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The serum proteins and transferrins of the ground squirrel subgenus Spermophilus
Comp. Biochera.Physiol., 1968, VoL 27, pp. 487 to 503. PergamonPress.Printedin Great Britain
T H E SERUM PROTEINS AND TRANSFERRINS OF T H E G R O U N D SQUIRREL SUBGENUS S P E R M O P H I L U S C H A R L E S F. N A D L E R Department of Medicine, Northwestern University Medical School, Chicago, Illinois (Received 15 April 1968)
Abstract--1. Serum proteins and transferrins from populations of Spermophilus townsendi, S. richardsoni, S. armatus, S. undulatus, S. columbianus and S. beldingi were analyzed by starch-gel electrophorasis. 2. Comparison of protein patterns demonstrated specific or infraspecific variation in nine fractions. Each species was easily identified by its proteins except for S. beldingi and certain slabspecies of S. townsendi which had similar patterns. 3. Eight molecular forms of transferrin, appearing as single or double bands, were observed among the six species. Data from an interbreeding population of S. undulatus indicate that one- and two-banded patterns result from homozygous and heterozygous expression of one or two transferrin alleles respectively. S. undulatus, S. townsendi, and S. richardsoni display transferrin polymorphism. 4. In the subgenus Spermophilus, serum proteins are of greatest systematic value at infraspecific levels where they both raise and answer questions concerning evolution within S. toumsendi, S. undulatus and S. richardsoni. INTRODUCTION
TrIE GROUNDsquirrel subgenus Spermophilus, genus Spermophilus, contains eight presently recognized North American species that are readily distinguished by a series of gross morphological, pelage and chromosomal characters (Hall & Kelson, 1959; Howell, 1938; Davis, 1939; Nadler, 1966). Gross morphological (Davis, 1939) and chromosomal evidence (Nadler, 1966) suggest the presence of two species groups. One consists of the desert-dwelling species S. townsendi, S. washingtoni and perhaps S. brunneus; this group is characterized by short ears and higher chromosome numbers. The second group is composed of the big-eared species S. richardsoni, S. armatus, S. beldingi, S. columbianus and S. undulatus which prefer a moister habitat and have lower chromosome numbers. Systematic relationships between the individual species comprising each group and among infraspecific categories are less well understood because traditional gross morphological features are often less definitive as taxonomic affinities become closer. In some instances use of additional taxonomic characters such as chromosomes has suggested considerable evolutionary divergence between taxa that display great pelage and cranial similarity; the finding of diploid numbers ranging from 2n36-46 in subspecies of S. townsendi and 2n34-36 in subspecies of S. richardsoni are examples of these contrasting, lines of evidence (Nadler, 1966). To provide further insight into some systematic problems in Spermophilus the serum proteins of S. undulatus, S. beldingi and S. colundn'anus were examined with 487
488
CHARLES F. NADLER
starch-gel electrophoresis (Nadler & Hughes, 1966). These species had generally similar protein patterns which confirmed a close relationship between them, although certain features suggested a closer affinity between S. undu[atus and S. columbianus. Polymorphism of the transferrins and several unidentified proteins were observed in S. undulatu~ and these fractions, when considered as population markers, suggested a possible divergence between sub-Arctic and Arctic populations. This initial, preliminary study demonstrated the potential value of certain protein fractions, especially transferrin, as taxonomic markers among specific and infraspecific categories in Spermophil~. T h e present investigation describes serum protein and transferrin patterns obtained by two-dimensional and conventional starch-gel electrophoresis from multiple population samples of S. townsendi, S. richardsoni and S. armatus. Additional samples of S. undulatus, S. behtingi and S. columbian~ are analyzed and compared with previous data (Nadler & Hughes, 1966). T h e proteins are discussed in relation to the present classification of ground squirrels (Hall & Kelson, 1959). MATERIALS AND METHODS Serum was analyzed from the following animals: Spermophilus townsendi townsendi (Bachman). Washington: (south of the Yakima River) Benton County, 7 miles W. of Prosser, 2 males and 3 females; 3 miles S. of Prosser, 2 females; Prosser, 3 males and 4 females; 15 miles E. of Prosser, 1 male and 1 female; 18 miles E. of Prosser, 3 females. Total, 19. SpermophiIus townsendi nancyae (Nadler, 1968). Washington: Benton County (north of the Yakima River), Richland, 22 males and 16 females; 3 miles N. of Richland, 3 females; 5 miles N. of Richland at S. boundary of Hanford Reservation, 3 males and 2 females; N. Yakima River near Prosser, 1 male and 3 females; 10-12 miles E. of Prosser, 3 males and and 1 female. Yakima County, Grandview, 1 male. Total, 55. Spermophilus townsendi canus (Merriam). Oregon: Hamey County, Narrows, 1 male and 2 females; Hines, 1 female. Total, 4. Spermophilus townsendi vigilis (Merriam). Oregon: Malheur County, 1½ miles S.E. of Vale, 4 males and 4 females; 5 miles W. of Ontario, 3 males and 2 females. Total, 13. Spermophilus townsendi moUis (Kennicott). Idaho: Cassia County, Burley, 2 males and 13 females. Total, 15. Spermophilus townsendi idahoensis (Merriam). Idaho: Elmore County, 4 miles E. of Mountain Home on Highway 30, 4 males and 5 females. Total, 9. Spermophilus beldingi oregonus (Merriam). Oregon: Hamey County, Bums, "4males and 4 females. Total, 8. Sperrnophilus columbianus columbianus (Ord). Montana: Lubrecht Forest 30 miles N.E. of Missoula, 3 females. Total, 3. Sperrnophilus armatus (Kennicott). Utah: Logan Canyon 20 miles N. of Logan, 2 males and 2 females. Total, 4. Spermophilus richardsoni richardsoni (Sabine). Montana: Gallatin County, Belgrade, 6 males and 1 female; Bozeman, 1 male and 7 females. North Dakota: Cass County, Mapleton, 1 male and 5 females. Total, 21. Spermophilus richardsoni elegans (Kermicott). Colorado: Larimer County, Cherokee Park 45 miles N.W. of Fort Collins, 7 males and 6 females. Total, 13. Spermophilus richardsoni aureus (Davis). Idaho: Clark County, 10 miles N.W. of Dubois, 10 males and 5 females. Total, 15. Spermophilus undulatus ablusus (Osgood). Alaska: Nome, 6 males and 4 females; 10 miles S.E. of Anchorage, 1 male and 4 females; Teklanika River Camp Ground at Mt.
SERUM PROTEINS AND TRA/qSFERRINS OF GROUND SQUIRREL SPERMOPHILUS
489
McKinley National Park, 3 females; 5 miles W. of Paxson Lake on the Denali Highway, 1 male and 2 females. Total, 21. Sperraophilus undulatus lyratus (Hall & Gilmore). Alaska: St. Lawrence Island, Gambell, 4 males and 6 females. Total, 10. Sperraophilus undulatus plesius (Osgood). Alaska: Chisana, 14 males and 22 females. Canada: British Columbia, 10 miles S. of junction of the Turnagain and Dall Rivers (Cassiar Mrs.), 3 males and 12 females; 5 miles W. of previous locality, 2 females. Total, 54. Blood was drawn from the heart with sterile syringes and the serum was frozen at - 2 0 to - 6 0 ° C without adding preservatives or antibiotics. Storage of frozen sera for periods up to 12 months did not alter the results. Serum proteins were analyzed by horizontal, two-dimensional, starch-gel electrophoresis (Poulik & Smithies, 1958) using a Trisdiscontinuous buffer (Poulik, 1957). The proteins were stained with Nigrosin (Nadler & Hughes, 1966). Two or three separations of each serum specimen were performed before recording the protein pattern of an individual animal as a scale indian ink drawing (Fig. 1).
B Tf
~
~ . ~ C
FIG. 1. A protein pattern from S. columbianus illustrating fractions of taxonomic significance. The labeled fractions correspond with the following characters: albumin (Character I), Fraction Group A (II), Fractions B and C (IV and V), transferrin or T f (III), Fraction D (VI), Fraction E (VII) and Fraction F (VIII). There are three arc fractions (IX) in this species. Fractions staining with the intensity of albumin or transferrin were drawn in black, those of intermediate staining intensity were stippled while the faintest fractions were drawn with open lines. The fractions with fastest and slowest mobilities were labeled as albumin and gamma globulin respectively. All other fractions except transferrin were arbitrarily given letter designations (A-F) as described previously (Nadler & Hughes, 1966). Transferrin (Tf) was identified by radioautography with Fe 5~. Interspecific and infraspecific differences in protein patterns were compared and confirmed by simultaneously running sera in a horizontal, one-dimensional separation; in the case of transferrin, differences in the mobilities and number of transferrin fractions were checked by repeated, simultaneous one-dimensional separations utilizing radioautography. RESULTS T h e influence of physiological or e n v i r o n m e n t a l factors on the s e r u m proteins was evaluated w h e r e v e r possible. A m o n g specimens o f S. townsendi, animals r a n g i n g in age f r o m several weeks to 1½ years had qualitatively similar p a t t e r n s ; no differences were attributable to sex, diet or the captive vs. wild state. Sera
490
CHARLES F. NADLSR
from S. undulatus and S. richardsoni were also uninfluenced by age or diet, the latter consisting of food from the natural environment or laboratory rat chow. Pregnant animals or those in hibernation were not examined. It is therefore concluded that the protein fractions discussed in this study are probably genetically controlled and of potential taxonomic value. The serum proteins of Spermophilus townsendi, S. richardsoni, S. armatus, S. undulatus, S. beldingi and S. columbianus display basically similar electrophoretic patterns (Figs. 1-18). There are, however, nine fractions or groups of fractions that exhibit qualitative variation at different taxonomic levels and these fractions, which were initially recognized in a limited study of S. beldingi, S. columbianus and S. undulatus (Nadler & Hughes, 1966), have taxonomic applications. Manifestations of the nine protein characters are described individually below and summarized for each taxon in Table 1. TABLE I~CoMPARISON OF PROTEIN CHARACTBI~
S . totonsendi
Character
townsouti
nancyae
?2
vigilis
P2
S. richardsoni
canus
mollis
idahoensis
richardsoni
elegans
aur~us
?2
3
4.
