d j u s t below t h e e e n t r o m e r i c region, d i s t a l to w h i c h is a b r o a d n e g a t i v e b a n d , w h i c h s t a i n s p o s i t i v e l y w i t h Cb a n d i n g (see below). T h e d i s t a l h a l f of t h i s a r m s t a i n s m o d e r a t e l y , 3 n a r r o w p o s i t i v e b a n d s b e i n g resolved. T h e b a n d i n g p a t t e r n of t h e a c r o c e n t r i e Y c h r o m o s o m e is v e r y d i s t i n c t i v e , t h e whole c h r o m o s o m e s t a i n i n g a u n i f o r m pale colour, e x c e p t for t h e tips of t h e long a r m s which stain darkly. C-banding. Most of t h e c h r o m o s o m e s i n t h e c o m p l e m e n t show a discrete b l o c k of h e t e r o c h r o m a t i n a t t h e i r c e n t r o meric region w h e n s t a i n e d w i t h t h e C - b a n d t e c h n i q u e (Figure 3), a n d t h e s e blocks c o r r e s p o n d to t h e n e g a t i v e l y s t a i n e d areas f o u n d a t t h e c e n t r o m e r e s w i t h G - b a n d i n g . T h e r e are some c h r o m o s o m e s , h o w e v e r , t h a t d e v i a t e f r o m t h i s simple generalization, a n d t o i d e n t i f y these, Q - b a n d ing was carried o u t p r i o r to t h e C - b a n d p r o c e d u r e (Figure 4). A m o n g t h e a u t o s o m e s , 1 m e t a c e n t r i c p a i r (No. 11) h a s n o d e m o n s t r a b l e C-band, a n d a n o t h e r (No. 8) h a s o n l y t h e s u g g e s t i o n of one. Some c h r o m o s o m e s , on t h e o t h e r hand, have extra centromeric heterochromatin. The Y
15.9. 1976
Specialia
c h r o m o s o m e , for e x a m p l e , consists a l m o s t e n t i r e l y of c o n s t i t u t i v e h e t e r o c h r o m a t i n while c h r o m o s o m e 12 a n d t h e X c h r o m o s o m e b o t h possess i n t e r s t i t i a l C - b a n d s halfw a y along t h e i r long arms, a n d c h r o m o s o m e 1 has a small C-band a t t h e distal t i p of t h e s h o r t arm. These v a r i a t i o n s in c o n s t i t u t i v e h e t e r o c h r o m a t i n c o n t e n t p r o v i d e a n add i t i o n a l m e a n s of i d e n t i f y i n g t h e s e c h r o m o s o m e pairs.
T h e P h y l o g e n e t i c S t a t u s of P h y l l o m e d u s i n e S t u d i e s of their S e r u m A l b u m i n s 1
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I t is also c o m m o n to find t h e C - b a n d s differ in size b e t w e e n homologues. This is p a r t i c u l a r l y t r u e of c h r o m o some p a i r No. 13, w h i c h s h o w e d a h e t e r o m o r p h i s m in a t least 4 o u t of t h e 9 a n i m a l s s t u d i e d (e.g. Figure 3). More w o r k is r e q u i r e d to d e t e r m i n e e x a c t l y h o w w i d e s p r e a d this p o l y m o r p h i s m is, a n d h o w m u c h o t h e r v a r i a t i o n t h e r e m i g h t be in t h e horse k a r y o t y p e .
