CHROMOSOMA Chromosoma (Bcrl) (1987) 95:339 344 :~ Springer-Verlag J987

Supernumerary chromosome variation and heterochromatin distribution in the endemic frog Leiopelma hochstetteri

David M. Green ~, James Kezer 2 and Ronald A. Nussbaum a LRedpath Museum. McGill Unix,ersily, Montreal, PQ, II3A 2K6 Canada 2 Department of Biology. University of Oregon, Eugene, OR 97403 USA ~ Museum of Zoology. Univcrsily of Michigan. Ann Arbor. MI 48109 USA

Abstract. Specimens of the endemic New Zealand frog Leio- erochromatic nature of supernumerary chromosomes in pe/ma hochslelleri from Tapu on North lshmd were found many species, it is possible that they may have arisen, in to have six, nine or ten supernumerary chromosomes in some cases, as free centromeres or centromeric fragments their karyotypes, In comparison with previously published (see Jones and Rees 1982). but there is little corroborating data, these results further indicate probable geographic evidence. Supernumerary chromosomes have been asso- variation in supernumerary chromosome number betwcen ciated, in various instances, with particular kinds of meiotic populations. Increased numbers of supernumeraries in these chromosome behaviour, decreased developmental rate, re- frogs is correlated with apparent decrease of centromeric duccd fitness or viability and/or increased C-values (Harvey heterochromatin in the five large metacentric chromosomes and lIcwitl 1979; Jones and Rees 1982). of the karyotype, as detected by C-banding. Meiosis was Earlier, we reported on the supernumerary chromo- abnormal in a male with a high number of supernumeraries. somes and heterochromatin of specimens of k. hochsletteri In lampbrush preparations from a single ['ernalc with one obtained from Dome Valley, near Warkworth north of supernumerary uniwtlent, the supernumerary often had a At, ckland, New Zealand (Fig. I), including comments on denser, beaded appearance in comparison with the regular a triploid female (Green et al. 1984a). The nutnber of super- bivalents. Evidence is consistent with the notion that these numerary chromosomes per individual in that samplc was supernumerary chromosomes may ha~e arisen from centro- low. All females except the triploid had one supernumerary meric fragments. while the males had none. The triploid had two supernu- meraries. The supernt, meraries were not unusually hetero- chromatic: C-bands were confined to the centromeres, as Introduction is ust,al among the regular set of chromosomes. All other chromosomes were similarly C-band positive at the centro- The three species comprising the endemic New Zealand gen- meres with an additional C-band observed in association us I,eiopeh,m arc often considered to be among the most with the prominent secondary constriction on chromo- primitive of living frogs (Bell 1982: Duclhnm:t and Trueb some 7. Stephenson et al. (1972) presented karyotype data 1986). The most widespread of these species, L. hoch.s'tetwri, from frogs taken fi'om rokatea Ridge on the Coromandel is found in damp seepages and stream banks in scattered Peninsula of the North Ishmd of New Zealand (Fig. 1). localities on the of New Zealand (Fig. 1). L. These specimens, all males, had either two or four super- hamilloni and L. archeyi are extremely restricted in range. numeraries. Another male from Dome Valley was without The three species are considered to be threatened in New supernumeraries. Two females from Dome Valley were ex- Zealand (Bell 1986), especially L. hamilloni. While L. hamil- amined by Morescalchi (1967). They had 1 and 12 supernu- toni and I,. archeyi have both been shown to have 2n= meraries, respectively. 18 chromosolnes. 12. hochstem, ri has 2n = 22 chromosomes We are now able to present, karyotypic data, including plus varying numbers of very small supernumerary, or B- C-banding information on heterochromatin distribution, chromosomes (Morescalchi 1967; Stephenson et al. 1972, from additional specimens of L. hochstetteri froln a third 1974: Green et al. 1984a). The regular karyotype, or A-set locality, "fapu, on the Coromandel Range (Fig. 1). In addi- of chromosomes, of L. hochstetteri consists of five pairs tion, we present further information from two more speci- of large metacentric chromosomes and six pairs of smaller mens of this rare frog from Dome Valley, including data telocentric chromosomes, one of which has a prominent from lampbrt,sh chromosomes obtained from the o6cytes secondary constriction. of a Dome Valley felnale. This information gives additional Supernumerary chromosomes, always considered to be insight into variation in heterochromatin distribution and mmsual in animals, are rare in anurans; only four species supernumerary chromosome numbers within the karyotype other than L. hochswtteri are reported in the literature to of tiffs species. possess them (Jones and Rees 1982) although there exist additional, unpublished examples (J.P. Bogart, personal communication). The origin of supernumerary chromo- Materials and methods somes in animal karyotypes has not been convincingly ex- Four frogs collected on the west side of the Tapu-Coroglcn plained (Jones and Rees 1982). Considering the highly het- Saddle of the Coromandel Rangc on thc North Island of O['['prinl request.~ to: D.M. Green New Zealand by Dr..Joan Robb in September, 1983 were 340