2
2
2
T+I (9[9)
T f 2-4 (2]21) Tf4 (19/21)
Tf 4 (9]13) Tf4-6 (4.]13)
Tf4 (15115)
I N u m b e r of albumins II Fraction Group A
?2
I I I Transferrin fractions
"If 1-3 (1119) Tf3 (18/19)
T f 1-3 (11/55) Tf3 (44/55)
T+3 (13/13)
I V Presence of Fraction B V Presence of Fraction C VI Presence of Fraction D V I I Presence of Fraction E V I I I Separation between fractions in Group F I X N u m b e r of arc fractions
+
-I-
+
+
+
+
+
+
-I-
+
+
_+
+
+
+
+
+
_+
+
+
+
+
+
+
+
+
+
-t-
+
+
+
+
+
+
+
+
+
+
+
+
+
-t-
+
+
+
1
1
1
1
1
1
1
1
1
T f 1-3 Tf3 (13]15) (4-]4) T f 3 (2115)
* S. u. plesius: Chisana Alaska. t S. u. p/edus: British Columbia, Canada. S. u. ablusus: Nome, Mt. McKinley and Anchorage, Alaska. § S. u. ablusus: Paxson Lake, Alaska. I] S. u. lyratus: St. Lawrence Island. ¶ S . u. kennicotti reported previously (Nadler & Hughes, 1966).
SERUM PROTEINS AND TRANSFERRINS OF GROUND SQUIRREL S P E R M O P H I L U S
491
Character I refers to the electrophoretic characteristics of the larger major albumin fraction and the smaller fraction migrating slightly slower than the large fraction. T h e latter fraction, although not identified chemically or immunologieally as albumin, is probably a second albumin and it is prominent in all species except S. undulatus where it is small and insignificant. In Table 1 S. undulatus is therefore listed as having one albumin (Figs. 13-19). S. beldingi has a homogeneous major fraction and the second, smaller probable albumin (Fig. 2). In S. richardsoni the subspecies richardsoni (Fig. 3), elegans (Fig. 4) and aureus (Fig. 5) are electrophoretically indistinguishable and have two large and widely separated fractions that are both presumably albumins. S. armatus (Fig. 6) also has two albumins that are less widely separated than those of S. richardsoni. A notched major albumin fraction, suggesting heterogeneity, plus a small minor fraction are found in S. colundn'anus (Fig. 1). Various albumin patterns are seen in subspecies of OF SOME SPECIES OF
Spermophilus S. undulatus
Sub-Arctic S. beldingi S. armatus S. columbianus ?2
?2
?2
Tf 3
Tf 3 ( - )
(8•8)
(4/4)
Tf 3 (3/3)
plesius*
plesiusf ablusus~ ablusus§ lyratus[I kennicotti¶
1
Tf 5 (14/36) Tf 5-7
1
1
Tf 7
Tf 7
(17117)
(18/18)
(16136)
_+
+
+
+
+
+
+
+
+
Arctic
[Tf 7 (6/36)] +_
1
1
'If 5-7 (1/3)
Tf 7
Tf 7 (2•3)
(10/10)
1
Tf 6 (28/36) T f 6-7 (8/36)
+
+
_+
+_
_+
+
+
+
+
+
+
+
+
+
+
+
+
--
+
+
.
.
.
.
.
.
+
+
+
.
.
.
.
.
.
1
1
2-3
1
1
1
1
1
1
492
CRAat~ F. NADt.ma
S. townsendi; townsen&" (Fig. 7), nancyae (Fig. 8), canus (Fig. 9) and vigilis (Fig. 10) have a single, large homogeneous albumin with the second smaller fraction migrating closely behind it on both paper and starch-gel. In striking contrast, moles (Fig. 11) and idahoens~ (Fig. 12) exhibit subdivision of albumin into three and four fractions respectively, features that permit differentiation of these taxa from any others. Character 11 refers to the number and configuration of fractions comprising Group A which migrates slightly slower than albumin. In all populations of
~ 0 e
A ~
B Tf
Q
aA B
Tf
Fzo. 2. A protein pattern from ,S. beldingi oregonus. A single Tf 3 fraction is present
FIO. 3. A protein pattern from S. richardsoni richardsoni with a single Tf 4 band. There
and a prominent gap separates the fractions comprising Group F.
are two large albumin fractions.
~ A
Tf m
C
..4~4
Fxo. 4. A protein pattern from S. richardsoni elegans. Except for the double Tf 4--6 bands the pattern is similar to ,S. r. richardsoni (Fig. 3) and S. r. aureus (Fig. 5) where two albumins are also observed.
Fzc. 5. A protein pattern from ,S. richardsoni aureus. A single T f 4 fraction is present. Fraction B migrates within the are fraction, a finding also seen in S. armatus (Fig. 6).
SERUM P R O T E I N S AND TRANSFF_.RRINS OF G R O U N D S Q U I R R E L 8PERMOPHILUS
493
S. townsendi (Figs. 7-12), S. richardsoni (Figs. 3-5), S. armatus (Fig. 6) and S. beldingi (Fig. 2) there are three fractions occupying positions, with reference to a
clock face, of 9, 12 and 3 o'clock. A second pattern containing three fractions located at 6, 9 and 12 o'clock is found in S. columln'anus (Fig. 1). Group A of S. undulatus (Figs. 13-18) populations has four fractions located at 12, 3, 6 and 9 o'clock. Character 111 consists of the number of transferrins and their comparative mobilities. Among the taxa analyzed in this study there are seven and probably eight electrophoretically distinct transferrin fractions. Each animal has one or
Q B
/
FIo. 6. A protein pattern from S. armatus illustrating two albumins migrating nearly together, Fraction B migrating within the arc fraction and a single transferrin, Tf 3(-).
•- O "
....
FIG. 7. A protein pattern from S. townsendi toumsendi. There is close similarity to the pattern of S. beldingi (Fig. 2) and both have a "If 3 of identical mobility. Two albumins migrating nearly together are present.
A
C3
y Fro. 8. A protein pattern from S. toumsendi nancyae illustrating double transferrins, Tf 1 and 3. Two albumins with nearly similar mobillties are present.
,7 Fro. 9. A protein pattern from S. toumsendi canus. This pattern is similar to the patterns of tourasendi (Fig. 7) axedvigilis (Fig. 10). A single Tf 3 is present.
494
CHARLESF. N~mL~n
two transferrins designated T f 1 through 7 in order of fastest to slowest mobility (Figs. 19 and 20). S. tom~sendi is characterized by T f ' s 1 and 3 which appear as single T f 1 or T f 3 patterns or a double-banded T f 1-3 configuration. T h e idahoensis population has a single-banded T f 1 pattern in all nine specimens; a single T f 3 band is observed in all four canus sera, and all thirteen specimens of vigilis. Both T f 1
0A
o
di~ ° ~
A
%_'V
o
m
/
FIG. 10. A protein pattern from S. toumsendi vigilis. A single T f 3 is present.
FIG. 11. A protein pattern from S. toumsendi moles. Three albumin sub-fractions characterize raollis. A double T f 1-3 pattern is present.
G
0A
~
A
Tfdi~
Fro. 12. A protein pattern from S. toumsendi idahoensis illustrating the albumin with four sub-fractions and the single transferrin, T f 1, band that characterize this subspecies.
FIG. 13. A protein pattern from an Arctic population of S. undulatus kennicotti studied previously (Nadler & Hughes, 1966). It is included for comparison with sub-Arctic populations of S. undulatus. Fraction Group A is characteristic of S. undulatus. The absence of Fraction D differentiates Arctic from sub-Arctic undulatus. A double T f 6-7 pattern is present. The fractior~ comprising Group F are continuous.
SERUM PROTEINS AND TRANSFERRINS OF GROUND SQUIRREL S P E R M O P H I L U S
495
and 3 are found in populations of townsendi, nancyae and mollis: a single T f 3 band is identified in eighteen of nineteen townsendi sera whereas one individual has a two-banded T f 1-3 pattern; nancyae exhibits T f 1-3 bands in eleven sera of the sample and a T f 3 band in the other forty-four animals; thirteen of the fifteen
. m ~=~Qo A
B
/-
I)
D F
FXGI14. A protein pattern from S. undulatus lyratus with single T f 7 pattern and a prominent Fraction D that are typical of sub-Arctic undulatus.
FzG. 15. A protein pattern from S. undulatus ablusus (Nome, Alaska). Patterns of ablusus from Anchorage and Mt. McKinley, Alaska are similar.
T f ...~.,~,
,-~'
"D
FzG. 16. A pattern from S. undulatus ablusus collected near Paxson Lake, Alaska that demonstrates T f 5-7 fractions.
FIG. 17. A pattern from S. undulatus plesius (Chisana, Alaska) exhibiting two transferrins (Tf 5 and 7), a moderately staining Fraction D and the absent gap in Fraction F.
mollis specimens show T f 1-3 bands while two animals have a single T f 3 pattern. T f 1 is randomly distributed among the multiple populations of tonmsendi and nancyae and is not confined to a single geographic sample. Both S. beldingi and S. columbianus display a single T f 3 pattern that is indistinguishable from the T f 3 seen in S. townsendi.
496
CHARLgSF. NADLER
A single transferrin, migrating slightly slower than the T f 3 of & beldingi and S. columbianus, is observed in all four sera of S. armatus and it is designated T f 3 ( - ). Analysis of a larger sample is desirable before clearly recognizing the transferrin of armatus as distinct from the other T f 3 bands.
B~'~ T ~
FIG. 18. A pattern from S. undulatus plesius (Chisana, Alaska) with a single Tf 7 fraction. Specimens of plesius from British Columbia, Canada display similar patterns. T f 4 characterizes all S. richardsoni populations and is the only transferrin seen in aureus. Four of thirteen specimens of elegans have a T f 6 band in addition to the T f 4 fraction found in all the specimens. All sera of richardsoni from Bozeman and Belgrade, Montana have a single T f 4 band whereas richardsoni from North Dakota exhibit a T f 2 in addition to T f 4 in sera from two of six animals. Three transferrins, T f 5, 6 and 7, are found in populations of Spermophilus undulatus (Fig. 20) examined in this and a previous study (Nadler & Hughes, 1966). S. undulatus kennicotti from two populations in the Brooks Range of Arctic Alaska previously showed a T f 6 fraction in all thirty-six specimens; in one population four of the fourteen animals had a second transferrin, T f 7, in addition to T f 6 (Figs. 13 and 20). The T f 6 of S. undulatus cannot be differentiated from T f 6 of S. richardsoni elegam by electrophoresis. Specimens of S. undulatus collected in sub-Arctic Alaska display single T f 5 and T f 7 patterns or double bands composed of both T f 5 and 7. Populations from St. Lawrence Island (lyratus) (Fig. 14), Nome (ablusus) (Fig. 15), Anchorage (ablusus) and Mt. McKinley National Park (ablusus) are indistinguishable and characterized by a single T f 7 band. Of three animals collected near Paxson Lake, which is in an area intermediate between the distribution of the subspecies ablusus and plesius, two have a single T f 7 and one animal has a T f 5 and 7 pattern (Fig. 16). Among the thirty-six specimens of S. undulatus plesius from Chisana, Alaska there are three different types of pattern (Fig. 20); fourteen have a single T f 5,
FIc. 19. Transferrins of the subgenus Spermophilus. (1) S. undulatus plesius (Tf 5); (2) S. richardsoni elegans (Tf4); (3) S. richardsoni richardsoni (Tf4); (4) S. armatus (Tf3 ( - ) ) ; (5) S. beldingi (Tf 3); (6) S. richardsoni richardsoni (Tf 2 4 ) and (7) S. townsendi mollis (Tf 1-3). Fe 59 radioautography was used to identify the transferrins. Note the widely separated albumins in S. richardsoni (2,3,6), the less widely separated albumins in S. beldingi (5), S. townsendi (7) and S. armatus (4). The albumin of S. undulatus appears homogeneous.