F r o g s ( H y l i d a e ) as E v i d e n c e d f r o m I m m u n o l o g i c a l
LINDA MAXSON 2
University o/ Illinois, Department o/Genetics and Development, 515 Morrill Hall, Urbana (Illinois 61801, USA), 22 March 1976. Summary. B a s e d on i m m u n o l o g i c a l c o m p a r i s o n s of t h e s e r u m a l b u m i n s of p h y l l o m e d u s i n e frogs w i t h b o t h hyline and b u f o n i d species, it is s u g g e s t e d t h a t p h y l l o m e d u s i n e frogs b e e r e c t e d to familial s t a t u s w i t h i n t h e s u p e r f a m i l y Bufonoidea. The N e o t r o p i c a l leaf frogs, P h y l l o m e d u s i n a e , consist of t h r e e g e n e r a ; Phyllomedusa (31 species), Agalychnis (8 species), a n d t h e m o n o t y p i c Pachymedusa. These frogs c o n s t i t u t e a d i s t i n c t p h y l e t i c line in t h e family Hylidae. All frogs t e s t e d in t h i s s u b f a m i l y are u n i q u e a m o n g o t h e r hylids in possessing large a m o u n t s of p o w e r f u l b r a d y kinin-like a n d p h y s a l a e m i n - l i k e p o l y p e p t i d e s in t h e i r skin 3. All species in t h i s s u b f a m i l y are also c h a r a c t e r i z e d b y h a v i n g v e r t i c a l pupils, diploid c h r o m o s o m e complem e n t s of 26, arboreality, a n d m o d e r a t e l y ossified skulls (with or w i t h o u t t h e d e r m i s co-ossified w i t h t h e skull). All m e m b e r s d e p o s i t t h e i r eggs on v e g e t a t i o n over w a t e r , into w h i c h t h e h a t c h l i n g t a d p o l e s drop. The a q u a t i c t a d poles are u n i q u e in h a v i n g a sinistral spiracle lying vent r a l l y on t h e midline 4. C o n s i d e r a t i o n of t h e a b o v e characteristics led DUELLMAN 5 tO recognize t h e s e t h r e e genera as a s e p a r a t e h y l i d s u b f a m i l y . DUELL~A/~'S decision has b e e n c o n f i r m e d b y r e c e n t b i o c h e m i c a l studies w h i c h h a v e s h o w n t h a t a t least some p h y l l o m e d u s i n e s , a n d no o t h e r hylids, e x c r e t e uric acid r a t h e r t h a n amm o n i a a n d urea as do m o s t a n u r a n s 6. Additionally, f u r t h e r studies on p h y l e t i c affinities of p h y l l o m e d u s i n e frogs h a v e s u g g e s t e d t h e s e frogs m a y be closely r e l a t e d to a t least some of t h e A u s t r a l i a n hylids (Litoria). 2 of 5 species of Litoria t e s t e d were f o u n d to possess large fibrous melanosomes, c o n t a i n i n g a n o v e l red p i g m e n t , w h i c h were f o r m e r l y i d e n t i f i e d as u n i q u e to N e o t r o p i c a l leaf frogs% My i n t e r e s t in p h y l l o m e d u s i n e frogs arose d u r i n g studies of a l b u m i n e v o l u t i o n in t h e a n u r a n s u p e r f a m i l y Bufonoidea s. These studies w i t h a l b u m i n s of b o t h h y l i d a n d b u f o n i d species s u g g e s t e d t h a t t h e p h y l l o m e d u s i n e frogs should be e r e c t e d as a p r o p e r family, p e r h a p s i n t e r m e d i a t e b e t w e e n t h e H y l i d a e a n d t h e Bufonidae. I n d e e d , earlier serological studies b y CEI s, i n v o l v i n g p r e c i p i t i n t e s t s w i t h s h o r t t e r m a n t i s e r a t o whole serum, led h i m to suggest the Phyllomedusinae might represent an independent p h y l e t i c b r a n c h ariziug f r o m s o m e u n d i f f e r e n t i a t e d H y l i d - B u f o n o i d stock. Materials and methods. A n t i s e r a t o pure a l b u m i n f r o m a single s p e c i m e n of Phyllomedusa trinitatus ~o was m a d e in 4 male N e w Z e a l a n d w h i t e r a b b i t s over a t h r e e - m o n t h i m m u n i z a t i o n schedule. The i n d i v i d u a l a n t i s e r a were t e s t e d for p u r i t y a n d pooled a c c o r d i n g to p u b l i s h e d procedures 1~. As sources of a l b u m i n , p l a s m a or skeletal muscle p r e s e r v e d in a p h e n o x y e t h a n o l solution ~ were used. M i c r o c o m p l e m e n t f i x a t i o n s t u d i e s w i t h t h e a l b u m i n s of
