TITE UNIVERSITY OF },{ANITOBA

KARYOTYPIC ANALYSIS OF THE SOMATIC CHROMOSOMES

OF NOTURUS GYRINUS (MITCHILT,) (ICTALURTDAE: TELEOSTET)

by

CATHERINE ARNOLD BROWN LEVIN

A TTTESTS

SUBMITTED TO TTM FACULTY OF GRADUATE STUDIES

TN PARTTAL FULFILLMENT OF TTTE REQUIREMENTS FOR THE DEGREE

OF MASTER OF SCIENCE TABLE OF CONTENTS

Pa ge

LTST OF TABTESOOC." iii

LIST OF FIGURES iV

ACKNOI^J'LEDGMENTS V

ABSTRACT. 1

INTRODUCTTON.. 1

METHODS 3

RESULTS " 10 Diploid CounLs 10

General Features of the Karyotype 11

Median Chromosomal Groups 19

Submedian Chromosomal Groups 27

Subterminal- Chromosomal Groups 24

DTSCUSSION"... 26

SU}tr\4ARY. 30

APPENDIX. 32

LITERATI]RE CITED 57

1l- LIST OF TABLES

Table Page

I. Variation in dipl_oid counts of mitotic metaphase chromosomes in Noturus gvrinus. 10

II. Arm lengths and arm ratios of chromosomal groups 16 III. Combined male and female arm length ratios for chromosomal groups o.. c o 18

t- 11 TIST OF FIGURES

Figure Pa ge 1. Total body length varíation in Noturus gYrínus specimens used in this studY. 5

2 Photoidiogram of Noturus gyrínus, female 13

J. PhoËoidiogram of Noturus gvrinus, male. T4

4" Coordinate karyogram of Noturus gyrinus chromosomes, based on arm length averages (RtC) 15 5. Partial coordinate karyogram of median ohromosomal groups¡ M-l, M-2, and ,M-3 20

6. Partial coordinate karyogram of submedian chromosomes. 22

7. Partial- coordÍnate karyogram of submedian chromos oma l- groups: SM-1, SM-2, SM-3, and SM-4 23 B. Partial coordinate karyogram of subterminal chromo- somal groups: ST-1 and ST-2 25

o Photoidiograms of four species 27

1V ACKNOWLEDGMENTS

I wísh to express my gratitude to Dr. K. W. Stewart, who patiently discussed, encouraged and supported my efforts to carry out this study.

I am also grateful to Dr" N. R. FosËer for allowing me to make full use of his laboratory facilities at the Academy of Natural Sciences of Philadelphía and to draw upon his excellent knowledge of fish systematics during the final phases of thís study. I am indebted to Drs. M. di

Berardino of the tr{oments Medical College of Philadelphia for suggesLions offered prior to the preparation of the karyotypes, R. J. Schultz of the University of Connecticut, M. Samoiloff of the University of Manitoba, and M. H. Levin of the University of Pennsylvania for reading the manu- scripË and makíng many important comments and criticisms. I am also indebted to the University of Manitoba, the Academy of Natural Sciences of Philadelphia, l,Ioments Medical College of Philadelphia, and Villanova

University for permítting me Ëhe use of library and other facilities.

To my husband, Michael, and daughter, Eleanor, upon whose under- standíng and personal sacrifice I so freely drew and wiLhout r,'rhom no portion of thís sËudy would have been completed, I offer my sincerest appreciation and heartfelt gratitude.

The present study r\Tas supported, in part, by grants to Dr. K. tr{. Stewart of the University of Manitoba from the President of the Univer- sity of Manitoba, the National Research Council of Canada, the Fisheries Research Board of Canada, and, in part, by grants to Dr. N. R. Foster of the Academy of Natural Sciences of Philadelphia from the National

Science Foundation (GB-6810 and GB-15159).

The typing of this thesis was done by Louise K. Ternay. ABSTRACT. A statistíca1 study of the metaphase chromosomesof the North

American catfish, Noturus gyrinus (Mitchi11) (tadpole ), has shown

that nine homolog groups can be identified among the 42 somatic

chromosomes of this species. Relative length measurements of chromo-

somal arms on a cellular base (RfC) and on a group base (RtG) formed

the basic data from which coordinate karyograms hTere constructed.

Three median (M-1, M-2 and M-3), four submedian (SM-l, SM-2, SM-3 and SM-4), and two subterminal (ST-1 and ST-2) homolog groups were identified beËween arm ratio isopleths 1.0-1.7, L.7-3.0, and 3.0-7.0, respectively.

No terminal chromosomes \,Iere identified between arm ratio isopleth 7.0 and co , nor \.{as sexual heterogamety evident. The N. gyrínus karyotype r,üas compared with three other known catfish karyotypes:

Clarius batrachus (Clariidae), Hypostomus plecostomus (Loricaridae), and Ictalurus punctatus (). Only M-1 and M-2 homolog

groups appeared common to all four karyotypes. Comparison of homolog groups suggests that the loricarid and ictalurid karyotypes have undergone extensive chromosomal reorganization by means of pericentric inversion while the clariid karyotype appears to have remained relatively unmodified from an ancestral form possessing 52-56 telocentríc chromosomes.

INTRODUCTION

Of the 413 families of teleosts listed by Greenwood et al. (L966), I92 species representing only 35 families and 11 orders of teleostean vertebrates have chromosome data for one or more species. Indeed, so few species have been examined karyologically that litt1e can be 2 concluded regarding the probable derivation of their chromosome complements" Most cytogenetic knowledge applicable to systematÍc problems ís derived from the studies of remarkably few \.^Torkers r¡/ho have concentrated on the following five fish families: Salmonidae,

íncluding Coregoninae and Thymallinae, (Svardson 1942, L945, 1958;

Kupka, L948; Boothroyd, 1959; Nogusa, L960; Simon, L963; Simon and

Dollar, L963; Rees, 1964, 1967; Karbe, L964; Ohno et a1., 1965,

L967b; Booke, 1968; Roberts, L97O); (Nogusa, L960; Post,

L965; Ohno and Atkin, 1966; Ohno et al., 1967a; Muramoto et a1., 7968; InIolf et a1. , 1969); Cyprinodontidae (Scheel , 7966a, 1966b, 1968; Setzer, 1968, I97O; Chen L970, L97l; Chen and Ruddle, L970);

Poeciliidae (Friedman and Gordon, L934: Inlickbom, L94L, 1943; Ohno and Atkin, L966; Schultz , 1967; Schultz and Ka11man, 1968); Centrachídae (Roberts , 1964; Becak et al. , 1966; Ohno and Atkin, L966) " In these families and very recently in Gasterosteidae (Chen and

Reisman, L970), adequate cytogenetic data is now avaílable to begin to infer phylogenetic relationships among species on the basis of compara- tive chromosome morphology" (Chen, L97I; Ohno 1970a, L970b.) Correlated cytogeneËic studies of relative DNA content and electrophoretic analysis of tissue protein components have furËher elucidated systematic relatíonships among lower . (Ohno and Atkin, L966; Taylor,

T966; Atkin and Ohno, L967; Ohno et al., I967a, I967b; Bender and Ohno,

L968; Booke, 1968; Klose et a1., 1968; Muramoto et al., L968; I^iolf et al., L969; Behnke, L970). Ohno (1970b) provides a detailed discussion of this subject.

The (Siluriformes) are among those orders of fishes which have received little attenËion cytogenetically. Nogusa (1960) Ja reports dipl-oíd counts of 58 rod-shaped (acrocentric) chromosomes foï

Parasilurus asotus (siluridae) (n = 29) and 56 rod-shaped chromosomes for Pelteobagrus nudíceps (Bagridae) (n = 28) but adequate material \^ras not provided f or karyol-ogical analysis of these two species. somatic chromosome data are avaílable, however, for a single species ín three other catfish families: clarius batrachus (clariidae), the walking catfish, having 2n = 52 (srivastava & Das , L96B); Hypostomus plecostomus (Loricaridae), the armoured catfish, having zn = 54 and rctalurus punctatus (rctaluridae), the channel catfish, havíng 2n = 56

(Muramoto et al., 196s). The chromosomes of these three species have been karyotyped according to conventional methods which sorts the chromosomes into metacentric-submetacentric, subtelocentric and acrocentric (telocentric) categories by inspection. Accordingly, C. batrachus is reported to have 6 metacentrics and 46 acrocentrics, while [. plecostomus and r. punctatus have karyotypic formulas of 24msm * L2st + 1Bt and 16msm * 22st + l8t, respecrively.

The purpose of this particular study is, first, to determine the diploid number and morphological characteristics of the chromosomes of a second species of rctalurid, Noturus gyrinus (Mitchi11) (radpole madtom) and, secondly, to examíne patterns of chromosomal morphology among known catfish karyotypes that would suggest possible derivation of their chromosome complements.

METHODS

Chromosome preparations were made from gi11 filament epithelial ce11s of actively-growing juvenile specímens following the method used by Stewart and Levín (1968) Ëo obtain permanent dry mounted. smears of 4 mitotic chromosomes. Fish, approximately 4.5 cm in body length (Fig. 1C), were given a dorsal intermuscular injection of 0"05-

0"10 ml- of 0.01% colchicine solutíon using a 27-gauge needle and

tuberculin syringe. In sma11 specimens, approximately 3"0 cm in body

length (Fig. 1D), a portion of the injection \,/as administered intraperitoneally. Injected specimens were placed in a r¡e11-aerated

tank 2-6hrs. at I6-20oC. The fish were killed by pithing and the fourth branchial arch removed and placed in fish Ringerrs. Mucous, blood and debris r,^lere caref ul1y removed from the gi11 1amellae. Cleaned arches \^rere transfered to 0.1 M potassium cyanide (KCN) for 30-90 sec, depending on the sLze of the arch, then transfered to tríp1e glass-distill-ed water for a 3-5 min dialysis period. i{hen the filaments were visibly swo11en, the arch was transfered to 45% acetic acid fixative for at least 15 min. Following fixation, a monolayer of ce11s r,{as prepared by t'painting" the tips of the gi11 filaments across a pre-cleaned glass microscope slide (Esco: 1mm thickness)"

The smear \^ras allowed to air-dry and Ëhen stained in Gíemsaf s staín. The stained slides were rinsed in dÍstilled \^rater and allowed to air dry after which they were mounted with Permount mounting medium and a /É0 Corning cover-glass.

All slides \¡rere examined for intact, flattened and well-spread metaphase chromosome complements. Suitable chromosome spreads were photographed under 100X oi1-immersion planapochromat lens at a photomagnification of 630X. Parametric measurements r¿ere made from ca2800X photographic enlargements.

Tndividual nuclei were karyoËyped by attaching double-stick tape to the back of photographic enlargements, cutting out the individual Figure 1 A-D. Total body length variaËion in Noturus

gyrinus specimens used in this study.