FIG. 20. Transferrins of S. undulatus. (1) S. undulatus ablusus (Tf7); (2) S. undulatus kennicotti (Tf 6-7); (3) S. undulatus kennicotti (Tf 6); (4) S. undulatus plesius (Tf 5-7) and (5) S. undulatus plesius (Tf 5).
SERUMPROTEINSANDTK~SVelmXNSOF ORotrsD SqUIl~a~T-SPERMOPHILUS
497
sixteen have a two-band T f 5-7 pattern (Fig. 17) and six specimens have a single T f 7 (Fig. 18). The T f 5 bands from sera obtained at Paxson Lake and Chisana are electrophoretically similar and the occurrence of this fraction in populations 125 miles apart suggests that T f 5 is not merely a local transferrin mutation. All seventeen S. undulatus plesius from two localities in British Columbia have a single T f 7 fraction that is identical to T f 7 of other sub-Arctic populations. The three different transferrin patterns (Tf 5, T f 5-7 and T f 7) identified in S. undulatus from Chisana, Alaska are of interest because they were derived from a single, continuous population distributed over an area 300 by 800 yards. In this apparently interbreeding natural population it is postulated that animals with single T f 5 or 7 fractions represent homozygous expression of each allele while the double T f 5-7 pattern is a heterozygous manifestation of two alleles, T f 5 and T f 7, occurring at a single locus. Although evidence of this sort is lacking in the other species it seems likely that their transferrins are under similar genetic control and it is also possible that all the molecular forms of transferrin in Spermophilus are due to allelic substitutions as a single locus. This latter possibility cannot be established without recourse to interspecific hybridization, a feat not yet achieved in Spermophilus. Characters I V and V consist of the presence or absence of Fractions B and C respectively. Their occurrence is highly variable and they may appear together or one or both may be randomly absent in S. undulatus (Figs. 13-18), S. tonmsendi (Figs. 7-12) and S. richardsoni (Figs. 3-5). Both Fractions B and C were observed in all sera of S. armatus (Fig. 6) and Fraction B always migrated within the arc fraction, a feature sometimes observed in S. richardsoni (Fig. 5). In S. beldingi (Fig. 2) Fraction B was inconsistently present whereas Fraction C was always found. All three S. columbianus (Fig. 1) had both Fractions B and C which substantiates a similar observation made on specimens from Idaho (Nadler & Hughes, 1966). The highly variable presence of these fractions plus a lack of knowledge regarding their structure or function make them of uncertain taxonomic value. Character V I is represented by a darkly staining Fraction, D, that migrates faster than gamma globulin on paper but slower on starch. Fraction D is present in all species. However, within S. undulatus Fraction D is absent in Arctic (Fig. 13) populations (Nadler & Hughes, 1966) but present in all sub-Arctic populations (Figs. 14-18). Character V I I consists of a small lightly or moderately staining fraction, Fraction E, which migrates slightly faster than Fraction D on both paper and starch-gel. Fraction E is consistently present in S. townsendi, S. richardsoni, S. armatus, S. beldingi and S. columbianus (Figs. 1-12); it is absent in all populations of S. undulatus (Figs. 13-18). Character V I I I refers to the configuration of the two fractions constituting Group F. A gap separating the two fractions, which is produced by differing migration rates on paper and starch-gel, is characteristic of S. townsendi, S. richardsoni, S. armatus, S. beldingi and S. columbianus (Figs. 1-12). The two fractions in Group F display no gap or separation in S. undulatus (Figs. 13-18).
498
CHAm.ESF. NADm
Character I X refers to the number of arc-like fractions. Three arc fractions are present in two S. columbianus specimens (Fig. 1) and two arcs in the third animal from Montana; all nineteen specimens reported from Idaho had three arcs (Nadler & Hughes, 1966). Never was more than one arc seen in S. totvnse~h, S. richardsoni, S. armatus, S. beldingi and S. undulatus. Certain protein characters alone or in combination are diagnostic for most species and some subspecies examined in this study (Table 1): S. columbianus is distinguished by Characters I, II and IX, S. richardsoni and S. arrnatus are identified by Characters I and III (Tf) and S. undulatus is differentiated by Characters II, III (Tf), VII and VIII. Within S. torvnsendi, the subspecies mollis and idahoer~is can each be identified by Character I and in part by the presence of T f 1 alone (idahoensis) or in combination with T f 3 (mollis); in contrast, to~nsendi, nancyae, canus and vigilis cannot be differentiated from S. beldingi unless T f 1 is present in addition to T f 3, which is the case in some tozvnsemdi and nancyae sera. S. bel&'ngi has the least unique protein pattern and shares some features with all species while lacking diagnostic fractions of its own. The subspecies of S. undulatus display no diagnostic features although when considered as a whole there is evidence for divergence between Arctic and sub-Arctic populations. Subspecies of S. richardsoni cannot be differentiated by their serum proteins. DISCUSSION A consideration of the validity and limitations of serum protein electrophoresis is necessary before the data from this study can be applied to an appraisal of systematic relationships. The suitability of electrophoretic methodology has been discussed by many investigators and a number of generally accepted conclusions are pertinent (Dessauer & Fox, 1964; Manwell & Kerst, 1966; Bianchi, 1967; Coates, 1967). Electrophoresis separates proteins according to net molecular charge and also by molecular size when certain supporting media such as starch or polyacrylamide gel are used. As a consequence, when homologous proteins from different species migrate with different mobilities in the same gel they may be considered to differ in their molecular structures (Bianchi, 1967). For this assumption to be valid, the proteins should be shown to be homologous by demonstrating chemical or functional similarity or by utilizing breeding data to ascertain whether the proteins in question are controlled by alleles at a single locus (Hubby & Lewontin, 1966; Bianchi, 1967). Environmental and physiological factors that can effect the proteins must also be excluded (Dessauer & Fox, 1964; Coates, 1967). In the interpretation of data derived from homologous protein comparisons, electrophoretically dissimilar fractions are recognized as having greater taxonomic significance than electrophoretically similar proteins because the latter may still differ structurally; a good example is the occurrence of structurally different hemoglobin D molecules with the same electrophoretic mobility (Lehmann & Huntsman, 1966). Most recent studies have considered these general principles and based taxonomic decisions on a comparison of presumably homologous specific proteins including hemoglobins, transferrins and enzymes from tissues
SERUM PROTEINS AND TRANSFERRINS OF GROUND SQUIRREL S P E I ~ I O P H I L U S
499
and serum (Goodman, 1963; Manwell & Kerst, 1966; Gorman & Dessauer, 1966; Baker & Hanson, 1966). In this study, the transferrins of ground squirrels appear to be homologous proteins from the standpoint of chemical function and in addition breeding data from a natural population of S. u~tulat~ indicate that the three transferrin bands are controlled by two alleles at a single locus; a similar mechanism seems likely in the other species examined. Comparison of the total protein pattern, while less valid than comparisons of single, homologous proteins, can provide apparently reliable taxonomic data especially when closely related species are analyzed (Coates, 1967). A close taxonomic relationship increases the chance that electrophoretically similar proteins of undetermined structure or function are in reality homologous proteins (Bianchi, 1967). In a study of three salamander species belonging to the genus Taricha serum proteins were analyzed by disc electrophoresis and qualitative differences, independent of environmental or physiological influence, were shown to give a valid reflection of evolution at specific and infraspecific levels (Coates, 1967). Goodman's (1963) studies of primates by starch-gel electrophoresis also gave similar, apparently reliable data. In the present study of ground squirrel proteins, analysis of a presumably homologous protein transferrin is combined with a comparison of certain unidentified proteins to assess evolutionary relationships in the manner utilized by Coates (1967) and Goodman (1963). Several areas for systematic speculation exist in the subgenus Spermophilus, which have been highlighted by different lines of taxonomic evidence, and it is against this background that serum protein data may be compared. Combined evidence from gross morphology and pelage, ecology zoogeography, parasitology and cytogenetics was reviewed by Nadler (1966) and the following conclusions were reached: (1) the subgenus contains two species groups, the big-eared and short-eared squirrels; (2) within the big-eared group, S. richardsoni and S. armatus are closely related ecologically, morphologically and cytologically; (3)'the relationship of S. beldingi to the other big-eared squirrels is obscure and (4) S. columbianus and S. undulatus are probably closely related although gross morphological and pelage features, parasites and zoogeography indicate a closer affinity than is suggested by their dissimilar chromosomes. When proteins from six of the eight species of the subgenus Spermophilus are compared (Table 1) there is no suggestion of a species grouping except perhaps in the case of 8. beldingi and S. toumsendi and in that instance a mass of morphological (Howell, 1938), ecological (Durrant & Hansen, 1954) and chromosomal (Nadler, 1966) data fail to indicate a close relationship between these forms. The protein data could be interpreted as showing a basic similarity between all species with perhaps the beldingi-toumsendi similarity serving as an indication of common ancestral relationship among all species of the subgenus. There is no support for raising the big-eared and short-eared species groups to subgeneric rank. The proteins of S. armatus and S. richardsoni have diverged to a degree equal to or surpassing that displayed by their gross morphological features (Howell,
500
Caamam F. NADI.ma
