MOLECULAR

PHYLOGENETICS

AND

Vol. 1, No. 3, September, pp.

EVOLUTION

193-201,

1992

Crocodilian Evolution: Insights from Immunological CARLA ANN HASS,* MICHAEL A. HowrwN,t

LLEWELLYN D. DENSMORE Ill,*

Data

AND LINDA R. MAXSON*

*Department of Biology and Institute of Molecular Evolutionary Genetics, 208 Mueller Lab, Pennsylvania State University, University Park, Pennsylvania 16802; flnstitute for Molecular Virology and Depaflment of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, Wisconsin 53706; and #Department of Biological Sciences, Texas Tech Universiv, Lubbock, Texas 79409 Received

May

The quantitative immunological technique of microcomplement fixation was used to examine serum albumin evolution among members of the order Crocodylia. The cross-reactivity of the albumin antisera and antigens employed in this study had been examined previously using the qualitative technique of immunodiffusion. The phylogenetic conclusions derived from these two data sets are highly congruent, including support of the families Alligatoridae and Crocodylidae, with the placement of Gavidis as the sister taxon of Tomistome. Both methods provide similar information on the relative amounts of amino acid sequence divergence between albumin molecules; however, the data obtained from microcomplement flxation comparisons are more discriminating than those derived from immunodiffusion. The estimated divergence times within the Crocodylia derived from the fossil record are examined in light of divergence times predicted by the microcomplement fixation-based albumin clock. The traditional phy logenetic placement of Gavialis outside the remaining extant crocodilians is inconsistent with all molecular data sets and we suggest that a careful reexamination of both the extant and the fossil morphological data is warranted. Q ~~JSZ Acrtdemio PWS, Inc.

INTRODUCTION

Immunological techniques have been employed to provide information on the phylogenetic relationships among living organisms since the beginning of this century (Nuttall, 1904). The two immunological techniques most widely used for assessing protein evolution and subsequently inferring phylogenetic relationships are immunodiffusion and microcomplement fixation (MC’F) and the most widely studied protein has been vertebrate serum albumin (Maxson and Maxson, 1990). In immunodiffusion tests, antiserum and antigen are placed in separate wells in a solid matrix (agar), through which they diffuse; a visible precipitate line forms where they meet. The position, intensity, and degree of spur formation between precipitate lines (obtained when an antiserum is run against two sepa-

8, 1992

rate antigens that each form a precipitate line at different positions) are estimated to obtain an antigenic distance (AD) between taxa (Goodman and Moore, 1971). These ADS are interpreted as a measure of the number of antigenic sites not shared by two taxa; the larger the spur, the larger the AD and the fewer the common antigenic sites. The scores obtained range from identical cross-reactivity (AD = 0) to no crossreaction (AD = 5). However, since albumin contains a minimum of 33 antigenic sites (Benjamin et al., 1984; Maxson and Maxson, 1986), immunodiffusion can provide only a qualitative estimate of the actual number of shared sites because this technique lacks the sensitivity to detect mutations in individual antigenic sites. In MC’F, antibody-antigen reactions are measured in very dilute solution, which allows only high-affinity antibodies to cross-react. Complement binds only to bound antibodies. In the homologous reaction (antiserum and antigen from the same organism), antibodies are available for all antigenic sites. In heterologous reactions (antiserum and antigen from different organisms), antibodies specific for an antigenic site that has been altered by amino acid replacements (AAR) in the heterologous species will no longer complex with that altered antigen and therefore complement cannot be bound (Maxson and Maxson, 1986). The relative amounts of bound and unbound complement are assayed by introducing a second antibody system (sheep red bl oo d ce11s and antibody to these cells) to the experiment. Any unbound complement from the experimental reactions will lyse these sensitized (antibody-coated) sheep red blood cells, releasing hemoglobin into solution. The amount of hemoglobin released is an inverse measure of the extent of the antibodyantigen reaction. The difference in antibody concentration between homologous and heterologous reactions allows the estimation of the number of AAR in albumins of the heterologous species (Maxson and Maxson, 1990). As the divergence time between organisms increases, the number of altered antigenic sites increases and fewer of the antibodies are able to bind to heterologous antigens. Therefore, the concentration of anti-

