Biology_of_Turtles

Auffenberg, 1978; Trachemys scripta: Moll & Legler, 1971; Gibbons, 1990; Gopherus polyphemus: Iverson, 1980; Chrysemys picta: Zweifel, 1989; Iverson & Smith, 1993; Apalone ferox: Iverson & Moler, 1997; Webb, 1956; Chelodina novaeguineae: Kennett et al., 1992). Some studies provide comparative information on reproductive behaviors and biology of two or more species (e.g.,

Chelydra serpentina, Apalone, Macrochelys, Trachemys), and some contain drawings or pictures of pre-copulatory and mating behavior of different species (e.g., Legler, 1955; Webb, 1961, 1962; Auffenberg, 1978; Bellairs, 1970; Bustard, 1972; LeBuff, 1990; Obst, 1986; Oliver, 1955; Dodd, 2001; Ewert, 1976; Harrel et al., 1996). Some provide comparative information on the biology and ecology of multiple species grouped by general habitat; for example, river turtles (Moll & Moll, 2004) and sea turtles (Lutz & Musick, 1997; Lutz et al., 2002). Still others summarize the information concerning multiple species over large geographic areas (Australia: Legler, 1985; Cann, 1998; Venezuela: Pritchard & Trebbau, 1984). Some species and groups have received more attention than others as a result of their accessibility (e.g., Pseudemys concinna: Seidel & Dreslik, 1996; Trachemys scripta: Seidel & Ernst, 2006; Terrapene carolina: Ernst & McBreen, 1991), distribution (e.g., Chelydra serpentina: Ernst et al., 1988; Gibbons et al., 1988; Lovich, 1993; Apalone: Webb, 1990), or conservation status (Swingland & Klemens, 1989; Van Abbema, 1997; Klemens, 2000).

The focus of this chapter is on the morphological diversity of chelonian reproductive structures and the effects these have on the range of reproductive strategies exhibited by turtles. Our goals are to provide a summary of information and ideas and to provide access to the literature in hope of stimulating further research. The literature cited throughout has been selected to demonstrate a point and to provide access to more detailed literature. No attempt has been made to cite every exemplary document that exists in the vast literature concerning chelonian morphology and reproductive ecology.

10.2General Morphology

The overall the anatomy of chelonians is consertative in that they have a shell encasing the soft parts of the body. However, the turtle shell—which incorporates the ribs and encases the girdles—is not just a two-part, ridged box connected around the body of the turtle by bridging bones. In some groups (e.g., Dermochelyidae, Trionyichidae), the dermally derived (thecal) portion of the shell is greatly reduced; in these, the pliable shell structure is provided by epithecal ossicles and connective tissue covered by epidermis (Zangerl, 1969). Among hard-shelled chelonians, the carapace is formed by thecal dermal elements and is more rigid than the plastron, with the exception of Kinixys in which the carapace is movable (Zangerl, 1969). Plastronal kinesis allows some chelonians to close the gap between the carapace and plastron, thereby providing increased protection of the soft parts. Kinesis occurs in both pleurodires (e.g., Pelusios) and cryptodires (e.g., Kinosternon, Sternotherus, Emydoidea, Emys, Terrapene, Cuora, Kinixys, Pyxis, Testudo) but is not universal, even among closely related species or between the sexes within a species (e.g., Testudo graeca) (Zangerl, 1969). Hinges occur in different places on the plastron and may have weak to strong mobility. For a hinge to work, the sutures between both the epidermal shields and the underlying bones must be aligned. Hinges may occur anterior to the bridge (Pelusios, Terrapene, Pyxis), both anterior and posterior (Kinosternon, Sternotherus), or on the bridge (emydids) (Zangerl, 1969).

