CHROMOSOMAL EVOLUTION AND THE MODE OF SPECIATION IN THREE SPECIES OF PEROMYSCLTS

Transcription

1 CHROMOSOMAL EVOLUTION AND THE MODE OF SPECIATION IN THREE SPECIES OF PEROMYSCLTS IRA F GREENBAV~I, ROBERT J BAKER, AND PALL R ~ ~ I S E Y Department of Bzolog~cal Sct~aces and Tlz~ Mi~sei~m, Texas Terlz L'nzz erszty, Li~bbock, Texas, and Departm~nt of Zoology, Louzszana Teclz L'~LZPYSL~~, Ruston, Loz~tstana Received May 13, 19ii Revised October li, 1977 The possibility that the Peromyscus maniculatus complex of deer mice arose via the process described as "centrifugal speciation" (Brown, 1957) has been discussed by Bowers et al., 1973, Lawlor, 1974, Baker et al., 1975, and Greenbaum et al., This study was designed to yield additional data on the cytogenetics of three species of this complex in order to evaluate the "centrifugal speciation" model as a viable explanation of the evolutionary origin of the complex. Peromyscus melanotis and P. polionotus are believed to be peripheral isolates from the stock that evolved into the species P. maniculatus (Blair, 1950, Bowers et al., 1973). P. maniculatus is a widespread species occurring in a variety of habitats, whereas P, melanotis is a high montane s~ecies found in coniferous forest and P, polionotus is a deep sand species found in grassy fields at lower elevations near sea level (see Hooper, 1968, for general distribution of these three species). It is probable that P. melanotis and P. polionotus are more closely related to P. maniculatus than they are to each other. Based on gross chromosome morphology, the karyotypes of P. polionotus and P. melanotis are more like each other than either is like the karyotypes of P. nzaniculatus. We have examined the G-banding and C-banding pattern of P. polionotus and compared it to the banding patterns of P. maniculatus and P. melanotis (Greenbaum et al., 1978) to determine whether this similarity in karyotype is reflected in homologous banding patterns or is a product of convergent evolution. If the chromosomal data for the species complex are to fit the centrifugal speciation model then the karyotypes of P. polionotus and P. nzelanotis must be most like the primitive condition and the karyotype of P. maniculatus must be the most derived. Additionally, the centrifugal speciation model would predict more chromosomal polymorphism and geographic variation in the "central stock" (P. maniculatus) and less in the peripheral isolates (P, polionotus and P, melanotis). Our study is designed to determine the chromosomal homologies in these species and thereby test the hypothesis that this group is the result of centrifugal speciation. All chromosomal preparations were from fibroblast tissue-cultured cells initiated from ear biopsies. Methods of tissue culture maintenance and chromosomal banding were as described by Greenbaum et al., The chromosome pair identification and numbering were based on the proposed standardized karyotype for Peromyscus (Committee for Standardization of Chromosomes of Perornyscus, 1977). In relating our banded chromosomes to the proposed standard we are relatively sure of our identifications except for pairs 13 and 19. All other chromosome pairs relate well to the standardized karyotype. Numbering of the specific pairs of C-banded chromosomes (Fig. 2) is tentative; however, based on C-band and morphological comparisons, we are confident that the polymorphic pairs have been properly labeled. Seven specimens of Peromyscus polionotus were examined for both G- and C-bands as follows: South Carolina, Aik-

2 CHROMOSOMAL EVOLUTION IN PEROMYSCUS 647 FIG.1. G-banded karyotype of a female Peromysrus polionotz~s. Chromosomes in parentheses are from other specimens of P. polionotz~s and illustrate intraspecific variability for pairs 18 and 19. en Co., 3 mi N 1-20 on S.C. 19 (2), Florida, Ocala Nat. Forest (3), Laboratory F, hybrids of above localities (2). Specimens of P. maniculatus and P. melanotis are the same as reported by Greenbaum et al., All specimens of P. polionotus examined in this study had a diploid number of 48. Complete G- and C-banded karyotypes with inserts showing intraspecific variation found in our study are presented in figures 1 and 2. IntraspeciJic variation.-c-banding revealed centric heterochromatin associated with most elements of P. polionotus and a variable number (2-6) of heterochromatic short arms (Fig. 2). The short arm of the X chromosome was heterochromatic and varied in size between individuals and between homologues in female specimens (see Fig. I). The 'L' chromosome was subtelocentric and heterochromatic. Intraspecific autosomal variation was detected involving three pairs (Figs. 1 and 2, pairs 16, 18, and 19). In each of these cases heterochromatin was involved in the observed polymorphism (Fig. 2). Polymorphism in chromosome pair 19 presents the only easily discerned intraspecific chromosomal variability detected in P.