New Zealand collected in March, ]976, by Joan Robb (UMMZ Nos. 146848 and 146850). At least 20 metaphase chromosome spreads per speci- men were examined under phase contrast optics. Photomic- rographs of good spreads were numerically analyzed with 1 the CHROMPAC III computer-assisted analysis system (Green et al. 1984b). The nomenclature used for chromo- somes is that suggested by Levan et al. (1964) and modified by Green et al. (1980). Schmid's (1978) C-banding method was used to demonstrate the presence of the heterochro- matic regions.

Results When examined unstained with phase contrast (Fig. 2a, b), the 11 pairs of chromosomes of the A-set of the Tapu speci- mens did not differ in any marked way from results ob- tained in previous studies of the karyotype of L. hochstet- teri. Neither were significant differences found upon mea- surement and numerical analysis of the A-chromosomes when compared with published data (Green et al. 1984a). All Tapu specimens had five pairs of large metacentric chro- mosomes and six pairs of smaller telocentric chromosomes, one of which, previously identified as the seventh chromo- some (Green et al. 1984a), had a conspicuous median sec- Fig. I. Map of North Island of New Zcaland showing the range ondary constriction. Some of the telocentric chromosomes of Leiopelma hochstetteri (cross-hatching) and localities mentioned possessed very small, but definable, short arms. However, in the text where chromosome information has been obtained. 1 the Tapu specimens had different numbers of supernumer- Dome Valley, Warkworth: 2 Tapu: 3 Tokatca Ridge ary chromosomes present than had previously been ob- served in L. hochstetteri (Table 1). One male and one female each had six supernumerary chromosomes, one male had examined. Chromosomes from one male and one female, nine supernumerary chromosomes and one male had ten deposited at the California Academy of Sciences (CAS supernumcrary chromosomes. All supernumerary chromo- Nos. 156252-3), were obtained using Bogart's (1981) corne- somes observed were strictly telocentric with one exception : al epithelium squash method while chromosomes from two one of the supernumerary chromosomes of the male with males, deposited at the Museum of Zoology, Univcrsity ten was metacentric (Fig. 2a). of Michigan (UMMZ Nos. ]80360-1) were obtained with The supernumerary chromosomes seen in L. hochstetteri Kezer and Sessions' (1979) gut epithelium and testis squash from Tapu were not all equal in size. If the combined length methods. Lampbrush chromosome preparations, using of the A-chromosomes is set to equal 100%, the largest methods described by Kezer et al. (] 960), were made from supernumeraries observed were about 1.4% of the length the o6cytes of two females from Dome Valley, Warkworth, of the A-set while the smallest supernumeraries were about

Fig. 2a, b. Unstained mitotic chromosome spreads obtained from corneal epithelium of Leiopelma hochstetteri from Tapu, New Zealand, viewed with phase contrast. Supernumerary chromosomes (arrows) are clearly visible, a Male with ten supernumerary chromosomes, one of which is metaccntric (as'terislr b female with six supernumeraries. Note that supernumerary chrmnosomes can be of different sizes. Bars represent 10 I,tm 341

