Comp. Bim'hem. Physiol. Vol. 72B, pp. I to 5. 1982 0305-0491/82/050001-05503.00/0 Printed in Great Britain. © 1982 Pergamon Press Lid

SERUM ELECTROPHORESIS AND SEA TURTLE CLASSIFICATION

WAYNE FRAIR Department of Biology, The King's College, Briarcliff, NY 10510, U.S.A.

(Received 13 August 1981)

Abstract--1. Electrophoresis and immunoelectrophoresis reveal that Caretta, Eretmochelys and Lepido- chelys share considerably similar blood serum proteins.

2. Proteins from Chelonia are more like those of Caretta and Lepidochelys than like Eretmochelys. 3. Dermochelys proteins are most distinct among living sea turtles and Dermochelys is classified either

in the same family or superfamily with other sea turtles.

I N T R O D U C T I O N

In past decades discussion regarding taxonomy of sea turtles has centered mostly on the enigmatic leather- back, Dermochelys, with its large size and lack of a hard shell. In more recent years the tendency has been to recognize a close affinity of the leatherback with other marine turtles (see Frair, 1979). In the past, elec- trophoretic studies involving sea turtles have been limited by the number of specimens or the number of species included (see Frair, 1969; Musquera et al., 1976; Smith et al., 1977; Michael, 1978; Bashtar, 1979). So this project was designed to add infor- mation gained from electrophoresis of a considerable number of specimens from all five types of sea turtles.

MATERIALS AND METHODS

Blood was obtained by aseptic cardiocentesis through the mid-ventral plastral seam. Serum was separated and usually frozen until used for electrophoresis which was per- formed according to basic procedures from Helena Labor- atories. Runs were in 25 cm-wide cellulose acetate plate at constant 180 V for 30 min using Tris-ethylenediaminetetra- cetic acid buffer at pH 8.8 and ionic strength of 0.05. Strips were cleared in methanol, glacial acetic acid and polyethyl- ene glycol (70: 30:4).

Most reactants used in immunoelectrophoresis (except in anti-DE runs) were serum pools, the antiserums being from among those utilized in the work of Frair (1979). To ab- sorb an antiserum I mixed a small volume of the absorbent serum with it, allowed time for reaction, centrifuged and poured off the supernatant which was mixed with more absorbent and the process continued until no more precipi- tate was observed after centrifugation.

The number of specimens followed by straight-line cara- pace lengths in cm were: Caretta c. caretta, 36 (7-100); Chelonia m. mydas, 35 (4.7-120); Dermochelys coriacea, 13 (9-165); Eretmochelys i. imbricata, 26 (4.9-89); Lepidochelys kempi, 16 (21-62); Lepidochelys olivacea, 22 (4.7-73). Using serums from the above, 340 electrophoretic patterns were employed in a comparative study which also included an additional 49 patterns from pools (each of 2-8 specimens, averaging 4) composed of serum from the above and at least an additional 6 specimens of Caretta and one of Che- lonia. More than 400 other plates were used in immuno- electrophoresis and protein identification.

Turtles were obtained from many locations but mostly eastern United States, Mexico and Surinam and deposited

as indicated in Frair (1979). In addition, the following are preserved with The American Museum of Natural History (AMNH), University of Utah (UU) or my (WF) numbers-- Caretta c. caretta: AMNH 107110, 107111, 107112; WF 104; Chelonia m. raydas: AMNH 107114, 118638; WF 106; Dermochelys coriacea: AMNH 118648; UU 11794, 11795; Eretmochelys i. imbricata: AMNH 118642; Lepidochelys olivacea: AMNH 118644, 118645, 118646.

R E S U L T S

From the serum electrophoregrams pictured in Fig. 1 it is apparent that all sea turtles have some similar- ly-migrating lines; also they have relatively short pat- terns as compared with human (and compared with many other turtles as well (Frair, 1964)). Although some intraspecific variability occurred, it more often was quantitative (heaviness of lines) rather than quali- tative (position of lines). With the procedure utilized consistent differences between sexes were not ob- served.

The sea turtle genus with the fastest-moving anodal line (albumin) is Chelonia, this being followed closely by Dermochelys. Other sea turtles tend to have simi- larly-positioned slower-moving leading albumin, a component which is important taxonomically. Preal- bumin lines were seen with a few specimens, for instance, three Chelonia possessed leading anodal triplets.