3 648 I. F. GREENBAUM ET AL FIG. 2. C-banded karyotype of a male Peromysczts polionotus. Chromosomes in parentheses are from other specimens of P. polionotus and illustrate the variation in pairs 16, 18 and 19. polionotus. In this pair the short arm is heterochromatic in the biarmed condition. The alternate form of this chromosome is an acrocentric which, based on G-banding pattern, is identical to the long arm of its biarmed homologue (Fig. 1). Although all three polymorphisms can be detected in C-banded preparations, in pairs 16 and 18 the amount of chromosomal material involved is not sufficient to enable detection of the polymorphism in standard karyotypes. These polymor- ~hisms are detectable onlv in our best G-banded karyotypes. In submetacentric pair 16, the polymorphism involves the presence or absence of short arm telomeric heterochromatin. In either condition, the pair remains distinctly biarmed. Pair 18 of P. polionotus is one of the smallest elements in the karyotype. The polymorphism in this pair involves small segments of C-band positive material that make up most of the second arm of this pair. It is very difficult to determine whether the form with extra heterochromatin is acrocentric or subtelocentric. Due to the nature of the chromosomal polymorphisms, it is very difficult to describe the intraspecific variation in P. po-

4 pp CHROMOSOMAL EVOLUTION IN PEROMYSCUS 649 FIG. 3. Composite partial G-banded karyotype of (left to right) P. nzaniculatus, P. polionotus and P. melanotis showing elements which are unchanged in the three species. FIG. 4. Composite partial G-banded karyotype of (left to right) P. maniculatzrs, P. polionotus and P. melanotis showing those elements which differ due to the involvement of segments of heterochromatin. lionotz~s in terms of numbers of biarmed autosomes. Based on the above information, the polymorphisms should range from 22 acrocentric autosomes (when pairs 18 and 19 are biarmed) to 26 acrocentric autosomes (when pairs 18 and 19 are homomorphic for the acrocentric condition). Pair 16 remains biarmed even when it lacks the telomeric heterochro- matin of the short arm. In our sample there were two individuals with 22, one with 23 and four with 24 acrocentric autosomes. Interspecijic homologies.-g-bands for P. polionotz~s are compared in Figures 3-7 with those for two other 5pecies in the P. maniculatus group (P. maniculatus bairdii and P. melanotis). Eleven pairs of

5 650 I. F. GREENBAUM ET AL ~ ~ FIG.5, Composite partial G-banded karyotype of (left to righti P. maniculatus, P. polionotzts and P. melanotis showing four of the six pairs that differ by pericentric inversion. These four pairs are biarmed in manicztlatus and acrocentric in both polionotus and melanotis. FIG.6. Composite partial G-banded karyotype of (left to right) P. maniculatus, P. polionotzts and P. melanotis showing two of the six pairs that differ by pericentric inversion. These are acrocentric in melanotis and biarmed in both maniculatus and polionotus. FIG.i. Composite G-banded pair 6 chromosomes of (left to righti P. maniculatzts, P. polionotzts and P. melanotis. autosomes have unchanged G-banding patterns in the three species (Fig. 3). The X chromosomes show homologous G- banding patterns in the euchromatic long arm. The Y chromosomes, which were entirely heterochromatic by C-band analysis, did not display distinct G-band markers. Five pairs of autosomes (pairs 16, li, 18, 19 and 2 1) possessing heterochromatic short arms in P. maniculatus have acrocentric counterparts in P. melanotis (Greenbaum et al., 1978). In P. polionotus, pairs li and 2 1 are acrocentric and identical to those of P. melanotis. Pairs 16, 18 and 19 are polymorphic in P. 90- lionotus and display a biarmed condition similar to that seen in P. maniculatus. Based on G-band analysis of pair 16, the biarmed condition of P. polionotus appears homologous to that of P. maniculatus. The biarmed condition of the P. polionotus chromosome 19 appears homologous to the 