0.87% of the A-set. The supernumerary chromosomes seen in fernales from Dome Valley exarnined by Green et al. (1984a) wcrc measured at about 0.98% of the A-set. The metacentric supernumerary observed in one male from Tapu was among the largest of the supernumeraries ob- served; each of its two arms was smaller (about 0.72% of the A-set) than the smallest telocentric supernumerary. The possibility of mitotic instability in inheritance of one or more of the B-chromosomes was investigated by counting the numbers of supernumeraries in many different chromosonac spreads of the same individual. In one male, 23 cells were examined. Of these, equal numbers of cells wcrc observed to have 5 supernumeraries visible as were observed to have 6 supernumeraries (6 being the maximum numbcr in this individual), l"ive of the 23 cells were ob- served to have fewer than 5 supernumeraries visible. In the male with 10 supernumeraries, many cells were observed where only 9 such chromosomes could be counted. How- ever, the modal number of supernumeraries per cell was the same as the maximum number of supernumeraries per cell in every individual, indicating that the observed varia- tion in number was probably artifactual. C-banding revealed the presence of strongly staining heterochromatic regions at the cenlromcres of all the telo- centric chromosomes and the supernumeraries of the Tapu specimens. Ilowever. the centromcres of the large metacent- ric chromosomes of these i,adividuals stained only weakly, or not at all (Fig. 3a, b). By contrast, results obtained from C-banding of chromosomes from Dome Valley frogs (Green et al. 1984a) showed that all their chromosomes, including the metacentrics, stained darkly at the centro- meres. Only one of the metacentric chromosomes of the Tapu frogs, chromosome 3, revealed dense C-band hetero- chromatin at its centromere. This band stained much less strongly than did bands on the telocentrics and supernu- lnerarJes. Fig. 3a, b. C-banded metaphtlse chromosome preparations from C-bands in addition Io those associated with the centro- gut epithelium of l, eiopelma hvchstetteri ['rom Tapu. New Zealand, meres of the chromosomes wcrc noted in the Tapu frogs. illustrate differential intensity of staining of centromeric hetero- The seventh chromosome had a prominent C-band asso- chromalin between metacentric and leh)centric chromosomes, a Male with six supernumerary chromosomes. Interstitial C-bands ciated with its secondary constriction (Fig. 3a, b), as was associated with the secondary constriction on chromosome 7 (ar- observed in Dome Valley frogs (,Green ct al. 1984a). In rows) arc hcteromorphic, b Male with nine supernumentries. Only the male with six supernumeraries, however, this band ap- one metz~centl"icchromosolne pair, 3, has appreciable centromeric peared to be heteromorphic in that one of the pair of sev- helerochromatin (arrows). Bars represent 10 I,tm enth chromosomes had a much more intensely stained band than the other (Fig. 3a). This frog also featured numerous weak C-bands on the "shoulders'" of the long arms of the chiasmata. All other bivalcnts had a single, terminal telocentric chromosomcs, proximal to the centromcres. The chiasma. A ring bivalent was not observed in meiotic prepa- male with nine supernumeraries did not have the hetero- rations of L. hochstetleri as illustrated by Stephenson et al. morphism in C-banding on chromosome 7. (1972). Similar ring bivalents, though, arc frequently ob- C-banding of the supernumerary chromosomes of the served in meiotic preparations fi'om more "'advanced" Tapu frogs was as observed before in Dome Valley speci- frogs such as B~[b or Rana (Morescalchi 1973). In other mens (Green et al. 1984a). Only the centromeres were het- meiotic spreads from the same frog, however, differing erochromatic (Fig. 3a, b)."l'he one small melacentric super- numbers of teloccntric A-chromosomes appeared to be numentry observed in one of the males likewise stained paired. A C-banded preparation (l'ig. 5) shows all of the only at its centromere. mctacentrics to be paired while some of the teloccntrics Meiotic chromosome preparations were obtained fi'om are paircd, but not others. In another figure, all of the the spermatocytes of one of the q'apu males. Meiosis in A-set chromosomes were paired. The supernumerary chro- this frog, which had nine supernumeraries and had been mosomes always appeared as tmivalents. inoculated with colchicine, was highly abnormal (Fig. 4a, l,ampbrush chromosomes were prepared from oacytes b). In some spreads, only the mctacentric chromosomes of two females fiom Dome Valley. Since these individuals appeared to be paired. Five large biwtlents were observed had not been treated with colchicine, we had not been able amol'lg numerous small univalent elements (Fig. 4a). One to secure mitotic chromosomes from them. As in other re- of the metacentric pairs formed a conspicuous ring bivalenL trades from this locality that we had examined earlier indicating the presence of two terminal, or near-terminal, (Green et al. 1984a), each of these two frogs had one super- 342