r e p r e s e n t a t i v e s of all t h r e e p h y l l o m e d u s i n e genera as well as w i t h o t h e r h y l i d and b u f o n i d species were p e r f o r m e d . R e s u l t s are r e p o r t e d as i m m u n o l o g i c a l d i s t a n c e units. F o r a n u r a n a l b u m i n s 1 u n i t of i m m u n o l o g i c a l d i s t a n c e bet w e e n 2 species r e p r e s e n t s r o u g h l y one a m i n o acid difference in t h e a l b u m i n s of t h e s e 2 speciesl~. Results and discussion. T h e T a b l e s u m m a r i z e s t h e results of t e s t s w i t h a n t i s e r u m to t ). trinitatus albumin. All available species of Phyllomedusa f o r m one i m m u n o logical cluster w i t h a r a n g e of 0-61 units. This is t h e o r d e r of m a g n i t u d e seen b e t w e e n species of Gastrotheca (Hylidae : v m p h i g n a t h o d o n t i n a e ) , b e t w e e n N o r t h A m e r i c a n Hyla species (HyIidae: Hylinae), a n d b e t w e e n species of Bu/o (Bufonidae) 8. Paehymedusa dachnicolor is of i n t e r e s t since, w h e n first d e s c r i b e d b y C o p e in 1864, t h e species was p l a c e d in t h e g e n u s Agalychnis. I t led a , s p o t t y h i s t o r y of ~cransfer f r o m Agalychnis t o Phyllomedusa u n t i l 1968 w h e n DUELLMAN p r o p o s e d its i n d e p e n d e n t generic s t a t u s . I m m u n o l o g i c a l l y Pachymedusa a l b u m i n a p p e a r s m o r e d i s t a n t f r o m P. trinitatus t h a n a n y species of Phyllomedusa b u t n o t q u i t e as d i s t a n t as b o t h species of Ngalychnis available for this s t u d y . T h u s a t t h e molecular level Pachymedusa's generic s t a t u s is also justified. The
1 This study was initiated in the Department of Biochemistry, University of California, Berkeley. Part of this work was supported by NSF grant No. DEB75-23543.. 2 The author gratefully acknowledges the helpful advice and discussion of A. C. WILSON. Thanks are also due to D. WAKE, H. COGGER,W. DUELI,M ~ and R. HEVERfor generously supplying frog samples. 8 j. M. CEI and V. ERSPA~ER, Copeia 74 (1966). 4 W. E. DUELLMAN,The Hylid Frogs o~ Middle America, Monograph of the Museum of Natural History (University of Kansas 1970), vols. 1 and 2. W. E. DVELLMAN,Univ. Kansas PubI. Mus. nat. Hist. 78, 3 (1968). V. H. SHOE~aAKERand L. L. MCCLANAHA~,JR. J. comp. Physiol. ]3 100, 331 (1975). 7 j. T. BAGNARAand W. FERRlS, Copeia 83, 592 (1975). 8 L. R. MAXSON, P h . D . thesis Univ. of California, ]3erkeley and California State University, San Diego (1973). 9 j. M. CEI, Bull. Serol. Mus. 30, 4 (1963). 10 A generous gift of G. E. DREWRY. Xl L. R. MAXSONand A. C. WILSON, Syst. Zool. 24, 1 (1975). 1~ L. KARIGand A. C. WII~SO~, Biochem. Genet. 5, 211 (1971). 13 L. R. MAXSONand A. C. WILSOn, Science 185, 66 (1974).
Specialia
Characterization of the Domestic Horse niques R. A.
(Equus caballus)
EXPERIENTIA 32/9
Karyotype Using G- and C - B a n d i n g Tech-
BUCKLAND,JUDITH M. FLETCHER a n d ANN C. CHANDLEY 1
Medical t~esearch Council, Clinical and Population Cytogene~ics Unit, Western General Hospital, Crewe Road, Edinburgh E H 4 2 X U (Scotland), 70 March 1976. Summary. O i e m s a b a n d i n g t e c h n i q u e s h a v e b e e n used to i d e n t i f y tile c h r o m o s o m e s of t h e d o m e s t i c horse. C y t o g e n e t i c s t u d i e s in t h e d o m e s t i c h o r s e (Equus caballus, 2 n = 6 4 ) h a v e b e e n c o n d u c t e d o n o n l y a l i m i t e d scale so far. I n e a r l y studies o n t h e k a r y o t y p e s of n o r m a l horses~,3, a n d of a n i m a l s s h o w i n g sex c h r o m o s o m e m o s a i c i s m 4-~, c h r o m o s o m e p r e p a r a t i o n s s t a i n e d o n l y w i t h orcein were used. A p p l i c a t i o n of t h e G- a n d C - b a n d ing t e c h n i q u e s to h o r s e c h r o m o s o m e s h a s r e c e n t l y e n a b l e d us to i d e n t i f y a n u m b e r of c o n s t i t u t i o n a l sex c h r o m o s o m e a b n o r m a l i t i e s in infertile m a r e s 8. As i t seems likely t h a t f u r t h e r c y t o g e n e t i c s t u d i e s o n t h e horse a n d o t h e r e q u i n e species will b e c a r r i e d o u t in t h e future, we p r e s e n t here a d e t a i l e d d e s c r i p t i o n of t h e k a r y o t y p e of E. caballus b a s e d o n G- a n d C - b a n d i n g . Materials and methods. P e r i p h e r a l b l o o d was o b t a i n e d f r o m 8 m a r e s a n d a stallion. T h e s e s a m p l e s were c u l t u r e d u s i n g a m o d i f i c a t i o n of t h e whole blood m i c r o t e c h n i q u e " a n d a i r - d r i e d c h r o m o s o m e p r e p a r a t i o n s m a d e in t h e s t a n d a r d way. G i e m s a b a n d s (G-bands) were p r o d u c e d b y t h e following p r o c e d u r e . C h r o m o s o m e p r e p a r a t i o n s were i n c u b a t e d in 2 • (SSC = 0.15 M NaCI: 0.015 