Not only do Caretta, Eretmochelys and Lepido- chelys have similarly-positioned leading albumins but also their total patterns, including position of the wide cathodal line and central region, are more simi- lar to one another than to either Chelonia or Dermo- chelys. The cathodal line of Chelonia is considerably more anodal than the cathodal line of the other hard- shelled specimens.

The Lepidochelys kempi and L. olivacea patterns are similar, but among sea turtles L. kempi was the most variable electrophoretically; whereas L. olivacea is more like the other sea turtles, particularly Caretta and Eretmochelys. L. olivacea characteristically has two close anodal lines (not obvious in Fig. 1), but they are not as clearly separated as the two heavy anodal lines of Chelonia.

.l~.J,, 72 l l~ A 1

WAYNE FRAIR

Caretta ci caretta

Chelonia m. mydas

Dermochelys cori acea

Eretmochelys i . imbricata

Lepidochelys kempi

Lepidochelys olivacea

Human

Terrapene c, carolina

Fig. 1. Photograph of cellulose acetate plate electrophore- grams of sea turtle serums.

Chelonia m. mydas

Caretta c. caretta

Lepidochelys olivacea

Eretmochelys i . imbricata

Dermochelys coriacea

Five L. olivacea hatchlings were dug from sand 53 days after the eggs were laid and they were bled 4 days later before being offered food. Their electro- phoretic patterns lacked the wide cathodal band seen for mature males and females.

Some preliminary tests have been run to identify proteins within the electrophoretic patterns studied here. lncluded have been lactic dehydrogenase enzymes, glycoproteins including haptoglobins and lipoproteins. I have seen a strong and similarly- migrating glycoprotein line especially in Che[onia, Oermochelys and Terrapene, whereas this line for Che- lydra is slightly anodal of them. Additional lines of these four and other turtles also show the presence of carbohydrates. Three lipoprotein lines have been identified especially for Caretta, Chelonia, Chelydra, Eretmochelys and Terrapene.

With immunoelectrophoretic strips each arc of a pattern was given a numerical value after careful vis- ual observation (with magnifying lens) of its width, density and length. A sum was obtained for values of all arcs in each pattern. For instance, sums for pat- terns of a single run as showed in Fig. 2 ranged from 34 for Chelonia to 12 for Terrapene. The sums for all the patterns of the reference antigens (Chelonia in Fig. 2) were added and this grand total divided by the grand total for each of the turtles. This procedure resulted in a final value of unity for the reference organism, slightly above unity for organisms tending to have weaker patterns, and the highest value for the organism with weakest patterns.

Also each pattern in a run was given a rank value, Number 1 for the heaviest (typically the reference antigen's pattern) to 7 for the weakest pattern. All values for a species were added to obtain a sum for that species. Each sum was divided by the sum for the

Chelydra s. serpentina

Terrapene c. caro l ina

Fig. 2. Immunoelectrophoresis employing an anti-('hehmia pool applied at upper edge of strip after electrophoresis

of serum from listed organisms.

reference organism, thus producing a series of final values with unity for the reference organism and higher values for organisms represented by weaker patterns. For each species the final value for ranking was averaged with the final value from width-density- length calculations to produce the numbers given in Table 1. With values obtained after absorptions the larger drops (and thus greater elevations of Table 1 values) are indicative of greater similarity among serum proteins of forms being considered.

Results from immunoelectrophoresis (Table 1) show Dermochelys to be the most diverse of sea turtles and Chelonia not closest to Eretmochelys as popularly believed. In many runs Caretta tested somewhat more like Chelonia than did Lepidochelys which usually was close, however. Also Dermochelys absorption did not indicate closest similarity of Dermochelys to Lepido- chelys. Both Caretta and Lepidochelys olit, acea gener- ally appeared more similar to Chelonia than did Eret- mochelys. Although Terrapene reacted well with anti- Chelonia, in many of the runs heavier arcs were seen for Chelydra (see Fig. 2). Both of the testudinids, Che- lydra and Terrapene, reacted similarly. My evalu- ations using immunoelectrophoresis I consider only rough approximations because of the difficulties attending efforts to quantify these reactions (see Wil- liams & Chase, 19713.

Table 1. Evaluation of immunoelectrophoretic strips

Anti Absorbent No. Runs CA* CH DE ER LE CS TC

CH 16 t.76+ 1.00 3.37 2.70 2.39 3.49 3.72++ CH CS 10 2.30 1.00 3.66 2.65 2 . 9 7 19.39§ 6.87 CH DE 6 2.77 1.00 5 .41" 3 .17 2.08 4.05 3.87 DE 3 2.28¶ 1.24 1.00 2.26 3.03

* CA. Caretta c, caretta: CH, Chelonia m. mydas; DE, Dermochelys coriacea; ER, Eret- mochelys i. imhricata: LE, Lepidochelys olivacea; CS, Chelydra s. serpentina; TC, Terrapene c. carolina,

t Values obtained by averaging the visual evaluation means with means for rank m each run.

Only 12 runs. Slight reaction in about half the runs.

¶ Patterns with CA, ER and LE were nearly identical.

Electrophoresis and sea turtle classification

Fig.

CA

ER

3. Dendrogram of suggested possible sea turtk relationships.

With antiserum to Chelonia absorbed using Che- lydra serum the quantity of precipitation for Terra- pene was 87Yo less, for Lepidochelys 72~o less, and Eretmochelys 64~o less. Dermochelys was similar with 68~o (losing less total precipitate than the above). Car- etta and Chelonia respectively with 68 and 55 showed the greatest drops in total precipitate (Chelonia start- ing highest, losing most and still retaining the heaviest pattern).

With antiserum to Chelonia absorbed using Dermo- chelys serum the reactions with Chelonia again showed loss of a greater total amount but a lower percentage of total precipitation (loss of 68Y0) than with any of the other forms so that it still retained the heaviest pattern. In terms of drop in quantity of pre- cipitate Caretta was second, but it had an 82~o drop (second only to Dermochelys). Others dropped 72-79Yo.

DISCUSSION

Cellulose acetate electrophoresis is a valuable taxo- nomic tool because it is a relatively simple procedure utilizing the whole complex of serum proteins and yielding a generalized pattern for each species. Simi- larities among sea turtle electrophoregrams generally confirm placement values determined using polyva- lent antiserum in immunoprecipitation tests (Frair, 1979).

Interestingly, Dermochelys and Chelonia have the fastest albumin among sea turtles--these being more like Terrapene, which also has a pattern more like certain sea turtles than does Chelydra (see Frair, 1972, Fig. 1). Even though this appears to be the case, con- siderable caution should be exercised about inferring close relationship between Terrapene and marine turtles on the basis of electrophoresis (immunoelec- trophoresis showing slightly greater reaction for Che- lydra than for Terrapene with anti-Chelonia).

Chelonia clearly differs from Eretmochelys, Caretta and Lepidochelys, but immunoprecipitation showed Chelonia most like Lepidochelys. The older belief that Eretmochelys is closest to Chelonia is not confirmed by electrophoresis, for Eretmochelys is closer to Lepi- dochelys (especially L. olivacea) and Caretta. Possibly Lepidochelys is closest to a sea turtle ancestry. The L. kempi could be more divergent than L. olivacea, a conclusion supported by Hendrickson (1980).

When immunoelectrophoretic results (Table 1) are compared with Frair (1979, Table 1) using anti-Der- mochelys it is seen that Chelonia has some likeness to Dermochelys, in the former case (only three tests) Che- Ionia reacting similarly with the leatherback and in the latter (considering the margin of error), Chelonia being grouped with the other hardshells in relation to the leatherback. With anti-Chelonia, results agree well except for the Caretta value in Frair (1979, Table 1, Column 2) which appears low. In reviewing that earlier data I find that all values are correct as listed but that two of the five runs employed pooled anti- Chelonia (as utilized in immunoelectrophoresis) and in both of these cases (and one of the other three involving a single antiserum) Caretta tested closer to Chelonia than did Dermochelys. In two of the five runs (which, incidentally, did not have as good curve values) Dermochelys was by a greater margin more like Chelonia than in the other three cases. For a concluding synthesis of all this evidence it seems best to leave Dermochelys outside the pale of hardshelled