Fig. 5. C-banded meiotic preparation from the testis of the same specimen as in Figure 4 showing paired (asterisks) and unpaired (arrow.s') telocentric chromosomes. Bar represents 10 lam

ary chromosome karyotype found by Morescalchi (1967) in a frog reported to be from Dome Valley. l:urther investi- gations will reveal if a geographic trend is real and whether it may continue to outlying parts of the species' range. While it has been suggested that supernumerary chro- mosomes are usually transcriptionally inactive (.Jones and Rees 1982), visual evidence from lampbrush chromosomes has been lacking. Our results indicate that the st, pernumer- ary chromosome in the lampbrush state (Fig. 6a, b) does have short lateral loops indicative of transcriptional activi- ty, though at a lower level than the A-chromosomes. Fig. 4a, b. Meiotic chromosomes at prometaphase from the colchi- The variation in the staining intensity of centromeric cine-treated testis o['a male Leiopehna hochstetteri with nine super- numerarics, a Onls, the five pairs of melacentric chromosomes (ar- heterochromatin in the large metacentric chromosomes of rows) appear as bivalents, one as a ring bivalent. The six pairs L. hochstetteri is unusual. Variation in C-band staining has of teloeentrie chromosomes are unpaired, as are the small supernu- been seen in association with heteromorphic nucleolar orga- naeraries, b All chromosomes are paired except the supernumeraries nizer regions (NORs)in various frogs (King 1980; Schmid and one pair of telocentrics (asterisk.s'). Bars represent 10 lain 1983). In one of the specimens of L. hochstetteri from Tapu, a C-band heteromorphism can be seen in the NOR-bearing chromosome7 (I:ig. 3a). /Iowever, the striking variation numerary chrornosome per cell, clearly discernible in the observed in L. hochstetteri involves only the metacentric lampbrush preparations (Fig. 6a, b). While the A-set lamp- chromosomes in apparent correlation with supernumerary brush chromosomes were paired as normal meiotic biva- chromosome number and/or with geographic distribution. lents, displaying chiasmata and lateral loops, the supernu- The Dome Valley frogs had strongly C-band positive cen- merary chromosome appeared as a more condensed univa- tromeric regions on all chromosomes while the Tapu frogs lent, with a dense, beaded axis and relatively few lateral had little or no C-banding at the centromeres of the meta- loops (Fig. 6a, b). centrics. Variation in heterochromatin banding has been exten- sively studied in many rodents. Subspecies of the house Discussion mouse, Mus musculus, have differing amounts of hetero- As of this study, 9 different cytotypes are known from chromatin in certain chromosomes (Dev et al. 1976). A sim- 18 specimens of L. hochstetteri. Diploid karyotypes with ilar sort of variation occurs in subspecies of the rat, Rattus 0, 1, 2, 4, 6, 9, 10 and 12 supernumeraries have been found. rattus (Yosida and Saga/ 1975). Patton (1977) found evi- Despite the small samples studied, there is an indication dence of three types of supernumerary chromosomes in the that the average number of supernumerary chromosomes pocket mouse, Perognathus baile:vi, and slight associated per individual differs between populations (Table i). Aver- variation in C-banding in the chromosomes of the A-set. age supernumerary number appears to increase with in- Patton, however, did not see any correlation between het- creasing latitude from Dome Valley to populations at Toka- erochromatin variation and supernumerary number. The tea and Tapu along the Corornandel Peninsula (Fig. 1). One supernumeraries of P. bailey/ include both totally hetero- observation is at odds with this pattern : a 12 supernumer- chromatic and, rarely, partially heterochromatic types. 343