1938) and chromosomes (Nadler, 1966) and it is not possible to confirm a close evolutionary relationship between these species by proteins alone. Among the six species examined, S. beldingi displays fewer unique protein fractions than any other taxon except for some S. townsendi subspecies. The occurrence of T f 3 in S. beldingi, S. columbianus and S. townsendi may indicate derivation from a common ancestral stock but these data do not lead to convincing new concepts concerning the taxonomic status of S. beldingi with respect to the other big-eared species. Evolutionary relationships of S. undulatus and S. columbianus were investigated by electrophoresis and a greater similarity was observed between the proteins of S. columbianus and sub-Arctic rather than Arctic populations of S. undulatus (Nadler & Hughes, 1966). Study of additional sub-Arctic sera from six new localities, plus examination of sera from three additional species of Spermophilus, now indicates a much greater degree of divergence between S. undulatus and S. columbianus; the two species differ with respect to Characters I, II, III (Tf), VII, VIII and IX. Previous analysis of the insular, sub-Arctic S. undulatus kodiacensis population indicated that Characters II and VIII were similar to those of S. columbianus (Nadler & Hughes, 1966), a finding now refuted by analysis of the other sub-Arctic populations. The apparent discrepancy may result from an initial analysis of a small, atypical population of S. undulatus or the insular form, kodiacensis, may be different from the mainland populations and truly share a closer protein relationship to S. columbianus. It is noteworthy, however, that the protein differences existing between S. undulatus and S. columbianus are now shown to be greater than those of any other two species in the subgenus. This is surprising because these species are quite similar in pelage and gross morphology (Davis, 1939), ectoparasite data suggest that sub-Arctic undulatus and columbianus arose from a single ancestral stock (Holland, 1958, 1963), and zoogeographic evidence suggests both species may have evolved following separation of the ancestral stock by continental glaciers during the Pleistocene (Rand, 1954). The dissimilarity of the proteins may now be combined with the previous observation of a dissimilarity of the chromosomes of S. undulatus (2n34) and S. columbianus (2n32) to provide an indication that evolution of gross morphological features is either proceeding at a slower, more conservative rate or that separation and divergence of the two species occurred at an earlier time than previously postulated. An examination of fleas (Siphonaptera) from Arctic and sub-Arctic populations of S. undulatus (Holland, 1958, 1963) initially raised the question of intraspecific divergence when it was noted that the former populations were parasitized by the same species of flea as Asian ground squirrels while sub-Arctic undulatus was parasitized by a different species of flea that was also found on ground squirrels from the western United States. Initial protein comparisons indicated that Arctic and sub-Arctic populations of undulatus differed with respect to Characters II, III and VI (Nadler & Hughes, 1966). New data from the larger sub-Arctic sample show only a difference in Character VI, manifested by an absence of this fraction in Arctic samples, and a difference in transferrins. Although study of transferrins
SERUM PROTEINS AND TRANSFERRINS OF GROUND SQUIRREL S P E R M O P H I L U S
501
from more Arctic populations is mandatory before drawing firm conclusions it is possible that Arctic populations are characterized by Tf 6 and sometimes Tf 7, whereas sub-Arctic undulatus is distinguishable by Tf 7 alone or in combination with Tf 5. Further investigation may strengthen the validity of the transferrins and Fraction D as population markers that now appear to correlate well with Holland's (1958, 1963) ectoparasite observations. If confirmatory evidence of a differentiation between Arctic and sub-Arctic populations can be obtained it will find practical application in clarifying questions pertaining to the zoogeography of Arctic mammals. Exploration of infraspecific relationships with protein electrophoresis is also valuable in S. richardsoni and S. townsendi. In S. richardsoni, the subspecies elegans, aureus, nevadensis and richardsoni are quite similar although the latter can be distinguished from elegans by differences in baculum morphology that led Burr (1960) to question whether elegans and richardsoni might not be distinct species. Chromosome analysis also revealed differences between richardsoni with 2n36 and the other subspecies which have thirty-four chromosomes (Nadler, 1966). Comparison of the proteins reveals indistinguishable patterns in elegans, aureus and richardsoni and all share a common Tf 4 band. The limited occurrence of Tf 2 and Tf 6 in populations of richardsoni and elegans respectively is most suggestive of transferrin polymorphism within local populations and is not of taxonomic significance. Thus, proteins fail to indicate a specific distinction for elegans and richardsoni and instead support the concept that these taxa are geographically isolated subspecies that exhibit differing degrees of evolutionary divergence with respect to chromosomes, baculum structure and proteins. Recently the chromosomes of S. townsendi were analyzed and striking variation was observed between subspecies (Nadler, 1968). The geographically contiguous taxa S. t. vigilis and S. t. canus had forty-six chromosomes, S. t. mollis and S. t. idahoensis, which are separated from each other by the Snake River barrier, had thirty-eight, and S. t. townsendi, which is isolated from the other forms by the Columbia River, had thirty-six chromosomes. A chromosomally homogeneous population, separated from to~onsendi by the Yakima River and having thirty-eight chromosomes, was recognized as a new subspecies, S. t. nancyae. The widely divergent chromosome patterns of populations classified as vigilis and canus, mollis and idahoensis, and townsendi led to speculation that each chromosomally distinctive group might represent a sibling species although morphologically identifiable intergradation between mollis (2n38) and vigilis (2n46) had been reported (Davis, 1939). When proteins from individual S. townsendi are compared the following taxonomic observations can be made: (1) a general similarity of patterns, with varying expression of a transferrin genome composed of Tf 1 and Tf 3, indicates that all populations are closely related to one another; (2) two populations, corresponding to mollis and idahoensis, each have distinctive albumins which supports the possibility raised by chromosomes that mollis (2n38) and vigilis (2n46) might be specifically distinct. It is conceivable that idahoensis is also a valid species but investigation
502
Cmucm F. N~mum
of natural intergradation with rnollis is prevented by the Snake River barrier; (3) sera from the geographically discontinuous Washington (townsendi, nancyae) and Oregon (canus, vigilis) populations are indistinguishable except for the occurrence of T f 1 in the former and its absence in the latter. Support for species status of these chromosomally different populations is lacking and (4) the greater frequency of "If 1 in nancyae compared to tomnsauti provides added evidence for the presence of a population (subspecific) divergence that was initially discovered by chromosome differences. A lack of major protein differences suggests these diverging taxa have not been isolated long enough to achieve species status. From the foregoing discussion it is apparent that protein differences occur at all taxonomic levels within the subgenus Spermophilus. At the species level where gross morphological differences are distinctive the electrophoretic contributions are least impressive. At the population and subspecies levels where morphological and cytological differences are less striking, proteins often display greater divergence. Here, proteins can serve as an effective means for clarifying evolutionary relationships and tracing the origin and composition of populations. Acknowledgements--The author wishes to thank Mr. C. F. Nadler, Jr., and Mrs. Nancy W. Nadler for their invaluable assistance during field work in Alaska and Canada. Additional specimens were obtained through the generous efforts of Dr. Robert L. Rausch, Dr. Francis H. Fay, Dr. Robert S. HotTman, Dr. Richard M. Hanaen, Dr. Kenneth S. Todd, Dr. Walter R. Quanstrom, Mr. Pat Magula, Mrs. Donna Grant, Mr. Russell Pengelly and Mr. Ken Small. I am especially indebted to Miss Kathleen E. Harris for her devoted technical assistance in the laboratory. This investigation was supported by National Science Foundation Grants GB-3251 and GB-S676X. REFERENCES BAI~R C. M. A. & HANSONH. C. (1966) Molecular genetics of avian proteins--VI. Evolutionary implication of blood proteins of eleven species of geese. Comp. Biochem. Physiol. 17, 997-1006. B I ~ e I U. (1967) Utility significance and limitations of electrophoresis, applied to particular taxonomic problems. Riv. Biol. N.S. 20, 237-24S. BtmT. W. H. (1960) Bacula of North American mammals. Misc. Pubis Mus. Zool. Univ. Mich. 113, 1-76. COAT~SM. (1967) A comparative study of the serum proteins of the species of Taricha and their hybrids. Evolution 21, 130-140. DAVISW. B. (1939) The Recent Mamnmls of Idaho, pp. 400. Caxton Press, Caldwell, Idaho. D~ssAtnm H. C. & Fox W. (1964) Electrophoresis in taxonomic studies illustrated by analys/s of blood proteins. In Taxonomic Biochemistry and Serology (Edited by LEo~rE C. A.), pp. 625-647. Ronald Press, New York. DUmO_NTS. D. & HANs~ R. M. (1954) Distribution patterns and phylogeny of some western ground squirrels. Syst. Zool. 3, 82-8S. GOODM~,~ M. (1963) Serological analysis of the systematica of recent Hominoids. Hum. Biol. 35, 377-436. GOnMAN G. C. & DmSAUm~H. C. (1966) The relationships of Anolis of the Roquet species group (Sauria: Iguanidae)--I. Electrophoretic comparison of blood proteins. Comp. Biochem. Physiol. 19, 845-853. HALL E. R. & KSLSONK. R. (1959) The Mammals of North America, Vol. 1, pp. 1-546. Ronald Press, New York.
SERUM PROTEINS AND TRANSFERRINS OF GROUND SQUIRREL SPERMOPHILUS
503
HOLLANDG. P. (1958) Distribution patterns of northern fleas (Siphonaptera). Proc. lOth int. Congr. Ent. 1, 645-658. HOLLAND G. P. (1963) Faunal affinities of the fleas (Siphonaptera) of Alaska: with an annotated list of species. In Pacific Basin Biogeography (Edited by GlU~SSXTTJ. L.), pp. 45-63. Bishop Museum Press, Honolulu. HOWELL A. H. (1938) Revision of the North American ground squirrels. N. Am. Fauna 56, 1-256. HI:BBYJ. L. & L~WONTXNR. C. (1966) A molecular approach to the study of genic heterozygosity in natural populations--I. The number of alleles at different loci in Drosophila pseudoobscura. Genetics 54, 577-594. LSHMANN H. & HUNTSMAN R. G. (1966) Man's Hemoglobins, pp. 1-331. Lippincott, Philadelphia. MANWXLL C. & IfamST K. V. (1966) Possibilities of biochemical taxonomy of bats using hemoglobin, lactate dehydrogenase, esterases and other proteins. Comp. Biochem. Physiol. 17, 741-754. NADLEa C. F. (1966) Chromosomes and systematics of American ground squirrels of the subgenus Spermophilus. ~. Mammal. 47, 579-596. NADI~R C. F. (1968) The chromosomes of Spermophilus townsendi (Rodentia: Sciuridae) and report of a new subspecies. Cytogenetics 7, 144-157. NADLER C. F. & Hucmm C. E. (1966) Serum protein electrophoresis in the taxonomy of some species of the ground squirrel subgenus Spermophilus. Comp. Biochem. Physiol. 18, 639-651. POULIK M. D. (1957) Starch-gel electrophoresis in a discontinuous system of buffers. Nature, Lond. 180, 1477-1479. POULIK M. D. & SMITHXESO. (1958) Comparison and combination of the starch-gel and filter-paper electrophoretic methods applied to human sera: two-dimensional electrophoresis. Biochem. y. 68, 636-643. RAND A. L. (1954)The ice age and mammal speciation in North America. Arctic 7, 31-35.