^ Mature adult , 72.7cm. B. Mature adult, 6"7cm"

C" Large juvenile , 5.Lrcm"

D. Sma11 juvení1e, 3.Bcm" A

l B c

i-- ti-' 6 chromosomes and temporarily af.fixing them to a piece of wire or synthetic screening. The screen facilitated manipulation of chromo- somes during inspection analysis. Chromosomes \.vere tentatively assigned to a homolog or homolog grorlp on the basis of over-all length and position of centromere. Each chromosome vras then quantitatively described by measuring the length of every chromatid arm with a divider and expressing the measure in arbitrary units wÍth the use of a finely divided rule as described by Ruddle (L964). Further classification of chromosome groups was based on the arm ratio (1ong arm/short arm) nomenclature of Levan et al.(1964) r¿hich can be sunnnarized as follows:

Term Centromeric position Arm ratio Chromosome designation

M median sensu stricto 1.0 me tacentric

m median region r.0 - r"7 me tacentric

SM submedian region 1.7 - 3.0 subme tacentric

ST subterminal region 3.0 - 7"0 subtelocentric

t termína1 region 7.0 - co te locentric

T terminal sensu stricto æ Ëe locentric

Length measurements of chromosomal arms form the basíc data for karyotypic analysis. Ruddle (1964) has provided the rationale for the notation and formulae employed here. The length of the long arm (1) together with that of the short arm (s) describes the over-all length of the chromosome (c) such that: c = 1 * s. The centromeric position or arm ratio (AR) is described by the length of the long arm relative to that of the short arm such that: AR = 1 / s. A single value for the long or short arm can be obtained by averaging the length of the sister chromatid arms. This value may be expressed in two forms: 7

(a) absolute measurement (AL) in arbitrary units and (b) relative or percentage measurements. The measurements represented by RLC (relative length on a ce11u1ar base) describes the arm length as a decimal or percentage of the combíned lengths of all the chromosomes in the cel1.

The sum of the RLC measurements is here termed the genome length. The measurement represent.ed by RLG (relative length on a group base) compares a particular arm on a decimal or percentage basis with the total chromosomal length of the particular restricted group of chromosomes to r,+hich the chromosome in question belongs. The sum of the RLG measurements is here termed the group lengËh" The notations and relationships to these systems of length representations have been adapted from Ruddle (I964) for N. gvrinus chromosomes as follows: 1ar.d(i) lnr,cd(i) = 42 Ãrr lar,d(i) + ¿sArd(i) i=lL i=l

sAL9 (i) sRtcS(i) = 42rr 42 + lsar,9çi¡ i=lÀrai,9(i) i=l

sALd(i) (for SM-3 sni,cd(i) = chromosomes) 44 f.r r- à te"a(t) + à'o"a(t) i=1 i=l where: l¿i.d(i) = the absolute length of the long arm of the ith chromosome in a particular male ce11. u/Vtr Ll¿l,'¿(i) + /.sdXe{Ð = rhe roral absolure lengrh of the chromo- i=l i=1 somes in a particular male ce11 having

42 chromosomes.

lnf,Cd(i) = the relative length of the long arm of rhe

ith chromosome of a male cell on the basis

of genome length.

snl,e9(i) = the relatíve length of the short arm of

the ith chromosome of a female ce11 on the basis of group length.

Because variation in measurements from cel1 to ce11 is a consequence of the coiling cycle of chromosomes, magnification, and photographic

enlargement, it is necessary to convert absolute measurements to relative

. measurements so that inter-nuclear comparisons can be made with a

standardized base. It is also advantageous to determine mean values

for the arm lengths of specifíc subgroups on an RLG base, Such means can be calculated as follows: r lmca(:) . à J-r-1 Toaa = N Tni,Cd where: = the mean long arm lengrh (RtG) for N number of male ce11s in a particular chromosomal group. (The group in questíon n may be stated in subscript.) \- L lni,Cdç¡)= the toral lengrhs (RT,c) of long arms of j=1 a particular chromosomal group in N number of cell_s. 9

The tabulation of arm length data on both RLC and RLG bases can

be presented in graphical form. RLC data are presented as a tl¡ro-

dimensional coordinate karyogram. In specific instances, both RLC and

RLG data are presented similarly as a partial coordÍnate karyogram. Coordinate karyograms are constructed by plotting the long and short

arm length of individual chromosomes against each other on the X and Y axes, respectively. Chromosomes wíth similar lengËh characteristics will distribuËe as clusters of points. Because a probability statement

regarding the separabÍlity of these clusters cannot be made, it will be

stated arbitrarily that t'if the clusters are tr¡ro or more standard

deviations apart then the clust.ers may be considered as representing

distinct chromosomal groups"t' (Ruddle, 1964.) The average values beËween male and female arm length daËa have been plotted and a line representing one standard devíation has been drawn to each side of the mean for x and Y values. Both Patau (ro0o1 and Ruddle (1964) poinred out that the coordinate karyogram offers a convenient means of recording and relating karyotypie information. specifically, the number and relationships of the various chromosomal groups can be readily established.

Secondly, standard deviations of coordinate values provide an immediate concept of arm length variation and permit a judgment regarding the degree of separability of the chromosomal groups. Finally, the arm ratio of a chromosomal group can be determined directly from the coordinate karyogram by dividing the long arm RT,c by that of the short.

Moreover, estimates of arm ratios are facilitated by including arm ratio isopleths which are lines drawn through the coordinate system describing coordinates of equal ratio values. Arm ratio isopleths

1.0, 1"7, 3.0, 7.0 andco have been included in Fig. 4. These particular 10

isopleths separate median, submedian, subterminal and terminal chromo-

somal groups as previously defined. Having classified the chromosomes

into groups on the basis of arm ratio, subgroups can then be designated on the basis of over-a11 length" The longest chromosomal group in each arm ratio category is termed 1, the next longest 2, and so on. Thus the longest metacentric chromosome is labeled metacentric-1, or simply, M-1, and so on.

RESULTS

Diploid Counts

One hundred and forty-five mitotic metaphase nuclei were photo- graphed from slides of eleven tadpole (five females and six males). The range and frequency of chromosome counts are ïecorded in

Table I. The number of nuclei photographed per specimen depended

Table I. Variation ín diploid counts of mitotic metaphase chromosomes in Noturus gyrinus.

Diploido C ounts Sex N .1 0 37 3B 39 40 4I 42 Tota 1

I 5 2302244 40 75

ê 6 1033t2 1 50 70

a Total 11 J J J 5 36 5 90 145

Pe rcent 2.L 2.1 2.L 3.4 24.8 3"4 62.7 100 primarily on the quality of the smear and the frequency of metaphase ce1ls produced by the specimen during the incubation period. The number of nuclei counted averaged 13 per specimen and ranged from 7 to 25. 11

Diploid chromosome numbers ranged frorn 36 to 42. The modal count, representing about two-thirds (62.r%) of the nuclei examined, T¡ras

2n = 42. I^Iith the exception of some cells which had 2n = 40, all other

counts occurred with such 1ow frequency (2.1 and 3"47.) that they were judged to represent ruptured ce1ls. Such hypoloid ce1ls revealed no consistent chromosomal variation. Although one female had an individual modal count of. 2n = 40, non- modal counts of this type were suff iciently numberous (24.g%) to r^rarrant

special consideration. Approximately 32% of. female counts and !7./" of male counts were of this particular non-modal type but no particular pattern of chromosome irregularity existed" Modification in arm number such as might be attributed to centric fusions or telomeric associations

r,rere not observed. Therefore, it is assumed that the relatively high frequency of 2n = 40 complements in male and female samples was due to random chromosome loss similar to that noted. in other non-moda1 counts. General Features of the Karvotvpe

TL'¡e 42 chromosomes of the tadpole madtom have been divided into níne subgroups on the basis of two karyotypic parameters: over-all

chromosome length and arm ratío. These groups are shown in phoËo- idiograms of f emale and male cells (rigs . 2 and. 3) and as a t\,ro

dímensional coordinate karyogram (Fig. a). Three metacentric groups, yl-z, M'-r, and M-3, can be readily separated by their over-a11 length. Submetacentric groups, SM-1, STI-2, SM-3, and SM_4, T,{ere initially placed Logether but subsequent analysis (discussed later) indicated that they are distinctive. There are tr^/o groups of subtelocentrics, sr-1 and sr-2. subgroup sr-l consists of only one pair of large homo- logs while subgroup sr-2 contains nine pair of smaller homologs. l2

Because their inherent lengths grade imperceptably into one another, pairing among ST-2 homologs is not meanÍngful. No telocentric chromo- somes are present in the tadpole madtom karyotype.

Mean RLC and arm ratio data f.or chromosomes from male and female madtoms are tabulated in Table II. Mean values for chromosome arms are specified by subgroup for both male and female samples and the number of chromosomes involved in each average is given. The number of chromo- somes in a particular subgroup times the number of cells employed in the study is represented by the number N. The standard deviatíons from the sample mean as well as the coefficients of variation have been calculated. The v values have been included because they permit variance comparisons between groups that are independent of differences in the magnitude of the group means.

The tadpole madtom karyotype can be characterized ín a number of

T¡/ays using RT,C data. First, the mean arm lengths within groups are consistent even for the sma1l number of cells involved. This is supported by the fact that ËT¡Io-l,ray tests of the differences between mean scores for arm length in independently collected samples from males and females are not significantly different at the 5 percent

1eve1. The standard deviation for the long arm is on the order of 10 percent of the mean lengËh and 15 percent for the shorter aïmo Ruddle

(1964) pointed out that increased variance of the short aïm measurements as compared to long arm measurements is probably due to experimental error that can be expected to increase as arm length decreases" rmprecision as well as chance distortion of the chromosome will be magnified in the short arm relative to that in the long arm. The data also indicate that the size of the coefficient of variaEion is a 13

Figure 2" Photoídiogram of Noturus gyrinus, female. # #w wwww, P#f,fY ffiffi WWWw #t IW & #'* & M-t M-2 Ån-3 #ffi#w 'w w,É Jq,¡ W'&;,WM ewe'ffi& w@ &ffiYt% MWv&wn sM-l SM-2 SM-3 5ñÅ-4 i"W *q w, w{ww&,e&'&ffi ST-I ffi6i6&e&effi@w Noturu.s gy.inus sT- 2 lO ¡.t (f emo le) L4

Figure 3. Photoidiogram of Noturug EJÉgs, mal-e. ffi wwww ewww M-t M-2 M-3 w* Ë,tiAã .EEã# ww sM-l sM- 2 SM-3 sM-4

.eff'.ffi-*ffiffi ,ffi & 5T- I #ffiæ&eww&ffiffi Noturus gyrinus (m o le) ST-2 ro u l5

Figure 4. Coordinate karyogram of Noturu.s gyrinus chromosomes, based on arm 1-ength averages (RtC) NoruRus cyRtNUs:2n=42 MEDIAN daçI CHROMOSOMES

SUEMEDIAN l.J CHROMOSOMES ¿3 d. ã ú t- d,2 o SUBTERMINAI -vl CHROMOSOME S

TERMINAT CHROMOSOME S (NONE)