Table I. Numbers of supernumerar 3. chromosomes known to bc fotmd in karyotypes of Leiopehna hochstetteri with the numbers of individuals, separated by sex, that haxe been found to possess Ihosc cytotypes

Number of Number o1" individuals and population supernumerary cllronlosofncs Males Females per karyotspe

0 2, Dome Valley ~~ 1 7, Dome Valley "-a 2 2, Tokatea b 1, l)omc Valley ': (Iriploid) 4 1, "l-okatea b 6 I, l'apu a 1, Tapu a 9 l. lapu a 10 1, Tapu a 12 t, l)ome Valley ~'

Data computed fiora all know sourccs ~ Morescalchi (1967) b Slephenson et al. (1972) Green ct al. (1985) a Present study

of U. caudimaculatus were relatively unaffected, and the supernumeraries had no euchromalic regions. The pattern ofheterochromatin varialion in L. hochstet- teri is interesting in that only ccntromeric heterochromatin seems to be involved and only certain morphologically dis- tinct chromosomes seem to be affected. The presence of increasing humors of supernumerary chromosomes appears to relate to meiotic behaviour of the A-set as well. It has been argued that in certain plants, for instance, supernu- merary chromosomes can affect pairing of the A-set in meiosis. Parker et al. (1981) suggested that supernumerary chromosomes affect chiasma frequency in the A-set in Hy- Fig. 6a, b. Lampbrush chromosomes Dora o6c),tes of a Female Leiopelma hochstelleri with one supernumerary chromosome (ar- pochoeris maculata (Family Compositae) and produce other rows), a Size and configuration of lampbrush supernumerar3, rela- effects which disrupt normal meiosis when the number of tive to a metaccntric A-chromosome bivalent in lampbrush condi- supernumeraries exceeds one. Patton (1977) also found ef- tion. b A lampbrush supernumerary shinning ils relalivcly dense, fects on meiotic chiasma formation among the A-chromo- beaded condition with few loops. Bars represent 10 I.im somes in the presence of supernumeraries in the pocket mouse P. bailcvi. Limited evidence from the present investi- gations of the karyology of L. hochstetteri seems to indicate Among other amphibians, differences in amount of cen- that supernumeraries may inlluence synapsis of bivalents tromeric heterochromatin have been noted in chromosomes in meiosis in L. hochstetteri. A single male without supernu- of other species, particularly subspecies of the red-legged meraries from Dome Valley from whom meiotic chromo- frog, Rana aurora (Green 1985), and of the crested newt, some preparations were made by Stephenson et al. (1972) 7)'iturus cristatus (Sessions 1984). In these species, all chro- showed no deviations from normally expected pairing of mosomes were affected and no supernumerary chromo- ;all normal chromosome homologs. However, our results somcs were present although the additional heterochroma- from a male frog from Tapu with nine supernumeraries tin differs markedly in its precise location in R. aurora and showed a case in which different elements exhibited varying 7". cristalus. degrees of asynapsis or chiasma failure. One pair of large An example pcrhaps more like that seen in L. hochstet- metacentrics also formed a circular bivalent with terminal teri with regard to heterochromatin and supernumerary chiasmata. The individual we examined may have been ab- chromosomes was studied in the Australian rodent Uromys errant but the colchicine used is unlikely to have been re- caudimaculalus (Baverstock et al. 1976). Animals from two sponsible tk)r the effects upon meiosis that we observed. disjunct parts of this species' range had great differences The origins of supernumerary chromosomes arc as yet in heterochromatin and supernumeraries. Northern animals ~sithout clear explanation. Two phenomena must be consid- had many