19 of P. maniculatus, whereas the acrocentric condition appears homologous to the 19 of P. melanotis. In pair 18, P. polionotus has a shorter heterochromatic second arm than is characteristic of our sample of P. maniculatus; however, the acrocentric condition of P. polionotus is identical to that of P. melanotis. In all three species, the respective euchromatic long arms of these five pairs are homologous based on G-banding patterns (Fig. 4). Pericentric inversions account for six interspecific difference~ (chromosome pairs 5, 10, 11, 13, 14 and 15) between P. maniculatus and P. melanotis (Greenbaum et al., 19i8). In each of these pairs the

6 CHROMOSOMAL EVOLUTION IN PEROMYSCUS 651 chromosomes are biarmed in P. maniculatzls and acrocentric in P. melanotis. Pairs 10, 11, 13 and 15 are acrocentric in P. polionotzis and identical to the acrocentrics of P. melanotis (Fig. 5). The other two pairs (5 and 14) are biarmed in P. polionotus and appear homologous to chromosomes 5 and 14 of P. maniczdlatzds (Fig. 6). Inspection of the G-banding patterns of pair 6 (Fig. 7) for the three species indicates that the long arm of P. mela~zotis is not identical to the long arm of P. ma~ziczllatz~sand the acrocentric of P. polionotzds (note only one trypsin resistant band proximal to the centromere in the P. melanotis chromosome 6 as opposed to two such bands in both P. ma~ziczllatz~s and P. polionotz~s).this interspecific variation might be explained by a small pericentric inversion and subsequent addition (or deletion) of heterochromatin; however, the exact nature of this variation is unclear. Evolution of the ma~zicz~latz~s group of the genus Peromyscus (Hooper, 1968) has received a great deal of attention. Blair (1950), based on general ecological, morphological, and physiological characters, concluded that P. ma~ziczllatz~s represents the evolutionary stock from which the species melanotis, polionotzls, sitkensis, selvi~ziand sejzlgis were derived via peripheral isolation. To this list of peripheral species a sixth, oreas, is sometimes recognized (Lawlor, 1974). Based on chromosomal, electrophoretic and breeding data, Bowers et al. (1973) suggested that the evolution of this superspecies complex might fit the centrifugal speciation model (Brown, 1957). "Centrifugal speciation" is an allopatric model which emphasizes the different rates of evolution in slowly evolving peripheral species as opposed to the more rapidly evolving central species. Therefore, in the majority of characters, a more primitive condition would be characteristic of the peripheral species, whereas a more derived condition would be found in the central stock. Centrifugal speciation ascribes the principal source of evolutionary change to the central stock and therefore predicts greater variation in the central species than in peripheral species. In the present case, P. maniczdlatz~srepresents the central species, whereas P. polionotzls and P. mela~zotis are peripheral isolates. In order to evaluate the model cytogenetically, it is critical to document the extent and nature of chromosomal variation in each of these species to determine whether P. ma~zicz~latzlsis more variable than the two peripheral species. Additionally, it is critical to ascertain the relative "primitive versus derived" status of the karyotype of each species. I~ztraspecific variation.-using standard karyotypic analysis, Te and Dawson (1971) described a chromosomal polymorphism in Peromyscz~s polio~zotzds in which the number of acrocentric autosomal elements varied from 24 to 26. They suggested that this polymorphism might be attributed to a single widely distributed inversion. Our data suggest that a heterochromatic short arm polymorphism, not an inversion, is res~onsible for the variation observed at the gross karyotypic level. Additionally, Te and Dawson (1971) re~orted minor variations in the presence of small second arms. Although variation in the relative degree of contraction in homologous elements may also account