A

Order--Testudines Batsch, 1788

Suborder--Cryptodira Cope, 1870

Superfamily--Chelonioidea Baur, 1893

Famlly--Cheloniidae Bonaparte, 1832

Su~famlly--Dermochelyinae Nopcsa, 1923

Genus--Der~oehelys Blainville, 1816

Subfamily--Cheloniinae Dollo, 1886

Tribe--Cheloniini Bonaparte, 1836

Genus--CheZonia Brongniart, 1800"

Tribe--Carettinl Zangerl & Turnbull, 1955

6enus--co~etta Rafinesque, 1814

Ere~moohel~8 Fitzinger, 1843

I~pidoohoZ~8 Fitzlnger, 1843

B

Order--Testudines Batsch, 1788

Suborder--Cryptodira Cope, 1870

$uperfamily--Chelonioidea Baur, ]893

Family--Cheloniidae Bonaparte, 1832

Subfamily--Cheloniinae Dollo, 1886

Genus--Che~onia Brongniart, 1800"

Subfamily--Carettinae Deraniyagala, 1953

Genus--Co~,etta Rafinesque, 1814

EretmocheZys Fitzinger, 1843

Lepidoohelys Fitzinger, 1843

Family--Dermochelyidae Baur, 1890

Genus--Dez~ooheZ~s Blainville, 1816

Fig. 4. Alternate classifications of living sea turtles.

* I prefer to accept the statement of Brongniart (1800), "Ce sont les tortues de mer", as a definition for Chelonia, this being in accord with personal communications from Harold F. Hirth and Roger Bour.

4 WAYNE FRA1R

sea turtles, while recognizing that it shares many pro- tein homologies with Chelonia and the other hard- shells.

In a report stressing some ecological strategies of sea turtles Hendrickson (1980) supported the fashion- able position that Chelonia is most similar to Eretmo- chelys; but he also discriminated between Chelonia and other sea turtles, including, for instance, the evi- dence that nesting female Chelonia dig deeper more distinct body pits than other sea turtles. Also he as well as others have said that Chelonia hatchlings singularly have light plastrons in contrast to darkly- pigmented carapaces. Al though Chelonia is individu- alized on the basis of many structural and behavioral attributes, I question that dis tr ibut ion of hatchling shield pigmentat ion should be included because I have observed at Bigisanti Beach in Surinam many Lepidochelys olivacea with dark carapaces and very light plastrons (see A M N H 118644-118646). Also, though less convincing, are observat ions of certain Caretta hatchlings with light plastrons.

Recently some biochemical studies utilizing isolated a lbumin and hemoglobin have yielded results consist- ent with conclusions of the present study that Dermo- chelys lies on the outskir ts but still within the precinct of other sea turtles (Chen et al., 1980; Chen & Mao, 1981). So on the basis of all available biochemical data, a tentative dendrogram may be constructed (Fig. 3).

In addi t ion to the biochemical results, including immunological values and electrophoretic patterns, there are impor tan t behavioral , morphological and physiological observat ions to be considered in erect- ing what would appear to be the most natural classifi- cation. Besides those discussed in this paper many others are considered in Frair (1979).

Based upon currently available information it appears that there are two best choices for classifi- cation of living sea turtles (Fig. 4), original proposers of most names being taken from Kuhn (1967). Wer- muth & Mertens (1977) was utilized for genus names except Brongniar t (1800) for Chelonia.

Recently Rhodin et al. (1981) discussed distinctness of Dermochelys based on its chondro-osseous mor- phology, but it appears that the main reason for dis- t inguishing Dermochelys at least in a separate family is allowance for fossil forms (see Gaffney, 1976; Zangerl, 1980). The Fig. 4B classification would not be contra- dicted by my data ; however, according to my per- sonal evaluation of phenetic evidence, the sea turtles consti tute a natural group which should be lumped together in a rank no higher than family. Even though Dermochelys has some dist inguishing features, the dif- ferences between it and other sea turtles are fewer than between certain members of the Testudinidae (Emydidae), for instance, emydine and batagurine tur- tles.

Acknowled,qements 1 am grateful to Paul J. Prol and Lora Sullivan for laboratory work, to Ruth J. Prol for her aid on the manuscript and to Chuck Crumly, Samuel B. McDowell and particularly George R. Zug for advice on

the manuscript. Curtis A. Williams and Stanley L. Arlton gave suggestions and Harold F. Hirth some literature aid. Photographic skill and material were contributed by David E. Carter and R. Max Maxwell.

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