2~I t r i s o d i u m citrate) a t 60 ~ for 1 h, r i n s e d in deionized w a t e r , d i p p e d in a s o l u t i o n of 1 % t r y p s i n in deionized w a t e r for 5-30 sec a t r o o m t e m p e r a t u r e , rinsed, s t a i n e d in 5 % G i e m s a ( G u r r ' s R 6 6 G i e m s a in buffer, p H 6.8) for 5 - 8 min, r i n s e d a n d m o u n t e d in D P X (cf. GALLIMORE a n d RICHARDSON10). Q u i n a c r i n e b a n d s (Q-bands) were o b t a i n e d b y t h e s t a n d a r d m e t h o d e m p l o y e d in t h i s l a b o r a t o r y n, a n d Cb a n d s b y t h e b a r i u m h y d r o x i d e m e t h o d 12. Results and discussion. T h e n o r m a l diploid c h r o m o s o m e n u m b e r of E. caballus is 2 n ~ 6 4 . T h e X c h r o m o s o m e is s u b m e t a c e n t r i c a n d t h e second l a r g e s t c h r o m o s o m e in t h e c o m p l e m e n t . T h e Y c h r o m o s o m e is s i m i l a r in size to t h e s m a I l e s t a u t o s o m e s . I n a d d i t i o n t h e r e are 26 m e t a c e n t r i c or s u b m e t a c e n t r i c a u t o s o m e s a n d 36 a c r o c e n t r i c a u t o s o m e s 2. G-banding. A s t a n d a r d G - b a n d e d k a r y o t y p e of t h e dom e s t i c h o r s e h a s b e e n e s t a b l i s h e d f r o m a s t u d y of 13 wellb a n d e d m e t a p h a s e spreads, 12 of w h i c h were f r o m t h e 8 mares. T h e l e n g t h of e a c h p a i r was m e a s u r e d f r o m p h o t o g r a p h i c e n l a r g e m e n t s a n d t h e c h r o m o s o m e s a r r a n g e d , acc o r d i n g t o l e n g t h , w i t h i n 3 groups, m e t a - or s u b m e t a c e n trics, a c r o c e n t r i c s a n d sex c h r o m o s o m e s . F i g u r e s 1 a n d 2 show G - b a n d e d k a r y o t y p e s of t h e s t a l l i o n a n d m a r e respectively. T h e c e n t r o m e r i c regions, w i t h t h e e x c e p t i o n of c h r o m o s o m e p a i r 11 a n d t h e Y c h r o m o s o m e , t a k e u p no stain, a f e a t u r e w h i c h t h e h o r s e k a r y o t y p e s h a r e s w i t h t h a t of c e r t a i n o t h e r species 13. T h e b a n d i n g p a t t e r n s of m a n y c h r o m o s o m e p a i r s are d i s t i n c t i v e e n o u g h to e n a b l e t h e m to b e i d e n t i f i e d w i t h reference t o F i g u r e s 1 a n d 2, a n d a d e t a i l e d d e s c r i p t i o n of these pairs was considered unnecessary. Chromosome p a i r s w h i c h h a v e p o o r b a n d i n g p a t t e r n s , h o w e v e r , are d e s c r i b e d to aid in t h e i r i d e n t i f i c a t i o n , a n d p a i r s w h i c h have similar banding patterns have their distinguishing f e a t u r e s d e s c r i b e d collectively. a) Meta- and submetacentrics. C h r o m o s o m e 5. M e t a centric, t h e s h o r t a r m h a v i n g 2 p o s i t i v e b a n d s , 1 n e a r t h e c e n t r o m e r e a n d 1 n e a r t h e d i s t a l e n d of t h e a r m . T h e long
Fig. 1. G-banded metaphase cell and karyotype from a male horse (2n = 64). Note the negatively stained centromeric region on most of the chromosomes.
a r m s t a i n s palely, a n d 3 v a g u e p o s i t i v e b a n d s c a n b e disc e r n e d in clear p r e p a r a t i o n s . T h e s e are spaced e v e n l y along the arm, the central band being the broadest. C h r o m o s o m e s 6, 7 a n d 9. T h e s e h a v e similar c e n t r o m e r e indices a n d b a n d i n g p a t t e r n s . All h a v e a b r o a d p o s i t i v e b a n d in t h e p r o x i m a l h a l f of t h e long a r m , b u t in c h r o m o some 6 t h i s is h a l f - w a y d o w n t h e long a r m , w h e r e a s in
15.9. !976
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c h r o m o s o m e s 7 a n d 9 it is o n l y a t h i r d of t h e w a y d o w n . T h e s e 2 p a i r s m a y be d i s t i n g u i s h e d b y t h e p r e s e n c e of a prominent positive band next to the centromeric region in t h e s h o r t a r m o f c h r o m o s o m e 7. C h r o m o s o m e s 12 a n d 13. T h e s e a r e v e r y s i m i l a r in size, b u t c h r o m o s o m e 12 is m e t a c e n t r i c w h e r e a s 13 is s u m b e t a c e n t r i c . B o t h h a v e b r o a d p o s i t i v e b a n d s o n e i t h e r side of t h e c e n t r o m e r e b u t t h o s e of c h r o m o s o m e 12 t e n d t o b e d a r k e r a n d t h e n e g a t i v e l y s t a i n i n g c e n t r o m e r i c r e g i o n of
Fig. 2. G-banded metaphase cell and karyotype from a female horse (2r~ = 64).