for some of the small variations observed by Te and Dawson (1971), it is probable that heterochromatin polymorphisms account for most of this variation (such heteromorphisms are seen in pairs 16 and 18, Fig. 2). Due to the nature of the variation, the amount of chromosomal polymorphism in P. polionotus has been underestimated. In only one of the three polymorphic pairs (pair 19, Figs. 1 and 2) can the acrocentric condition be easily distinguished from its biarmed homologue. The karyotype presented by Te and Dawson (1971) in their Figure 2 appears, based on our data, to be heteromorphic for pair 19. The poly-

7 652 I. F. GREENBAUM ET AL morphism in pair 16 (Figs. 1 and 2) of P. Polionotus does not involve an acrocentric condition and, therefore, is not detectable without banding. The second arm of chromosome pair 15 is quite small and, in some cases, mostly heterochromatic, so that both conditions appear essentially acrocentric. Such a situation might be detected as a minor variation in the second arm. Based on standard karyotypes the number of acrocentrics will appear to range from 24 to 26, even though the total range of variation is more accurately described as 22 (when pair 18 is homomorphic for the biarmed condition) to 26 acrocentric autosomes. The range of four to 20 acrocentrics found in P. ma~zicz~latz4s (Bowers et al., 1973; Greenbaum et al., 1978) is far greater than the range reported for any of the peripheral species. It is unlikely that extensive chromosomal polymorphisms exist in P. melanotis (Bowers et al., 1973; Greenbaum et al., 1978), no variation has been reported for P. sitkensis (Hsu and Arrighi, 1968; Thomas, 1973), and the number of acrocentrics in P. polio~zotz~s ranges only from 22 to 26. An exception to the low level of chromosomal variability in peripheral species is the variation reported for P. oreas which has from eight to 22 acrocentric autosomes (Thomas, 1973; see also Lawlor, 1974). However, P. oreas may be conspe- cific with P. ma~zicz~latz~s or it may have been isolated from the P. ma~ziculatus complex much later than either P. polionotus or P. melanotis. This extensive chromosomal variability, therefore may have been characteristic of the stock that gave rise to P. oreas and not the result of newly evolved variability in oreas after isolation from the central species. Chromosomally, P. ma~zicz~latz4sdoes seem to be more variable than the peripheral species (especially P. mela~zotis and P. polionotz4s). In agreement with our findings, Bowers et al. (1973) have reported more electrophoretic variation in P. manicz~latz~s than in P. mela~zotis. Primitive verszls the derived condition of karyotypes.-bowers et al. (1973) speculated that the primitive karyotype for the Peromyscz4s ma~ziculatuscomplex might have been characterized by a large number (near 30) of acrocentric autosomes. The high number of acrocentric autosomes in the peripheral isolate species of the manicz~latz~sgroup was cited as evidence of the more primitive nature of these peripheral species. Six pairs of chromosomes (10, 11, 13, 15, 17 and 2 1) differ between the karyotype of P. ma~zicz4latus and the karyotypes of P. polionotzls and P. melanotis. Of these, 17 and 21 differ by the presence of a C-band positive second arm in P. ma~zicz4latz4s and the absence of a corresponding second arm in P. Polionotz4s and P. melanotis. The other four pairs represent inversion differences. Independent events as an explanation for the four sets of homologous inverted elements in P. polionotzds and P. mela~zotis is highly unlikely and unparsimonious. Bowen (1968) has suggested that P. manicz4latz4s bairdii gave rise to P. polionotzds, whereas P. mela~zotis is morphologically more similar to P. maniculatus rzifinus (Bowers, 1974). Blair (1950) and Lawlor (1974) also interpreted the origins of melanotis and polionotz~s from maniculatzds as separate events. Additionally, electrophoretic data (Avise, pers. comm.) support an independent evolution of P. polio~zotusand