C-band positive regions in the A-set, mostly as ered in this context: the origination of supernumeraries and telomeric or interslitial bands, alld had no supernumeraries. the accumulalion of pre-existing supernumeraries. Certainly Southern animals had few heterochromatic rcgions in the supernumeraries have accumulated in certain, but not all, A-set yet had six to nine totally hetcrochromatic supernu- populations of L. hochstetteri. Among Dome Valley speci- merary chromosomes. Unlike the chromosomes of L. hoch- mens. supernulneraries were found only in females (Ta- slelleri, however, the centromeres of the A-chromosomes ble J). Meiotic accumulation mechanisms have been pro- 344 posed, pertaining in particular to certain plants (Jones and Bogart JP (1981) Chromosomc studies in Sminthillus from Cuba Rees 1982). Progressive origination of supernumeraries can- and Eleutherodactylus from Cuba and Puerto Rico (Anura, not be ruled out in L. hochstetteri, however. The different Leptodaetylidae). Life Sci Contrib R Ontario Museum sizes of supernumeraries indicate that they are not all identi- 129:1-22 cal and thus probably do not represent proliferation of a Dev VG, Miller DA, Tantravani RR, Schreck RR, Roderick TH, Erlanger BF, Miller OJ (1976) Chromosome markers in Mus single chromosome. The metacentric supernumerary chro- musculus: differences in C-banding between the subspecies M. mosome found in one male has no counterpart in other m. inusculus and M. m. molossinus. Chromosoma 53:335 individuals examined. It may have arisen separately, or be 344 the product of fusion of two very small telocentric supernu- Duellman WE, Trueb L (1986) The biology of amphibians. meraries, or be an iso-chromosome. McGraw-ttill Book Co. New York, p 670 It is possible that at least some of the supernumeraries Green DM (1985) Differentiation in anaount of centromeric hetero- in the Tapu frogs may have arisen as centromeric frag- chromatin between subspecies of the red-legged frog, Rana aur- ments, especially as tYagments from the large metacentrics. ora. Copeia 1985:1071-1074 This sort of mechanism has been discussed by Patton (1977) Green DM, Bogart JP, Anthony EH, Genner DL (1980) An inter- active, microcomputer based karyotype analysis system for phy- and Jones and Rees (1982). The loss of centromeric frag- Iogcnetic cytotaxonomy. Comput Biol Med 10: 219-227 ments preferentially from the large metacentrics, with their Green DM, Kezer J, Nussbaum RA (1984a) Triploidy in Hochstet- accompanying heterochromatin, is an explanation consis- ter's fiog Leiopelma hochstetteri fi'om New Zealand. NZ J Zool tent for both the possible origination of the supernumerary 11:457-461 chromosomes of L. hochstetteri and the observed patterns Green DM, Myers PZ, Reyna DL (1984b) CHROMPAC 111: an of heterochromatin distribution seen in this species. How- improved package for anicro-colnputer assistcd analysis of kar- ever, the apparent increase in limctional kinetochores repre- yotypes. J Hcred 75:143 sented by the supernumerary chromosomes awaits explana- lIarvey AW, Hewitt GM (1979) B-chromosomes slow development tion. in a grasshopper. Heredity 42:397 401 Jones RN, Rees I! (1982) B-Chromosolnes. Academic Prcss, New Wc have made a number of assertions concerning the York, p 266 distribution, effect and origin of the supernumerary chro- Kezer J, Sessions SK (1979) Chromosome variation in the pletho- mosomes of L. hochstetteri based on the information at dontid salamander Aneidcs fcrreus. Chromosoma 71:65 80 hand. Obviously, however, the volume of that information Kezer J, L~on PE, Sessions SK (i980) Structural differentiation is limited by the relatively small number of specimens avail- of the meiotic and mitotic chromosomes