The evolution of the turtle shell occurred in concert with a number of other anatomical changes that had physiological and behavioral consequences (Zangerl, 1969). For example, changes occurred in the shape and articulations of the bones of the neck (Zangerl, 1969); also, muscles of the head and jaw region altered over time, thus allowing some diversity in foraging (Schumacher, 1973; Shah, 1963) while retaining one of two general patterns of neck articulation (pleurodires, cryptodires). Further, alterations of size and shape of limbs and feet that occurred with the evolution of the shell in conjunction with specializations associated with habitat use affected patterns of locomotion (Walker, 1973, 1979). As a result, some chelonians have paddle-shaped flippers well suited to an aquatic life (Cheloniidae, Carettochelyidae, Trionychidae), whereas others have expansible and

webbed hind feet and reduced webbing on their forefeet (many Emyididae, Chelidae, Pelomedusidae, Geomydidae) suited for aquatic and semi-aquatic habitats. Others have elephantine feet (Testudinidae, some Emydidae) specialized for terrestrial locomotion.

10.3Reproductive Structures

The reproductive structures of turtles include the gonads (testes, ovaries) and associated ducts to carry sperm (epididymis, vas deferens) or ova (oviducts), and the cloaca containing the clitoris or penis and associated structures.

10.3.1 Gonads

The development of the urinogenital system in reptiles, including reproductive organs and associated ducts of turtles, was reviewed by Fox (1977) and Raynaud and Pieau (1985). Miller and Limpus (2003) provide a detailed description of the ontogeny of the gonads of marine turtles; a similar pattern occurs in other cryptodires (Fox, 1977). No similar description exists for pleurodires, but it is assumed that the process is similar.

The gonads develop on the germinal ridges on the ventro-medial surfaces of the mesonephros (Sternotherus: Risley, 1933a; Chrysemys picta marginata: Allen, 1906; Fox, 1977). As differentiation continues, testis and ovary become distinguishable by the histological differences in the relative amounts of the cortex (thin layer in males) or medulla (dense, non-tubular in females) during the last half of embryonic development (Fox, 1977). By hatching, the gonads of most species are distinguishable based on their microscopic structure (e.g., Caretta caretta: Mrosovsky & Yntema, 1980; Chelonia mydas: Miller & Limpus, 1981, 2003). Vestigial oviducts (Müllerian ducts) occur in many species of male chelonians (e.g., Emydoidea blandingii: Nicholson & Risley, 1941; Mauremys caspica leprosa: Stefan, 1959, 1963; Fox, 1977), at least for a while after hatching. Conversely, the embryonic expression of the vas deferens (Wolffian duct or mesonephric duct) may persist in the female but tends to degenerate to become part of the peritoneal wall as the turtle ages (Fox, 1977; Wibbels et al., 1999).

10.3.1.1 Testis

Details of the structure of the testis and the spermatogenetic cycle have been described—at least in part—for a number of species in six families (Emydidae, Chelydridae, Cheloniidae, Kinosternidae, Testudinidae, Trionychidae) (Table 10.1; Gist, 1999; Girling, 2002). Testes have been illustrated in varying detail in drawings and photographs (e.g., Agassiz, 1857; Bojanus, 1819; Thomson, 1932; Noble & Noble, 1940; Ashley, 1962; Miller & Limpus, 2003; Owens, 1980, 1997; Lance & Rostal, 2002; Hamann et al., 2003; Rostal, 2005). The testes are located toward the dorsal-midline of the body cavity, attached mediolaterally to the kidneys (Emydoidea blandingii, Trachemys scripta, Glyptemys insculpta, Chelydra serpentina: Fox, 1977). The hatchling testis shows clear immature seminiferous tubules within the medulla (e.g., Caretta caretta: Mrosovsky & Yntema, 1980; Chelonia mydas: Miller & Limpus, 1981, 2003). The testis does not become functional until puberty; however, it does increase in size as the turtle grows. Maturation is a slow process and may require a decade in some species (Caretta caretta: Limpus & Limpus, 2003). During puberty, the testis enlarges and the epididymis extends from the peritoneal wall until it is pendulous (Miller & Limpus, 2003).