4 TONG ARM RLC Table TI. Arm lengths and arm ratios of chromosomal groups.

M-1 I 10 4.04 0. 16 3.96 3.10 0.18 5 .81 1.31 0 .04 3 .05 M-1 ð 10 3 "67 0.24 6,54 3 .00 0.18 0.00 I "23 0 .08 6 .50 vr-2 a 20 2.3L 0.35 15.15 1 .98 0.33 L6.67 1. 18 0.13 TL "02 M-2 ð 20 2.46 o,23 9 .35 L.99 0.30 15 .08 r.25 0. 15 12 .00 o M-3 + 20 L "20 0"06 5 .00 L.20 0 .06 5 .00 1"00 0 .00 0 .00 M-3 ê 20 r.26 0 .03 2 "38 L.26 0"03 2.38 1 .00 0"00 0 .00 SM- 1 I 10 s.34 0.08 1 .50 2.78 0. 18 6.47 1"93 0.12 6.22 SM- 1 é 10 5 "34 0.25 4.68 2.86 0.23 8"04 7.87 0,74 7 o "49 SM-2 + 20 4 "46 0 .55 12.33 2.L0 0.23 10 .95 2.15 0 .40 18.60 SM-2 4 20 4.63 0 .48 70.37 2.30 0"40 t7 .39 2.t9 0 .48 2\.92 SM-3 I 20 3 .60 0.31 8.61 L 0. 15 8 "77 "82 2.t2 0.25 7I.7 9 H SM-3 ð 20 3.22 0.36 11"18 1 .68 0.36 13 .53 1 .98 0.22 11.11 o\ SM-4 o 10 + 2 "52 0,10 4 .00 1 .06 0 .06 5 "45 2 "44 0 .05 2.49 SM-4 ð 10 2.58 0.20 7 ,69 7.04 0.11 11"00 2.47 0"02 8.10 ST- 1 I 10 5 "56 0.24 4.32 1"56 0. 16 r0.26 3 .60 0 .48 13 .33 ST- 1 & 10 5"50 0 .48 8.73 0 L.66 .08 4.82 3 .31 0 ^22 6 "6s ST-2 o+ 90 3.23 0.55 17 0 .03 .87 0.27 31 .03 3 .93 0 .85 2L "63 ST-2 90 3 0 .50 L5,62 O.BB 0 ê "20 "22 25.00 3.75 0 "72 L9 "20 a RCT, = Mean relative length expressed as a percentage of the genome length. b Standard deviation c V = Coefficient of variation d ÃR = Mean arm raËio T7 function of (a) mean arm ratio (AR) and (b) the number of homologs (N) ín the group. For example, the single pair of M-l homologs has about the same mean total length (7 percent) as a single pair of ST-1 homologs. However, the coefficíent of variation for the ST-1 short arrn which has a mean arm ratio equal to 3.StO.A (Table II) is consider- abLy greater t.han that of the longer M-l short arm i¿hich has a mean aïm ratio equal to 1.gtO.f Similarly, the si-ze of the V score reflects the number of homologs in the group. Chromosomes in groups M-1, SM-l, SM-4, and ST-1 have only one homolog and give the lowest V scores

(about 10 percent) . Chromosomes in groups M-2, M-3 , Sl'4'-2, and SM-3 have three homologs and, with the exception of group M-3, give higher V scores (about 20 percent). Low variability among M-3 chromosomes is probabl-y due to their very smalJ. size and near-median centromere. A bias Loward perceiving both arms as equal measures combined with Ímperceptible length varíations tends Ëo obscure real differences that undoubtedly exist. Therefore, the arm lengËhs and arm ratios for

M-3 chromosomes are likely more variable than the data suggest.

Correspondíng differences within groups composed of larger chromosomes, such as group ST-2, are more accurately reflected in their V scores" As previously noted, V scores for long arms in extremely subcentric groups such as ST-2 tend to be lower (Vg = L7.03; Vd = 15"62) than for short arms (V9 = 31.03; Vd= 25.00). No consistent differences between male and female V scores r,uere noted among the 9 subgroups.

Values for mean aïm ratios for each group have been calculated directly from absolute arm length data. These values together with standard deviations and coefficients of variation are given in Table IÏt.

Because no significant differences in arm length means beËween male and 18

Table III" Combined male and female arm length

ratios for chromosomal groups "

Group AR vb

M-1 20 7.27 0.06 4.78

I,1,-2 40 1,.22 o.L4 11.51

M-3 40 1.00 0.0 0.0

SM- 1 20 1" 90 0. 13 6.86

SM-2 40 2.L7 0.44 20.26

SM-3 40 2.05 0.24 tI.45

SM-4 20 2.46 0.04 5.30

ST-I 20 3 "46 0.35 9 "99 ST-2 180 3"84 0. 78 20 "42

s = standard deviation,

b V : coefficient of variation female samples were found, absolute values for both sexes have been combíned. The standard deviations again are comparatively low, ranging between 5 and 20 percent of the mean. As in t.he case of arm length measures, groups with very sma11, equal-armed chromosomes (M-3) and groups with only one homolog have typicall_y loi¿ variability (M-1,

SM-1, SM-4, and ST-1). Multiple homolog grorlps with similar centromeric position in each homolog have somewhat higher scores (M-2 and sM-3).

The highest v values are obtained in homogeneous groups in which the arm ratio varies substantially between homologs (sM-2), and in groups with sma11 arms having (ST-2) high experimenral error " T9

Median Chromosomal Groups

A partial coordinate karyogram of groups M-l, M-2, and M-3 is

shown in Fig. 5. It can be seen that three distinct clusters of points are grouped within the metacentric arm ratio range (1.0-1.7). The clusters are separable not only in terms of over-alI length but also on the basis of arm ratio. Arm ratio ísopleths have been constructed for each subgroup based on data from Table TII. The fit is sufficiently cl-ose to a straight line in each group so that ít is unnecessary to

carry ouL a regression anal-ysis. These data suggest that the arm ratio for each group is isomorphic over a change of chromosomal length by a factor of L.2, L.4, and 2"0 for M-L, M-2, and M-3, respectively.

Group M-1 consists of two homologs whose mean aïm ratio is 1"27!0.06

(cf. Table III). They are the largest and most asymmetrical meta- centrics in the genome" Because M-l chromosomes have the same over-a11 length as SM-2 chromosomes (about 7 percent), good preparations are required to achieve arm ratio separation. Group M-2 contains four homologs sufficíently similar in total length (about 5 percent) and arm ratio (7.22t0.74) to make pairing meaningless. In some cells, however, t!üo M-2 homologs \^7ere def ínitely targer than the other two and this variation in over-all length is reflected in V scores on the order of 15 percent (cf" Table II). So few cells exhibÍted size differences that consistent pairing within this subgroup would be unreliable without the aid of additional chromosomal markers. Group M-3 consists of the four smallest homologs in the genome and are, therefore, easily classified by inspection. As has been noted, their very sma1l size prevents precise arm ratio descrimination. Hence, distribution of all M-3 chromosomes along the 20

Figure 5. Partial coordinaLe karyogram of median chromosomal groups: M-l, M'2, and M-3"

27

1.0 RIC arm ïatio ísopleth probably represents a mole perfect fit to this isopleth than is truly the case.

Submedian ChromosomaL Groups

The submetacentric chromosomes rlere most difficult to classify' High variability in absol-ute length and arm ratio from ce11 Ëo cell made consistent pairing questionable. Fígures 6 and 7 show the same data plotted as RIC and RLG val-ues, resPectively'

RLC values (Fig. 7) distributed along the 2.2 mean arm ratio isopleth make descrimínation of subgroups inconclusive. Some suggestion of separability of distaL homologs from adjacent groups can be seen in the clusLerÍng of points at each end of the isopl-eth. Further evidence of the distinctiveness of the distal cl-usters is shown in their mean arm raËios which, when calculated separately (cf. Table II), are 1.90t0.13 and 2.46t0.04 for SM-l and SM-4 chromosomes, respectively.

On the other hand, SM-2 and SM-3 homol-ogs have similar arm ratios

(2.I7!0.44 and 2.05+0.24, respectively) with relatÍvely large standard deviations so that the real-ity of tvro distinct seLs of homologs can only be demonstrated in terms of total length differences. The partial

RLG karyogram (Fíg. 7), has an over-all length difference of about 1 peïcent, sepaïâting each of the four sets of homologs. According to convention, t'once chromosomal groups have been shown to have little overlap, as in this instance, it is permissible to classify the chromo- somes of individual- cells inËo homologs for the purpose of determíning mean arm lengths. It should be pointed out that the degree of differ- entiation that must exist in order to decide whether homologs may be classified with confidence is really undefined." (nuadle , L964) 22

Figure 6. Partial coordinate karyogram of submedian chromosomes. NoruRUs GYRINUS t2n=42 ås

íå 8. s 8 L., Èt *Þ áÞ æ@ 8ou ã ôA &, ôa I $ '$ ;â s & oo$ o oo ttl /,, SUBMEDIAN CHROMOSOMES oct

0.5

0 2.o 2.5 4.0 4 IONG ARM RTC 23

Figure 7. Partial coordinate karyogram of submedian chromosomal groups: SM-l, SM-2, SM-3, and SM-4.

24

Subterminal Chromosomal Groups

The tadpole madtom karyotype contains 20 subterminal- chromosomes only two of vihich are sufficiently different in total length to be paired separately. The remaining 18 subterminal homologs form a continuum for

total chromosome 1-ength. Figure 8 illustrates the dispersal of sub-

telocentric chromosomes along mean Rl,c arm ratio isopl-eths 3"5 and 3"8 for ST-1 and ST-2 homologs, respectively. ST-l- homologs are readily separated from ST-2 homologs by a genome length facËor of about 1 percent and easily distinguished from other large elements, such as M-l, SM-l, and sM-2 chromosomes, by theír relatively large mean arm ratio. sr-2 homologs can not be paired on any normalized basis. Partial pairing in some cell-s can be achieved with satisfaction but there rnrere so few of

this type that pairing patterns in ST-2 chromosomes must be regarded as unreproducible. The partíal coordinate karyogram (Fig. 8) emphasizes the difficulty of resolving distinct homologous pairs within this group. Because there is no suggestion of discontinuities which míght refer to homolog differences, Ëhe data r¡rere not normaLized on an RLG base.

NevertheLess, distinctl-y different chromosomes exist in this population if one considers total chromosome length. The shorËest chromosomes have an RT,c val-ue of 2.7 whíle the longest have values of 5.8. I^ihen one considers that the standard deviations of mean length (RT,C) are on the order of.2o percent of the average length values, length differences between the 1-ongest and shortest chromosomes represenËs a true length difference. Therefore, if only the longest and shortest chromosomes r¡rere present, they would be easily separable. similarly, the arm ratios for ST-2 chromosomes grade from 3"0 to 6.5. Real differences among ST-2 chromosomes do indeed exist but the differences are very slíght from one 25

Figure 8. Partial coordinate karyogram of subterminal chromosomal groups: ST-1 and ST-2. \t\

t!i',

Norunus GYRTNUs:2n=42

d, E d,

4 ST-2 o I

8å @#8 æ a I ö ê æ & o=1 ¡=d

4.0 4.5 IONG ARM RI,C ¿o set of homologs to another and normal variation tends to obscure them even further. The data suggest that the ability to paír ST-2 hornologs on the basis of either arm length or centromeric position is illusory and that these homologs must be considered indeterminate.