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194

HASS

serum in the complement fixation reaction must be increased in order to obtain the same number of antigen-antibody complexes and thus “fix” the same amount of complement. This difference in antiserum concentration required to fix equivalent complement in the homologous and heterologous reaction is a measure of the number of altered antigenic sites. MC’F reliably estimates AAR between molecules up to about 15% sequence divergence, approximately equal to 100 immunological distance units (ID) for the albumin molecule (Maxson and Maxson, 1986). Beyond this degree of sequence divergence, MC’F ceases to be quantitative but remains a qualitative measure of protein sequence similarity up to approximately 30% sequence divergence (200 ID). MC’F is a very sensitive technique and is able to detect individual amino acid replacements in a molecule (Cocks and Wilson, 1972). In order to test the congruence of these two techniques in the measurement of AAR in serum albumin between taxa, we used MC’F to study the same antisera and antigens that had been used in an immunodiffusion study of crocodilian albumins (Densmore, 1983). We then compared our reciprocal ID matrix to the reciprocal AD matrix. In addition to providing information about these two techniques, the data obtained from MC’F allow the investigation of relationships among extant crocodilians and the estimation of divergence times within the order Crocodylia as well as a comparison of these times to those derived from the fossil record of this group. MATERIAL

AND METHODS

To compare the results from immunodiffusion and MC’F experiments, we used a subset of samples (Appendix) from the animals that were examined by Densmore (1983). Each extant genus, with the exception of Osteolaemus, was represented by an antiserum. MC’F experiments were performed as described in Maxson and Maxson (1990). All results are reported as IDS, with one ID approximately equal to one amino acid substitution between the albumins compared (Benjamin et al., 1984; Maxson and Maxson, 1986; Prager and Wilson, 1992). The data were corrected to compensate for any significant nonrandom variation in antibody reactivity by the method of Cronin and Sarich (1975). Trees were constructed from averages of reciprocal data both by the modified distance Wagner method of Hutchinson and Maxson (1987) and by the neighbor-joining method of Saitou and Nei (1987) using both raw and corrected data. Midpoint rooting was employed because there is no appropriate taxon to use as an outgroup to the Crocodylia that is within the technical range of MC’F. In addition, one-way IDS were determined for taxa within the genera examined to give a more complete picture of albumin evolution within the Crocodylia.

ET

AL.

A Mantel

test (Mantel,

1967; as applied by Schnell to determine the correspondence between the albumin AD values and the ID values. In this test, two square difference matrices (the matrix of ADS and the matrix of IDS), with all possible reciprocal comparisons between antisera, are compared to determine statistically the degree of association between corresponding values. In the case of distances obtained through immunological techniques, the matrix may not be symmetric because the distance measured using the antiserum against species A to species B may be different than that from the antiserum against species B to species A. The Mantel test is an appropriate statistical test to use because the distance values reported between all possible pairs of taxa are not independent values and therefore statistical tests which assume independence are inappropriate (Schnell et al., 1985). The reciprocal matrix of raw ID values (Table 1) was compared to the adjusted reciprocal matrix of AD values presented by Densmore (1983, Table VIII). The relationship between AD and ID was calculated using a simple linear regression. This line was not adjusted to pass through zero because AD values of zero can be obtained for taxa which do have sequence differences when measured by MC’F. et al., 1985) was performed

RESULTS Microcomplement

Fixation

The seven antisera used in the MC’F analysis had an average titer of 1500 and an average slope of 350. While this slope is typical for albumin, the titers of the antisera, ranging from 700 to 2600, were lower, on average, than those previously reported in MC’F studies (Maxson and Maxson, 1990). Low antibody titers make it more difficult to measure higher IDS between taxa because the antibody concentration used becomes so great that residual binding by low-affinity antibodies can dominate the reaction. However, the experimental curves generated for all antisera used were the normal “bell-shaped” curve obtained in MC’F reactions and we did not see the type of curve that indicates immeasurable cross-reaction (Maxson and Maxson, 1986). The reciprocal matrix for the seven antisera examined is shown in Table 1. The standard deviation from reciprocity of this matrix (Maxson and Wilson, 1975) is 15.9%; the deviation from reciprocity for the corrected matrix is 10.5%. IDS from the antiserum against Crocodylus albumin to Caimun and Melarwsuchus could not be measured and therefore the one-way IDS from those taxa to Crocodylus were not included in the above calculations. The correction factor (CF) calculated for each antiserum is shown in Table 1. Again, the one-way ID values for Crocodylus were not used in