Testes of mature turtles are oval in cross section but may be cylindrical or flattened; typically, they have a smooth surface with seminiferous tubules visible within the tissue (Moll, 1979; Owens, 1980, 1997; Miller & Limpus, 2003). The histological structure of the adult testis reveals the thin epithelium of the cortex on the tunica albuginea. The seminiferous tubules are contained within the vascularized connective tissue of the medulla. Leydig’s cells occur throughout the stroma in

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Table 10.1

Descriptions of the Spermatogenetic Cycle of Turtles

See also reviews by Lofts (1968, 1969), Moll (1979), Licht (1984), Owens (1997), Fox (1977), Gist (1999), Kuchling (1999), and Hamann et al. (2003).

the tissue between the testicular tubules. These interstitial cells secrete testosterone and lipoidal substances (Pellegrini, 1925a, 1925b) under pituitary hormonal control (Licht, 1974; Fox, 1977; Crews & Garrick, 1980). Interstitial cells enlarge prior to spermatogenesis then regress as the reproductive cycle ends. During their active period, they stimulate secondary sexual characteristics such as changes in coloration (Cooper & Greenberg, 1992) and courtship behavior (Moore & Lindzey, 1992). Spermatogonia occur inside the basement membrane of the seminiferous tubules; during the breeding season, primary spermatocytes are also visible in this area. Sertoli cells are situated inside the seminiferous tubules next to the basement membrane (Sternotherus odoratus: Risley, 1938; Terrapene carolina: Altland, 1951). Sertoli cells facilitate spermatogenesis via adenohypophysial stimulation (Lofts, 1968). The cells may fill the tubule lumen at the end of the reproductive period (Mauremys caspica: Lofts & Boswell, 1961). Sertoli cells eliminate remaining sperm during regression of the seminiferous tubules (Fox, 1952).

Licht (1984) defined four phases of the spermatogenetic cycle for reptiles: regenerative, progressive, cumulative, and regressive. The spermatogenetic cycle can be further divided into five phases (Moll, 1979) or eight (McPherson & Marion, 1981; Wibbels et al., 1990). The differences in the definitions of phases result from different emphasis being placed on particular characteristics of spermatogenesis. At the cellular level, spermatogenesis appears to follow a general pattern of sperm production that is regulated by pituitary hormones (Licht, 1974; Crews & Garrick, 1980; Owens 1997).

Mature testes change size in reflection of spermatogenesis: size increases as spermatogenesis increases and regression occurs as activity decreases (e.g., Kinosternon subrubrum hippocrepis,

K. flavescens, Sternotherus odoratus, S. carinatus: Mahmoud & Klicka, 1972; Clemmys: Lofts & Boswell, 1961; Trachemys: Moll & Legler, 1971; Chrysemys picta: Gibbons, 1968a; Ernst, 1971a;

Terrapene ornata: Legler, 1960a; Testudo hermanni: Kuchling, 1982a, 1982b; Mauremys caspica leprosa: Combescot & Guyon, 1955). Seminiferous tubules increase in diameter as they fill with sperm (Mauremys caspica: Lofts & Boswell, 1961; Sternotherus odoratus: Risley, 1938b); seminiferous tubules may regress following the reproductive season (Trachemys: Moll & Legler, 1971) or may contain sperm throughout the year (Macrochelys temminckii: Dobie, 1971; Chelydra serpentina: Conant, 1938). Testis size and pattern of enlargement as well as the timing of sperm in the seminiferous tubules may vary within a species exhibiting temperate and tropical patterns of reproduction (Moll & Legler, 1971; Moll, 1979). The temperate pattern shows “peak Leydig cell activity during gametogeneic quiescence” (Moll, 1979), whereas in the tropical pattern, the period of quiescence is short and the peak Leydig cell activity does not necessarily occur during the quiescent period (Moll, 1979).

The epididymis is attached to the peritoneal wall near the testis and is located between the rete testis and the vas deferens. The epididymis is a low ridge until puberty, but as puberty progresses it becomes more convoluted and extended from the peritoneal wall (Miller & Limpus, 2003; Limpus

&Limpus, 2003). During the reproductive cycle, the epididymis becomes enlarged and turgidwhite in appearance; it regresses but remains obvious during the non-reproductive period (Miller

&Limpus, 2003). The vas deferens provides for sperm transport between the epididymis and the ostia leading into the cloaca near the proximal end of the penile groove; the histology of this has not been described.

