HEMOGLOBIN is the major oxygen-transport protein in vertebrates. Typically, it occurs as a tetramer consisting of two alpha subunits and two beta subunits (globins) attached to a heme (iron porphyrin), which is the oxygen binding site. Hemoglobin is one of the most widely examined proteins, and numerous studies have compared variation in molecular structure among families and orders of fish, amphibians, reptiles, birds, and mammals (Dessauer, 1970; Goodman, 1982). Nearly all electrophoretic studies of turtle hemoglobins report two major bands (a fast Hb A and a slow Hb D; Rucknagel and Braunitzer, 1988) and up to seven minor hemoglobin components. Although most work on turtles has compared the properties of hemoglobin in distantly related species, variation among closely related taxa has been virtually overlooked. Sullivan and Riggs (1967) presented the most comprehensive taxonomic analysis of turtle hemoglobin using starch-gel electrophoresis to define polymorphism among eight families. However, they drew relatively few systematic conclusions, perhaps because of small sample sizes and questionable resolution of some bands. Nevertheless, Sullivan later (1974) stated that electrophoretic patterns of turtle hemoglobin often reflect phylogenetic relationships. Lykakis (1974) conducted a very limited phylogenetic analysis of turtles based on hemoglobin variation among the families Testudinidae (three species), Bataguridae (one species), and Emydidae (one species).

In 1989, Seidel and Adkins reported on relationships of emydid turtles based on myoglobin polymorphism. The present analysis was designed to examine hemoglobin variation in the family Emydidae (sensu stricto, Shaffer et al., 1997). The emydids are aquatic or semiaquatic turtles native to the New World, except for Emys orbicularis, which occurs in Eurasia and northern Africa (Iverson, 1992). Electrophoresis by isolectric focusing was employed, which provided relatively high resolution of protein bands (up to 0.02 pH units) separated by their surface charge (isoelectric point, pI). This technique has proven to be especially effective in detecting polymorphism of hemoglobin and myoglobin in vertebrates (Oshima et al., 1982) and turtles in particular (Seidel and Adkins, 1989; Seidel et al., 1999; King and Heatwole, 1999).

Materials and Methods

For electrophoretic analysis of hemoglobins, 35 emydid taxa (representing all 10 genera of the family Emydidae), six species of the family Bataguridae, and one species in the family Testudinidae were examined. I collected blood samples from the following specimens: family Emydidae, Chrysemys picta picta (2), Chrysemys picta belli (2), Chrysemys picta dorsalis (1), Chrysemys picta marginata (2), Clemmys guttata (3), Clemmys insculpta (7), Clemmys marmorata (2), Clemmys muhlenbergii (4), Deirochelys reticularia (2), Emydoidea blandingii (2), Emys orbicularis (2), Graptemys barbouri (1), Graptemys flavimaculata (1), Graptemys pulchra (1), Graptemys pseudogeographica (1), Graptemys versa (2), Malaclemys terrapin (2), Pseudemys concinna (2), Pseudemys gorzugi (1), Pseudemys texana (2), Terrapene carolina (2), Terrapene coahuila (2), Trachemys decorata (1), Trachemys decussata (1), Trachemys dorbigni (2), Trachemys gaigeae (8), Trachemys scripta scripta (8), Trachemys scripta elegans (13), Trachemys scripta troostii (3), Trachemys callirostris (4), Trachemys venusta (3), Trachemys emolli (1), Trachemys yaquia (2), Trachemys stejnegeri (2), Trachemys terrapen (1); family Geoemydidae, Rhinoclemmys diademata (1), R. areolata (1), R. punctularia (3), R. pulcherrima (2), Sacalia bealei (1), Cyclemys dentata (1); family Testudinidae, Kinixys sp. (2). For most of the emydid genera, males, females, and juveniles were sampled. Species designations for Trachemys follow Seidel (2002). Turtles in the families Geoemydidae (sensu McCord et al., 2000; formerly Bataguridae) and Testudinidae, collectively Testudinoidea, are considered closest relatives of the Emydidae (Shaffer et al., 1997).

Blood (0.5–4.0 ml) was collected from the forelimb (Avery and Vitt, 1984) in a heparinized syringe. Whole blood samples were stored at 4 C for less than one week before separating plasma and cells by centrifugation. Preparation of hemoglobin followed the procedure of Moo-Penn and Schmidt (1977). Packed erythrocytes were rinsed with physiological saline, lysed with CCl₄ and cell debris removed by centrifugation. For removal of partially oxidized hemoglobins, KCN was added to the hemolysate and then flushed with CO. In addition to analysis of whole (tetrameric) hemoglobin, monomeric fragments were analyzed for some genera. Blood sample aliquots were treated with sulphydryl reagent, p-chloromercuribenzoate (PCMB), following the method of Oshima et al. (1982). PCMB is commonly used to separate hemoglobin into alpha and beta globin chains (monomers), but its activity depends on the number and position of sulphydryl groups in each subunit. It can also produce fragments (electrophoretic bands) caused by conjunction of PCMB with cysteine residues or by dimer (alpha and beta) formation (Oshima et al., 1982).