1 The authors wish to acknowledge the assistance of Mr. PETER ROSSDALE and his veterinary colleagues from Newmarket, Suffolk, for supplying us with the blood samples. 2 K. BENIRSCHKE, L. ]~. BROWNHILL and M. M. BEATH, J. Reprod. Fert. 4, 319 (1962). 8 J. M. TRUJILLO, C. STENIUS, L. C. CHRISTIAN and S. OHNO, Chrolnosoma 13, 243 (1962). 4 p. IK. BASRUR, H. KANAGAWA and J. P. W. GILMAN, Can. J. comp. Med. 33, 297 (1969). S. BORNSTEIN, Acta vet. scan& 8, 291 (1967). 6 R. 3OUTERS, M. VANDEPLASSCHEand A. DE MOOR, Equipe Vet. J. 4, 150 (1972). N. GLUHOVSCHI, M. BISTRICEANO, A. Suclu and M. BRATU, Br. Vet. J. 126, 522 (1970). 8 a . C. CHANDLEY, J. M. FLETCHER, P. D. ROSSDALE, C. K. PEACE, S. W. RICKETTS, R. J. MCENERV, J. P. THORNE, R. V. SHORT and W. R. ALLEN, J. Reprod. Fert. Suppl. 23, 377 (1975). 9 D. A. HUNGERFORD, Stain Technol. dO, 333 (1965). 10 p. H. GALLIMORE and C. R. RICHARDSON, Chromosoma 41, 259 (1973). 11 H. J. EVANS, K. E. BUCKTON and A. T. SUM~ER, Chromosoma 35, 310 (1971). 1s A. T. SUMNER, Expl Cell Res. 75, 304 (1972). 13 H. J. EVANS, R. A. BUCI~LANOand A. T. SUMNER, Chromosoma d2, 383 (1973).
Fig. 3. Horse metaphase chromosomes and karyotype, stained with the C-band technique. All but 2 pairs contain eentromeric heterochromatin. Note also that pair 13 is heteromorphie for its heterochromatin content (arrow).
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Fig. 4. Male horse cell which was Q-banded prior to C-banding to identify the pairs which Iack centromeric heterochromatin. Tile figure shows these to be pairs 8 avd 11. Note that the homologues of pair 13 (double arrows) have the same heterochromatm content in this individual.
t h i s c h r o m o s o m e is larger. T h e s e c h r o m o s o m e pairs m a y be u n e q u i v o c a l l y d i s t i n g u i s h e d f r o m one a n o t h e r b y Cb a n d i n g , as c h r o m o s o m e 12 h a s a n i n t e r s t i t i a l C - b a n d o n its long a r m (see below a n d F i g u r e s 3 a n d 4). b) Acrocentric chromosomes. C h r o m o s o m e s 14 a n d 15. C h r o m o s o m e 14 h a s a n e g a t i v e b a n d h a l f w a y d o w n t h e a r m , a n d d i s t a l to t h i s it s t a i n s darkly. 3 p o s i t i v e b a n d s m a y u s u a l l y be resolved in t h i s region. P r o x i m a l t o t h e negative band the chromosome stains palely with 3 dark, n a r r o w b a n d s . C h r o m o s o m e 15 also h a s a series of n a r r o w p o s i t i v e b a n d s d o w n its l e n g t h , b u t t h e s e o n l y n u m b e r 5, a n d are e v e n l y s p a c e d on a u n i f o r m pale s t a i n i n g b a c k ground. C h r o m o s o m e 17. T h e r e is a n e g a t i v e b a n d a t h i r d of t h e w a y d o w n t h e arm. 2 n a r r o w p o s i t i v e b a n d s m a y b e resolved p r o x i m a l l y to this, a n d 2 b r o a d p o s i t i v e b a n d s distally. C h r o m o s o m e 18. T h e r e is 1 n a r r o w p o s i t i v e b a n d i m m e d i a t e l y below t h e c e n t r o m e r i c region a n d 2 b r o a d e r ones in t h e m i d d l e of t h e c h r o m o s o m e . D i s t a l to t h i s is a n e g a t i v e b a n d , w h i c h in t u r n is b o u n d e d b y a b r o a d positive b a n d . C h r o m o s o m e s 19 a n d 20. B o t h h a v e p o s i t i v e b a n d s a t h i r d of t h e w a y d o w n t h e i r arms, w i t h b r o a d n e g a t i v e b a n d s d i s t a l to these, t h e tips of t h e c h r o m o s o m e s b e i n g dark. H o w e v e r , t h e s e f e a t u r e s are all m o r e p r o n o u n c e d i n c h r o m o s o m e 20 a n d i t is n o t i c e a b l y smaller. C h r o m o s o m e 23. T h i s c h r o m o s o m e h a s a poor b a n d i n g p a t t e r n . T h e d i s t a l h a l f of t h e l o n g a r m s t a i n s d a r k l y as does a n a r r o w region r o u n d t h e c e n t r o m e r e . C h r o m o s o m e s 25 a n d 27. T h e s e b o t h s t a i n d a r k proxim a l l y a n d pale distally. T h e d a r k region m a y be resolved i n t o 2 b a n d s w h i c h are b o t h t h e s a m e size in c h r o m o s o m e 27. T h e m o r e p r o x i m a l b a n d in c h r o m o s o m e 25 is t h e s a m e size as these, b u t t h e m o r e d i s t a l is smaller.