Turtles do not have spermatheca; instead, sperm are stored in the epididymis until passed to the female during coitus. Some turtles may have sperm in the epididymis year-round even though spermatogenesis occurs seasonally (Moll, 1979).

10.3.1.2 Ovary

Details of the structure of the ovary and the changes that occur throughout the reproductive cycle have been described in few turtle species from three families (Cheloniidae, Geoemydidae, Kinosternidae) (Table 10.2; Girling, 2002). The hatchling ovary shows a compact (homogeneous) medulla separated by the tunica albuginea from the columnar epithelium of the cortex. Germinal

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Table 10.2

Descriptions of the Ovarian Cycle and Changes in the Structure of the Oviducts in Turtles

oocytes appear in the cortex as large cells typically with an obvious space within the cell membrane (Miller & Limpus, 1981, 2003) until the turtle begins puberty. During puberty, the stroma of the ovary increases in size, providing the support for the first development of the oocytes in late puberty and regular follicular development in adulthood.

The mature ovary is comprised of stroma, pre-vitellogenetic follicles, maturing follicles, atretic follicles, corpus lutea, and corpus atretica; the relative numbers of each depend on the maturity of the individual and its phase in its reproductive cycle. Ovaries have been illustrated in varying detail in drawings and photographs (e.g., Agassiz, 1857; Bojanus, 1819; Thomson, 1932; Noble & Noble, 1940; Ashley, 1962; Moll, 1979; Miller & Limpus, 2003; Rostal, 2005; Owens, 1980, 1997). The morphology of the ovary has not been described for pleurodires; it is presumed to be essentially the same as in cryptodires.

In general, the descriptions of the morphology of the ovary of the adult green turtle (Chelonia mydas: Aitken et al., 1976; Owens, 1980; Miller & Limpus, 2003), the soft-shelled turtle (Lissemys punctata punctata: Sarkar et al., 1996), and Blanding’s turtle (Emydoidea blandingii: Nicholson & Risley, 1941) represent the ovaries of all chelonians. The ovary is a “membranous, curtain-like structure with a relatively short attached border” (Chelonia mydas: Aitken et al., 1976). In smaller species, the regressed ovaries appear to be more compact but stretch, becoming curtain-like during the reproductive period. Regardless of species, the expression of the curtain-like stroma is greatest in mature, reproducing individuals. Follicles are present on both sides of the ovarian stroma. Various aspects of the structure of follicles have been described for several species (Testudo: Loyez, 1906; Clemmys guttata, Graptemys geographica, Emydoidea blandingii, Sternotherus odoratus,

Chrysemys picta: Thing, 1918) and illustrated by Agassiz (1857). The ovarian cycle—including vitellogenesis, ovulation, the retention of corpora lutea, and their eventual regression as corpora albucantia—is controlled by hormones (Licht et al., 1979, 1980; Licht, 1980; Owens, 1980, 1997). Owens (1997) and Hamann et al. (2003) provide integrated reviews of the role of hormones in the reproductive ecology of sea turtles; based on the existing information, the general pattern holds true for other chelonians, especially for those species that produce multiple clutches in a breeding season (Lofts, 1968, 1969; Moll, 1979; Licht, 1984; Owens, 1997; Kuchling, 1999).

The ovarian cycle is divided into four phases (Moll & Legler, 1971; Moll, 1979):

Enlargement of follicles

Ovulation and preoviposition (intrauterine) period

Oviposition

Follicle regression and quiescence (between reproductive seasons)

Toward the end of puberty, the turtle ovary is stimulated to initiate vitellogenesis, wherein multiple follicles enlarge but do not ovulate (Limpus & Limpus 2003). These follicles become atritic; as they regress, they are invaded by blood vessels and eventually reduce in size. As they become atritic, the color changes from the yellow-cream color of a maturing follicle to become orange and eventually red-orange as the invading blood vessels proliferate. They eventually become small reddish lumps (corpora atretica) in the stroma.

In reproductively quiescent turtles, no enlarged follicles occur in the stroma but previtellogenetic follicles are abundant. Previtellogeneic follicles contain an oocyte surrounded by a layer of cuboidal granulosa that, in turn, is surrounded by the vacuolated cells of the gonadal mound. Previtellogenetic follicles appear yellow-cream in color; several different size classes may be evident but all are much smaller than a follicle in vitellogenesis. Small white corpora lutea may be visible in the stroma. The expanded stroma contains many arteries and veins (Aitken et al., 1976).

In reproducing individuals, follicles undergo vitellogenesis and enlarge. In temperate freshwater species, follicular enlargement may occur before or after brumation, depending on geographic location, age, or species (Table 10.3). For Chelonia mydas, vitellogenesis requires a year or two, possibly longer, before ovulation (Limpus & Nicholls 1988, 2000). In species that produce multiple

Table 10.3

Examples of the Timing of Follicular Development in Relation to Hibernation in Several Species of Turtles

clutches, enlarged follicles will not be the same size (Gibbons & Tinkle, 1969). The largest will be ovulated in the first clutch; follicles for the second clutch will increase in size while the eggs are in the oviduct or immediately following oviposition. In general, the number and diameter of enlarged follicles correlates with the number and the size of the eggs to be deposited in the clutch; a larger number of follicles yields a larger egg count in the clutch and larger diameter follicles yield larger diameter eggs. However, one or more follicles may enlarge but not be ovulated; these will regress to become corpora atretica.

Following ovulation, the stroma and theca surrounding the follicle collapse to form a corpus luteum. This is the primary site of steroidogenesis converting pregnenolone and cholesterol to progesterone (Callard et al., 1976, 1991; Klicka & Mahmoud, 1972, 1973; Licht & Crews, 1976). The corpus luteum is a disc-shaped structure comprised of the theca externa and theca interna plus a thick layer of granulose cells; the granulose cells go through luteinization to become granulose lutein cells (Cyrus et al., 1978). The supporting ovarian tissue swells and at least in some species becomes vesicular (e.g., Cheloniidae: Miller & Limpus, 2003). The theca interna begins to invade the granulose lutein layer with collagenous fibers, fibroblasts, and blood vessels (Cyrus et al., 1978). Corpora lutea have been reported in Trionyx (Agassiz, 1857), Terrapene ornata, Emydoidea blandingii (Legler, 1958), Chrysemys picta (Powell, 1967), Trachemys scripta (Moll & Legler, 1971), Chelydra serpentina (Klicka & Mahmoud, 1973), and Geochelone gigantea (Swingland & Coe, 1978). The corpora lutea remain large until the uterine eggs are oviposited, after which they regress to become corpora albicantia. Given the function of the corpora lutea, it is likely that they occur in all chelonians.

As regression progresses, the corpora lutea decrease in size, first becoming semi-white then white as connective tissue replaces gransulosa lutein cells. Eventually, the structure becomes a small scar (corpora albicantia) on the ovarian stroma. The scar remains visible for the current breeding season and may be retained for several years, at least in some species (Cheloniidae: Miller & Limpus, 2003). When corpora albicantia remain as small scars embedded in the stroma, they provide clear evidence that the turtle has reproduced (Apalone spinifera: Moll, 1979; Chelonia: Miller & Limpus, 2003; Caretta caretta: Limpus & Limpus, 2003). However, corpora albicantia may disappear within a short time following ovulation (Trachemys: Moll & Legler, 1971; Chrysemys picta: Ernst, 1971a). The ovaries of more species should be examined carefully to look for the presence of small scars (approx 1 to 2 mm) that carry over between reproductive episodes.

At the end of the reproductive season, any remaining follicles begin to regress. Following regression, the ovaries enter quiescence and remain essentially quiescent between reproductive seasons. It is assumed that the quiescent period allows the female to regain the energy expended in maintaining her system and required for the production of her eggs. In reproductively quiescent

also lined with ciliated and non-ciliated cells, as well as glandular epithelial cells (Palmer & Guillette, 1988). The glandular structure of the uterine tube suggests that it produces albumen from the numerous mucosal glands (Palmer & Guillette, 1988; Aitken & Solomon, 1976). This region has internal longitudinal folds; externally, it appears “pleated” transversely (Gopherus polyphemus: Palmer & Guillette, 1988). The oviductal isthmus appears to be transitional between the uterine tube and the uterus; this short section may be aglandular (Gopherus polyphemus: Palmer & Guillette, 1988) or contain active glandular cells, at least during ovulation (Lissemys p. punctata: Sarkar et al., 1995).

The endometrial glands of the uterine section secrete the fibers of the eggshell (Gopherus polyphemus: Palmer & Guillette, 1988). The epithelial lining of the shell-forming region contains ciliated and non-ciliated columnar cells (Aitken & Solomon, 1976; Palmer & Guillette, 1988; Motz & Callard, 1991; Sarkar et al., 1995). The shell-forming region in turtles sequentially secretes both the supporting shell membrane and the calcareous matrix of the shell (Aitken & Solomon, 1976). In the shell-secreting region, the crystalline structure of the eggshell is deposited onto the strands of the membrane matrix. Differences in the structure of the eggshells produced may reflect differences in the glandular structure of the uterine region (Girling, 2002). The heavily folded vagina is lined with ciliated epithelial cells, and the muscularus layer is thick. The short, muscular vagina keeps the eggs from entering the cloaca until oviposition (Palmer & Guillette, 1988). The vagina may or may not contain glands; if present, the glands support sperm storage (Sever & Hamlett, 2002).

Details of the impact from changes in hormonal levels on oviductal tissues throughout the reproductive cycle have been described in few turtle species from three families: Emydidae, Testudinidae, Trionychidae (Table 10.5; Girling, 2002). Plasma levels of estradiol seem to control the changes in the oviductal tissues (Girling, 2002). Secreted by the corpora lutea, progesterone helps to maintain the function of the oviduct while the eggs are being coated with albumen and covered by the shell material. The openings of the two oviducts enter the cloaca from the ventral medio-lateral aspect (Emydoidea blandingii: Nicholson & Risley, 1941), the dorsal-lateral aspect (Trionyx euphraticus: Salih, 1965), or the ventro-lateral aspect (Emys (Testudo) europaea: Bojanus, 1819; Testudo graeca: Thomson, 1932; Chelonia mydas, Caretta caretta, Eretmochelys imbricata: Miller, unpublished).

10.3.3 Cloaca and Associated Structures

The cloaca is an expandable tube that has a multi-ridged, longitudinally folded epithelium surrounded by longitudinal and transverse muscles, and connects between the sphincter of the colon and the sphincter at the anus. The cloaca of the female is relatively short, whereas the cloaca of the male is much longer. The increased length allows for the penis to be accommodated inside the cloaca. The bladder, any ancillary bladders, ureters, and the oviducts or vas deferens enter the cloaca near the proximal end, usually from the ventrolateral aspect (Kuchling, 1999).

Kuchling (1999) described the cloaca as being partially divided into “an upper and lower chamber” by septae and stated that “the vasa deferentia and ureters of each side open into the lower chamber” called the sinus urogenitalis. Older descriptions of the cloaca show some disagreement in terminology and observed structures (Bojanus, 1819; Thomson, 1932). However, these descriptions agree that there is a small vestibule formed by the ostia of the oviducts and folds of the cloacal wall in the floor of the cloaca. There has been no modern detailed description of the cloaca of turtles that allows comparison within and among taxa.

All chelonians have urinary bladders (Minnich, 1982) comprised of transitional epithelium that allows expansion and has at least a few muscle fibers that facilitate contraction. The bladder is filled by urine carried through ureters from the kidneys; the ureters connect at the base of the bladder in the short muscular neck area between the bladder and the cloaca (Thomson, 1932; Noble & Noble, 1940; Ashley, 1962). The bladder empties into the cloaca through a short urethra that connects between the two structures. The urinary bladder may be singular, as in sea turtles, or bi-lobed, as in most freshwater species.