T H E SERUM PROTEINS AND TRANSFERRINS OF T H E G R O U N D SQUIRREL SUBGENUS S P E R M O P H I L U S C H A R L E S F. N A D L E R Department of Medicine, Northwestern University Medical School, Chicago, Illinois (Received 15 April 1968)
Abstract--1. Serum proteins and transferrins from populations of Spermophilus townsendi, S. richardsoni, S. armatus, S. undulatus, S. columbianus and S. beldingi were analyzed by starch-gel electrophorasis. 2. Comparison of protein patterns demonstrated specific or infraspecific variation in nine fractions. Each species was easily identified by its proteins except for S. beldingi and certain slabspecies of S. townsendi which had similar patterns. 3. Eight molecular forms of transferrin, appearing as single or double bands, were observed among the six species. Data from an interbreeding population of S. undulatus indicate that one- and two-banded patterns result from homozygous and heterozygous expression of one or two transferrin alleles respectively. S. undulatus, S. townsendi, and S. richardsoni display transferrin polymorphism. 4. In the subgenus Spermophilus, serum proteins are of greatest systematic value at infraspecific levels where they both raise and answer questions concerning evolution within S. toumsendi, S. undulatus and S. richardsoni. INTRODUCTION
TrIE GROUNDsquirrel subgenus Spermophilus, genus Spermophilus, contains eight presently recognized North American species that are readily distinguished by a series of gross morphological, pelage and chromosomal characters (Hall & Kelson, 1959; Howell, 1938; Davis, 1939; Nadler, 1966). Gross morphological (Davis, 1939) and chromosomal evidence (Nadler, 1966) suggest the presence of two species groups. One consists of the desert-dwelling species S. townsendi, S. washingtoni and perhaps S. brunneus; this group is characterized by short ears and higher chromosome numbers. The second group is composed of the big-eared species S. richardsoni, S. armatus, S. beldingi, S. columbianus and S. undulatus which prefer a moister habitat and have lower chromosome numbers. Systematic relationships between the individual species comprising each group and among infraspecific categories are less well understood because traditional gross morphological features are often less definitive as taxonomic affinities become closer. In some instances use of additional taxonomic characters such as chromosomes has suggested considerable evolutionary divergence between taxa that display great pelage and cranial similarity; the finding of diploid numbers ranging from 2n36-46 in subspecies of S. townsendi and 2n34-36 in subspecies of S. richardsoni are examples of these contrasting, lines of evidence (Nadler, 1966). To provide further insight into some systematic problems in Spermophilus the serum proteins of S. undulatus, S. beldingi and S. colundn'anus were examined with 487
488
CHARLES F. NADLER
starch-gel electrophoresis (Nadler & Hughes, 1966). These species had generally similar protein patterns which confirmed a close relationship between them, although certain features suggested a closer affinity between S. undu[atus and S. columbianus. Polymorphism of the transferrins and several unidentified proteins were observed in S. undulatu~ and these fractions, when considered as population markers, suggested a possible divergence between sub-Arctic and Arctic populations. This initial, preliminary study demonstrated the potential value of certain protein fractions, especially transferrin, as taxonomic markers among specific and infraspecific categories in Spermophil~. T h e present investigation describes serum protein and transferrin patterns obtained by two-dimensional and conventional starch-gel electrophoresis from multiple population samples of S. townsendi, S. richardsoni and S. armatus. Additional samples of S. undulatus, S. behtingi and S. columbian~ are analyzed and compared with previous data (Nadler & Hughes, 1966). T h e proteins are discussed in relation to the present classification of ground squirrels (Hall & Kelson, 1959). MATERIALS AND METHODS Serum was analyzed from the following animals: Spermophilus townsendi townsendi (Bachman). Washington: (south of the Yakima River) Benton County, 7 miles W. of Prosser, 2 males and 3 females; 3 miles S. of Prosser, 2 females; Prosser, 3 males and 4 females; 15 miles E. of Prosser, 1 male and 1 female; 18 miles E. of Prosser, 3 females. Total, 19. SpermophiIus townsendi nancyae (Nadler, 1968). Washington: Benton County (north of the Yakima River), Richland, 22 males and 16 females; 3 miles N. of Richland, 3 females; 5 miles N. of Richland at S. boundary of Hanford Reservation, 3 males and 2 females; N. Yakima River near Prosser, 1 male and 3 females; 10-12 miles E. of Prosser, 3 males and and 1 female. Yakima County, Grandview, 1 male. Total, 55. Spermophilus townsendi canus (Merriam). Oregon: Hamey County, Narrows, 1 male and 2 females; Hines, 1 female. Total, 4. Spermophilus townsendi vigilis (Merriam). Oregon: Malheur County, 1½ miles S.E. of Vale, 4 males and 4 females; 5 miles W. of Ontario, 3 males and 2 females. Total, 13. Spermophilus townsendi moUis (Kennicott). Idaho: Cassia County, Burley, 2 males and 13 females. Total, 15. Spermophilus townsendi idahoensis (Merriam). Idaho: Elmore County, 4 miles E. of Mountain Home on Highway 30, 4 males and 5 females. Total, 9. Spermophilus beldingi oregonus (Merriam). Oregon: Hamey County, Bums, "4males and 4 females. Total, 8. Sperrnophilus columbianus columbianus (Ord). Montana: Lubrecht Forest 30 miles N.E. of Missoula, 3 females. Total, 3. Sperrnophilus armatus (Kennicott). Utah: Logan Canyon 20 miles N. of Logan, 2 males and 2 females. Total, 4. Spermophilus richardsoni richardsoni (Sabine). Montana: Gallatin County, Belgrade, 6 males and 1 female; Bozeman, 1 male and 7 females. North Dakota: Cass County, Mapleton, 1 male and 5 females. Total, 21. Spermophilus richardsoni elegans (Kermicott). Colorado: Larimer County, Cherokee Park 45 miles N.W. of Fort Collins, 7 males and 6 females. Total, 13. Spermophilus richardsoni aureus (Davis). Idaho: Clark County, 10 miles N.W. of Dubois, 10 males and 5 females. Total, 15. Spermophilus undulatus ablusus (Osgood). Alaska: Nome, 6 males and 4 females; 10 miles S.E. of Anchorage, 1 male and 4 females; Teklanika River Camp Ground at Mt.
SERUM PROTEINS AND TRA/qSFERRINS OF GROUND SQUIRREL SPERMOPHILUS
489
McKinley National Park, 3 females; 5 miles W. of Paxson Lake on the Denali Highway, 1 male and 2 females. Total, 21. Sperraophilus undulatus lyratus (Hall & Gilmore). Alaska: St. Lawrence Island, Gambell, 4 males and 6 females. Total, 10. Sperraophilus undulatus plesius (Osgood). Alaska: Chisana, 14 males and 22 females. Canada: British Columbia, 10 miles S. of junction of the Turnagain and Dall Rivers (Cassiar Mrs.), 3 males and 12 females; 5 miles W. of previous locality, 2 females. Total, 54. Blood was drawn from the heart with sterile syringes and the serum was frozen at - 2 0 to - 6 0 ° C without adding preservatives or antibiotics. Storage of frozen sera for periods up to 12 months did not alter the results. Serum proteins were analyzed by horizontal, two-dimensional, starch-gel electrophoresis (Poulik & Smithies, 1958) using a Trisdiscontinuous buffer (Poulik, 1957). The proteins were stained with Nigrosin (Nadler & Hughes, 1966). Two or three separations of each serum specimen were performed before recording the protein pattern of an individual animal as a scale indian ink drawing (Fig. 1).
B Tf
~
~ . ~ C
FIG. 1. A protein pattern from S. columbianus illustrating fractions of taxonomic significance. The labeled fractions correspond with the following characters: albumin (Character I), Fraction Group A (II), Fractions B and C (IV and V), transferrin or T f (III), Fraction D (VI), Fraction E (VII) and Fraction F (VIII). There are three arc fractions (IX) in this species. Fractions staining with the intensity of albumin or transferrin were drawn in black, those of intermediate staining intensity were stippled while the faintest fractions were drawn with open lines. The fractions with fastest and slowest mobilities were labeled as albumin and gamma globulin respectively. All other fractions except transferrin were arbitrarily given letter designations (A-F) as described previously (Nadler & Hughes, 1966). Transferrin (Tf) was identified by radioautography with Fe 5~. Interspecific and infraspecific differences in protein patterns were compared and confirmed by simultaneously running sera in a horizontal, one-dimensional separation; in the case of transferrin, differences in the mobilities and number of transferrin fractions were checked by repeated, simultaneous one-dimensional separations utilizing radioautography. RESULTS T h e influence of physiological or e n v i r o n m e n t a l factors on the s e r u m proteins was evaluated w h e r e v e r possible. A m o n g specimens o f S. townsendi, animals r a n g i n g in age f r o m several weeks to 1½ years had qualitatively similar p a t t e r n s ; no differences were attributable to sex, diet or the captive vs. wild state. Sera
490
CHARLES F. NADLSR
from S. undulatus and S. richardsoni were also uninfluenced by age or diet, the latter consisting of food from the natural environment or laboratory rat chow. Pregnant animals or those in hibernation were not examined. It is therefore concluded that the protein fractions discussed in this study are probably genetically controlled and of potential taxonomic value. The serum proteins of Spermophilus townsendi, S. richardsoni, S. armatus, S. undulatus, S. beldingi and S. columbianus display basically similar electrophoretic patterns (Figs. 1-18). There are, however, nine fractions or groups of fractions that exhibit qualitative variation at different taxonomic levels and these fractions, which were initially recognized in a limited study of S. beldingi, S. columbianus and S. undulatus (Nadler & Hughes, 1966), have taxonomic applications. Manifestations of the nine protein characters are described individually below and summarized for each taxon in Table 1. TABLE I~CoMPARISON OF PROTEIN CHARACTBI~
S . totonsendi
Character
townsouti
nancyae
?2
vigilis
P2
S. richardsoni
canus
mollis
idahoensis
richardsoni
elegans
aur~us
?2
3
4.