P. melanotis from P. maniczdlatz4s. If it is true that both P. polionotzls and P. melanotis evolved independently from a pre-manicz~latz4s stock, the most logical explanation for the presence of the acrocentric condition of these six pairs of chromosomes in the two peripheral species is that this condition for these pairs was present in the karyotype of the evolutionary stock prior to the divergence of P. polionotz4s and P. mela~zotis. Another explanation for the inversion data is that the inversions in chromosomes 10, 11, 13 and 15 occurred in a melanotispolionotzds ancestor after this ancestor separated from the pre-manicz~latz~stock. We are unaware of data which support a common ancestor for melanotis and poli-

8 CHROMOSOMAL EVOLUTION IN PERO.VYSCUS 653 onotzls after their divergence from the ma- ~zicz4latz~s stock. Pair 6 is polymorphic in P. maniczdlatzds. P. polionotz4s has only an acrocentric condition for pair 6 and P. mela~zotis displays only a biarmed condition similar (but not identical) to that of P. maniczllatz~s (Fig. 7). As mentioned above, both a pericentric inversion and addition (or deletion) of heterochromatin may have been involved in the evolution of this chromosome pair. It is not reasonable from the available data to infer a primitive condition for this chromosome pair. Chromosome pairs 16, 18 and 19, which may be variable for heterochromatic short arms in both P. polionotus and P. maniculatzds, display only the acrocentric condition in P. melanotis. Pairs 5 and 14. which are biarmed in P. polio~zotz~s and P. maniczdlatus, differ from the acrocentric condition of P. melanotis by an inversion. These data indicate that P. polio~zotzds diverged later from the premaniculatus stock than P. melanotis, and therefore displays the manicz4latz4s condition for these pairs. We feel that chromosomally P. polionotus and P. maniculatus display the derived condition for pairs 5 and 14. Electrophoretic data (Avise, pers. comm.) support the contention that polionotz4s is a more recent derivative from maniczdlatus than is mela~zotis. These conclusions may be shown diagrammatically as in Figure 8. Centrifugal speciation in the P. maniculatus complex.-the karyotypic data, especially the more primitive nature, the similarity of the two peripheral species (P. polionotz~s and P. melanotis) and the greater degree of intraspecific variability in the central stock (P. manicz~latz~s) fit the conditions predicted by the centrifugal speciation model. They also agree with the data presented by Bowers et al. (1973). The centrifugal speciation model does not require synchronous speciation of the peripheral isolates. It only requires that once isolated, a peripheral species evolve more slowly than the central stock. Two points are relevant to the number of acmelanotin maniculatur Inr. in 10.11,13.15 CI in C+ ln FIG.8. Proposed phylogenetic tree for the three species of the Pe~onzyscus nzanirzrlatzrs-group studied, based on the cytogenetic data. rocentric chromosomes in the karyotype of species evolved from the pre-ma~ziculatz~s stock. If, through time, there has been a gradual decrease in the number of acrocentric elements in the ma~ziczdlatzds evolutionary line, the number of acrocentrics in peripheral species will reflect the chronology of their divergences. Peromyscz~s melanotis, with 30 acrocentric elements, would be an earlier derivative from the pre-ma~zicz~latzdstock than P. polionotz~s and P. oreas would be the most recent of the peripheral isolates. In addition, P. polionotus and, to a greater extent, P. oreas would display chromosomal variability due to a later separation from the rapidly evolving P. maniczdlatzds central stock. Lawlor (1974) presented arguments to refute both the centrifugal model of speciation for the ma~zicz4latus complex and the primitive nature of a large number of acrocentrics for the Peromyscz~s karyotype. Against centrifugal speciation, he argued that not all the peripheral species exhibit similarity by having large numbers of acrocentric elements. It is not necessary, however, for gross karyotypes of the peripheral isolates to resemble each other and the karyologically primitive condition in maintaining a similar number of