ALBUMIN

TABLE Reciprocal

ID Matrix

EVOLUTION

1

for Seven Species in the Order Crocodylia Antiserum

CF:

CM 1.57

ME 0.97

PA 1.10

AL 0.87

CR 0.92

GA 0.99

TO 0.86

Cuimun (CM) Melanosuchus (ME) Paleosuchus (PA) A&g&or (AL) Crocodylw (CR) Gavinlis (GA) Tomistoma (TO)

0 20 35 52 81 74 76

28 0 53 78 131 125 110

38 38 0 60 124 122 116

88 92 90 0 114 93 76

-= --a 138 127 0 90 76

122 116 114 83 80 0 5

128 117 116 81 78 12 0

Antigen

a No cross-reaction.

these calculations. As the CFs indicate, most of the antisera provide relatively good reciprocal estimates of ID (between a CF of 0.9 and 1.1). However, the antiserum to Caimun consistently underestimates ID; the antisera to Alligator and Tomistoma appear to slightly, but not consistently, overestimate ID. All trees generated, both by the modified distance Wagner method and by the neighbor-joining method using raw and corrected reciprocal data, gave the same topology (Fig. l), although the branch lengths varied slightly. Osteoluemus was included in this tree based upon one-way ID values. Two major groups are defined; one, corresponding to the Alligatoridae, includes the three caiman genera and Alligator, and the other, corresponding to the Crocodylidae and Gavialidae,

- - - - - Ostso/aemus I I Crocodylos

46

I

I

24

Melanosuchos

I

24

Paleosuchus

FIG. 1. Neighbor-joining tree generated from the raw ID data _ in the reciprocal matrix. The Prager and Wilson (1976) percentage standard error is 7.34 and the Fitch and Margoliash (1967) standard deviation is 10.7%. The topology shown is the same as that of the tree constructed from the corrected data (Prager and Wilson percentage standard error, 8.63; Fitch and Margoliash standard deviation, 13.5%). Osteolaemus was added after the tree was constructed; its placement was based upon its one-way cross reaction to Crocodylus.

IN CROCODILIANS

195

contains Crocodylus, Osteolaemus, and the gharials. Within each major group two separate lineages can also be identified. The three caiman genera cluster, with Caiman and Melunosuchus as sister taxa (2 ID = 24). Alligutor clusters distantly with this group at a mean ID of 77. Crocodylus and Osteolaemus cluster, with a one-way ID of 36. The gharials, Tomistomu and Guuialis, are the most closely related genera (2 ID = 8). They, in turn, cluster with the crocodile lineage (2 ID = 81). The IDS between the Alligutorlcaiman group and Crocodyluslgharial group average 108 units. However, the IDS between Alligator and the gharials average consistently lower (jz ID = 83) than those seen between the caimans/AlZigcztor and Crocodylus (3 ID = 119) or the caimans and gharials ($ ID = 111). This discrepancy in cross-reactivity was also noted in the immunodiffusion trefoil analysis (Densmore, 1983) and may be due to a change (reversal) in an antigenic site which makes the albumins of Alligutor and the gharials more similar. This suggestion is supported by the reciprocity of this reaction; the antiserum against Alligator and the two antisera against the gharials estimate the same degree of sequence divergence. The consistency of the other IDS in this data set strongly supports an Alligutorlcaiman lineage distinct from the crocodiles and the gharials. The table of one-way IDS provides additional information on crocodilian relationships (Table 2). The albumins of all the subspecies of Caiman crocodilus are indistinguishable from the antiserum to Caimun c. crocodilus. Caiman lutirostris is the most distant of the Cuimun species and is approximately equidistant from both Cu. crocodilus and Melanosuchus. Paleosuchus albumin is the most divergent of the caiman genera and the albumins of the two species, P. palpebrosus and P. trigonatus, are distinct from one another (ID = 15). Comparisons of all caiman representatives (reciprocal and one-way IDS) to Alligator show that these lineages are distant (2 ID = 78) from one another. However, the three IDS measured from the antiserum against Alligator to the caimans for which antisera were available are consistently higher (f ID = 93) than the reciprocal values (3 ID = 60). The two species of Alligator examined, mississippiensis and sinensis, appear very closely related, separated by only 5 ID. Within the genus Crocodylus, all the species examined have albumins that are distinct from that of Cr. pulustris, with IDS ranging from 3 to 20. IDS between the gharials and all the crocodile species are very consistent (2 ID = 79), suggesting a relatively constant rate of albumin evolution since these lineages diverged. One-way distances were also obtained from Tomistomu to all the caiman taxa available (except P. trigonatus). These IDS were also very consistent, although there is somewhat more variation as the limit of the quantitative resolving power of MC’F is approached.