2
2
2
T+I (9[9)
T f 2-4 (2]21) Tf4 (19/21)
Tf 4 (9]13) Tf4-6 (4.]13)
Tf4 (15115)
I N u m b e r of albumins II Fraction Group A
?2
I I I Transferrin fractions
"If 1-3 (1119) Tf3 (18/19)
T f 1-3 (11/55) Tf3 (44/55)
T+3 (13/13)
I V Presence of Fraction B V Presence of Fraction C VI Presence of Fraction D V I I Presence of Fraction E V I I I Separation between fractions in Group F I X N u m b e r of arc fractions
+
-I-
+
+
+
+
+
+
-I-
+
+
_+
+
+
+
+
+
_+
+
+
+
+
+
+
+
+
+
-t-
+
+
+
+
+
+
+
+
+
+
+
+
+
-t-
+
+
+
1
1
1
1
1
1
1
1
1
T f 1-3 Tf3 (13]15) (4-]4) T f 3 (2115)
* S. u. plesius: Chisana Alaska. t S. u. p/edus: British Columbia, Canada. S. u. ablusus: Nome, Mt. McKinley and Anchorage, Alaska. § S. u. ablusus: Paxson Lake, Alaska. I] S. u. lyratus: St. Lawrence Island. ¶ S . u. kennicotti reported previously (Nadler & Hughes, 1966).
SERUM PROTEINS AND TRANSFERRINS OF GROUND SQUIRREL S P E R M O P H I L U S
491
Character I refers to the electrophoretic characteristics of the larger major albumin fraction and the smaller fraction migrating slightly slower than the large fraction. T h e latter fraction, although not identified chemically or immunologieally as albumin, is probably a second albumin and it is prominent in all species except S. undulatus where it is small and insignificant. In Table 1 S. undulatus is therefore listed as having one albumin (Figs. 13-19). S. beldingi has a homogeneous major fraction and the second, smaller probable albumin (Fig. 2). In S. richardsoni the subspecies richardsoni (Fig. 3), elegans (Fig. 4) and aureus (Fig. 5) are electrophoretically indistinguishable and have two large and widely separated fractions that are both presumably albumins. S. armatus (Fig. 6) also has two albumins that are less widely separated than those of S. richardsoni. A notched major albumin fraction, suggesting heterogeneity, plus a small minor fraction are found in S. colundn'anus (Fig. 1). Various albumin patterns are seen in subspecies of OF SOME SPECIES OF
Spermophilus S. undulatus
Sub-Arctic S. beldingi S. armatus S. columbianus ?2
?2
?2
Tf 3
Tf 3 ( - )
(8•8)
(4/4)
Tf 3 (3/3)
plesius*
plesiusf ablusus~ ablusus§ lyratus[I kennicotti¶
1
Tf 5 (14/36) Tf 5-7
1
1
Tf 7
Tf 7
(17117)
(18/18)
(16136)
_+
+
+
+
+
+
+
+
+
Arctic
[Tf 7 (6/36)] +_
1
1
'If 5-7 (1/3)
Tf 7
Tf 7 (2•3)
(10/10)
1
Tf 6 (28/36) T f 6-7 (8/36)
+
+
_+
+_
_+
+
+
+
+
+
+
+
+
+
+
+
+
--
+
+
.
.
.
.
.
.
+
+
+
.
.
.
.
.
.
1
1
2-3
1
1
1
1
1
1
492
CRAat~ F. NADt.ma
S. townsendi; townsen&" (Fig. 7), nancyae (Fig. 8), canus (Fig. 9) and vigilis (Fig. 10) have a single, large homogeneous albumin with the second smaller fraction migrating closely behind it on both paper and starch-gel. In striking contrast, moles (Fig. 11) and idahoens~ (Fig. 12) exhibit subdivision of albumin into three and four fractions respectively, features that permit differentiation of these taxa from any others. Character 11 refers to the number and configuration of fractions comprising Group A which migrates slightly slower than albumin. In all populations of
~ 0 e
A ~
B Tf
Q
aA B
Tf
Fzo. 2. A protein pattern from ,S. beldingi oregonus. A single Tf 3 fraction is present
FIO. 3. A protein pattern from S. richardsoni richardsoni with a single Tf 4 band. There
and a prominent gap separates the fractions comprising Group F.
are two large albumin fractions.
~ A
Tf m
C
..4~4
Fxo. 4. A protein pattern from S. richardsoni elegans. Except for the double Tf 4--6 bands the pattern is similar to ,S. r. richardsoni (Fig. 3) and S. r. aureus (Fig. 5) where two albumins are also observed.
Fzc. 5. A protein pattern from ,S. richardsoni aureus. A single T f 4 fraction is present. Fraction B migrates within the are fraction, a finding also seen in S. armatus (Fig. 6).
SERUM P R O T E I N S AND TRANSFF_.RRINS OF G R O U N D S Q U I R R E L 8PERMOPHILUS
493
S. townsendi (Figs. 7-12), S. richardsoni (Figs. 3-5), S. armatus (Fig. 6) and S. beldingi (Fig. 2) there are three fractions occupying positions, with reference to a
clock face, of 9, 12 and 3 o'clock. A second pattern containing three fractions located at 6, 9 and 12 o'clock is found in S. columln'anus (Fig. 1). Group A of S. undulatus (Figs. 13-18) populations has four fractions located at 12, 3, 6 and 9 o'clock. Character 111 consists of the number of transferrins and their comparative mobilities. Among the taxa analyzed in this study there are seven and probably eight electrophoretically distinct transferrin fractions. Each animal has one or
Q B
/
FIo. 6. A protein pattern from S. armatus illustrating two albumins migrating nearly together, Fraction B migrating within the arc fraction and a single transferrin, Tf 3(-).
•- O "
....
FIG. 7. A protein pattern from S. townsendi toumsendi. There is close similarity to the pattern of S. beldingi (Fig. 2) and both have a "If 3 of identical mobility. Two albumins migrating nearly together are present.
A
C3
y Fro. 8. A protein pattern from S. toumsendi nancyae illustrating double transferrins, Tf 1 and 3. Two albumins with nearly similar mobillties are present.
,7 Fro. 9. A protein pattern from S. toumsendi canus. This pattern is similar to the patterns of tourasendi (Fig. 7) axedvigilis (Fig. 10). A single Tf 3 is present.
494
CHARLESF. N~mL~n
two transferrins designated T f 1 through 7 in order of fastest to slowest mobility (Figs. 19 and 20). S. tom~sendi is characterized by T f ' s 1 and 3 which appear as single T f 1 or T f 3 patterns or a double-banded T f 1-3 configuration. T h e idahoensis population has a single-banded T f 1 pattern in all nine specimens; a single T f 3 band is observed in all four canus sera, and all thirteen specimens of vigilis. Both T f 1
0A
o
di~ ° ~
A
%_'V
o
m
/
FIG. 10. A protein pattern from S. toumsendi vigilis. A single T f 3 is present.
FIG. 11. A protein pattern from S. toumsendi moles. Three albumin sub-fractions characterize raollis. A double T f 1-3 pattern is present.
G
0A
~
A
Tfdi~
Fro. 12. A protein pattern from S. toumsendi idahoensis illustrating the albumin with four sub-fractions and the single transferrin, T f 1, band that characterize this subspecies.
FIG. 13. A protein pattern from an Arctic population of S. undulatus kennicotti studied previously (Nadler & Hughes, 1966). It is included for comparison with sub-Arctic populations of S. undulatus. Fraction Group A is characteristic of S. undulatus. The absence of Fraction D differentiates Arctic from sub-Arctic undulatus. A double T f 6-7 pattern is present. The fractior~ comprising Group F are continuous.
SERUM PROTEINS AND TRANSFERRINS OF GROUND SQUIRREL S P E R M O P H I L U S
495
and 3 are found in populations of townsendi, nancyae and mollis: a single T f 3 band is identified in eighteen of nineteen townsendi sera whereas one individual has a two-banded T f 1-3 pattern; nancyae exhibits T f 1-3 bands in eleven sera of the sample and a T f 3 band in the other forty-four animals; thirteen of the fifteen
. m ~=~Qo A
B
/-
I)
D F
FXGI14. A protein pattern from S. undulatus lyratus with single T f 7 pattern and a prominent Fraction D that are typical of sub-Arctic undulatus.
FzG. 15. A protein pattern from S. undulatus ablusus (Nome, Alaska). Patterns of ablusus from Anchorage and Mt. McKinley, Alaska are similar.
T f ...~.,~,
,-~'
"D
FzG. 16. A pattern from S. undulatus ablusus collected near Paxson Lake, Alaska that demonstrates T f 5-7 fractions.
FIG. 17. A pattern from S. undulatus plesius (Chisana, Alaska) exhibiting two transferrins (Tf 5 and 7), a moderately staining Fraction D and the absent gap in Fraction F.
mollis specimens show T f 1-3 bands while two animals have a single T f 3 pattern. T f 1 is randomly distributed among the multiple populations of tonmsendi and nancyae and is not confined to a single geographic sample. Both S. beldingi and S. columbianus display a single T f 3 pattern that is indistinguishable from the T f 3 seen in S. townsendi.
496
CHARLgSF. NADLER
A single transferrin, migrating slightly slower than the T f 3 of & beldingi and S. columbianus, is observed in all four sera of S. armatus and it is designated T f 3 ( - ). Analysis of a larger sample is desirable before clearly recognizing the transferrin of armatus as distinct from the other T f 3 bands.
B~'~ T ~
FIG. 18. A pattern from S. undulatus plesius (Chisana, Alaska) with a single Tf 7 fraction. Specimens of plesius from British Columbia, Canada display similar patterns. T f 4 characterizes all S. richardsoni populations and is the only transferrin seen in aureus. Four of thirteen specimens of elegans have a T f 6 band in addition to the T f 4 fraction found in all the specimens. All sera of richardsoni from Bozeman and Belgrade, Montana have a single T f 4 band whereas richardsoni from North Dakota exhibit a T f 2 in addition to T f 4 in sera from two of six animals. Three transferrins, T f 5, 6 and 7, are found in populations of Spermophilus undulatus (Fig. 20) examined in this and a previous study (Nadler & Hughes, 1966). S. undulatus kennicotti from two populations in the Brooks Range of Arctic Alaska previously showed a T f 6 fraction in all thirty-six specimens; in one population four of the fourteen animals had a second transferrin, T f 7, in addition to T f 6 (Figs. 13 and 20). The T f 6 of S. undulatus cannot be differentiated from T f 6 of S. richardsoni elegam by electrophoresis. Specimens of S. undulatus collected in sub-Arctic Alaska display single T f 5 and T f 7 patterns or double bands composed of both T f 5 and 7. Populations from St. Lawrence Island (lyratus) (Fig. 14), Nome (ablusus) (Fig. 15), Anchorage (ablusus) and Mt. McKinley National Park (ablusus) are indistinguishable and characterized by a single T f 7 band. Of three animals collected near Paxson Lake, which is in an area intermediate between the distribution of the subspecies ablusus and plesius, two have a single T f 7 and one animal has a T f 5 and 7 pattern (Fig. 16). Among the thirty-six specimens of S. undulatus plesius from Chisana, Alaska there are three different types of pattern (Fig. 20); fourteen have a single T f 5,
FIc. 19. Transferrins of the subgenus Spermophilus. (1) S. undulatus plesius (Tf 5); (2) S. richardsoni elegans (Tf4); (3) S. richardsoni richardsoni (Tf4); (4) S. armatus (Tf3 ( - ) ) ; (5) S. beldingi (Tf 3); (6) S. richardsoni richardsoni (Tf 2 4 ) and (7) S. townsendi mollis (Tf 1-3). Fe 59 radioautography was used to identify the transferrins. Note the widely separated albumins in S. richardsoni (2,3,6), the less widely separated albumins in S. beldingi (5), S. townsendi (7) and S. armatus (4). The albumin of S. undulatus appears homogeneous.