DÏSCUSSION

Muramoto et a1.(1968) provided evidence that loricarid and ictalurid specíes are diploid and have acquired varying degrees of genetíc redundancy by means of 1) unequal exchange during mitosis, 2) unequal crossing-over during meíosís and 3) regional duplication of chromosomal segments. Consequently, it is supposed that pericentric inversions, divergence of duplicated gene loci and deletion of non-vita1 chromosomal material has been the primary mechanisms by which modified diploid counts and chromosome structure have evolved from an ancestral siluriform prototype consisting of 50-54 acrocentric chromosomes"

(Ohno et al., 1967a.)

While more parametric studies of siluriform karyotypes are needed

Ëo elucidate phylogenetic relationships within and between catfish families, comparíson of known catfish karyotypes on the basís of apparent chromosome homology can be considered. Chromosome complements of three previously karyotyped catfish together with that of \" gyrínus are presented in Fig. 9. Chromosomes of C. batrachus are classífied according to its investigators, Photoidiograms of H, plecostomus and L, punctatus are tentatively re-grouped according to over-411 length measurements and arm ratio. Initial karyotyping was presumably done by

ínspection of many nuclei and it is recognized that confident subgroup- ing would require parametric analysis of more chromosome sets than are immediately avail-able . 27

Figure 9. Photoidiograms of four catfish species. A. Clarius batrachus (after Srivastava & Das, 1968), 2n = 521' kf. = 6m * 46t.

B. HvposËomus plecostomus (modified from Muramoto et al., l-968) . 2n = 54¡' tentative kf = 10m * 12sm * 14st + l8t. C. Ictalurus puncËatus (modified from Muramoto et a1. , 1968). 2n = 56; tentatíve kf = 10rn t 12sm * 16st + l8t. D. Noturus gyrinus. 2n = 42, kf = 10m 4 12sm * 2ost.

:k kf = karyotypic f ormula. CLARIIDAE ICTALURIDAE kf' Clorius bo lrochus 2n=52 kf lctolurus punctotus 2n=56

óm X X trrt co x 2.500 lOm Kt'K &,þt,?&w íMwiM&,

l2 sm 't,, Ii ¡r 46r nå0 nnntñâánn & fr'gu,$ 1g|e$t&Wþè, t&

ß n n ñ n n a i rt fi I o 0 rrn ló st & & fr,& *¡W', : U'ft'&1,'ï\,:&'w & & þ'

fìñAì¡gônñaAlâÀ ---- l8r 6': '*6ø 's & & e # ffi'& *. e ft,rrtr '&, &'ffi '& íb

. LORICARIDA ICTALURIDAE gyposromus I kf plecosromus 2n=54 kf Not u rus syrinus 2n= 42

ro m &w ew lOm Ww:* ffi-% We Æ,w we WWffi"w w&¡ew _ti ,"1 12 "rt¡ I sm Y'ffi WffieW &,w&æg"ø' 12 sm Jrè '"# 'l

r4st &'e, 20 st e e' ffi w fuu'f'effiffi**'&åw*"þ ffiit$ ffi, w %,&w'W,& & e&' &'W's& ì8 t &*:ø" *u*'*w ffiffi&& wewffi &tu ffiø',&W

1., .¡1 28

0f the four species available for karyologíca1 comparison, C. bqtrachus appears to have undergone the least chromosomal reorganLza- tion. The larger pair of metacentrics and two medium-sized metacentrics are thought to be homologous to M-1 and M-2 groups in N. gyrinus. These six metacentrics probably arose very early in catfish evolution because they appear in representatives of widely divergent siluriform families. The derivation of two paírs of minute metacentrics (M-3) ís obscure.

Their presence in both taxonomically primitive and advanced groups, such as lctaluridae and Loricaridae, and their absence in C, batrachqg suggest one of two possibilities. Either an O1d Inlorld clariid ancestor diverged very early from the main line of catfish evolution, prior to the f ormation of the M-3 subgroup, or silurif orm families of New trnlorld origin (tentatively including Tctaluridae) have retained the four M-3 homologs while all or some 01d I,riorld form have either lost these chromosomes or incorporated them into existing linkage groups. A dÍp1oid number of. 52 and the presence ot 46 telocentrics in the C. batrachus karyotype tends to support the hypothesis that clariid chromosome complements more closely resemble that of the ostariophysian protoEype than do other taxonomically primitive and advanced New I^Iorld catfish groups, Perciform fishes are another exampl-e where taxonomicall-y advanced species have retained relatively unmodified chromosome numbers and structure. Thus, it is 1ikely that the ancestral karyotype from r¿hich C. batrachus evolved with littl-e structural modif ication consÍsted of 52-56 telocentric chromosomes. Nogusa (f000¡ reports a diploid count of 56 rod-shaped chromosomes for Pelteobagrus nudiceps. This species belongs to Bagridae which is generall-y regarded as a taxonomically 29 primitive 01d l^iorld catf ish f amily'

The chromosomes of H. plecostomus, I. punctatus and N. gyrinus show little morphological resemblance to those of C. batrachus. However, the karyotype of I. punctatus can be equated to that of C. batrachus to the extent thaË a steady progression of pericentric inversions among 46 telocentrics could provide the 46 submetacentric, subLelocentric and telocentric chromosomes observed in I. punctatus. The inversion process presumably continued further in the Noturus line where, ín the case of

N.. gyrinus, no truly telocentric chromosomes are evident. Seven pairs of telocentrics appear to have either been lost in N. gyrinus or incor- porated into existing linkage groups, I,rihatever the mechanism, consíder- able genetic consolídation and chromosomal- reorganization seem to have accompanied the divergence of Ictalurus and Noturus lines. A similar pattern of chromosomal reotganízation and diversificatíon of diploid numbers has been proposed for another group of ostaríophysians.

According to Ohno et a1. (1968) "it appears that an ancestor of the family Cyprinidae I order ] had 48 acrocentrics and a DNA content of about 20% that of mammals. Without substantial change in the DNA content, modification of this karyotype mainly by perícentric inversíon subsequently occurred to many members of this family. They came to possess 50 to 54 chromosomes, nearly half of which are meta- centric Iincluding submetacentric] ." In the case of siluriform groups, however, Muramoto et a.1 . (1963) poínt out that "dipl-oid members of Ostariophysi Lui". Siluriformes] undervrent extensive chromosomal ïeaïrangement as well as a sËeady increase in DNA content by regional duplication of chromosomal segments.'l 30 It is not possíble to deduce the degree of genetic divergence between L punctatus and H. plecostomus so1e1y on the basis of chromosome morphology, The presence of an additional paír of sub- telocentrics accounts for the difference in diploid number between them, Their karyotypes appear to be quite similar in other respects. Spectrodensitometric studies (Muramoto et a1., 1968) indicate that the nuclear DNA content of H. plecostomus is 51% thaË of mammals while that of L punctatus is only 30%, As a consequence of tandem duplication of

DNA segments, it is possible that parametric studies of chromosome length would show the genome length of H. plecostomus to be sígnificantly greater than that of I. punctatus,

SUMMARY

A study has been made of the somatíc chromosomes of Noturus gyrinus

(Mirchill). The diploid number is 42 and sexual heterogamety tnTas not evident, Parametric analysis of over-a1l- chromosome length and arm ratio has permitted separation of chromosomes into nine homolog groups. Three median groups consisting of one pair of M-1 homologs, tvlo paír of M-2 homologs and two paír of M-3 homologs have been identified on an RLC base. Separation of four submedian homolog groups was possible only on an RLG base. Submedian groups consisted of one pair of SM-l homologs, two paír of SM-2 homologs, t\^Io pair of SM-3 homologs and one paír of

SM-4 homologs. Ten pair of subtermína1 chromosomes have been identifíed onLy one of which (ST-1 homologs) was separable on an RLC base. The remaining eighteen subterminal chromosomes (ST-2 homologs) could not be separated by either base method. Homologous paíring within the ST-2 31 subgroup r¡Ias theref ore regarded as indeterminate. Possible derivation of four catfish karyotypes vras considered. Clarius batrachus (Clariidae) appeared to have the least modífied karyotype consisting of 6 metacentrícs (M-1 and M-2 groups) and 46 telocentríc chromosomes. The chromosomes of N. gyrinus resembled those of Tctalurus punctatus (Ictaluridae) and Hypostomus plecostomus (Loricaridae) in that all three had 10 metacentric chromosomes (M-1,

M-2, M-3 groups) and L2 submetacentric chromosomes (SM-1, SM-2, SM-3,

SM-4 groups). The N. gyrinus karyotype contained four more subterminal chromosomes than both H. plecosLomus and I. punctaËus but was lacking eighteen terminal chromosomes found in the other two species.

Hypostomus plecostomus and T. punctatus karyotypes appeared to differ by one subterminal pair of chromosomes. Comparison of homolog groups suggests that the loricarid and ictaluríd karyotypes have undergone extensive chromosomal reorganization by means of pericentric inversion whíle the cl-aríid karyotype appears to have remaíned relatively unrnodified from an ancestral form possessÍ-ng 52-56 telocentric chromo- somes. 32

APPENDTX A variety of karyological symbols and nomenclature are used to

describe diploid sets of chromosomes. The symbols, as reported by workers cited in the Appendíx, are listed and defined below.

Svmbol Karyol-ogical description

a acrocentric chromosomes; subterminal or terminal centromere; rod-shaped chromosomes .

m metacentric chromosomes; median or near-medían centromere; V-shaped chromosomes.

msm metacentric and submetacentric chromosomes; median or submedian centromere; bí-armed chromosomes.

sm submetacentric chromosomes; submedian centromere; J-shaped chromosomes.

sn supernumerary chromosomes; centromere presumed diffuse or lacking; dot-shaped chromosomes,

st submetacentric chromosomes; submedian centromere; J-shaped chromosomes.

t telocentric chromosome; terminal or near-terminal centromere; rod-shaped chromosomes .

F fundamental chromosome; including sma11 metacentric, submetacentric and acrocenËric chromosome presumably derived by pericentric inversion.

L large metacentric or near-metacentric chromosomes presumably derived by centric fusion.

S satellited F chromosomes.

I^l morphologically ídentifiable female sex chromosomes; female heterogametic.

X morphologically identifiable female sex chromosomes; female homogametic.

Y morphologicall-y identifiable male sex chromosomes; male heterogame tic .

Z morphologically identifiable male sex chromosome; male homogame tic .

( ) specifíed sex chromosome noË morphologícally identifiable" Annotated chromosome numbers in fish and fish-like chorda tes .

No" Taxon Chromosome Tissues Reference 2n I nlfn De scrip.tion examined

Urochorda ta : Ciona intestína1is 2B 28a, minute 5 tes tis aylor, 7967. ç.. intestinalis 6 tkin & Ohno, 1967 . ç.. intestinalis 2B 28a, mínute 6 testis hno et al., 1968. Styela plic.ata 32 T6 32a 10 testis ay1or, 1967.

Cephalochordata: L,) F. Branchios toma belcher! J¿ L6 32a testis ogusa, 1960. B. lancgolatum L7 o et al", 1968.

Cyclostomi: ,) !g!atretus burgeri 4B /, 48a testis ogusa , 1960. E. okínoseanus 46 23 46a te s tis ogusa, 1960. E. 48 4Ba testis aylor 1967. "t""tii , E. 4B 4Ba, large 78 no, É 91., "tt"tff L968. Entosphenus reissneri 4-96 94-96a tes tis ogusa, 1960. Lampetra planerí 4-96 a, minute 3B no et al., 1968. Elasmobranchíí:

..'gDasvatis akaiei 84 42 64a + 20m Lestis ogusa , 1960. Mustelus manazo 72 36 LZm + 60a tesLís ogusa , 1960. Taxon Tissues Reference De s cript ion

Teleostei: Division I Superorder ELOPMORPHA Order Anguilliformes Suborder Anguilloidei Family: Anguillidae Anguí1la anguilla 38 cornea ick eL a1., L962. A. iaPonica 3B cornea ick et al., 1962. A. ros tra ta 3B cornea ick et al. , Family: Echelidae 1962. Echelus uropterus 50 25 46a * 4sm te s tis ogusa , 1960 Family: Muraenidae Gr/mnothorax kidako 42 2L 30a * I2m testis ogusa, 1960 Muraena pardalis 40 20 26a I 14m tes tis ogusa, 1960 Supe rorder CLUPEOMORPHA Order Clupeiformes Suborder Clupeoídei Family Clupeidae Clupea harengus 52 ce 11 berts, 1966 cu1t. Q. Pa 1la sa i 52 60 2B gi11, no et al., spleen, 1967b. 1iver, tes tis 9. pallasaí 52 60 2B gi11, lose et al s p1e en, 1968. liver, tes tis Taxon Tissues Reference De scr ipt ion

C. pallasai 52 44a * 6m 28 Ohno et al., 1968 Hypomesis pretiosus s01 60 2L gi11, Ohno et al., 1iver, L967b. spleen, I tes tis H. pretiosus 501 60 2L gi11, Klose et a1., spleen, 1968. liver, tes tis Sprinchus starksi s0t 60 24 gi11, Ohno et al., spleen, L967b. 1iver,

J testis S. starksi 50i 60 24 gi11, Klose gL al., sp leen, 1968. liver, tes tis Family Engraulidae M. iaponicus 4B 24 4Ba tes tis Nogusa , L960. Engraulis mordax 48 48 43 gi1l, Ohno et a1. , spleen, 1967b. 1iver, testis E. mordax 4B 48 44 gi11, I(lose et a1., spleen, 1968. liver, testís E. mordax 4B 48 40 hno et a1", 1968. Chromosome No Tissues Taxon Reference De s crip tion o

Divisíon III Supe rorder PROTACANTHOPTERYGII Order Salmoniformes Suborder Salmonoidei Family Salmonidae Subfamily Salmoninae Onchorhynchus gorbuscha 52 embryo Simon , 1963, 0. ketA 74 embryo Makino, L937. Q. keta 50 te s tis Nogusa , 1960, O. keta 74 embryo Simon , L963. O. kisutch 60 embryo Simon, L963. L\) 0. kisutch B-60 104 87 gi11, Ohno et a1., { spleen, 1967b, 1iver, tes tis O. kisutch 8-60 104 B7 gi11, Klose et al., spleen, 1968, 1iver, testis -L O. kisutch 78: 87 Ohno et al", 1968. 0. masou 50 te s tis Nogusa, 1960. 54 tes tis Nogusa, 1960. 9.. "er&e. O. rhodurus 50 tes tis Nogusa, 1960. O. tshawvtscha 68 embryo Simon , L963. Salmo alpinus BO I6a embryo, Svardson, L942 testis 1945. Salmo clarki 64 106 22a * 42m embryo Simon & Do11ar 1963 Chromosome N Tissues Taxon Reference Descríptíon examined

Þ.. irideus (=eaird""ri) 60 right, 1955. !.. irÍdeus 60 embryo Lieder, 7956. Þ. i ride us 104 52 90a * 14m tes tis Nogusa, 1960. q. irideus. 60 104 L6a * 44m embryo Simon & Do11ar 1963 S. irideus 58- 65 embryo, Ohno et â1. , liver, L96s kidney, spleen, tes tis [. irideus 59-64 80 spleen, Ohno & Atkin, te s tis 1966. irideus sB-64 L04 BO gi11, Klose et al", spleen, 1968. liver, tes tís S. Írideus 58-64 104 36m BO Ohno et al., 1968. S . sa 1a.r 60 embryo Prokofieva, L934 S. salar 60 L2a embryo, Svardson , L942, testis L945 S. salar 56 embryo Boothroyd, 1957 , 1958, 1959 . S. salar 60 embryo Rees, L964. S. salar 54; 72 ce11. Roberts , L970. 55; cult. 56 S. trutta 84 embryo Prokofieva, L934 S. trutta (=fario) 84 embryo Pomini , L939. Taxon omosome No. Tissues Reference De scrip tion examined

Þ. trut ta B4 embryo Svardson , L942. g. trut ta 80 L6a embryo, Svardson , 1945. testis t. trut ta BO embryo Rees , 1964. S. trut ta 77-82 00 gi11, Klose et al., spleen, t968. 1iver, tes tis S. trutta 8ot Ohno et a1., L968. (, Salvelínus fontinalis BO embryo Prokofieva, 7934, \o S. fontína1is B4 I6a embryo Svardson, 1945. S. fontinalis B4 embryo hlright, 1955. S. fontinalis 50 testis Nogusa, 1960. S. namaycush ð4 embryo lnlahl , 19 60 . Subfamily Coregoninae Coregonus albula B2 embryo, Svardson , L942. tes tis C. a lbu1a BO 96 l6a embryo, Svardson, L945, tes tis C. artedii BO 06 6m*10sm*54a embryo Booke, 1968 C. asperi ca36 Kupka, 1948 C. asperi 36 20 Ohno et al. 1968. C. clupeaformis BO 9i11, Klose et al spleen, T968. 1íver, tes tis osome No Taxon Reference De scr ip t ion

ç.. c lupeaf-ormis BO t0B 20m*Bsm*52a embryo Booke , 1968. ç.. c lupeaformis BO Ohno et al., 1968. c. exiguus ca72 Kupka , I94B " ç.. exiguus 96 Karbe , 1964, q.. exiEuus 80+ 10ot 90 gi11, Klose et a1., tes tis , 1968. spleen, liver ç.. hovi BO 9B 10m*Bsm*62a embryo Booke, 1968 +r " O C. lava re tus 80 96 L6a embryo, Svardson , 1942, tes tis 794s. ç.. lava re tus 96 96 Karbe , L964. C. lava re tus BO 96 Viktorovsky, L964 " ç.. lava re tus ca 80 90 gi11, Klose et al., spleen, 1968. 1íver, tes tis C. macrophthalmus 90 Klose et al., 1968. na sus 96 96 Karbe, 1964. oxyrhvnchus 96 96 Karbe, 1964. pe 1ed 80 92 Viktorovsky, 1964 " pidschian 96 96 arbe, L964, reighardi 80 I04 L2m*12sm*56a embryo ooke, 1968. schínz i i ca72 upka, L948. s chinz i i 96 rbe, 1964. osome No. Tissues Taxon Reference De s cr ipt ion examined

C. wartmanni 7Bt3 BargetzL, 1960. C. wartmanni 96 Karbe, 1964. C. wartmanni ca 80 Ohno et a1., 1968 C. zenithicus BO 98 10m + 8sm * 62a embryo Booke, 1968. P. coulteri 82 r00 10m + 8sm * 64a o embryo Booke. 1968. r. LYI^'-t.indraceum 7B 100 L2r.l + 10sm * 56a embryo Booke, 1968. Subf anrily Thyma l1inae Thymallus thvmallus LO2 130 2Ba embryo, Svardson , 1945. tes tis T. thvmallus 60 Klose et a 1. , N 1968. H Family Osmerídae Hvpomesus pretiosus 52 Bm 2L Ohno et al., 1968 Osmerus eperlanus 58 10m f 48a embryo, Svardson , 1945. testis Suborder Galaxioidei Family Salangidae Sa langichthys mícrodon 2B testis Nogusa , L960. Suborder Esocoidei Family Esocidae E. lucius 48 embryo Prakken et a1 1955 . Family Umbridae Umbra limi 22 11 testis Foley , L926. Superorder 0STARIOPHYST Order Cypriniformes Suborder Characoidei Family Characidae Taxon Tissues Reference De s criptíon e>

Cha lceus macrolepidotus 32m 4 22st 54 testis, Muramoto et al., kidney, 1968. spleen, gi 11 Serrasalmus hollandi 64 48 tes tis , Muramoto et a1", kidney, 1968. spleen, gi 11 Suborder Cyprinoidei Famí1y Cyprinidae F. 50 25 6m * 44sm testis Nogusa, L960. * I Abramis brama 50 B0l 36 gi1 1 l^Iolf et a1. , 1969. AcheiloglaËhus cyanos trLgma 44 22 4m * 40a testis Nogusa, 1960. A. limbata 44 22 4m * 40a tes tis Nogusa, 1960.

A. rhombea 46-48 23-24 I 2m I 44or46a tes tis Nogusa, 1960. Barbus barbus 100 r44: 49 gi 11 Inlolf et a1., 1969. B. fasciatus 52 22 gi11, Ohno et a1., spleen, 1967 a . tes tis B. semífaciolatus 52 26 4m * 48a tes tis Nogusa , L960.