196

HASS ET AL.

TABLE One-Way Immunological

Distances

2 to Additional

Crocodilian

Taxa

Antiserum Antigen

CM

ME

PA

AL

CR

GA

TO

0

29

28 31 31 25 21 0 53 42

38 37 35 36 44 38 0 15

88 -102

-a

122

--a 138

116 114

128 126 116 118 134 117 116

52

78

60

0 5

127

83

81

131

124

114

0 16 19 16 8 20 8 8 14 6 3 36

80 78 84 79 82 80

86

78 78 66 83 82 84 79 82 76 83 80 65

90 76

0 5

12 0

Caimans Caiman c. crocodilus Caiman c. apaporiensis Caiman c. fuscus Caiman c. yacare Caiman latirostris Melanosuchus niger Paleosuchus palpebrosus Paleosuchus trigonatus

2 0

1 18 20 35

95 101 92 90

Alligators Alligator Alligator

mississippiensis sinensis

49

Crocodiles Crocodylus Crocodylus Crocodylus Crocodylus Crocodylus Crocodylus Crocodylus Crocodylus Crocodylus Crocodylus Crocodylus Osteolaemus

81

palustris acutus cataphractus intermedius johnsoni moreletii niloticus n. mindorensis porosus rhombifer siamensis tetraspis

146

160

-a

74 76

125 110

122 116

s120

76 80

Gharials Gavialis gangeticus Tomistoma schlegelii

93 76

A blank indicates that the reaction was not performed. a No cross-reaction.

Note.

TABLE Mean Estimates

Caiman Melanosuchus

Paleosuchus Alligator Crocodylus

Gavialis Tomistoma

of Immunological

Distance between Measurements

3 Genera Using Both Reciprocal of ID

CM

ME

PA

AL

CR

(O-18)

26 of:4.8 (N = 6)

36.3 2 4.5 (N = 7) 44.3 t 7.8 (N = 3) (15)

17 k 24.6 (N = 5) 85 2 9.9 (N = 2) 75 f 21.2 (N = 2) (5)

119.2 f 20.3 (N = 6)

0

(3-20)

and One-Way GA

TO

114.4 k 16.5 (N = 16) 83.2 + 7.1 (N = 4) 81 -c 4.1 78.9 f (N = 9) uv = 0 8.5 f (N = 0

4.9 12) 5 2)

Note. The value given is the mean f one standard deviation and N is the number of ID comparisons used. Distances from the antiserum for Crocodylus to the caiman/Alligator lineage are averaged, while the antisera to the ghariala are grouped relative to the cairnan lineage and Alligator separately. Values for Osteolaemus are not included because it was not represented by an antiserum. Values on the diagonal show the range of ID within a genus for polytypic genera.