FIG. 20. Transferrins of S. undulatus. (1) S. undulatus ablusus (Tf7); (2) S. undulatus kennicotti (Tf 6-7); (3) S. undulatus kennicotti (Tf 6); (4) S. undulatus plesius (Tf 5-7) and (5) S. undulatus plesius (Tf 5).
SERUMPROTEINSANDTK~SVelmXNSOF ORotrsD SqUIl~a~T-SPERMOPHILUS
497
sixteen have a two-band T f 5-7 pattern (Fig. 17) and six specimens have a single T f 7 (Fig. 18). The T f 5 bands from sera obtained at Paxson Lake and Chisana are electrophoretically similar and the occurrence of this fraction in populations 125 miles apart suggests that T f 5 is not merely a local transferrin mutation. All seventeen S. undulatus plesius from two localities in British Columbia have a single T f 7 fraction that is identical to T f 7 of other sub-Arctic populations. The three different transferrin patterns (Tf 5, T f 5-7 and T f 7) identified in S. undulatus from Chisana, Alaska are of interest because they were derived from a single, continuous population distributed over an area 300 by 800 yards. In this apparently interbreeding natural population it is postulated that animals with single T f 5 or 7 fractions represent homozygous expression of each allele while the double T f 5-7 pattern is a heterozygous manifestation of two alleles, T f 5 and T f 7, occurring at a single locus. Although evidence of this sort is lacking in the other species it seems likely that their transferrins are under similar genetic control and it is also possible that all the molecular forms of transferrin in Spermophilus are due to allelic substitutions as a single locus. This latter possibility cannot be established without recourse to interspecific hybridization, a feat not yet achieved in Spermophilus. Characters I V and V consist of the presence or absence of Fractions B and C respectively. Their occurrence is highly variable and they may appear together or one or both may be randomly absent in S. undulatus (Figs. 13-18), S. tonmsendi (Figs. 7-12) and S. richardsoni (Figs. 3-5). Both Fractions B and C were observed in all sera of S. armatus (Fig. 6) and Fraction B always migrated within the arc fraction, a feature sometimes observed in S. richardsoni (Fig. 5). In S. beldingi (Fig. 2) Fraction B was inconsistently present whereas Fraction C was always found. All three S. columbianus (Fig. 1) had both Fractions B and C which substantiates a similar observation made on specimens from Idaho (Nadler & Hughes, 1966). The highly variable presence of these fractions plus a lack of knowledge regarding their structure or function make them of uncertain taxonomic value. Character V I is represented by a darkly staining Fraction, D, that migrates faster than gamma globulin on paper but slower on starch. Fraction D is present in all species. However, within S. undulatus Fraction D is absent in Arctic (Fig. 13) populations (Nadler & Hughes, 1966) but present in all sub-Arctic populations (Figs. 14-18). Character V I I consists of a small lightly or moderately staining fraction, Fraction E, which migrates slightly faster than Fraction D on both paper and starch-gel. Fraction E is consistently present in S. townsendi, S. richardsoni, S. armatus, S. beldingi and S. columbianus (Figs. 1-12); it is absent in all populations of S. undulatus (Figs. 13-18). Character V I I I refers to the configuration of the two fractions constituting Group F. A gap separating the two fractions, which is produced by differing migration rates on paper and starch-gel, is characteristic of S. townsendi, S. richardsoni, S. armatus, S. beldingi and S. columbianus (Figs. 1-12). The two fractions in Group F display no gap or separation in S. undulatus (Figs. 13-18).
498
CHAm.ESF. NADm
Character I X refers to the number of arc-like fractions. Three arc fractions are present in two S. columbianus specimens (Fig. 1) and two arcs in the third animal from Montana; all nineteen specimens reported from Idaho had three arcs (Nadler & Hughes, 1966). Never was more than one arc seen in S. totvnse~h, S. richardsoni, S. armatus, S. beldingi and S. undulatus. Certain protein characters alone or in combination are diagnostic for most species and some subspecies examined in this study (Table 1): S. columbianus is distinguished by Characters I, II and IX, S. richardsoni and S. arrnatus are identified by Characters I and III (Tf) and S. undulatus is differentiated by Characters II, III (Tf), VII and VIII. Within S. torvnsendi, the subspecies mollis and idahoer~is can each be identified by Character I and in part by the presence of T f 1 alone (idahoensis) or in combination with T f 3 (mollis); in contrast, to~nsendi, nancyae, canus and vigilis cannot be differentiated from S. beldingi unless T f 1 is present in addition to T f 3, which is the case in some tozvnsemdi and nancyae sera. S. bel&'ngi has the least unique protein pattern and shares some features with all species while lacking diagnostic fractions of its own. The subspecies of S. undulatus display no diagnostic features although when considered as a whole there is evidence for divergence between Arctic and sub-Arctic populations. Subspecies of S. richardsoni cannot be differentiated by their serum proteins. DISCUSSION A consideration of the validity and limitations of serum protein electrophoresis is necessary before the data from this study can be applied to an appraisal of systematic relationships. The suitability of electrophoretic methodology has been discussed by many investigators and a number of generally accepted conclusions are pertinent (Dessauer & Fox, 1964; Manwell & Kerst, 1966; Bianchi, 1967; Coates, 1967). Electrophoresis separates proteins according to net molecular charge and also by molecular size when certain supporting media such as starch or polyacrylamide gel are used. As a consequence, when homologous proteins from different species migrate with different mobilities in the same gel they may be considered to differ in their molecular structures (Bianchi, 1967). For this assumption to be valid, the proteins should be shown to be homologous by demonstrating chemical or functional similarity or by utilizing breeding data to ascertain whether the proteins in question are controlled by alleles at a single locus (Hubby & Lewontin, 1966; Bianchi, 1967). Environmental and physiological factors that can effect the proteins must also be excluded (Dessauer & Fox, 1964; Coates, 1967). In the interpretation of data derived from homologous protein comparisons, electrophoretically dissimilar fractions are recognized as having greater taxonomic significance than electrophoretically similar proteins because the latter may still differ structurally; a good example is the occurrence of structurally different hemoglobin D molecules with the same electrophoretic mobility (Lehmann & Huntsman, 1966). Most recent studies have considered these general principles and based taxonomic decisions on a comparison of presumably homologous specific proteins including hemoglobins, transferrins and enzymes from tissues
SERUM PROTEINS AND TRANSFERRINS OF GROUND SQUIRREL S P E I ~ I O P H I L U S
499
and serum (Goodman, 1963; Manwell & Kerst, 1966; Gorman & Dessauer, 1966; Baker & Hanson, 1966). In this study, the transferrins of ground squirrels appear to be homologous proteins from the standpoint of chemical function and in addition breeding data from a natural population of S. u~tulat~ indicate that the three transferrin bands are controlled by two alleles at a single locus; a similar mechanism seems likely in the other species examined. Comparison of the total protein pattern, while less valid than comparisons of single, homologous proteins, can provide apparently reliable taxonomic data especially when closely related species are analyzed (Coates, 1967). A close taxonomic relationship increases the chance that electrophoretically similar proteins of undetermined structure or function are in reality homologous proteins (Bianchi, 1967). In a study of three salamander species belonging to the genus Taricha serum proteins were analyzed by disc electrophoresis and qualitative differences, independent of environmental or physiological influence, were shown to give a valid reflection of evolution at specific and infraspecific levels (Coates, 1967). Goodman's (1963) studies of primates by starch-gel electrophoresis also gave similar, apparently reliable data. In the present study of ground squirrel proteins, analysis of a presumably homologous protein transferrin is combined with a comparison of certain unidentified proteins to assess evolutionary relationships in the manner utilized by Coates (1967) and Goodman (1963). Several areas for systematic speculation exist in the subgenus Spermophilus, which have been highlighted by different lines of taxonomic evidence, and it is against this background that serum protein data may be compared. Combined evidence from gross morphology and pelage, ecology zoogeography, parasitology and cytogenetics was reviewed by Nadler (1966) and the following conclusions were reached: (1) the subgenus contains two species groups, the big-eared and short-eared squirrels; (2) within the big-eared group, S. richardsoni and S. armatus are closely related ecologically, morphologically and cytologically; (3)'the relationship of S. beldingi to the other big-eared squirrels is obscure and (4) S. columbianus and S. undulatus are probably closely related although gross morphological and pelage features, parasites and zoogeography indicate a closer affinity than is suggested by their dissimilar chromosomes. When proteins from six of the eight species of the subgenus Spermophilus are compared (Table 1) there is no suggestion of a species grouping except perhaps in the case of 8. beldingi and S. toumsendi and in that instance a mass of morphological (Howell, 1938), ecological (Durrant & Hansen, 1954) and chromosomal (Nadler, 1966) data fail to indicate a close relationship between these forms. The protein data could be interpreted as showing a basic similarity between all species with perhaps the beldingi-toumsendi similarity serving as an indication of common ancestral relationship among all species of the subgenus. There is no support for raising the big-eared and short-eared species groups to subgeneric rank. The proteins of S. armatus and S. richardsoni have diverged to a degree equal to or surpassing that displayed by their gross morphological features (Howell,
500
Caamam F. NADI.ma