C h r o m o s o m e 26. This c h r o m o s o m e s t a i n s a l m o s t uniformly, t h e r e b e i n g suggestions of p o s i t i v e b a n d s in t h e m i d d l e ot t h e a r m a n d a t t h e d i s t a l t i p of t h e c h r o m o somes. C h r o m o s o m e s 28-31. C h r o m o s o m e 28 h a s 2 p o s i t i v e b a n d s , while c h r o m o s o m e 29 h a s o n l y one, w h i c h is proxim a l to t h e c e n t r o m e r e . C h r o m o s o m e 30 h a s 1 d i s t i n c t p o s i t i v e b a n d a t its distal tip, a n d c h r o m o s o m e 31 s t a i n s palely, h a v i n g a n a r r o w p o s i t i v e b a n d j u s t below t h e c e n t r o m e r i c region. c) Sex chromosomes. T h e s h o r t a r m of t h e s u b m e t a c e n t r i c X c h r o m o s o m e h a s 2 n a r r o w p o s i t i v e b a n d s , one a t t h e c e n t r o m e r i c region a n d t h e o t h e r h a l f w a y a l o n g its length. T h e long a r m h a s a b r o a d i n t e n s e l y s t a i n e d b a n d j u s t below t h e e e n t r o m e r i c region, d i s t a l to w h i c h is a b r o a d n e g a t i v e b a n d , w h i c h s t a i n s p o s i t i v e l y w i t h Cb a n d i n g (see below). T h e d i s t a l h a l f of t h i s a r m s t a i n s m o d e r a t e l y , 3 n a r r o w p o s i t i v e b a n d s b e i n g resolved. T h e b a n d i n g p a t t e r n of t h e a c r o c e n t r i e Y c h r o m o s o m e is v e r y d i s t i n c t i v e , t h e whole c h r o m o s o m e s t a i n i n g a u n i f o r m pale colour, e x c e p t for t h e tips of t h e long a r m s which stain darkly. C-banding. Most of t h e c h r o m o s o m e s i n t h e c o m p l e m e n t show a discrete b l o c k of h e t e r o c h r o m a t i n a t t h e i r c e n t r o meric region w h e n s t a i n e d w i t h t h e C - b a n d t e c h n i q u e (Figure 3), a n d t h e s e blocks c o r r e s p o n d to t h e n e g a t i v e l y s t a i n e d areas f o u n d a t t h e c e n t r o m e r e s w i t h G - b a n d i n g . T h e r e are some c h r o m o s o m e s , h o w e v e r , t h a t d e v i a t e f r o m t h i s simple generalization, a n d t o i d e n t i f y these, Q - b a n d ing was carried o u t p r i o r to t h e C - b a n d p r o c e d u r e (Figure 4). A m o n g t h e a u t o s o m e s , 1 m e t a c e n t r i c p a i r (No. 11) h a s n o d e m o n s t r a b l e C-band, a n d a n o t h e r (No. 8) h a s o n l y t h e s u g g e s t i o n of one. Some c h r o m o s o m e s , on t h e o t h e r hand, have extra centromeric heterochromatin. The Y
15.9. 1976
Specialia
c h r o m o s o m e , for e x a m p l e , consists a l m o s t e n t i r e l y of c o n s t i t u t i v e h e t e r o c h r o m a t i n while c h r o m o s o m e 12 a n d t h e X c h r o m o s o m e b o t h possess i n t e r s t i t i a l C - b a n d s halfw a y along t h e i r long arms, a n d c h r o m o s o m e 1 has a small C-band a t t h e distal t i p of t h e s h o r t arm. These v a r i a t i o n s in c o n s t i t u t i v e h e t e r o c h r o m a t i n c o n t e n t p r o v i d e a n add i t i o n a l m e a n s of i d e n t i f y i n g t h e s e c h r o m o s o m e pairs.
T h e P h y l o g e n e t i c S t a t u s of P h y l l o m e d u s i n e S t u d i e s of their S e r u m A l b u m i n s 1
1149
I t is also c o m m o n to find t h e C - b a n d s differ in size b e t w e e n homologues. This is p a r t i c u l a r l y t r u e of c h r o m o some p a i r No. 13, w h i c h s h o w e d a h e t e r o m o r p h i s m in a t least 4 o u t of t h e 9 a n i m a l s s t u d i e d (e.g. Figure 3). More w o r k is r e q u i r e d to d e t e r m i n e e x a c t l y h o w w i d e s p r e a d this p o l y m o r p h i s m is, a n d h o w m u c h o t h e r v a r i a t i o n t h e r e m i g h t be in t h e horse k a r y o t y p e .
F r o g s ( H y l i d a e ) as E v i d e n c e d f r o m I m m u n o l o g i c a l
LINDA MAXSON 2
University o/ Illinois, Department o/Genetics and Development, 515 Morrill Hall, Urbana (Illinois 61801, USA), 22 March 1976. Summary. B a s e d on i m m u n o l o g i c a l c o m p a r i s o n s of t h e s e r u m a l b u m i n s of p h y l l o m e d u s i n e frogs w i t h b o t h hyline and b u f o n i d species, it is s u g g e s t e d t h a t p h y l l o m e d u s i n e frogs b e e r e c t e d to familial s t a t u s w i t h i n t h e s u p e r f a m i l y Bufonoidea. The N e o t r o p i c a l leaf frogs, P h y l l o m e d u s i n a e , consist of t h r e e g e n e r a ; Phyllomedusa (31 species), Agalychnis (8 species), a n d t h e m o n o t y p i c Pachymedusa. These frogs c o n s t i t u t e a d i s t i n c t p h y l e t i c line in t h e family Hylidae. All frogs t e s t e d in t h i s s u b f a m i l y are u n i q u e a m o n g o t h e r hylids in possessing large a m o u n t s of p o w e r f u l b r a d y kinin-like a n d p h y s a l a e m i n - l i k e p o l y p e p t i d e s in t h e i r skin 3. All species in t h i s s u b f a m i l y are also c h a r a c t e r i z e d b y h a v i n g v e r t i c a l pupils, diploid c h r o m o s o m e complem e n t s of 26, arboreality, a n d m o d e r a t e l y ossified skulls (with or w i t h o u t t h e d e r m i s co-ossified w i t h t h e skull). All m e m b e r s d e p o s i t t h e i r eggs on v e g e t a t i o n over w a t e r , into w h i c h t h e h a t c h l i n g t a d p o l e s drop. The a q u a t i c t a d poles are u n i q u e in h a v i n g a sinistral spiracle lying vent r a l l y on t h e midline 4. C o n s i d e r a t i o n of t h e a b o v e characteristics led DUELLMAN 5 tO recognize t h e s e t h r e e genera as a s e p a r a t e h y l i d s u b f a m i l y . DUELL~A/~'S decision has b e e n c o n f i r m e d b y r e c e n t b i o c h e m i c a l studies w h i c h h a v e s h o w n t h a t a t least some p h y l l o m e d u s i n e s , a n d no o t h e r hylids, e x c r e t e uric acid r a t h e r t h a n amm o n i a a n d urea as do m o s t a n u r a n s 6. Additionally, f u r t h e r studies on p h y l e t i c affinities of p h y l l o m e d u s i n e frogs h a v e s u g g e s t e d t h e s e frogs m a y be closely r e l a t e d to a t least some of t h e A u s t r a l i a n hylids (Litoria). 2 of 5 species of Litoria t e s t e d were f o u n d to possess large fibrous melanosomes, c o n t a i n i n g a n o v e l red p i g m e n t , w h i c h were f o r m e r l y i d e n t i f i e d as u n i q u e to N e o t r o p i c a l leaf frogs% My i n t e r e s t in p h y l l o m e d u s i n e frogs arose d u r i n g studies of a l b u m i n e v o l u t i o n in t h e a n u r a n s u p e r f a m i l y Bufonoidea s. These studies w i t h a l b u m i n s of b o t h h y l i d a n d b u f o n i d species s u g g e s t e d t h a t t h e p h y l l o m e d u s i n e frogs should be e r e c t e d as a p r o p e r family, p e r h a p s i n t e r m e d i a t e b e t w e e n t h e H y l i d a e a n d t h e Bufonidae. I