ALBUMIN

EVOLUTION

Relationship of ID to Trefoil Antigenic Distances The results of the trefoil analyses of crocodilian serum albumins were presented both as raw data and as adjusted ADS (Densmore, 1983), the latter derived from analysis using a set theory algorithm (Goodman and Moore, 1971). These AD values were further adjusted to account for differences in “strength” of the antisera, resulting in a table of corrected ADS (Table VIII in Densmore, 1983). These adjusted ADS were interpreted as estimates of relative difference between clusters of antigenic sites on the albumin molecules being compared. The ID matrix used is presented in Table 1; the two values that could not be obtained using the antiserum to Crocodylus were replaced by the respective reciprocal distances to form a complete reciprocal ID matrix for this analysis. The Mantel test found that the two reciprocal matrices (AD and ID) covary significantly at an alpha level of 0.01 (t = 4.001), indicating that there is a very strong correlation between the AD and ID values. Although it is not appropriate to use parametric statistics to determine the correlation of these two data sets, a least-squares regression line was calculated for AD and ID to provide predictive information. ID values above 100 were excluded from this calculation because ID values become increasingly qualitative beyond 100 ID (Maxson and Maxson, 1986). Figure 2 shows adjusted ADS and IDS plotted for all pairs of taxa examined with IDS less than 100 (ZV = 26). Again, the correlation between these two data sets was high (r = 0.78). The slope of the line is 16.8, indicating that one AD unit covers a wide range of ID and thus cannot provide

197

IN CROCODILIANS

data as discriminating as MC’F can provide, although general patterns of relationships should be congruent. DISCUSSION

Evolution of the Crocodylia Relationships among the Crocodylia. The phylogenetic inferences about the Crocodylia obtained using MC’F are highly congruent with conclusions from trefoil analyses (Densmore, 1983). The genera Caiman and Melanosuchus cluster and are the sister group of Paleosuchus. The caiman taxa in turn cluster with AZZigator. Within the crocodile lineage, all the species of Crocodylus examined are distinct immunologically from Cr. palustris. Earlier studies summarized by Densmore and Owen (1989) suggested that, at the molecular level, Cr. cataphractus is the most distinct of all species of Crocodylus. Our data indicate that both Cr. cataphractus and Cr. moreletti are quite distant from Cr. palustris. Current work examining the level of mitochondrial DNA sequence variation should provide the data necessary to clearly define phylogenetic relationships within this genus White and Densmore, unpublished data). Osteolaemus is the sister genus of Crocodylus, but is almost twice as distinct genetically from the antiserum to Cr. palustris as the most distant species of Crocodylus. Overall, our data on generic relationships are in complete agreement with other molecular analyses (Densmore and Owen, 1989; Densmore and White, 1991). The phylogenetic placement of Gavialis and Tomistoma has been the most contentious point of disagreement between molecular and morphological data bearing on crocodilian systematics. While most morphological analyses place Gavialis as the sister group 120to the remaining extant Crocodylia (Frey et al., 1989; Norell, 1989; Tarsitano et al., 1989), all molecular data sets indicate that the gharial genera are sister taxa (Densmore, 1983; Densmore and White, 1991; Densmore and Dessauer, 1984; Gatesy and Amato, 1992). aoThe trefoil immunodiffusion data for serum albumin (Densmore, 1983) could not distinguish between Gavialis and Tomistomu; we also have found these taxa to be very closely related (2 ID = 8). This level of molecular divergence is comparable to that measured between the two species of Alligator and less than that obtained between the two species of Paleosuchus or between the * 20dL most divergent species of Crocodylus. A Gatesy and Amato (1992) presented sequence data 0 I I I I I from the 12s rRNA mitochondrial gene for four taxa 5 0 1 2 3 4 (Gavialis, Tomistoma, Cr. rhombifer, and Cu. crocodiAD Zus). A comparison of 258 bp of this gene found Gavialis and Tomistoma to be the most similar, but no outgroup FIG. 2. Correlation between AD and ID. Values for both comparisons of a reciprocal pair are shown for all ID values less than 100 was included in this analysis nor were the data eval(N = 26). ID data are from this paper; AD data are from Densmore uated statistically. We have added the 12s rRNA (1983, Table VIII). The slope of the best-fit least-squares regression sequence from Alligator and three outgroup taxa line is 16.8. The product moment correlation coefficient for these two [a mammal, Procyon Zotor; a lizard, Ameiva auberi data sets is 0.78.