1938) and chromosomes (Nadler, 1966) and it is not possible to confirm a close evolutionary relationship between these species by proteins alone. Among the six species examined, S. beldingi displays fewer unique protein fractions than any other taxon except for some S. townsendi subspecies. The occurrence of T f 3 in S. beldingi, S. columbianus and S. townsendi may indicate derivation from a common ancestral stock but these data do not lead to convincing new concepts concerning the taxonomic status of S. beldingi with respect to the other big-eared species. Evolutionary relationships of S. undulatus and S. columbianus were investigated by electrophoresis and a greater similarity was observed between the proteins of S. columbianus and sub-Arctic rather than Arctic populations of S. undulatus (Nadler & Hughes, 1966). Study of additional sub-Arctic sera from six new localities, plus examination of sera from three additional species of Spermophilus, now indicates a much greater degree of divergence between S. undulatus and S. columbianus; the two species differ with respect to Characters I, II, III (Tf), VII, VIII and IX. Previous analysis of the insular, sub-Arctic S. undulatus kodiacensis population indicated that Characters II and VIII were similar to those of S. columbianus (Nadler & Hughes, 1966), a finding now refuted by analysis of the other sub-Arctic populations. The apparent discrepancy may result from an initial analysis of a small, atypical population of S. undulatus or the insular form, kodiacensis, may be different from the mainland populations and truly share a closer protein relationship to S. columbianus. It is noteworthy, however, that the protein differences existing between S. undulatus and S. columbianus are now shown to be greater than those of any other two species in the subgenus. This is surprising because these species are quite similar in pelage and gross morphology (Davis, 1939), ectoparasite data suggest that sub-Arctic undulatus and columbianus arose from a single ancestral stock (Holland, 1958, 1963), and zoogeographic evidence suggests both species may have evolved following separation of the ancestral stock by continental glaciers during the Pleistocene (Rand, 1954). The dissimilarity of the proteins may now be combined with the previous observation of a dissimilarity of the chromosomes of S. undulatus (2n34) and S. columbianus (2n32) to provide an indication that evolution of gross morphological features is either proceeding at a slower, more conservative rate or that separation and divergence of the two species occurred at an earlier time than previously postulated. An examination of fleas (Siphonaptera) from Arctic and sub-Arctic populations of S. undulatus (Holland, 1958, 1963) initially raised the question of intraspecific divergence when it was noted that the former populations were parasitized by the same species of flea as Asian ground squirrels while sub-Arctic undulatus was parasitized by a different species of flea that was also found on ground squirrels from the western United States. Initial protein comparisons indicated that Arctic and sub-Arctic populations of undulatus differed with respect to Characters II, III and VI (Nadler & Hughes, 1966). New data from the larger sub-Arctic sample show only a difference in Character VI, manifested by an absence of this fraction in Arctic samples, and a difference in transferrins. Although study of transferrins
SERUM PROTEINS AND TRANSFERRINS OF GROUND SQUIRREL S P E R M O P H I L U S
501
from more Arctic populations is mandatory before drawing firm conclusions it is possible that Arctic populations are characterized by Tf 6 and sometimes Tf 7, whereas sub-Arctic undulatus is distinguishable by Tf 7 alone or in combination with Tf 5. Further investigation may strengthen the validity of the transferrins and Fraction D as population markers that now appear to correlate well with Holland's (1958, 1963) ectoparasite observations. If confirmatory evidence of a differentiation between Arctic and sub-Arctic populations can be obtained it will find practical application in clarifying questions pertaining to the zoogeography of Arctic mammals. Exploration of infraspecific relationships with protein electrophoresis is also valuable in S. richardsoni and S. townsendi. In S. richardsoni, the subspecies elegans, aureus, nevadensis and richardsoni are quite similar although the latter can be distinguished from elegans by differences in baculum morphology that led Burr (1960) to question whether elegans and richardsoni might not be distinct species. Chromosome analysis also revealed differences between richardsoni with 2n36 and the other subspecies which have thirty-four chromosomes (Nadler, 1966). Comparison of the proteins reveals indistinguishable patterns in elegans, aureus and richardsoni and all share a common Tf 4 band. The limited occurrence of Tf 2 and Tf 6 in populations of richardsoni and elegans respectively is most suggestive of transferrin polymorphism within local populations and is not of taxonomic significance. Thus, proteins fail to indicate a specific distinction for elegans and richardsoni and instead support the concept that these taxa are geographically isolated subspecies that exhibit differing degrees of evolutionary divergence with respect to chromosomes, baculum structure and proteins. Recently the chromosomes of S. townsendi were analyzed and striking variation was observed between subspecies (Nadler, 1968). The geographically contiguous taxa S. t. vigilis and S. t. canus had forty-six chromosomes, S. t. mollis and S. t. idahoensis, which are separated from each other by the Snake River barrier, had thirty-eight, and S. t. townsendi, which is isolated from the other forms by the Columbia River, had thirty-six chromosomes. A chromosomally homogeneous population, separated from to~onsendi by the Yakima River and having thirty-eight chromosomes, was recognized as a new subspecies, S. t. nancyae. The widely divergent chromosome patterns of populations classified as vigilis and canus, mollis and idahoensis, and townsendi led to speculation that each chromosomally distinctive group might represent a sibling species although morphologically identifiable intergradation between mollis (2n38) and vigilis (2n46) had been reported (Davis, 1939). When proteins from individual S. townsendi are compared the following taxonomic observations can be made: (1) a general similarity of patterns, with varying expression of a transferrin genome composed of Tf 1 and Tf 3, indicates that all populations are closely related to one another; (2) two populations, corresponding to mollis and idahoensis, each have distinctive albumins which supports the possibility raised by chromosomes that mollis (2n38) and vigilis (2n46) might be specifically distinct. It is conceivable that idahoensis is also a valid species but investigation
502
Cmucm F. N~mum
of natural intergradation with rnollis is prevented by the Snake River barrier; (3) sera from the geographically discontinuous Washington (townsendi, nancyae) and Oregon (canus, vigilis) populations are indistinguishable except for the occurrence of T f 1 in the former and its absence in the latter. Support for species status of these chromosomally different populations is lacking and (4) the greater frequency of "If 1 in nancyae compared to tomnsauti provides added evidence for the presence of a population (subspecific) divergence that was initially discovered by chromosome differences. A lack of major protein differences suggests these diverging taxa have not been isolated long enough to achieve species status. From the foregoing discussion it is apparent that protein differences occur at all taxonomic levels within the subgenus Spermophilus. At the species level where gross morphological differences are distinctive the electrophoretic contributions are least impressive. At the population and subspecies levels where morphological and cytological differences are less striking, proteins often display greater divergence. Here, proteins can serve as an effective means for clarifying evolutionary relationships and tracing the origin and composition of populations. Acknowledgements--The author wishes to thank Mr. C. F. Nadler, Jr., and Mrs. Nancy W. Nadler for their invaluable assistance during field work in Alaska and Canada. Additional specimens were obtained through the generous efforts of Dr. Robert L. Rausch, Dr. Francis H. Fay, Dr. Robert S. HotTman, Dr. Richard M. Hanaen, Dr. Kenneth S. Todd, Dr. Walter R. Quanstrom, Mr. Pat Magula, Mrs. Donna Grant, Mr. Russell Pengelly and Mr. Ken Small. I am especially indebted to Miss Kathleen E. Harris for her devoted technical assistance in the laboratory. This investigation was supported by National Science Foundation Grants GB-3251 and GB-S676X. REFERENCES BAI~R C. M. A. & HANSONH. C. (1966) Molecular genetics of avian proteins--VI. Evolutionary implication of blood proteins of eleven species of geese. Comp. Biochem. Physiol. 17, 997-1006. B I ~ e I U. (1967) Utility significance and limitations of electrophoresis, applied to particular taxonomic problems. Riv. Biol. N.S. 20, 237-24S. BtmT. W. H. (1960) Bacula of North American mammals. Misc. Pubis Mus. Zool. Univ. Mich. 113, 1-76. COAT~SM. (1967) A comparative study of the serum proteins of the species of Taricha and their hybrids. Evolution 21, 130-140. DAVISW. B. (1939) The Recent Mamnmls of Idaho, pp. 400. Caxton Press, Caldwell, Idaho. D~ssAtnm H. C. & Fox W. (1964) Electrophoresis in taxonomic studies illustrated by analys/s of blood proteins. In Taxonomic Biochemistry and Serology (Edited by LEo~rE C. A.), pp. 625-647. Ronald Press, New York. DUmO_NTS. D. & HANs~ R. M. (1954) Distribution patterns and phylogeny of some western ground squirrels. Syst. Zool. 3, 82-8S. GOODM~,~ M. (1963) Serological analysis of the systematica of recent Hominoids. Hum. Biol. 35, 377-436. GOnMAN G. C. & DmSAUm~H. C. (1966) The relationships of Anolis of the Roquet species group (Sauria: Iguanidae)--I. Electrophoretic comparison of blood proteins. Comp. Biochem. Physiol. 19, 845-853. HALL E. R. & KSLSONK. R. (1959) The Mammals of North America, Vol. 1, pp. 1-546. Ronald Press, New York.
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HOLLANDG. P. (1958) Distribution patterns of northern fleas (Siphonaptera). Proc. lOth int. Congr. Ent. 1, 645-658. HOLLAND G. P. (1963) Faunal affinities of the fleas (Siphonaptera) of Alaska: with an annotated list of species. In Pacific Basin Biogeography (Edited by GlU~SSXTTJ. L.), pp. 45-63. Bishop Museum Press, Honolulu. HOWELL A. H. (1938) Revision of the North American ground squirrels. N. Am. Fauna 56, 1-256. HI:BBYJ. L. & L~WONTXNR. C. (1966) A molecular approach to the study of genic heterozygosity in natural populations--I. The number of alleles at different loci in Drosophila pseudoobscura. Genetics 54, 577-594. LSHMANN H. & HUNTSMAN R. G. (1966) Man's Hemoglobins, pp. 1-331. Lippincott, Philadelphia. MANWXLL C. & IfamST K. V. (1966) Possibilities of biochemical taxonomy of bats using hemoglobin, lactate dehydrogenase, esterases and other proteins. Comp. Biochem. Physiol. 17, 741-754. NADLEa C. F. (1966) Chromosomes and systematics of American ground squirrels of the subgenus Spermophilus. ~. Mammal. 47, 579-596. NADI~R C. F. (1968) The chromosomes of Spermophilus townsendi (Rodentia: Sciuridae) and report of a new subspecies. Cytogenetics 7, 144-157. NADLER C. F. & Hucmm C. E. (1966) Serum protein electrophoresis in the taxonomy of some species of the ground squirrel subgenus Spermophilus. Comp. Biochem. Physiol. 18, 639-651. POULIK M. D. (1957) Starch-gel electrophoresis in a discontinuous system of buffers. Nature, Lond. 180, 1477-1479. POULIK M. D. & SMITHXESO. (1958) Comparison and combination of the starch-gel and filter-paper electrophoretic methods applied to human sera: two-dimensional electrophoresis. Biochem. y. 68, 636-643. RAND A. L. (1954)The ice age and mammal speciation in North America. Arctic 7, 31-35.