n d e e d , earlier serological studies b y CEI s, i n v o l v i n g p r e c i p i t i n t e s t s w i t h s h o r t t e r m a n t i s e r a t o whole serum, led h i m to suggest the Phyllomedusinae might represent an independent p h y l e t i c b r a n c h ariziug f r o m s o m e u n d i f f e r e n t i a t e d H y l i d - B u f o n o i d stock. Materials and methods. A n t i s e r a t o pure a l b u m i n f r o m a single s p e c i m e n of Phyllomedusa trinitatus ~o was m a d e in 4 male N e w Z e a l a n d w h i t e r a b b i t s over a t h r e e - m o n t h i m m u n i z a t i o n schedule. The i n d i v i d u a l a n t i s e r a were t e s t e d for p u r i t y a n d pooled a c c o r d i n g to p u b l i s h e d procedures 1~. As sources of a l b u m i n , p l a s m a or skeletal muscle p r e s e r v e d in a p h e n o x y e t h a n o l solution ~ were used. M i c r o c o m p l e m e n t f i x a t i o n s t u d i e s w i t h t h e a l b u m i n s of
r e p r e s e n t a t i v e s of all t h r e e p h y l l o m e d u s i n e genera as well as w i t h o t h e r h y l i d and b u f o n i d species were p e r f o r m e d . R e s u l t s are r e p o r t e d as i m m u n o l o g i c a l d i s t a n c e units. F o r a n u r a n a l b u m i n s 1 u n i t of i m m u n o l o g i c a l d i s t a n c e bet w e e n 2 species r e p r e s e n t s r o u g h l y one a m i n o acid difference in t h e a l b u m i n s of t h e s e 2 speciesl~. Results and discussion. T h e T a b l e s u m m a r i z e s t h e results of t e s t s w i t h a n t i s e r u m to t ). trinitatus albumin. All available species of Phyllomedusa f o r m one i m m u n o logical cluster w i t h a r a n g e of 0-61 units. This is t h e o r d e r of m a g n i t u d e seen b e t w e e n species of Gastrotheca (Hylidae : v m p h i g n a t h o d o n t i n a e ) , b e t w e e n N o r t h A m e r i c a n Hyla species (HyIidae: Hylinae), a n d b e t w e e n species of Bu/o (Bufonidae) 8. Paehymedusa dachnicolor is of i n t e r e s t since, w h e n first d e s c r i b e d b y C o p e in 1864, t h e species was p l a c e d in t h e g e n u s Agalychnis. I t led a , s p o t t y h i s t o r y of ~cransfer f r o m Agalychnis t o Phyllomedusa u n t i l 1968 w h e n DUELLMAN p r o p o s e d its i n d e p e n d e n t generic s t a t u s . I m m u n o l o g i c a l l y Pachymedusa a l b u m i n a p p e a r s m o r e d i s t a n t f r o m P. trinitatus t h a n a n y species of Phyllomedusa b u t n o t q u i t e as d i s t a n t as b o t h species of Ngalychnis available for this s t u d y . T h u s a t t h e molecular level Pachymedusa's generic s t a t u s is also justified. The
1 This study was initiated in the Department of Biochemistry, University of California, Berkeley. Part of this work was supported by NSF grant No. DEB75-23543.. 2 The author gratefully acknowledges the helpful advice and discussion of A. C. WILSON. Thanks are also due to D. WAKE, H. COGGER,W. DUELI,M ~ and R. HEVERfor generously supplying frog samples. 8 j. M. CEI and V. ERSPA~ER, Copeia 74 (1966). 4 W. E. DUELLMAN,The Hylid Frogs o~ Middle America, Monograph of the Museum of Natural History (University of Kansas 1970), vols. 1 and 2. W. E. DVELLMAN,Univ. Kansas PubI. Mus. nat. Hist. 78, 3 (1968). V. H. SHOE~aAKERand L. L. MCCLANAHA~,JR. J. comp. Physiol. ]3 100, 331 (1975). 7 j. T. BAGNARAand W. FERRlS, Copeia 83, 592 (1975). 8 L. R. MAXSON, P h . D . thesis Univ. of California, ]3erkeley and California State University, San Diego (1973). 9 j. M. CEI, Bull. Serol. Mus. 30, 4 (1963). 10 A generous gift of G. E. DREWRY. Xl L. R. MAXSONand A. C. WILSON, Syst. Zool. 24, 1 (1975). 1~ L. KARIGand A. C. WII~SO~, Biochem. Genet. 5, 211 (1971). 13 L. R. MAXSONand A. C. WILSOn, Science 185, 66 (1974).