198

HASS

(Hedges et al., 1991); and a turtle, Sternotherus odoratus] to their data set. A bird sequence could not be included due to an apparently large deletion in all the bird 12s sequences that we have examined. These data were aligned by eye (Cabot and Beckenbach, 1989), highly variable regions were eliminated, and the resulting sequence (158 bp total length) was subjected to a neighbor-joining analysis with 2000 bootstrap replications (Hedges, 1992). The results of this analysis support a GavialislTomistoma sister group relationship in 83% of the bootstrap replicates and place Crocodylus as their sister taxon in 85% of the replicates (Hass and Maxson, unpublished data). In the face of such overwhelming molecular evidence for the sister group relationship and recent divergence of Gauialis and Tomistoma, why does the morphological evidence place Gauialis outside all living crocodilians? Buffetaut (1985), in a reinterpretation of the paleontological and morphological data, concluded that Gauialis is descended from Tomistoma-like ancestors. He hypothesized that many of the unique features of Gavialis have resulted from the pronounced elongation of that taxon’s jaws and a sit-and-wait foraging strategy. However, most other workers studying morphology have not followed this reinterpretation. Tarsitano et al. (1989) used morphological data to place Gauialis outside the other eusuchians and they state that this conflict between the morphological and molecular data could be due to the ability of “similar genotypes (to) give rise to rather different morphologies.” We know this to be true in other groups. Perhaps the most striking example is that of humans and great apes, which despite major morphological differences (reflected in their placement in two separate families) are over 98% similar at the molecular level (Wilson, 1976). This marked uncoupling of morphological and molecular evolution is not unique (Avise, 1990) and has been speculatively attributed to differences in expression of regulatory genes (Wilson, 1976; Avise, 1990) and/or heterochrony (Raff et al., 1991) among other mechanisms. A number of the morphological characters used to determine the placement of Gauialis are features of the skull. In particular, the braincase floor of adult crocodilians is verticalized (to varying degrees) in all species except Gavialis. However, as Tarsitano et al. (1989) pointed out, all juvenile eusuchian crocodilians have a flat ventral floor of the braincase (juvenile Gavialis have not been studied). It is possible that the morphology of Gavialis represents a paedomorphic condition that led to a misinterpretation of the extant and fossil data. Paedomorphosis is a common theme in evolution (Wake, 1966; Gould, 1977) and can lead to incorrect phylogenetic conclusions. Norell(1989) has investigated some morphological characters in this light but ontogenetic change in all characters that are key to the placement of Gavialis must be investigated in the additional crocodilian lineages.

ET AL.

Time

frame

for evolution

within

the Crocodylia.

One of the strengths of albumin immunological data has been its estimation of divergence times between taxa. Although the albumin clock is not a perfect chronometer, it can provide information which is often not otherwise available, particularly for groups with a poor fossil record. The crocodilians are exceptional among reptile lineages in that they do possess a relatively continuous fossil record throughout the Tertiary. However, the fossil morphological data are clouded, in general by the morphological conservatism of this group, and in particular by convergence and parallel evolution in skull morphology (Taplin and Grigg, 1989). Therefore, we have chosen to evaluate the major lineage divergences in this group by comparing the fossil evidence to divergence times predicted by a “standard” calibration of the rate of albumin evolution derived from diverse vertebrate groups (Maxson, 1992). This calibration, with approximately 100 ID accumulating in 60 million years, can be used to explore divergence times within the crocodilians. The approximate times of divergence between the major crocodilian lineages as predicted by the fossil record are shown in Fig. 3. If all these estimates were compatible with the estimates derived from the albumin clock, they would lie along the dashed line. There are two “classes” of discrepancies between divergence times predicted by the fossil record and those suggested by the albumin clock. The estimates falling to the left of the line indicate times of appearance in the fossil record that postdate the expected appearance based upon the albumin data. Points in this area do not conflict with the use of albumin immunological data as estimators of divergence; our data predict that older fossils should exist. However, the points to the right of this line indicate that the taxon appears in the fossil record before expected based upon the albumin data. The two points in this region (Fig. 3) represent fossils assigned to the two extant species of Alligator (Malone, 1979) and to the genus Gavialis (Taplin and Grigg, 1989). These fossils are interpreted to show the existence of these taxa much earlier than our molecular phylogeny predicts. Indeed, for both these taxa, the fossils suggest that these extant lineages date to the Oligocene or Eocene, rather than emerging in the Pliocene. If the molecular data are accurate, and the estimates of sequence divergence in the albumin molecule can be used to estimate divergence time, and most of the divergence times within the Crocodylia are consistent with these estimates, how do we explain these discrepancies? One possible explanation is that these fossils have been incorrectly assigned and do not represent extant lineages. This has occurred in other vertebrate groups. Perhaps the most well-known example is that of Ramapithecus [Sivapithecus (Groves, 1989)], a fossil taxon which, until recently, was unquestionably assigned to the hominid lineage and believed to repre-