B. tetxazona 50 34m 20 gi11, Ohno et al., spleen, 7967 a . tes tis It tetrazona 50-52 Bender & Ohno, L968. 20 Þ.. tettazona 50 .L 34m Ohno et al., 1968. Þ.. tetrazona 50 901 20 gi 11 I{o1f et a1., 1969. Chromosome No. Tissues Taxon Reference De scription

Carassius aLlratus 96-L04 64m 50 spleen, Ohno & Atkin, L966. tes tis C . aura tus 100 12m + 36sm * 52a kidney Ojima et al", 1966 " C. auratus 100 I2m + 36sm * 52a kidney Ojima & Hitotsumachi, L967 . C. auratus <104 52 gi11, Ohno et al., 1967 a . spleen, tes tis C. auratus 104 52 Ohno et al., 1968. C. auratus <104 t66! 53 gi 11 I^Iolf et a1., 1969, +r C. carpio <104 50 gi11, Ohno et a1., I967a UJ spleen, tes t is C. carpio 104 46m f, * 18st * 36t 50 Ohno et a1r, 1968. C. carpío <104 168 l 52 gi 11 olf et a1., L969. Ctenopharyngodon ide 11us 4B 24 4Ba testis Nogusa 1960 , " Gnathopogon elongatus 50 25 50a testis Nogusa , 1960. Hemibarbus longiros tris 50 25 50a tes tis Nogusa 1960 , " Ilemigrammocvpr i s rasborella 48 24 48a tes tis Nogusa , L960. Ishikauia steenackeri 48 24 2m* 46a testis Nogusa, 1960. Labeo chrysophekadion 50 14m * lSst * 18a 40 tes tis , ramoto et al., kidney, L968. spleen, gi 11 l,euciscus cephalus J 50 881 3B gi 11 olf et gl. , 1969 , Moroco perenurus 27 tes tis ogusa L960. M. steindachneri 54 27 8m4 46a tes tis ogusa 1960. Opsarííchththvs unc iros tr i s 66 JJ 66a tes tis ogusa 1960. Pseudogobio esocinus 52 26 52a tes tis ogus a 1960. Pungtungia herzL 50 4m -l- 46a tes tis ogusa 1960. Tissues Taxon Reference Descript ion o

Rhodeus ocellatus 44 22 2m * 42a or tes tis Nogusa , L960. 4m J * 40a 70 | Rutilus rutilus 50 t(J- 28 gi 11 Wolf et al", L969. SarcochíeLíchthv s variegab:s 50 25 50a tes tis Nogusa, 1960" Tinca tinca 4B B0f 30 gi 11 I,rlolf et a1., L969 " Zaeco platvpus 4B 24 48a tes tis Nogusa, 1960. Z. temmincki 50 4m 46a = testis Nogusa , 7960 " Famíly Cobitidae Acanthophthalmus khu11i 50 14m 4 l2st*24a 2B testis, Muramoto et a1., kidney, 1968, spleen, N N gi 11 Barbatula oreas 48 4m* 44a Makino, I94I. Botia marcracantha 9B 28m .l- 7Oa 26 testis, Muramoto et al., gi11, 1968. sp leen, kidney Cobiris biwag 54 27 4m * 50a testis Nogusa 1960. Lefua echigonia 50 25 50a tes Nogusa 1960 tis " Misgurunus an$rlllicaudat u s 52 52a Makino l94L Order Siluriformes " Family Ictaluridae Ictalurus punctatus 56 6msm422 s t*l8a 30 tes tis , Muramoto et a1., spleen, 1968. kidney, gi 11 Noturus gyrinus 42 0m*12sm*20st gí 11 Levin,MSc thesis (Le72) Famí1y Bagridae " Pelteobagrus nudiceps s6 to 56a tes tis ogusa , L960 Tissues Taxon Reference De scr ip tion e

Family Siluridae Parasilurus asotus 5B 29 5Ba tes tis Nogusa, 1960. Family Clariidae Clarius batrachus 52 6m * 46a kidney Srivastava & Das, 1968. Family Lorícariidae Hypostomus plecos tomus 54 4msm*12s tfl8a 51 testis, Muramoto et al., gi11, 1968. sp leen , kidney

Supe rorde r A THERINOMORPHA N Order Atheriniformes (Jl Suborder Cyprinodontoidei Family Cyprinodontidae Aplocheilus blocki 48 24 testis Scheel , L966 b, 1968. A. davi 4B 24 te s tis Scheel , 1966 b, 1968. A. lineaLus 50 25 tes tis Scheel , 1966 b, 1968. A. panchax 36 1B tes tis Scheel, 1966 b, 1968, Aphvosemion arnoldi 36 1B tes t is Scheel, L966 b, 1968. A. australe 30 15 testis Scheel , 1966 b, L968. A. Þertholdi 42 2L tes tis Scheel , L966 b, 1968. A. bivittatum 40 20 tes tis Scheel , 1966 a, 1968. Chromosome N Taxon Ref erenc e De scr ipt ion

¿r. ca lliurium J¿ 16 tes tis Scheel , 1966 b, 1968 " ftô f i lamentosum JO 18 tes tis Scheel, 1966 b, L968, guine es e 38 L9 ^ tes tis Scheel, 1966 b, 1968. A. gulare 32 16 tes tis Scheel , 1966 b, 1968. 20 ^ louessense 10 tes tis Schoel , 1966 b, 1968. A. nigerianum -10 18 testis Scheel , 1966 b, N 1968. o\ A. s ioestedti 46 23 tes tis Scheel, L966 b, L968. A. spurrelli 26 1B tes tis Scheel , 1966 b, 1968. Cvprínodon variegatus 4B 24 2m * 46a o'i 1 l vin & Foster, tes tis unpub 1 is hed Epiplatys annulatus 50 25 testis Scheel , L966 b, 1968. E. bifasciatus 40 20 testis Scheel, 1966 b, 1968. q. chaperi 50 25 tes tis Scheel, 1966 b, 1968. E. dageti 50 25 tes ti s Scheel, 1966 b, 1968. E. fasciolatus 38 L9 tes t is Scheel, 1966 b, 1968. E. sexfaseiatus 48 24 tes t is Scheel, 1966 b, 1968. E. spilargvreius J+ 77 tes tis Scheel, 1966 b, 1968 Tissues Taxon Ref De scr ipti on e

Fundulus catenatus 46 etzer, F. catenatus 46 2L + 44F ovarian ehen, 197L. cu1t. chrvsotus 36 Setzer, 1968. T. chrysotus 34 14T, + 20F ovarian Chen, I97I (2s + 16A) culË. E. c ingula tus 46 gi 11 Setzer, 1968. c ingu 1a tus 46 4B 2L + 44F ovarian Chen,197L. (2s + 42A) cult. F. conf luentus 48 48 4BF(2S + 46A) ova ria n Chen,797L. cult. F. diaphanus 4B 52 Xsm; Ym gi 11 Chen & Ruddle, 7970. F. diaphanus 48 52 4BF(2S + 42A ovarian Chen, L97I. + 4SM) cult. E. grandíS 48 gi 11 Setzer, 1968. E. grandis 48 50 48F(4s + 44A) ovarian Chen, I97I" cult . T" heteroclitus 36 embryo Moenkhaus, L904 q. heteroclitus 4s embryo Pinney,1918. F. he teroc litus 4B ova r ian Chen, T970. cult. , gi1l, kidney, tes tís , s p leen F. heteroclitus 48 4B ovarian Chen & Ruddle, cul- t . L970. F. heteroclitus 4B 4B 4BF(2s + 46A) ovarian Chen, L97I" cult. F. kansae 4B gi 11 Setzer , 7968. Taxon Ref erence De s cript ion

F. kansae 48 4B 48F(2S + 46A) ovarian Chen, 7971, cu1t. T. lineola tus 46 48 2L*44F (25+42A) ovarian Chen, I97L. cult. q. 1uc ia e JZ 52 16L+16F (25+2SWr2A) ovarian Chen, I97I. cu1t. F. ma ialis 48 ovarian Chen, I970. cult. , gi11, kidney, testis, s p leen q. maj a lis 4B 50 ovarian Chen & Rudd1e,

cult " L970. q" ma jalis 48 50 4BF(2s + 46A) ova ria n Chen, L97I. cult. q. nota tus 40 gi 11 Setzer, L968, T970. q. nota tus 40 52 8L+32F (25+26A +2}/F]2SM) ova rian Chen, I97L. cult. E. not ti 46 gi 1l Setzer , 1968. F. notti 46 4B 2L+44F (25+42A) ova r ian Chen, L97L" cult. E. olivaceus 4B gi 11 Setzer , L968, r970. T. olivaceus 48 52 48F (25+44A+2M) ovar ia n Chen, L97L. cu1t. q. parvipinni s t¡8 gi 11 Setzer, 1968. E. parvipinnis 4B 50 Xsm; (v)a spleen Chen & Ruddle, 7970. E. parvipínnis 4B 48 48F (2s+46A) ovarian Chen, I97L. cu1t. Tissues Taxon Reference Des cr ip t ion e>camined

E. pulvereus 48 50 4BF (4s+44A) ovarian Chen, I97I. cu1t. F. rathbuni 48 50 48F (25+44A+2SM) ovarian Chen,197L. cul-t. F. sciadieus 44 50 4L+40F (2S+36A ovarian Chen, L97L. +2sM) cult " F. seminolís 48 gi 11 Setzer, 1968. F. seminolis 48 4B 48F (2S+46A ) ova ría n Chen, I97L

cult " F. similis 48 gi 11 Setzer, L968" f. similis 4B 50 4BF (2s+46A) ovarian Chen, I97I. cu1t. F. stellifer 4B 48F Chen,1977. F. waccamensis 48 52 48F (25+44A+2SM) ovarian Chen, I977. cult. F. zebrÍnus 48 gi 11 Setzer , 1968. F. zebrinus 48 4BF Chen, 1-971.

Ga rma ne 1 1a pulchra 489 2sm*46a 3 gi11, Levin & Foster, 2 (X1) u+2 (X2) ¿ testis 1972. 47¿ ¿.J 1m*2sm*44a; Ym+(X1) a+(Xz), Jordanella floridae 4B 24 2sm*46a gi11, Levin & Foster, tes tis 1972. Nothobranchius guentheri 38 L9 tes tis Scheel , 1966 b, 1968. N. rachovii 18 tes tis Scheel , L966 b, L968, Pachvpanchax playf a íri 48 24 tes tis Scheel , 1966 b, 1968. Taxon Tis s ues Reference De scr ipËi on o

Famíly Poeciliidae Gambusia affínis (=holbrooki) 35-36 tes tis Geiser , 1924, G. affinis 4B 24 tes tis Roberts , 1965. Lebístes reticulatus 46 Winge , 1922. L. reticulatus 46 Vaupel , 7929. L. reticulatus 46 tes tis Iríki ,1932 " Lima dominicensis 46 tes tis I^Iickbom , L94L. @rrícolor) !. vittata 46 tes tis InIíckbom, !94L. Poecilia (=Ileterandría ) 46 gi 11 Schultz & f ormosa (¡ Ka11man, 1968. O P. (=Poec!1igp-rs.) 48 gil 1 Schultz, L967. 1a tidens P. lucida 48 gi11, Schultz , 196I, testis 1967 . g.( =Mo 1 1íenis ia ) 36 tes tis Meyer,1938. sphenops P. sphenops_ 23 46 tes tis I^Iickbom, L94I , t943. ?. sphenops melanistica 3B T9 tes tis inlÍckbom, L94L, 7943. P" sphenops 46 gÍ 11 Schultz & Kallman, 1968. !. occidentalig 48 tes tis Schultz, I96L" !. velifera 46 23 te s tis !,Iickbom, I94L, 1943. Pha 1 loce ros caudinnculat.us 46 23 testis trniickbom, L94l , L943. Phallichtys pittieri 46 tes tis I,Iickbom, L947. Taxon Reference De scription