9 654 I. F. GREENBAUM ET AL biarmed or acrocentric chromosomes so long as similarity in the euchromatic portions of the genome is maintained. As discussed above, asynchronous divergence of the peripheral isolates should result in progressively decreasing numbers of acrocentric elements and increasing intraspecific variability with increased association to the central stock. SUMMARY Analysis of G- and C-banding patterns of Peromyscus polionotus reveals intraspecific variation in three chromosome pairs (numbers 16, 18 and 19). All three cases involve segments of heterochromatin. Chromosomal homologies (based on G-banding patterns) between P. polionotus, P. maniculatus and P. melanotis suggest that the ancestral karyotype for the three species was composed of a large number of acrocentrics (probably near 30). The data indicate that P. maniculatus and P. polionotus have increased the number of biarmed chromosomes in their karyotypes by additions of heterochromatin and pericentric inversions. Four inversion products are unique to P. maniculatus, whereas two inversions are shared by P. maniculatus and P. polionotus. Peromyscus maniculatus and P, polionotus also share derived heterochromatic additions. These data indicate that P. maniculatus and P. polionotus had a common ancestor after P. melanotis diverged from the line. Chromosomal data for these three s~ecies are discussed in light of the centrifugal speciation model. As predicted by this model, P. maniculatus has the most derived karyotype and the most intraspecific variation. We thank Dr. Oscar Ward for assistance in determining the numerical designations of chromosomes according to the standard Peromyscus karyotype. John Avise, James Mascarello, John C. Patton and Oscar Ward offered valuable criticisms of earlier drafts of this manuscript We thank R. K. Barnett, R. A. Bass, R. L. Honeycutt, M. A. Johnson, and T. L. Yates for their assistance. This study was made possible through a Graduate student-faculty research grant from the Graduate School, Texas Tech University, and by National Science Foundation grant DEB BAKER, R. J., J. H. BOWERS, AND MICHAELH. SMITH Reply to comments on "Chromosomal evolution in Peromysczrs." E\rolution 29:189. BLAIR,W. F Ecological factors in the speciation of Peromyscus. E\rolution 4: BOWEN,W. Ur Variation and evolution of gulf coast populations of beach mice, Peromysczrs polionotus. Bull. Fla. State Mus. 12:l-91. BOWERS,J. H Genetic compatibility of Peromysczls maniculatzrs and Peromysczrs melanotis, as indicated by breeding studies and morphometrics. J. Mammal. 55: BOWERS,J. H., R. J. BAKER, AND M. H. SMITH Chromosomal, electrophoretic, and breeding studies of selected populations of deer mice (Peromysczts maniczilatzrs) and black-eared mice (P. melanotis). Evolution 27: BROWN,W. L., JR Centrifugal speciation. Quart. Rev. Biol. 32: COMMITTEEFOR STANDARDIZATION OF CHROMO- SOMES OF PERO~MYSCUS Standardized karyotype of deer mice, Peromysczrs (Rodentia). Cytogenet. and Cell Genet. 19:3843. GREENBAUM, I. F., R. J. BAKER, AND J. H. BOW- ERS Chromosomal homology and divergence between sibling species of deer mice: Peromysczrs maniczrlatzrs and P. melanotis (Rodentia, Cricetidae). Evolution 32: HOOPER,E. T Classification. Pp , In J. A. King (ed.), Biology of Peromysczrs (Rodentia), Spec. Publ. 2, American Society of Mammalogists, ii pp. ~IsL~, T. C., AND F. E. ARRIGHI Chromosomes of Peromysczrs (Rodentia, Cricetidae) I. Evolutionary trends in 20 species. Cytogenetics 7: LAWLOR, T. E Chromosomal e\~olution in Peromysczrs. Evolution 28: TE, G. A,, AND W. D. DAWSON Chromosomal polymorphism in Perom)~sczrs polionotzrs. Cytogenetics (Basel) 10: THOMAS, B Evolutionary implications of karyotypic variation in some insular Peromysczrs from British Columbia, Canada. Cytologia 38: