Cnemidophorus tesselatus

TAYLOR, HARRY L., COLE, CHARLES J., HARDY, LAURENCE M., DESSAUER, HERBERT C., TOWNSEND, CAROL R., WALKER, JAMES M. & CORDES, JAMES E., 2001, Natural Hybridization Between the Teiid Lizards Cnemidophorus tesselatus (Parthenogenetic) and C. tigris marmoratus (Bisexual): Assessment of Evolutionary Alternatives, American Museum Novitates 3345, pp. 1-1 : 1-

Cnemidophorus tesselatus

Sexually mature and reproductive adults of C. tesselatus usually exhibit the following characteristics: oocytes are present, the ovarian follicle cell layer is complete and well organized, connective tissue is not conspicuous, vacuoles are few, and the follicle is well vascularized. The distal oviduct contains a thin mucosa with poorly developed folds. The middle oviduct is ciliated and contains well­developed alveolar glands, which are restricted to this section of the oviduct. The proximal oviduct has a thick (normal) mucosa and well­developed folds. The mesonephric tubules are 20–30 µm in diameter ( figs. 15C , 18A–D ).

The ovary of a normal, reproductive C. tesselatus contains developing follicles with a well­developed tunica granulosa that includes active pyriform cells ( fig. 14E, F ). The zona radiata (formed by microvilli) is usually evident and probably indicates the active transport of components (such as phosvitin and lippovittelin) for the later syn­

thesis of yolk granules ( fig. 14G ). The increase in the surface area that results from the presence of the microvilli facilitates transport of substances between the oocyte and the follicle cells (Balinsky, 1975; Anderson and Beams, 1960). The cytoplasm is granular but homogeneous in distribution in young oocytes and becomes layered in older oocytes ( fig. 14I, K ). Yolk deposition begins when the oocyte is 2.3 mm (largest nonyolked oocyte measured) to 3.1 mm (smallest yolked oocyte measured) in diameter (table 6, fig. 14G, H ); as vitellogenesis progresses the theca granulosa regresses with the loss of pyriform cells but retention of the small granulosa cells ( fig. 16G ). Atretic oocytes have stopped development for some unknown reason. A previtellogenic oocyte that is atretic shows irregular or vacuolated cytoplasm ( fig. 14A, B ). The theca granulosa is often hypertrophied, and enlarged granulosa cells move through the disrupted zona radiata or zona pellucida into the oocyte cytoplasm ( fig. 14C, D ). As atresia progresses the basement lamina of the theca granulosa becomes a hyaline layer known as the glassy membrane ( fig. 14C, D ). At ovulation the follicle wall appears thicker because the thin, stretched wall of the mature follicle is now shrunken due to the collapse of the follicle; cellular debris may be present in the lumen, but neither oocyte nor yolk will be present ( fig. 14J ). As the corpus luteum ages the lumen becomes filled with luteal cells ( fig. 14K, L ). The fully developed corpus luteum is easily distinguished from an advanced atretic follicle by the absence of yolk, absence of the glassy membrane, and the presence of vacuolated luteal cells ( fig. 14L ). Corpora lutea regress rapidly to a distinctive, triangular collapsed structure, composed primarily of connective tissue and usually located between other follicles ( fig. 14I ). Perhaps this is typical of lizard corpora lutea (Miller, 1948: pl. 13d, e for Xantusia ; Goldberg, 1970: fig. 12 for Sceloporus ).

Two individuals exhibited gonadal abnormalities. One of these (AMNH R­146636), a large individual (95 mm snout–vent length), had very small ovaries lacking enlarged oocytes; no sperm were seen in the reproduc­ tive tract. The second (AMNH R­146640) was the smallest C. tesselatus in the sample

(snout–vent length = 65 mm) and contained a few abnormal follicles (many irregular spaces and an incomplete theca granulosa); no sperm were found in her reproductive tract. All other representatives of C. tesselatus contained 3–22 follicles; seven individuals were undergoing vitellogenesis in follicles 1.4–8.5 mm in diameter (table 6).

Salient features of the reproductive systems of representative individuals of C. tesselatus , all C. tesselatus X C. tigris marmoratus hybrids , and all male C. tigris marmoratus collected at the hybridization site are described below with date of collection in parentheses. The first series are representatives of C. tesselatus .

AMNH R­146629 (June 11, 1998): The left ovary was 3 X 7 mm. Both oviducts were swollen; the left was 16 X 3.5 mm, the right was 14 X 4 mm. The largest oocyte had not begun vitellogenesis but the cytoplasm is differentiated into at least three zones, and the granulosa and zona radiata are well developed (as in fig. 14A, I ). Several oocytes show pyriform cells ( fig. 16H ), which indicate the onset of yolk deposition in Sceloporus (Goldberg, 1970) . At least two corpora lutea were present ( fig. 14I ). The small size and triangular shape of the corpora lutea suggest that ovulation and oviposition occurred from at least two weeks (Miller, 1948: pl. 12 for the viviparous Xantusia ) to several

months earlier (Goldberg, 1970, for the ovoviviparous Sceloporus jarrovi ); however, the sequence and timing of corpus luteum degeneration in Cnemidophorus is not well known. Two atretic follicles were present. No sperm were found.

AMNH R­146636 (June 12, 1998): The left ovary and oviduct were smaller than those of the other C. tesselatus examined; however, this individual is 95 mm snout–vent length, clearly of adult size; the mean snout– vent length for the other 25 specimens examined is 89.6 (65–109 mm). The mesonephric kidney and adrenal were well developed and vascularized. The ovarian epithelium (cortex) was very thin and contained some oocytes and a well­developed lip ( fig. 15E ). The ovary consisted of a thin layer (3– 4 cells thick) over a thinner layer of smooth muscle, all of which were on the surface of the adrenal away from the mesonephros ( fig. 15E ). Follicles were not present, the ovary was probably just beyond the indifferent stage of development, and the oviduct was tiny. This animal, although of large size for a mature female, is equivalent to a hatchling in its reproductive anatomy. The karyotype and protein electrophoresis confirmed the identity of this specimen. No sperm were found .

of an oocyte; note the layered cytoplasm of the adjacent oocyte (AMNH R­146629, slide 28, row 1, section 4). J. A recently ovulated follicle that will become a corpus luteum; note triangular shape and point of rupture (AMNH R­146620, slide 31, row 1, section 1). K. Corpus luteum (left) and normal follicle (right; AMNH R­146618, slide 1, row 2, section 13). L. Detail of corpus luteum (AMNH R­ 146621, slide 1, row 2, section 9). Slides stained with Mallory triple connective tissue technique are designated MT; all other slides were stained with Harris hematoxylin and eosin. Abbreviations for this and the following figures are as follows: A, adrenal gland; AC, adrenal cells; AF, atretic follicle; Al, alveolar gland; BGE, boundary of germinal epithelium; BV, blood vessel; CL, corpus luteum; CT, connective tissue; C1, outermost layer of pre­yolk cytoplasm of oocyte; C2, middle layer of pre­yolk cytoplasm of oocyte; C3, innermost layer of preyolk cytoplasm of oocyte; D, discharging pyriform cell; Dod, distal oviduct; E, epididymis; FC, follicle cell; FR, site of follicle rupture due to ovulation; GM, glassy membrane of an atretic follicle; Gr, granulosa cells of follicle; I, interrenal cells; L, lumen; LC, luteal cells; Le, Leydig cell layer; Lip, lip of germinal epithelium; LRV, left renal vein; M, mesonephros; ML, mesonephros, large tubules; Mod, middle oviduct; MS, mesonephros, small tubules; Mu, mucosa; Mus, muscularis; N, nucleus; O, ovary; Od, oviduct; OF, ovarian follicle; Og, oogonia of germinal epithelium; Oo, oocyte; P, pyriform cell in theca granulosa; S, serosa; Sp, spermatozoa; ST, seminiferous tubules; TE, theca externa of ovarian follicle; TG, theca granulosa of ovarian follicle; TI, theca interna of ovarian follicle; V, vas deferens; Y, yolk of oocyte; ZR, zona radiata. Scale bar, 0.1 mm, except E– H and L, which are 0.01 mm.

Female C. tesselatus X C. tigris marmoratus Hybrids

row 1, section 5). E. Germinal epithelium with lip (AMNH R­146636, slide 11, row 1, section 3). F. Germinal epithelium with developing oocytes; section did not pass through lip (AMNH R­146618, slide 1, row 1, section 15). G. One follicle containing two oocytes. The theca interna completely separates the two oocytes and a single theca externa encompasses the follicle (AMNH R­146618, slide 4, row 2, section 2). Scale bar, 0.1 mm, except B, which is 0.01 mm.

AMNH R­146637 (June 12, 1998): The left oviduct was enlarged but lacked eggs .

The left ovary contained 14 enlarged follicles ( fig. 14F ) up to 1.4 mm in diameter and one corpus luteum. Macroscopically, the ovary showed one reddish spot, possibly the corpus luteum detected later by histological examination. The germinal epithelium contained oogonia and a distinctive lip ( fig. 15D ). The two largest oocytes had not begun vitellogenesis. No sperm were found.

AMNH R­146638 (June 12, 1998): The left ovary and oviduct were enlarged and the largest oocyte was yolked. No other oocytes were yolked and no recognizable corpora lutea were visible. No sperm were present in the oviduct .

AMNH R­146639 (June 12, 1998): The left ovary and oviduct were enlarged and the ovary contained three enlarged and yolked oocytes. The largest nonyolked oocyte was 1.9 mm in diameter, almost as large as those containing yolk, and larger by 0.5 mm than a yolked oocyte in AMNH R­146637. There were at least four corpora lutea present, all flattened or triangular, and up to 1.4 mm wide. No sperm were found.

AMNH R­145142 (May 12, 1999): Two oocytes 8.5 mm in diameter were depositing yolk when preserved. Several previtellogenic follicles exhibited secretory activity of the theca granulosa and conspicuous pyriform cells ( fig. 14E ) similar to those seen in Ctenosaura (del Carmen et al., 1996). Near the end of vitellogenesis, the granulosa was still evident as a distinctive cell layer ( fig. 14H ). The distal oviduct had a very thin mucosa and very thin folds near the infundibulum, but near the junction with the middle oviduct the mucosa was thickened ( fig. 17F ). The middle oviduct contained normal alveolar glands ( fig. 17G, H ). No sperm were found .

AMNH R­145143 (May 14, 1999): This specimen had an early atretic follicle with disorganized cytoplasm ( fig. 14A, B ) and follicles that were undergoing vitellogenesis

( fig. 14G ). The oviducts were enlarged; the folds of the distal oviduct were shallow near­ er the infundibulum ( fig. 17E , left) and deep­ er away from the infundibulum ( fig. 17E , right). No sperm were found.

AMNH R­146612 (June 10, 1996): This specimen contained a yolked follicle (6.4 X 6.7 mm) and a corpus lutuem (0.5 mm diam.). The mesonephric tubules were small (0.02 mm diam.). Other ovarian and oviductal structures appeared normal. No sperm were found .

AMNH R­146613 (June 10, 1996): The ovary contained follicles of several sizes (as in fig. 16E ), including yolked and atretic follicles ( fig. 16F ). No sperm were found .

AMNH R­146614 (July 20, 1997): This specimen contained two small corpora lutea and very small follicles (table 6). Like all others collected in July 1997 ( fig. 21 , table 6), this individual was postreproductive .

AMNH R­146618 (July 20, 1997): This specimen contained a recently ovulated follicle ( fig. 14K ), several normal follicles with germinal epithelium ( fig. 15F ), and two oocytes in one follicle (future twins that might share the same eggshell; fig. 15G ). This follicle would have been available for the 1998 reproductive season. No sperm were found .

AMNH R­146620 (July 21, 1997): A large (7.0 X 3.7 mm) yolked atretic follicle ( fig. 14C ) and a recent corpus luteum with visible rupture site ( fig. 14J ) suggest that one of the two large oocytes ovulated and the other aborted, perhaps within two weeks (Miller, 1948) prior to collection. No sperm were found .

AMNH R­146621 (July 21, 1997): An atretic follicle ( fig. 14D ) and two corpora lutea ( fig. 14L ) were present. No sperm were found .

follicles containing oocytes (AMNH R­146619, slide 3, row 1, section 3, MT). F. Follicle edges of a normal yolked follicle (upper right), atretic yolked follicle (lower right), and normal nonyolked follicle (left; AMNH R­146613, slide 6, row 2, section 9, MT). G. Previtellogenic follicle (right) and vitellogenic follicle (left) showing differences in cytoplasm, yolk, and theca development (AMNH R­146613, slide 6, row 2, section 6, MT). H. Theca granulosa of normal developing oocyte (AMNH R­146629, slide 19, row 1, section 3). Scale bar, 0.1 mm, except C, D, G, H, which are 0.01 mm.

AMNH R­146640 (June 12, 1998): This specimen contained enlarged oviducts but

tiny (unrecognizable macroscopically) ovaries. Many spaces were present around follicle cells and the theca granulosa was almost absent; however, there were no follicle cells in the cytoplasm of the oocytes, which appeared normal ( fig. 18C ). The adrenal and mesonephric tubules appeared normal (not enlarged) for a female ( fig. 18C ). No sperm were found.

Other specimens examined did not vary conspicuously from the above descriptions. Important to the question of hybridization frequency, none of the specimens of C. tesselatus had sperm in the oviducts.

During development the ovary is attached to the surface of the adrenal. In its earliest stages (during and soon after the indifferent stage) the ovary appears as a thickened epithelium, almost always with a distinctive lip on one edge. This ovarian lip makes the extremely young ovary more easily recognizable (see Hardy and Cole, 1981: fig. 3 , top right corner, fig. 4 , lower right corner, and fig. 6, lower right corner; Hardy and Cole, 1998: fig. 3B , top center, and fig. 2 ). In some samples the ovary is so small that only a few oocytes are present. The scarcity of oocytes ( figs. 15F , 16B, E ) and small size of the immature ovary make it a difficult organ to recognize. Thus, one must focus on the shape of the ovarian lip, plus the presence of oocytes, in order to recognize the small ovary ( fig. 15A–E ).

Vitellogenesis in C. tesselatus begins when the follicles are between 3.1 mm (smallest yolked oocyte measured) and 2.3 mm (largest nonyolked oocyte measured) in diameter (using largest dimension of oocyte [table 6]); however, if vitellogenesis is well under way for a normal complement of eggs (2–3 per ovary), then vitellogenesis might be delayed for other oocytes even though they are within the minimum size range for initiation of vitellogenesis.

The two oocytes in one follicle ( fig. 15G ) could have been independently derived from two different oogonia; a mutation in one oogonium but not in the other would result in these two oocytes being genetically different (one preserving the mutation). Alternatively, they could be the daughter cells, fol­ lowing endomitotic duplication, of a single oogonium, and mutation in the oogonium

would be contained in both daughter oocytes.

Rupture of the follicle during ovulation would probably result in both oocytes moving as a unit, held together by the theca interna, into the oviduct and thence becoming incorporated within a single eggshell. Development of each oocyte to hatching would produce two hatchlings from a single shell—apparent twinning. However, if the oocytes separated after ovulation but before eggshell formation so that each had its own eggshell, then the potential twinning event would never be detected.

The number of normal yolks produced by a mature female in a season (about five, see below) is probably determined by the energy required for yolk synthesis and the available food supply. How the yolk is packaged is independent of the food supply and reflects the anatomical constraints of the lizard (is the egg too large to be laid?). Containment of both embryos in the same eggshell would probably result in a much larger egg (if both had a normal yolk reserve); this could be detrimental to the mother—the only significant evolutionary consequence of the apparent twinning.

All except 1 of the 26 adult C. tesselatus females had developing follicles and oocytes, and 4 were nearly ready for oviposition (oocyte diameters greater than 6 mm). One female (AMNH R­146636; snout–vent length = 95 mm) was nonreproductive, evidently because of developmental failure. This specimen had a snout–vent length large enough that one would expect it to contain enlarged oocytes, but it had no follicles, only a thin ovarian epithelium (almost an indifferent stage of embryonic development; fig. 15E ), no corpora lutea, no increase in collagen, no evidence of atretic follicles, and a tiny oviduct, whereas the smallest female (AMNH R­146640; snout–vent length = 65 mm) had at least 10 enlarged oocytes (maximum size = 0.3 X 0.5 mm) in follicles that appeared normal ( fig. 18C ). With the exception of AMNH R­146636, these specimens of C. tesselatus appeared to be typical females in all details.

Cnemidophorus tesselatus X C. tigris mar­

Figures

Fig. 15. Germinal epithelium and adjacent adrenal gland of hybrid females (A–C) and C. tesselatus females (D–F). A. Germinal epithelium with well­defined lip, margin, and oogonia (AMNH R­146689, slide 3, row 2, section 6, MT). B. Detail of epithelium in figure 15A (AMNH R­146689, slide 3, row 2, section 6, MT). C. Germinal epithelium, adrenal gland, and mesonephros (AMNH R­146691, slide 13, row 2, section 9). D. Germinal epithelium showing boundary and lip (AMNH R­146637, slide 41,

Fig. 18. Mesonephros of hybrid females (A, B), C. tesselatus (C, D), hybrid males (E–G), and a C. tigris male (H). A. Mesonephros showing small and large tubules (AMNH R­145147, slide 8, row 2, section 6). B. Adrenal gland with large and small mesonephric tubules (AMNH R­145146, slide 11, row 1, section 2). C. Ovary and adrenal with small mesonephric tubules; no large tubules present (AMNH R­146640, slide 4, row 1, section 1). D. Mesonephros with small tubules; no large tubules present (AMNH R­146629, slide 10, row 1, section 4). E. Large tubules (= epididymis) and small mesonephric tubules; debris does not include sperm (AMNH R­145148, slide 8, row 1, section 6). F. Epididymis and small mesonephric tubules (AMNH R­146695, slide 25, row 3, section 6). G. Vas deferens and small mesonephric tubules (AMNH R­146692, slide 12, row 2, section 6, MT). H. Large mesonephric tubules and epididymis (packed with sperm) and small mesonephric tubules (AMNH R­ 146653, slide 10, row 2, section 4). Scale bar, 0.1 mm, except G, which is 0.01 mm.

Fig. 14. Atretic and normal follicles and corpora lutea of C. tesselatus. A. Developing oocyte during vitellogenesis (right) and atretic follicle (left; AMNH R­145143, slide 19, row 1, section 1). B. Atretic follicle with disorganized cytoplasm but intact nucleus (AMNH R­145143, slide 19, row 1, section 1). C. Atretic follicle after vitellogenesis had begun; notice the glassy membrane (AMNH R­146620, slide 4, row 2, section 5). D. Atretic follicle (left) and normal follicle (right), both before vitellogenesis (AMNH R­146621, slide 4, row 1, section 2). E. A normal follicle showing the structure of the follicle wall and discharging pyriform cells (AMNH R­145142, slide 106, row 1, section 3). F. Detail of the tunica interna showing discharging pyriform cells prior to the formation of yolk granules (AMNH R­ 146637, slide 41, row 1, section 4). G. Detail of a follicle during vitellogenesis, showing a welldeveloped zona radiata, vascularization, and the tunica granulosa (AMNH R­145143, slide 28, row 1 section 2). H. Near the end of vitellogenesis the tunica interna is reduced to the tunica granulosa (AMNH R­145142, slide 49, row 1, section 2). I. Older corpora lutea showing the triangular shape and absence

Fig. 16. Ovary structure of hybrid females (A–D) and C. tesselatus females (E–H). A. Ovary with abnormal follicle (AMNH R­145146, slide 5, row 1, section 4). B. Ovary with empty follicle and abnormal follicular epithelia (AMNH R­145147, slide 16, row 1, section 1). C. Detail of follicle wall of empty follicle (AMNH R­145147, slide 16, row 1, section 1). D. Empty follicle with blood supply (AMNH R­146681, slide 4, row 1, section 2). E. Ovary with oogonia and several sizes of developing

Fig. 17. Oviduct structure of hybrid females (A–D) and C. tesselatus females (E–H). A. Distal oviduct showing thin mucosa and absence of folds (AMNH R­145147, slide 8, row 1, section 1). B. Middle oviduct with alveolar glands (AMNH R­145147, slide 8, row 1, section 1). C. Middle oviduct showing poorly developed alveolar glands (AMNH R­145146, slide 11, row 2, section 2). D. Middle oviduct (AMNH R­146691, slide 9, row 2, section 6, MT). E. Distal oviduct showing mucosa and variation in fold development (AMNH R­145143, slide 5, row 2, section 6). F. Distal and middle oviducts; note the swollen (active) posterior part of the distal oviduct and the distinctive appearance of the middle oviduct. The proximal oviduct is not visible (AMNH R­145142, slide 136, row 1, section 2). G. Middle oviduct with mucosa and well­developed alveolar glands (AMNH R­145142, slide 80, row 1, section 2). H. Middle oviduct showing serosa, mucosa, and organization of the alveolar glands (AMNH R­145142, slide 85, row 1, section 4). Scale bar, 0.01 mm, except F, which is 1 mm, and H, which is 0.1 mm.

Fig. 21. A monthly comparison of size distributions and reproductive status of individuals of Cnemidophorus tesselatus collected at the Arroyo del Macho hybridization site in 1996–1999.

Fig. 3. Ventral view of the same lizards arranged in the same sequence as in figure 2.

Fig. 4. Representatives of Cnemidophorus tesselatus from the Arroyo del Macho hybridization site. A. AMNH R­146615 (91 mm SVL); B. AMNH R­146618 (83 mm SVL); C. AMNH R­146619 (85 mm SVL); D. AMNH R­146623 (86 mm SVL); E. AMNH R­146627 (89 mm SVL); F. AMNH R­ 146626 (90 mm SVL).

Fig. 2. Dorsolateral views of three representative whiptail lizards (Cnemidophorus) from Arroyo del Macho. A. Diploid unisexual C. tesselatus, AMNH R­146638, body length 97 mm. B. Triploid C. tesselatus X C. tigris marmoratus hybrid male, AMNH R­146693, body length 100 mm. C. Diploid bisexual C. tigris marmoratus male, AMNH R­146653, body length 94 mm. All photographed June 19, 1998.