Following hemoglobin treatments, samples were stored at −60 C prior to electrophoretic analysis. Focusing was performed on ultra thin (0.1 mm) polyacrylamide gels pH 5,6–9 (Serva 1987–1992 Catalogs). Gels were placed in a cooling cell (2 C) and focused for 2–5 h at constant power (3.5 watts, maximum 1700 volts) using an LKB 2103 power supply and Bio-Rad electrodes. Immediately after focusing, gels were fixed in trichloroacetic acid and stained with Serva Blue W (a tri-phenylmethane dye). Gels were scored for presence or absence of bands and comparisons between gels were based on replicates and a commercially prepared standard (Protein Mixture 9, Serva). The general staining procedure elucidated highly concentrated and clearly resolved protein bands, which were subsequently identified as hemoglobins by the Benzidine Method (Smith, 1976). Isoelectric points (pIs) were estimated by direct comparisons to known pIs of the protein standard and polymorphism was evaluated phenotypically. Because pH of the bands was not measured directly, the assigned pI of each electromorph is presented as a relative value. Variable forms of hemoglobin were identified by the isolectric points of their electromorphic bands. All taxa which appeared to share the same Hb electromorph, or showed variants separated by < 0.3 isoelectric points, were compared to each other at least once on adjacent lanes of a gel.

Results

Hemoglobin was variable among the geoemydid and testudinid turtles (outgroup) examined and all of the major forms (densely concentrated bands) appeared different from emydid hemoglobins. In all of the emydid blood samples, two major hemoglobin bands were evident. The more cathodal and less concentrated electromorphs (here referred to as Hb A) ranged from 7.6–8.3 pI, whereas the more anodal electromorphs (Hb D) ranged from 5.2–6.1 (Table 1, Fig. 1). Very little intraspecific variation of hemoglobin structure was detected.

Table 1.  Variant Forms of Hemoglobin (Defined by Isoelectric Points) in Emydid Turtles

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The most common form of Hb A (pI 8.3) appeared in all Pseudemys, Graptemys, Malaclemys, and Trachemys. The next most common form (pI 7.9) was observed in all Clemmys, Emys, and Trachemys scripta. Chrysemys (pI 7.8) and Terrapene (pI 8.1) had unique forms of Hb A, whereas Deirochelys and Emydoidea shared an electromorph (pI 7.6). The most common form of Hb D (6.0) again appeared in all Pseudemys, Graptemys, Malaclemys, and Trachemys as well as in Terrapene. Emys orbicularis, Clemmys marmorata, and C. guttata shared a common electromorph (pI 5.6); although unique forms of the Hb D were observed in Deirochelys (5.9), Chrysemys (5.7), Clemmys insculpta (5.3), C. muhlenbergii (6.1), and Emydoidea (5.2). The only example of intraspecific variation was observed in Trachemys scripta. The nominate subspecies, T. s. scripta and juvenile T. s. troostii and T. s. elegans have hemoglobins A and D identical to Pseudemys, Graptemys, and other Trachemys. In contrast, adult T. s. troostii and T. s. elegans have hemoglobin electromorphs identical to those in Emys, Clemmys guttata, and C. marmorata. Nevertheless, separation of adult T. s. elegans and T. s. troostii hemoglobin (PCMB treated) into monomeric globins (or related fragments) indicated similarities to other Trachemys. Focusing of these fragments revealed two distinct anodal bands (pI 4–5) shared by all Graptemys and Trachemys.

Discussion

Presence of two major forms of emydid hemoglobin (A and D) and absence of intraspecific polymorphism reported here is similar to results found in other studies of turtle hemoglobin (Sullivan and Riggs, 1967; Sullivan, 1974). Also similar to present results, Dessauer (1970) found that adult Trachemys scripta elegans have different electromorphic bands compared to juveniles. Ramirez and Dessauer (1957) reported two major hemoglobin bands in Trachemys scripta, a fast anodal band (pI 5.7) and a slow cathodal band (pI 7.2). Two similar electromorphs (pI 5.7 and pI 7.3) were also described for Chrysemys picta (Rucknagel and Braunitzer, 1988). Rucknagel and Braunitzer (1988) determined that the two major forms of turtle hemoglobin (A and D) are controlled by three genetic loci: one for the alpha chain of Hb A, one for the alpha chain of Hb D, and one which codes for the beta chain of both. Therefore, if turtles vary in both forms (A and D), it could be the same amino acid substitution, resulting from a single mutation at the beta locus.

For phylogenetic interpretation of variation in emydid hemoglobins, each electromorphic pattern, Hb A and D collectively, can be considered a single character (Hemoglobin A/D, Table 1) with multiple character states. Unquestionably this is a conservative estimate of hemoglobin divergence in the family Emydidae. Because none of the hemoglobin electromorphs observed in emydids were present in the outgroup (Geoemydidae and Testudinidae) the two shared character states, Hb 8.3/6.0 and Hb 7.9/5.6, are interpreted as synapomorphies. Similarly, Seidel and Adkins (1989) reported that the two variant forms of myoglobin in emydid turtles are apomorphic.

Although intraspecific variation was observed in Trachemys scripta, the hemoglobin character state Hb 8.3/6.0 occurred in all species of Trachemys examined. Presence of this synapomorphic pattern also in Malaclemys, Graptemys, and Pseudemys (Table 1, Fig. 1) was not surprising. These four genera have similar nucleotide sequences of the mitochondrial 16-S gene (Bickham et al., 1996), similar morphology (Seidel and Jackson, 1990), and identical myoglobins (Seidel and Adkins, 1989). A close relationship between Trachemys and Graptemys was especially evident from monomeric globins/fragments. The different and unique Hb pattern observed for the closely related genus Chrysemys is consistent with its basal divergence proposed by Seidel and Jackson (1989). No variation in hemoglobin was observed among Emys, Clemmys guttata, and C. marmorata (synapomorphy Hb 7.9/5.6), whereas C. insculpta and C. muhlenbergii each have unique forms of Hb D. This appears to provide additional support for the hypothesis that the genus Clemmys sensu McDowell (1964) is not monophyletic (see Bickham et al., 1996; Burke et al., 1996; Ernst, 2001; Holman and Fritz, 2001; Feldman and Parham, 2002). In conclusion, present results add to the growing set of data for analysis of phylogenetic relationships in the turtle family Emydidae.