ALBUMIN 140 :’ I I

120 I

2.

+s

:’ 0

:.,

:

: I

10

-li=r4

20

30 Time

40

1

I

60

60

of divergence

IN CROCODILIANS

199

instance, fossils assigned to Gaviulis may represent other lineages which were morphologically similar, perhaps due to paedomorphosis, or may represent a lineage that did not leave modern descendants. Additional fossil material and a better understanding of ontogenesis in extant taxa may shed new light on this conflict. However, the overwhelming concordance of all molecular data sets, showing a low level of divergence between Tomistomu and Gaviulis, strongly suggests that the placement of Gauialis outside the remaining extant eusuchians is incorrect. Correlation of Different Immunological Techniques

:

,.:. :.

o

EVOLUTION

1

I

I

1

70

60

SO

100

(mybp)

FIG. 3. Estimates of divergence time between crocodilian groups from the fossil record plotted against mean IDS between these groups (from Table 3). The numbered points represent the following divergences: (1) separation of the alligator (including caiman) and crocodile lineage in the late Cretaceous/early Tertiary (75-55 mybp); (2) separation of the GavialislTomistomn lineage from the crocodile lineage based upon the appearance of Tomistoma in the Eocene (35-56 mybp); (3) separation of Alligator from the caiman lineage in the Eocene; (4) separation of Gavialis and Tomistoma based upon the appearance of Gaviulis in the Oligocene (24-35 mybp) of Columbia; (5) divergence of the two extant Alligator species in the Upper Miocene or Lower Oligocene (30-20 mybp); (6) divergence of Paleosuehus from the other caiman genera. Times of divergence were derived from Steel (19731, Malone (1979), and Taplin and Grigg (1989).

sent the earliest known hominid, with fossils that were accurately dated to at least 14 mybp (Pilbeam, 1986). Such an ancient hominid was in direct conflict with immunological data (the first molecular evidence) dating the hominid-pongid divergence at approximately 3-5 million years ago (Sarich and Wilson, 1967). Present understanding of hominid phylogeny, a result of the reanalysis of fossils of Ramupithecus and new fossil finds, places Ramapithecus as a sister taxon to the orangutan and thus outside the group composed of the African apes and humans (Andrews and Cronin, 1982; Lipson and Pilbeam, 1982; Kappelman et al., 1991). Recent fossils bearing on the African ape-hominid divergence have not seriously challenged the original estimated divergence time based upon molecular data (Andrews, 1990; Conroy et al., 1992; but see De Bonis et al., 1990). The oldest unequivocal fossil Homo dates to 2.4 million years (Hill et al., 1992) and the oldest hominid fossil (Australopithecus afarensis) is placed at 3.7 to 4.4 million years of age (Fleagle et al., 1991). Likewise, substantial additional molecular data have not significantly changed the 3-5 million year estimate of the time of the pongid-hominid divergence (Gonzalez et al., 1990). We suggest that the contradictory information provided by the crocodilian fossils also may be due to improper taxonomic allocation. For