of the salamander able of this rare frog. Our hypotheses concerning apparent Ambystoma macrodactylum. Chromosoma 81 : 177 197 geographical differentiation in numbers of supernumerars~ King M (1980) C-banding studies on Australian hylid frogs: Sec- chromosomes present per individual could be proven false ondary constriction structure and the concept of cuchromatin transformation. Chromosoma 80:191-217 if additional animals with other numbers of supernumera- Levan A, Fredga D, Sandberg AA (1964) Nomenclature for eentro- ries are found: higher numbers from Dome Valley and merit position on chromosomes. Hereditas 52:201-220 lower numbers from Tapu, for example. This would also Morescalchi A (1967) The karyotype of two specimens of Leio- provide information on the possible occurrence of karyo- pelma hochslcllcri Fitz. (Amphibia Salientia). Caryologia types with very high numbers of supernumerary chromo- 21 : 37-46 somes, such as described by Morescalchi (1967). If these Morescalchi A (1973) Amphibia. In: Chiarelli A, Capanna E (eds) other karyotypes are present, the apparent correlation of Cytotaxonomy and vertebrate cvolution. Acadcmic Press, New supernumeraries with heterochromatin distribution could York, pp 233-283 be tested. Is the observed variation related to geographic Patton JL (1977) B-chromosome syslems in the pocket mouse, Perognathus bailcyi : Meiosis and C-band studies. Chromosoma divergence or to gain of supernumeraries? At present, these 60:1-14 cannot be separated. Other populations of L. hochstezteri, Parker .IS, Ainsworth CC, Taylor S (1981) The B-chromosome on or in the East Cape region, for system of Hypochocris maculata 11. B-effects on meiotic A- example, have not been examined karyotypically. The lamp- chromosome behaviour. Ch romosoma 67:123-143 brush behavior of the supernumerary chromosomes were Schmid M (1978) Chromosome banding in amphibia [. Constitu- more than one is present is also unknown. We hope that tive heteroehromatin and nucleolus organizer regions in Bufo further studies may be possible to address these questions. and Hyla. Chromosoma 66:361-388 Schmid M 0983) Chromosome banding in Amphibia. Vii. Analy- Acknowledgements. We arc gratelifl to l.)r. Joan Robb for providing sis of the structure and variability of NORs in Anura. Chromo- the specimens used in this study and to Dr. R.C. Drewes for making soma 87 : 327-344 available two of those sent to thc Cali['ornia Academy of Sciences. Sessions SK (1984) Cylogenetics and evolution in salamanders. Dr. W.F. Grant kindly providcd use of his computer and Drs. Ph.D. dissertation. University of California, Bcrkeley S.K. Sessions and C.ll. Daugherty offered valuable comments Stcphenson EM, Robinson ES, Stepheuson NG (1972) Karyotype upon the manuscript. This study was supported in part by NSI~RC x,ariation within the genus Leiopclma (Amphibia: Anura). Can Canada grants A2198 and UO-526 to I)MG. J Genet Cytol 14:691-702 Stephenson EM., Robinson ES. Stephenson NG (1974) Inter-specif- ic rclationships of Lciopehna (Amphibia: Anura). Further References karyological evidence : Experientia 30 : 1248-1250 Baverstock PR, Watts CHS, Hogarth JT (1976) I Ieterochromatin Yosida l'II. Sagai T (1975) Variation of C-bands in the chromo- variation in the Australian rodent Uromys caudimaculatus. somes of several subspecies of Rallus rattus. Chromosoma Chromosoma 57 : 397-403 50:283-300 Bell BI) (1982) The mnphibian fauna ofNew Zeahmd. In: Newman DG led) New Zealand herpetology. New Zealand Wildlife Ser- vice, Wellington, pp 27 89 Bell BD (1986) The conservation status of New Zealand wildlife. Received I"ebruar) 18, 1987 / in rcvised form May 19, 1987 Occ Publ N-Z Wildlifc Serv 12 : 1--103 Acccpled by H. Macgregor