Xiphophorus testis Fríedman & Gordon, (=3.1elypsssi-1se-) 1934. couchianus X. he1lerí 4B tes tis Ralston, \934. X. helleri 24 testis Friedman & Gordon , 1934. X. helleri 4B 48a ¿J spleen, Ohno & Atkin, tes tis t966,

X. helleri 48 48a ß-n Ohno et a 1. ,

1968 " II A. ma cula tus 4B tes ti s Ralston , 1934. F X. macula tus ¿4 testis Friedman & Gordon , 1934. Å. montezumae 25 tes tis Friedman & Gordon , L934. E. varia tus 24-25 testis Friedman & Gordon, 1934" E. xiphidurn 25 tes t is Friedman & Gordon, L934. Suborder Atherinoidei Family Atherinidae Menidia notata (=nenidia) a36 embryo Moenkhaus , 1904. Superorder ACANTHOPTERYGII Order Gasterosteiformes Suborder Gasterosteoidei Family Gasterosteidae Apeltes guadracus 46 23 ð78 / :I^Isrn; tes tis , Chen & 977 9:trnlsm,Za gi11, Reisman , L970. s p leen ula ea inc ons ta ns 46 23 54 testis, Chen & gi11, Reísman , \970, spleen Chromosome No. Tissues Taxon Reference Description ø

Gasterosteus aculeatus 42 embryo Swarup, L959. G. aculeatus 42 2t 54 tes tis , Chen & Reisman gi1-1, r970. s pleen G whea t land i 42 2T 52 9:(x¡a; d:Ya testis, Chen & Reisman gi1-1, L970. s pleen Pungitius pungitius 42 testis Makino , I934a, L934b, !. Pungitius 2L 70 tes tis , Chen & Reisman gi11, 7970. L.n t\) s p leen Order Scorpaeniformes Suborder Scorpaenoidei Famí1y Synancejídae Inimicua iaponicus 50 25 50a te s tis ogusa, 1960 Suborder Cottoídeí Family Cottidae Cottus bairdii a3G38 tes tís Hann, 1927 . 9. polLux 48 24 48a tes tis ogusa , 1960. Order Perciformes Suborder Percoidei Family Serranídae Coreoperca kavramebari 4B 24 48a testis Nogusa , L960 Family Centrarchidae Acantharchus pomotis 4B te s tis Roberts , 1964. Ambloplites rupestrís a48 testis aker, 1956, A, rupestris 48 t.estis berts , 1964. Centrarchus macropterus a48 tes tis Baker, L9s6. C, macropterus 4B tes tÍs berLs , L964. Chaenobrvttus gul-osus 4B te s tis erts , 1964. E. zonatum, 48 tes tis be rts , 1964. Taxon Description Reference

Enneacanthus chaetodon 4B tes ti s berts 1964. [. gloriosus 48 tes tis Roberts 7964. E. obesus 4B tes t is oberts 1964. Lempomis auritus 48 tes tis Roberts L964. L. cvanelLus (N.C.) 4B testis Roberts 1964. !. cyanellug (l^I.Va.) 46 tes tis ber ts 1964 " ce 11 cul-t. L. cyanellus, 46 2m* 44a sp leen , Becak et a1., 47 1m* 46a testís, L966. 48 48a kídney (,\JI !. cvane l-l-us 46-48 31 tes tis , no & Atkín, sp1-een 7966. !. cvane lLus 46-48 3ù35 no et al., L968, !. gibbosus 4B testis, berts , 7964. ce 1l- cu1t. L. macrochirus, ca44 tes tis ríght , 1940, L. macrochirus 48 tes tis aker,7956. L. macrochirus 48 tes Ëís , oberts, 1964. ce 11 cult. L. marginatug 4B tes tis ber ts , 1964. T,, megalotis 48 testis oberts , 1964, L, microl-ophus 48 testis, oberts , 1964. ce 11 cu1t. Mícropterus dolomieui 46 testis, oberts, 1964 cel-1 cult. Chromosome No Tissues Taxon Reference Description o

M. sa lmoides ca48 testís ker, 7956. M. sa lmoides 46 testis, oberts, 1964, cel-1 cu1t. Pomoxig annularis 40-50 te s tis Baker, L956. P. nigromaculatus 48 tes tis , Roberts , L964. cel-1 cult. Family Sillaginidae Sillago sihama 4B 24 48a tes tis Nogusa , L960. Fami ly Cichlída e Symphvsodon 60 44m * I6a 35 s p1een, Ohno & Atkin, aeq uifa sc ia ta tes tis L966. S. aequifasciaLa 60 Ohno et a1., 1968. Ti 1a pía mossambíca 44 22 gi11, Natarajan & tes tís , Subra hmanyam, 19 68 oogonia Suborder Mugiloidei Fami 1y Mugí lidae Cí1ias pulchel1a 48 24 48a tes tís Nogusa, l-960. Suborder Ca 1-1-ionymoidei Family Ca llionymidae Ca llionymus richardsoni 38 l9 3Ba te s tis Nogusa, 1960. Suborder Gob ioide i Family Gobiidae No. Taxon Chromosome Tissues Reference Description e

Acanthogobius f lavimanus 44 22 44a tes ti s ogusa , L960. Boleophtha lmus pec t iniros tris 46 23 46a tes tís ogusa, 1960, Chaenogobíus isaza 46 23 46a testís ogusa , L960. C. urotaenia 44 22 44a testis ogusa, 7960. Gobius abei 46 23 46a testis ogusa , L960. G. similis 44 22 44a testis ogusa, 1960. Mogrunda obscura 62 31 62a tes tis Nogusa, 1960. Perioptha lmus cantonens is 46 23 46a tes tis ogusa, 1960. Tridentiger obscurus 44 22 44a tes tis Nogusa, Le6o. $ Suborder Anabantoídei Family Anabantidae Betta splendens 42 Bennington, 7936. B. splendens 42 36a Les tis Svardson & ickbom, L942" Macropodus opercularis 2L testis Svardson & ickbom, L942, Order Pleuronectif ormes Suborder Pleuronectoídei Family Bothidae Para lichthlzs olivaceus 46 23 46a te s tis ogusa , 1960. Xystreurys liolepis 4B 48a 23 testis, Ohno & Atkin, s pleen L966. X. liol-epis 4B 48a L%23 no et a1,, 7968. Family Pleuronectidae I(areius bicolora tus 4B 24 48a Le s tis Nogusa , 1960, Taxon Tissues Reference Description esmined

Limanda vokohamae 4B 48a te s tis Nogusa , L960. Pleuronichthvs verËica 1is 4B 48a t9 testis, Ohno & Atkin, spleen 7966. P. verticalis 4B 48a 19.23 Ohno et a1., 7968. Order Tetradontif ormes Suborder Balistoidei Famil-y Ba listidae Novodon modestus 40 20 40a tes tis Nogusa , 1960, Rudaríus ercodes 36 1B 36a testis Nogusa , 1960. Stephanolepis cirrhifer 34 L7 34a tes tis Nogusa, 1960. Crossopte xygli: Lepidosiren paradoxa 3B 3W spleen, Ohno & Atkin, testis 7966. 57

LITERATURN CTTED

AtkÍn, N. B. and S. Ohno. 1967. DNA values of four primitive

chordates . Chromosoma 23:10-13.

Baker, W. D. L956. A study of the chromosome numbers of some centrarchid fishes. Unpublished master's thesis, Dept, ZooI.,

North Carolina State Co11-ege, Raleígh. Bargetzi, J. P. 7960. Application de máthodes d'analyse biochimique ã un problàme taxonomique: les Cor6gones de lac de Neuchâtel. Schweíz Z. Hydrol. 22: 64L-758. Becak, W., M. L. Becak and S. Ohno. 7966. Intra-individual

chromosomal polymorphism in green sunfish (Lepomis cyanellus) as evidence of somatic segregation. cytogenetics 5t 373-320. Behnke, R. J. lg7O. The application of cytogenetic and biochemical

systemaËics to phylogenetic problems in the family salmonidae. Trans. Amer. Fish. Soc. 99(1) z 237-248. Bender, K. and S. Ohno. 1968. Duplication of the autosomally inherited $-phosphogluconate dghydrogenase gene locus in tetraploid species of Cyrinid fish. Biochem. Genet. 2: 101-107' Bennington, N. L. 1,936. Germ ce11 origin and spermato-genesis in

rhe Siamese fighring fish, Betta splendens. J. Morph. 60: L03-725. Booke, H. E. 1968. Cytotaxonomic studies of the coregonine fishes

of the Great T,akes, USA: DNA and karyotype analysis. J. Fish. Res.

Bd. Canada, 25 (8) 2 1667 -7687 .

Boothroyd, E. R. 7957. The chromosomes of spring and fa11-run Atlantic salmon. Proc' Genet, Soc. Canada 2z 4I. 58

1958. The chromosomes of Canadian populatíons of Atlantic

salmon, Salmo salar L. Proc" Xth Intern'1. Congr. Genet. 2: 29-30.

. L959. Chromosome studies on three Canadian populations of

Atlantic salmon (Sa1mo salar L.) . Canadian J. Genet. Cyto1. 1:

161-172. BrÍght, W. M. L940. Spermatogenesis in sunfish. Trans. Kent. Acad. Sci. B:37-38.

Chen, T. R. 7970. Fish chromosome preparations air-dríed displays of cultured ovarian cell-s in two killifishes (Fundulus). J. Fish

Res. Bd. Canada: 27 (1): 158-161.

. L97I. A comparative chromosome study of twenty species of

the genus Fundulus (Teleosteí: Cyprinodontidae)" Chromosoma

(Ber1.) 32: 426-453.

and H. M. Reisman. L970. A comparative chromosome study of the North American species of sticklebacks (TeleosEei:

Gasterosteídae). CyËogenetics 9: 32L-332.

and F. H. Ruddle. 1970. A chromosome study of fourspecies and a hybrid of the ki11ifísh genus Fundulus (Cyprinodontídae).

Chromosoma 292 255-267 " Foley, J. O. L926. The spermatogenesis of Umbra limi, with special

reference to the behavior of the spermatogonial chromosomes and the first maturation division. Biol. 8u11. 60: ll7-L47.

Friedman, B. and M. Gordon. 1934. Chromosome numbers ín Xíphophorin

físhes. Amer. Nat . 67: 446-455. Geiser, S. W. L924. Sex-ratios and spermatogenesis in the top-

minnow, Gambusia holbrooki Grd. Biol.8u11.472 175-2L9" 59

Greenwood, P. H., D. E. Rosen, S. H. Inleítzman and G. S. Myers , L966' Phyletic studies of teleostean fishes, with provisíona1 classifica- rion of living forms. 8u11. Amer. Mus. Nat. Hist. 131 (4):34L-455" Hann, H. I^I. L927. The history of the germ cells of Cottus bairdii

Girard. J. Morph . 43(2) 2 427 -498. Iriki, S. 1932. Prelíminary notes on the chromosomes of Pisces, I. Aplocheílus latipes and Lebistes retic!É4!uq-" Proc. Imp. Acad. Tokyo. B: 262-263. Karbe, T,. L964. Die Chromosomenverhaltnisse beí den Coregonen des

Bodensees und einiger weiterer volalpiner Seen, ein Beítrag zum

Problem der spezíation in der Gattung coregonus. z. zool" syst. Evo1" 2z 18-40. K1ose, J., U. I¡Iolf , H. Hítzeroth, H. Ritter, N. B. Atkin, and S. Ohno' L968" DuplicatÍon of the LDH gene loci by polyploidízation of the fish order Clupeiformes" Humangenetik 5: 190-196.