There have been some attempts to evaluate the degree of congruence between the data obtained from these two immunological methods and those obtained from other immunological techniques, including precipitin tests, radioimmunoassay, and ELISA (see Maxson and Maxson, 1990, for a description). Prager and Wilson (1971) reported a high correlation between MC’F and precipitin tests using the same antisera for bird egg white lysozyme, which is a relatively small protein (less than 150 amino acids). Percentage crossreactivity measured by ELISA was also found to be correlated with that MC’F data set (Hoffmann and Van Regenmortel, 1984). However, albumin, nearly four times larger than lysozyme (approximately 580 amino acids), has been the protein of choice for estimating phylogenetic relationships within the tetrapods (Maxson and Maxson, 1990; Maxson, 1992). Goodman and Moore (1971) compared one-way albumin distances between humans and diverse primate taxa and found a strong correlation of increasing ID measured by MC’F, quantitative precipitin values, and spur size measured by trefoil immunodiffusion. This was also true for their experiments using an antiserum to human transferrin. However, they used one-way distances, which are generally less accurate estimators of actual divergence than reciprocal measurements. Schwaner and Dessauer (1982) studied the evolution of another serum protein, transferrin, in natricine snakes using trefoil analyses and reported a strong correlation between that data and MC’F data from the same taxa (George and Dessauer, 1970; Mao and Dessauer, 1971; Gartside and Dessauer, 1977). They also calculated a linear regression for the distances measured by the two techniques and found that a wide range of ID (m = 14.4) corresponded to a single AD. The y-intercept of their line was lower (8.76 vs 22.7), reflecting the faster rate of evolution for transferrin. Their study further suggests that these two techniques provide congruent estimates of sequence divergence irrespective of the protein being investigated. However, in that study, as in the majority of studies, most of the distances were not measured reciprocally. Without two complete reciprocal matrices, the Mantel test cannot be performed and therefore the correlation cannot be determined on a sound statistical basis.

200

HASS ET AL.

This comparative analysis has shown that both MC’F and immunodiffusion studies using trefoil analysis provide congruent estimates of AAR between albumin molecules. However, the advantage of MC’F is that this method enables us to count AAR between molecules, thus (1) providing quantitative estimates of protein sequence divergence and (2) permitting estimation of divergence times of lineages with a poor and/ or incomplete fossil record.

Appl.

APPENDIX Taxa Examined in Both the Immunodiffusion Microcomplement Fixation Tests

and

All taxa were obtained from zoos or wildlife parks [see Densmore (1983) for more complete information]. Taxa against which antisera were raised are indicated by an asterisk. The LM number is the number in the Linda Maxson, Penn State University Frozen Tissue Collection; the HCD number is the corresponding designation in the Louisiana State University Museum Frozen Tissue Collection. *Alligator mississippiensis (LM 2424; HCD 2588); Alligator sine&s (LM 2436; HCD 4216); “Cairnan crocodilus crocodilus (LM 2429; HCD 2781); Caiman c. apaporiensis (LM 2442; HCD 3380); Caiman c. fuscus (LM 2441; HCD 2785); Caiman c. yacare (LM 2440; HCD 3367); Caimun Zatirostris (LM 2439; HCD 3417); “Melanosuchus niger (LM 2428; HCD 3369); “Paleosuthus palpebrosus (LM 2426; HCD 3149); Paleosuchus trigonatus (LM 2450; HCD 4222); Crocodylus acutus (LM 2446; HCD 3147); Crocodylus catuphractus (LM 2437; HCD 3362); Crocodylus intermedius (LM 2443; HCD 3416); Crocodylus johnsoni (LM 2447; HCD 3414); Crocodylus moreletii (LM 2435; HCD 3365); Crocodylus niloticus (LM 2451; HCD 3372); Crocodylus nouaguineae mindorensis (LM 2438; HCD 3381); *Crocodylus palustris palustris (LM 2430; HCD 3419); Crocodylus porosus (LM 2444; HCD 3370); Crocodylus rhombifer (LM 2448; HCD 3375); Crocodylus siamensis (LM 2445; HCD 3371); Osteolaemus tetraspis tetraspis (LM 2431; HCD 3376); “Tomistoma schlegelii (LM 2433; HCD 3361); “Gavialis gangeticus (LM 2449; HCD 4220). ACKNOWLEDGMENTS We thank Diana Weedman for laboratory assistance. R. D. Maxson offered helpful comments. This work was supported in part by the NSF Systematic Biology Program.

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