Kupka, E. L948" Chromosomal Verschiedenheíten bei Schweizerischen Coregenen. Rev. Suisse ZooL. 55: 285-293. Levan, 4., K. Fredga and A. A. Sandberg' 1964" Nomenclature for centromeric position on chromosomes. Hereditas 52r 20I-220" Levin, C. B. and N. R. Foster. L972. Cytotaxonomic studies in CyprinodonËinae: multiple sex chromosomes in Garmanella pulchra" Not. Nat. Acad. Nat. Sci. Philadelphia. (ittpress.)

T,ieder, U. 7956" Chromosomenstudien an knochenfischen. IV. Die

chromosomenverhaltnisse bei der regenbogen-und bachforelle und ihren bastarden. Zeítschrift fur Fishcherei 4: 589-594" Makino, S. 7934a" The chromosomes of the sticklebacks Pungitius tr/mensis and P. pungitius" Cytologí.a 5: 155-168. 60

7934b. Notes on the chromosomes of some freshwater teleosts. Jap. J. Gener. 9: 100-103. . 7937. Notes on the chromosomes of some teleost fishes. Zoo7" YIag. (Japan) 49: 75-76.

. L94L. The chromosomal relation between the two a1lied species of the loach (Cobitidae, Pisces)" Cytologia L2z 79'82, Meyer, H. 1939. Investigations concerning the reproductíve behavíor of Molliensia t'f ormosat'. J. Genet. 36: 329-366.

Moenkhaus, W. J. L904" The development of the hybrÍds between Fundulus heteroclitus and @!!is !e!g¡!g, with special ref erence to the behavior of the maternal and paternal chromatin. Amer. J. Anar. 3: 29-65. Muramoto, J., S.Ohno, and N. B. Atkin. 1968. 0n the diploid state

of the fish order Ostariophysi. Chromosoma 24 (1): 59-66. Natarajan, R. and K. Subrahmanyam. 1968. A prelimínary study on the

chromosomes of Tilapía mossambica (Peters). Curr. Sci. 392

262-263.

Nogusa, S. 1960. A comparative study of the chromosomes Ín fishes

with particular considerations on and evolutíon. Mem. Hyogo. Univ. Agrí" 3: l-62. Ohno. S. L970a. The enormous diversity in genome sizes of fish as a reflection of naLurets extensive experiments with gene duplica- tion. Trans. Amer. Fish. Soc. 99(1): 120-130.

. I970b. Evolutíon by Gene DuplicaËíon. Springer-Verlag,

Berlin. xv 160. 6t

and N. B. Atkin. L966" Comparative DNA value and chromosome

complements of eight species of fishes. Chromosoma 183 455-466. J. Muramoto, L. Christian, and N. B" Atkin. L967a" Diploid-

tetraploíd rel-atíonship among old world members of the fish family

Cyprinidae. Chromosoma 232 1--9. J. Klein and N. B. Atkin. L967b " Diploid-tetraploíd relationship in clupeiod and salmonoíd fish. Chromosomes -, -, Today 2z L29-747.

_, C. Stenius, E. Faisst and M. T. Zenzes" 7965. Post-zygotíc chromosomaL rearrangements ín rainbo\^/ trout (Salmo írideus, Gibbons). Cytogenetics 4: ll7-729. _, U. I^Iolf , and N. B. Atkin. 1968. EvoluËion from fish to

mammals by gene duplícation. Hereditas 59(1) z 169-L87" Ojima, Y. S. Hítosumachi, and S. Makino. 1966. Cytogenetic sËudies

in lower vertebrates I" A preLiminary report on the chromosomes of the Funa (Carassius auratus) and the goldfish (a revísed study). Proc. Japan Acad" 42(L)z 62-66"

_, and S. Hítotsumachi. 1967. Cytogenetic studies in lower vertebrates: IV. A note on Ëhe chromosomes of the carp (Cyprinus carpio)in comparison with those of the funa and the gold-fish (Carassius auratus). Jap. J. Genet" 42(3)z 163'L67.

Patau, K. 1960. The ídentification of individual chromosomes,

especially in Man. Am. J. Human Genet. L2z 250'276. Pinney, E. 1918. A study of the relation of the behavior of the chromatin to development and heredity in teleost hybrids. J" Morph. 312 225-292. 62 Pominí, P. 1939" Fenotípi e genotipí nei salmo ítaliani. Scientia Genetica 1: 206-218. Post, Alfred. 1965. Vergleichende Untersuchunger der Chromosomenzahlen bei Susswasser-Teleostern. Z. Zoo7. Syst. Evolutionsforsch 3(ll2):

47 -93 .

Prakken, R., J" Bekendam and G. A. Pieters, 1955. The chromosomes of Esox lucius L. Genetica 27'.484-489,

Prokofiovâ, A. L934" On t.he chromosome morphology of certaÍn pisces. Cytologia 5: 498-506.

Ralston, E. M. 1934. A study of the chromosomes of Ãi-pltgphorus, Platypoecilus, and of Xiphophorus-Pl-atvpoecilus hybrids duríng spermatogenesis. J. Morph " 562 423-433. Rees, H. L964. The questíon of polyploidy in the Salmonidae.

Chromosoma 15; 275-279.

1967. The chromosomes of Sal-mo sa1ar. Chromosoma 2L:

472-474.

Roberts, F. L. L964. A chromosome study of twenty species of Centrarchidae. J. Morph. 115: 401-418.

" L965. A chromosome study of Gambusia affinis Holbrooki.

Copeia L965(2) z 238-239 " . L966. Ce11 culture of fibroblasts from Clupea harengres gonads. Nature 212 (5070) z L592-I593"

7970. Atl-antic salmon (Salrno salar) chromosomes and speciation. Trans. Amer. Fish. Soc. 99(L)z 105-111. Ruddle, F. H. L964" Quantitation and automation of chromosomal data with special reference to the chromosomes of the Hamshire pig (Sus acrofa). In Cytogenetics of CelLs in Culture, R. J. C. Harris, ed. Acad. Press, N. Y. Symp. Int. Soc. for Ce11 Biol.3: 273-305. Scheel, J. J. 1966a. Notes on phenotype, distribution and systematícs

of Aphyosemion bívittatug (Loennberg), with remarks on the chromo-

some number ín the Rivulinae. Ichthyologíca 952 26I-278. 1966b. Taxonomic studies of African and Asian toothcarps

(Rivulinae) based on chromosome numbers, haemoglobin patterns

some morphological traits, and crossing experiments. Vidensk.

Medd. Dansk Naturh. Foren. L2Bz L23'I48. 1968. RivulÍns of the Old l^iorld. T. F. H. PublicaLions Inc.: Jersey City. Schultz, R. J. L96L. Reproductíve mechanisms of unisexual and bi- sexual sEraíns of the viviparous fish Poecil-iopsis. Evolution 15:

302-325. L967. Gynogenesis and triploidy in the vivíparous fish Poeciliopsis. Science I57 (3796)r 1565-1567.

and K. D. Kallman. 1968. Triploíd hybrids between the all- female teleost Poecilia formosa and Poecilia sphenops. Nature 2L9 (5151) z 280-282. Setzer, P, Y. 1968. A karyological analysis of members of Ëhe genus Fundulus. M" A. Thesís, Univ. of Texas.

. L970. An analysis of a natural hybrid s\^7arm by means of

chromosome morphology. Trans. Amer. Fish Soc. 99(1) | 139-146. Sick, K., M. üIestergaard, and O. Frydenberg. 1962 " Haemoglobin pattern and chromosome number of American, European, and Japanese

eels (Angui1La), NaËure 193: 1-001-1002. 64

Simon, R. C. 7963. Chromosome morphology and speciatíon in the five North American species of Pacific salmon (Oncorhynchus). J"

Morph " 172. 77-97 . and A. M. Dollar. 1963" Cytological aspects of speciation

in Ëwo North American teleosts. Can. J. Genet. Cyto1. 5: 43-49.

Srivastava, M. D" L" and Bhagwan Das. L968. Somatic chromosomes of

Clariuq Þa¡fqçLr"s (t.) (Cl-ariídae: Teleostomi) . Caryologia 2L(4) z

349-352 " Stevrart, K. W. and C. B. Levin. 1"968. A method of obtaining permanent

dry mounted chromosome preparations from teleost fish. J. Fish.

Res. Bd. Canada 25(5): 1091-1093. Svardson, G. 7942. Somatic pairing in Salmo and Coregonus and its hypothetical explanation. Arkiv. For Zoologí Band 33(9): I-6"

L945. Chromosome sËudies on Salmonidae. Rep. Swedísh State Inst. Freshw. Fishery Res. Drottningholm 23: 1-151. L95B" Speciation in freshwaËer fish, as illustrated by

Coregonus. Proc. XVLh Intern. Congr. Zoo7. 15: L37-L4I.

and T. irÏickbom. 1942. The chromosomes of two species of

Anabantidae (Teleostei) with a ner^I case of sex reversal.

Hereditas 2B: 21,2-2L6 " Swarup, H. 7959. Production of tríploidy in Gasterosteus aculeatus. J. Genet. 561 I29-L42. Taylor, K. M. 7967. The chromosomes of some lower chordates.

Chromosoma 27: 181-188. Vaupel, J" 1929. The spermatogenesis of Lebistes reticulaËus. Jour. Morph. and Phys " 472 555. 65

Viktorovsky, R. M. 7964" Chromosome sets of Coregonus peled and C" lava¡elgq baunti. Tsitologiya 6: 636-638.

Wahl, R. I^I . L969. Chromosome morphology ín lake trout Salvelinus

namaycush. Copeia 1960(1) ? L6-19.

Wickbom, T. 794L. The sex chromosomes of Cyprinodontidae and of

teleosts in general wiLh a list of new chromosome numbers of Cyprínodontidae. Arkiv. For ZooLogi Band 33(10): l-6. L943. Cytological studies on the family Cyprinodontidae. Hereditas 29: L-24. trriinge, O. 1922. One-sided mascul-ine and sex-línked inheritance in T,ebisËes reticulatus. Jour. Genet. L2z L45-L62. I,{olf , U., and H. Rítter, N" B. Atkins, and S. Ohno. 1969" Polyploidízation in the fish famí1y Cyprinídae, order Cvpriníformes.

I" DNA content and chromosome sets in various species of Cyprínídae.

Humangenetlk 7 z 240-244. hlrighË, J" E. 1955. Chromosome numbers in trouË" Progr. Fish-Cult.

L7z 172-L75.