Reproduction of the Parents in the Laboratory

The following details pertain to the clutch of three eggs and laboratory hybrids on which we focus for most of this paper, from a cross of A. inornata arizonae (♀) × A. t. marmorata (♂). The female arizonae was AMNH R-148431 and the male marmorata was AMNH R-153156 (see appendix, Specimens Examined, for locality data).

The female lived in captivity for nearly six years, from 30 July 1994 to 21 June 2000, and the male also for nearly six years, from 13 July 1996 to 8 May 2002. During the four years that they were living in the laboratory simultaneously, they were caged with each other for only part of the time. The female was probably a yearling when captured; she was first measured five months later at 60 mm snout-vent length.

The female produced 10 clutches of eggs in captivity in the absence of conspecific males, with a total of more than 28 eggs (1–4 per clutch). The absolute total number of eggs is unknown because one clutch was eaten by a cagemate before we found the eggs. Only 7 of the 28 eggs hatched, from two full clutches. The first successful clutch was of 3 eggs and these were the 3 hybrids on which this paper focuses. The second was a clutch of 4 eggs, all of which hatched into abnormal offspring with unusually short snouts and 2 of which also had unusually large eyes; all of these were weak and died within 17 days of hatching.

The successful clutch of three eggs that produced the hybrids described here was laid on 19 May 1999, when the mother had a snout-vent length of 70 mm and weight of 7.8 g. The clutch weighed 1.8 g, or 23% of the mother's weight. Egg dimensions were as follows (in mm): 8.9 × 13.5; 9.1 × 13.6; and 8.1 × 15.9. At 11:00 A.M., when the spent mother emerged from her damp burrow, the presumed father mated with her. Following incubation at room temperature, one egg hatched in 69 days, on 26 July 1999, and the other two in 70 days, on 27 July 1999. All three offspring appeared to be normal, and the light spots on their sides suggested immediately that they were hybrids, as hatchlings of the maternal species do not have spots on the body.

During her tenure in captivity, at various times the mother had been caged with other females of A. i. arizonae and with at least five different males of A. t. marmorata, but the only male with which she was observed mating was the one we refer to as the presumed father. From March through June 1999 the father was mating frequently with the only female in his cage, but that was not the mother, which had been housed alone in a different cage beginning 18 August 1998. However, because of this male's frequent mating activity at this time, we added the mother to the cage, on 11 May 1999, eight days before oviposition of the hybrid clutch, and apparently just in time.

The three hybrid offspring were the following: (1) AMNH R-148432, which hatched on 26 July 1999; its sex was not apparent while it was alive, and it died under the water dish in its cage on 13–15 November 1999, apparently from an accident when a caretaker (not in Acknowledgments) returned the cleaned dish to the cage; (2) AMNH R-153157, which hatched on 27 July 1999, which appeared to be a male, and had attained a snout-vent length of 78 mm when sacrificed for study on 15 August 2002 at more than three years of age; and (3) AMNH R-153158, which appeared to be a female and had attained a snout-vent length of 81 mm when sacrificed for study on 13 August 2003 at more than four years of age.

Upon hatching, the three hybrids were caged together but with no other lizards for about three months, until 1 November 1999, at which time they were maintained separated thereafter to ensure that no mating could occur. None of these lizards ever laid eggs, but the apparent female (AMNH R-153158) developed a large mass in the abdomen, the histology of which is described below.

Identification of the Hybrids: Genetics

KARYOTYPES: Clearly resolved karyotypes of most of the species in all the species groups of Aspidoscelis were published previously (e.g., Lowe et al., 1970; Cole et al., 1988), including the species discussed in this paper. Each species group has a diagnostically distinct karyotype, and A. inornata belongs to the A. sexlineata species group while A. tigris belongs to the tigris group (for phylogenetic relationships, see Reeder et al., 2002).

The haploid set of A. inornata (including A. i. arizonae, n = 23) consists of a single large Set I metacentric chromosome (with a slightly elongate satellite on one arm set off by a sub-terminal secondary constriction) + 12 smaller Set II intermediate-sized telocentric or subtelocentric macrochromosomes + 10 Set III microchromosomes. Sex chromosomes are not morphologically recognizable.

The haploid set of A. tigris (including A. t. marmorata; n = 23) consists of three large Set I biarmed (metacentric or submetacentric) macrochromosomes + eight smaller Set II biarmed (submetacentric) intermediate-sized macrochromosomes + 12 Set III microchromosomes. The second largest chromosome in Set I has a dotlike satellite on one arm set off by a nearly terminal secondary constriction, which often is difficult or impossible to see with standard microscopy. The third largest chromosome is the sex chromosome (XX female, XY male; Cole et al., 1969; Bull, 1978), the X and Y differing in centromere position, although both are submetacentric. In both A. inornata and A. tigris the secondary constrictions on large macrochromosomes are the nucleolar organizer regions (Ward and Cole, 1986).

As expected, the karyotype of the laboratory hybrid examined (AMNH R-153158) was diploid (2n = 46), consisting of one normal haploid complement each from the maternal A. inornata and the paternal A. tigris (fig. 1). The individual examined was the adult-sized apparent female, and, as expected, had the X sex chromosome of A. t. marmorata. The karyotype confirmed that this laboratory-hatched lizard was indeed a hybrid, probably between the A. inornata arizonae and A. tigris marmorata adults in whose cage the clutch of eggs appeared. This karyotype also is identical to that reported previously for one of the unisexual, parthenogenetic, and clonal species, A. neomexicana (see review in Cole et al., 1988), which had an origin in nature resulting from hybridization between A. t. marmorata (♀) × A. inornata (♂).

FIGURE 1.

Karyotype of laboratory hybrid of A. i. arizonae (♀) × A. t. marmorata (♂), AMNH R-153158, adult-sized intersex individual that superficially resembled a female. Upper row represents the haploid complement of A. t. marmorata (with 3 large Set I metacentric and submetacentric macrochromosomes including the X + 8 biarmed Set II macrochromosomes + 12 Set III microchromosomes). Lower row represents the haploid complement of A. i. arizonae (with 1 Set I macrochromosome including its characteristic NOR and satellite [arrow] + 12 subtelocentric Set II macrochromosomes + 10 Set III microchromosomes). Line represents 10 microns.

ALLOZYMES: Protein electrophoresis of allele products representing 21 nuclear gene loci, with emphasis on those that specifically demonstrated the hybrid origin of A. neomexicana, provided additional evidence on the parentage of the laboratory hybrid examined. For these analyses, on each gel we compared one specimen each of A. i. arizonae from the maternal parent's population (AMNH R-153168), one of A. t. marmorata from the paternal parent's population (AMNH R-153163), one of A. neomexicana from New Mexico (AMNH R-151740), and one of the laboratory hybrids (AMNH R-153157), the adult-sized apparent male.

The allozyme data (table 1; fig. 2) confirm the differences previously reported for A. inornata versus A. tigris and the hybrid nature of both the unisexual A. neomexicana and the laboratory hybrid. For 8 loci (footnote b, table 1), all individuals examined were homozygous for the same allele (i.e., no variation observed), as expected for these lizards. Of the 13 loci showing variation (table 1), 6 were simply as expected, needing no further discussion, showing the normal allelic differences between A. inornata and A. tigris, with both the laboratory hybrid and A. neomexicana being heterozygous for the two respective parental alleles (LDH1, sSod, sAAT: fig. 2A; mAAT, ADA, and MPI: fig. 2B). The same is basically true for the remaining 7 loci, which are consistent with expectations based on Cole et al. (1988) and Dessauer et al. (2000), but the allozymes observed in these individuals merit further discussion, as follows.

IDDH (previously referred to as Sord): One would predict that all individuals compared here would be homozygotes for one and the same allele (no variation), because A. t. marmorata is usually homozygous for the b-allele at this locus. However, an alternative a-allele of lower frequency does occur in A. t. marmorata (see Dessauer et al., 2000), and the individual examined for this report happened to be a heterozygote having both the a-allele and b-allele.

TABLE 1.

Genotypesa at 21 Gene Loci^b in Samples of Aspidoscelis^c

FIGURE 2.

Electrophoretic phenotypes representing products of four gene loci of four individuals of Aspidoscelis. A. sAAT, a dimeric enzyme, from skeletal muscle homogenates. B. MPI, a monomeric enzyme, from erythrocyte hemolysates. C. ESTD, a dimeric enzyme, from skeletal muscle homogenates. D. PEPB, a dimeric enzyme, from kidney homogenates. Letters below gels identify allozymes based on alleles present (table 1). Lanes for individual lizards are labeled beside each gel (with genotype) as follows: MAR, A. t. marmorata; I × M, laboratory hybrid of A. i. arizonae × A. t. marmorata; INO, A. i. arizonae; and NEO, A. neomexicana. Anode for each is to the right.

sIDH (previously referred to as Icd-1): One would predict the results obtained (table 1) except that the A. inornata would have the a-allele, as found in the heterozygous state in A. neomexicana and laboratory hybrid. However, A. inornata is known to have alternative alleles of lower frequency (Cole et al., 1988), including the b-allele seen here.

ESTD (previously referred to as Es-D): A. inornata and A. tigris were known to normally have the same allele, consistent with the laboratory hybrid being homozygous for their common allele (fig. 2C). The alternative allele seen in the heterozygous state in the field sample of A. neomexicana is its orphan allele at this locus, known to characterize A. neomexicana, but having never been observed in either of its parental species.

PEPA: The a-allele observed is typical for A. inornata, as is the b-allele for A. tigris from the Lordsburg area, and as found in the heterozygous laboratory hybrid. The c-allele, which is typical for A. neomexicana, is found at times in A. tigris from the Rio Grande Valley, perhaps the area of origin of A. neomexicana (see Cole et al., 1988).

PEPB: All alleles observed fit the prediction (fig. 2D). The c-allele seen in the heterozygous state in the field sample of A. neomexicana is its orphan allele at this locus, known to characterize this species, but which has not been observed in either of its parental species.

PEPD: This locus has not been analyzed before in A. neomexicana, in which we observed heterozygosity for ab, but the other results are not surprising. Considering that this locus is the most variable known in A. tigris (see Dessauer et al., 2000), it would not be surprising if the ancestral A. t. marmorata had contributed the slower b-allele observed here in A. neomexicana.

GPI: The a-allele and b-allele both occur as variants in both A. inornata and A. t. marmorata (Dessauer and Cole, 1989), and the a-allele is known in marmorata from the Lordsburg area (Dessauer et al., 2000), so it is not surprising that it occurred in the laboratory hybrid.

In summary, the allozyme data are fully consistent with the karyotype data and indicate that the laboratory hybrid did have the A. inornata × A. tigris hybrid origin predicted, even though this hybrid differed from A. neomexicana in allele combinations at 5 loci analyzed, including the 2 orphan alleles previously known for neomexicana.

Identification of the Hybrids: External Morphology

COLOR AND PATTERN: The variation in colors and patterns of the whiptail lizards discussed here is difficult to quantify, but A. i. arizonae and A. t. marmorata are so different they can be readily identified even while running on the ground. Consequently, we use general descriptions based on field notes and specimens noted in life. The A. i. arizonae are from the vicinity of Willcox, Cochise County, Arizona; the A. t. marmorata from the vicinity of Lordsburg, Hidalgo County, New Mexico; and the clonal A. neomexicana from throughout its range.

Aspidoscelis inornata arizonae (Maternal Species, fig. 3): The dorsum of this lizard is dark gray to brown (bluish gray atop head) usually with seven conspicuous light yellow stripes (but the vertebral stripe may be less conspicuous than the others and may be broken). There are no spots or bars on the body. The arms are uniform grayish brown, but there may be a light beige line on the outer edge of the lower arm. The thighs are brown with a trace of a light yellowish beige reticulation, the lower legs grayish brown. The tail is basically tan at the base, with a rapid transition to bright blue. The ventral surfaces are pale blue to nearly white (in females) or bright blue (males, especially in the breeding season).

FIGURE 3.

Parents of the Aspidoscelis hybrids shown in fig. 4. Upper. A. i. arizonae, as a very old female, AMNH R-148431, SVL = 69 mm. Lower. A. t. marmorata, male, AMNH R-153156, SVL = 82 mm.

Aspidoscelis tigris marmorata (Paternal Species, fig. 3): The dorsum often is “unstriped (a few light yellow stripes or portions or traces thereof may be visible), with a dark brown to black ground color and a pattern (reticulate, marbled, or, especially laterally, cross-barred) of light yellow or beige, including some light spots; some individuals have more prominent wavy vertebral and paravertebral stripes” (Cole et al., 1988: 13). Moreover,

FIGURE 4.

Three Aspidoscelis of hybrid origin. Upper. Laboratory hybrid, intersex, AMNH R-153158, SVL = 81 mm. Middle. Laboratory hybrid, male, AMNH R-153157, SVL = 78 mm. Lower. A. neomexicana from nature, AMNH R-122946, SVL = 77 mm. Parents of upper two are in fig. 3. Ancestry of lower one was of similar parents but the reciprocal cross.

Aspidoscelis neomexicana and Laboratory Hybrid, Adult-sized Apparently Female, AMNH R-153158 (fig. 4): The laboratory hybrid noted in detail and typical Aspidoscelis neomexicana were so similar that the notes for both are included here. The extract below quotes a description of the color notes of A. neomexicana; these apply equally to the laboratory hybrid of A. i. arizonae × A. t. marmorata, except where differences of the hybrid are noted in brackets:

In addition, the hybrid, unlike A. neomexicana but somewhat like its paternal parent had a very pale orange wash across the entire throat on which there were many dark gray to pale black dots or small spots.

What is most striking about the colors and patterns of the laboratory hybrids is that they appeared much more similar to A. neomexicana than to either of their parents (figs. 3, 4). This is no surprise, given that A. neomexicana had a hybrid origin of A. t. marmorata × A. inornata.

SCALATION AND MULTIVARIATE STATISTICS: The maternal parent (A. i. arizonae) of the laboratory hybrids had lower scores than the paternal parent (A. t. marmorata) for all seven meristic characters (table 2). Hybrids were either intermediate to their parents or they equaled one of the parents in GAB, FP, COS, SDL, and PSC scores (see Materials and Methods and table 2 for details on recording the characters abbreviated here with capital letters). All three hybrids had smaller GUL scores than those expressed by the maternal parent, as did two hybrids for ILS. The two hybrids that attained adult size resembled the larger, paternal parent (A. t. marmorata) in SVL (table 2).

TABLE 2.

Counts of meristic characters and SVL for three laboratory hybrids and their maternal (Aspidoscelis inornata arizonae) and paternal (A. tigris marmorata) parents and descriptive statistics for samples of A. inornata arizonae, A. tigris marmorata, and the related A. neomexicana. Means ± SE are subtended by sample size and (range limits); M = maternal parent of hybrids; P = paternal parent of hybrids.

We used two principal components analyses (PCAs) to provide unbiased representations of meristic variation. PCA does not use a priori information on specific affiliations (i.e., specimen identification is not recognized), and we coupled PC scores to particular specimens only after the analyses were completed. As determined from character loadings (table 3), both PCAs were identical in having the greatest contributions to PC1 made by GAB, FP, COS, ILS, SDL and GUL, while PC2 was based primarily on the PSC character.

Our first PCA model (PCA1; N = 39 specimens) addressed the question of how the laboratory hybrids compared with each other, with their parents, and with field samples of their parental taxa from the same localities where their parents were collected. Although the three hybrids were intermediate to their individual parental specimens on the first principal component axis (PC1), where 69.5% of the variation was summarized, the hybrids most closely resembled their paternal parent, A. t. marmorata on PC2, where 12.1% of the variation was summarized (fig. 5). Considering all samples rather than just the individual parents, however, the laboratory hybrids were most similar to A. i. arizonae, perhaps expressing matriclinous inheritance.

TABLE 3.

Principal component loadings and standardized canonical variate coefficients used in multivariate analyses of three laboratory hybrids, their Aspidoscelis tigris marmorata and A. inornata arizonae parents, geographically representative samples of the parental species, and the related A. neomexicana.

Our second PCA model (PCA2; N = 50 specimens) was identical to PCA1 except that a geographically relevant sample of A. neomexicana was added to the mix in order to determine whether the laboratory hybrids resembled this parthenogenetic species of similar hybrid origin. Because the laboratory hybrids originated from a reciprocal cross between the same parental species that gave origin to A. neomexicana (evidence summarized in Cole et al., 1988), this permitted us to assess differences in the pattern of multivariate variation between A. i. arizonae (♀) × A. i. marmorata (♂), the source of the laboratory hybrids, and A. t. marmorata (♀) × A. inornata (♂), the source of the lineage represented by contemporary A. neomexicana. The first principal component summarized 63.0% of the meristic variation in this analysis, with PC2 summarizing 14% of the remaining variation (table 3). The three hybrids were intermediate to their individual parental specimens on both PC1 and PC2 (fig. 6; table 4). The tendency of the laboratory hybrids and a hybridization-derived species to exhibit matriclinous resemblances was demonstrated by the products of the two reciprocal crosses–the laboratory hybrids most closely resembled their maternal A. i. arizonae, and the A. neomexicana most closely resembled their maternal A. t. marmorata on PC1 (fig. 6).

FIGURE 5.

Pattern of morphological variation expressed by the distribution of scores on the first two principal components extracted from a correlation matrix of seven meristic characters of three laboratory hybrids, 18 specimens of A. i. arizonae (including the maternal parent of the hybrids), and 18 specimens of A. t. marmorata (including the paternal parent of the hybrids). All samples represent populations in the vicinities of those from which the parents of the hybrids were collected. Note that the three hybrids are intermediate to their individual parents on PC1.

We also wondered how these laboratory hybrid specimens might be identified if they had been found in the field by a collector who prepared no material for genetic analyses. Because the three laboratory hybrids had the basic colors and pattern of A. neomexicana, their true identity might not have been recognized in the field. However, meristic counts would have aroused suspicion. Scores for laboratory hybrids were below the lower range limits in our reference sample of A. neomexicana (table 2) for the following characters: GAB (two hybrids); FP (all three hybrids); COS (all three hybrids); and ILS (two hybrids). These low counts presumably reflect genetic effects that had been derived from A. i. arizonae, their maternal parent. Therefore, we did a canonical variate analysis (CVA), with the hybrids included in the model as unknowns. The reference samples of A. i. arizonae, A. t. marmorata, and A. neomexicana were used as a priori groups. Although not uniformly obvious from the CV coefficients in table 3, GAB, COS, FP, SDL, and ILS had higher correlations (communalities) with CV1 while GUL and PSC were most highly correlated with CV2. The CVA assigned hybrid AMNH R-153157 to the A. i. arizonae a priori group (P = 1.0) and hybrids AMNH R-153158 and 148432 to the A. neomexicana a priori group (P = 0.88 and P = 0.54, respectively). Nevertheless, collectively, the hybrids appeared to occupy a position intermediate to their two parental taxa and A. neomexicana on CV1 (fig. 7). Consequently, a field collector in the absence of genetic data might have identified these hybrids incorrectly, as hybrids between A. neomexicana (♀) × A. i. arizonae (♂), considering both color pattern and meristics.

FIGURE 6.

Pattern of morphological variation expressed by the distribution of scores on the first two principal components extracted from a correlation matrix of seven meristic characters of three hybrids, 18 specimens of A. i. arizonae (including the maternal parent of the hybrids), 18 specimens of A. t. marmorata (including the paternal parent of the hybrids), and 11 specimens of the unisexual A. neomexicana. All samples represent populations in the vicinities of those from which the parents of the hybrids were collected. Note that the three hybrids are intermediate to their individual parents and that A. neomexicana most closely resembles its maternal progenitor species, A. t. marmorata on PC1.

As a check on the distinctiveness of the laboratory hybrids as an independently recognizable entity, we included them as a fourth a priori group in a follow-up CVA (plot not reproduced here). The a priori hybrid group was as distinctive as the A. i. arizonae, A. t. marmorata, and A. neomexicana a priori groups, as there was 100% classification success for the members of each group, and each hybrid had an assignment probability of 1.0. Finally, we compared the list of characters of our laboratory hybrids (table 2) with the same characters for the type specimen of “Cnemidophorus perplexus” (USNM 3060) as reported by Wright and Lowe (1967). The specimens are quite similar, differing primarily in appearance of the vertebral light stripe and ILS, the latter of which could reflect different methods of counting by different investigators. This illustrates once again the difficulties of accurately identifying perplexing individuals of whiptail lizards in the absence of genetic data. For example, Taylor and Walker (1996) and Walker (1997) suggested that USNM 3060 is an unusually large individual of A. neomexicana, rather than a hybrid as suggested by Wright and Lowe (1967), all in the absence of genetic data.

TABLE 4.

Multivariate statistics for three laboratory hybrids and their maternal (Aspidoscelis inornata arizonae) and paternal (A. tigris marmorata) parents and descriptive multivariate statistics for samples of A. inornata arizonae, A. tigris marmorata, and the related A. neomexicana. Means ± SE are subtended by range limits; M = maternal parent of hybrids; P = paternal parent of hybrids.

As discussed above, Aspidoscelis neomexicana shows matriclinous inheritance, in resembling A. t. marmorata. This stronger multivariate resemblance of A. neomexicana to A. t. marmorata was also evident in a previous analysis (Cole et al., 1988: fig. 5). That analysis differed from the present one in two respects, neither of which should negate the conclusion of matriclinous inheritance: (1) their reference sample for inornata was of A. i. llanuras Wright and Lowe, 1993, from the vicinity of Lordsburg, Hidalgo County, New Mexico, rather than A. i. arizonae; and (2) in addition to the sample of A. neomexicana from the Lordsburg area (the same sample we used here), they also used a sample of A. neomexicana from the northern periphery of its range in the Rio Grande Valley, New Mexico. In fact, the morphological data analyzed for the two subspecies of A. inornata (arizonae vs. llanuras) are very similar (compare Cole et al., 1988; Wright and Lowe, 1993; and table 2 here).

FIGURE 7.

Pattern of morphological distinctiveness expressed by the distribution of canonical variate scores derived from a canonical variate analysis of seven meristic characters of three a priori groups: 18 specimens of A. i. arizonae (including the maternal parent of the laboratory hybrids), 18 specimens of A. t. marmorata (including the paternal parent of the hybrids), and 11 specimens of A. neomexicana. All samples represent populations in the vicinities of those from which the parents of the hybrids were collected. The three laboratory hybrids were included in the CVA as unassigned, for classification to the a priori group that each most closely resembled. Note the position of the hybrid group intermediate to A. i. arizonae, A. t. marmorata, and A. neomexicana clusters. This suggested that the hybrid group itself is distinctive, which was verified by a follow-up CVA (not illustrated, but see text).

INTERNAL ANATOMY: The maternal parent of the laboratory hybrids (AMNH R-148431) was of normal size and external morphology for an individual of A. i. arizonae. The left and right oviducts were swollen and of normal appearance for a reproductive female, although neither contained eggs. The left ovary contained one yellowish ovum 3.4 mm in diameter that appeared to be undergoing vitellogenesis; all the other ova that were visible macroscopically in this ovary were white and the largest one was 1.5 mm in diameter. The right ovary contained three yellowish ova that were undergoing vitellogenesis and were 3.5, 3.7, and 3.3 mm in diameter (from anteriormost to posteriormost). The next largest ovum in the right ovary was white and 1.7 mm in diameter. Both ovaries appeared normal. The stomach was full and there was no evidence of any internal abnormalities. This animal was reproductive (she had produced offspring in the laboratory) and appeared normal in every respect; therefore, no organs were removed for histological study.

FIGURE 8.

Gross morphology of the adult-sized, apparently female (but intersex) laboratory hybrid (AMNH R-153158) of A. i. arizonae × A. t. marmorata. A. Ventral view of the viscera through the opened body wall; the organs have not been disturbed. The arrows indicate a boundary between the left adrenal gland and the left ovary. B. The left kidney and dorsal body wall visible with the adrenal/ovary mass displaced to the right. C. Adrenal/ovary mass displaced to the left. D. Remaining viscera after removal of the adrenal/ovary mass and a suspected testis. Scale bars: 5 mm. Abbreviations in figures 8–17 are explained in Materials and Methods.

The paternal parent (AMNH R-153156) contained testes and epididymydes of normal appearance on both sides, as expected for an individual of A. t. marmorata. The anterior end of the left testis was 4 mm posterior to the posterior end of the right testis, and seminiferous tubules were visible macroscopically in both. The left testis and epdidymus were removed for histological study.

The adult-sized, apparently female hybrid (AMNH R-153158) appeared to be in good health when sacrificed and preserved. Examination of the abdominal viscera via incisions in the ventral abdominal wall revealed a greatly enlarged mass (23 × 16 mm) that dominated the abdominal cavity (fig. 8A). The mass, when displaced to the right, but still intact, revealed the left metanephric kidney against the dorsal body wall (fig. 8B). When the mass was displaced to the left the right metanephric kidney could be seen (fig. 8C). Also visible on the left side, approximately adjacent to the anterior end of the left metanephric kidney, was a small oval structure to be identified later as having malelike structures (discussed below as specimen AMNH R-153158B; fig. 8C). Consequently, histological examination revealed that this individual was an intersex. The large mass was removed also (discussed below as specimen AMNH R-153158A); no other gonadlike structures were visible in the body cavity (fig. 8D).

The smallest hybrid offspring (AMNH R-148432) was in bad condition, as it had died in its cage and was partly decomposed when found. The stomach was thin-walled and full of cricket parts, but the intestine distal to the pyloric sphincter was empty. Two small structures (provisionally thought to be possibly a gonad and adrenal gland) on the right side were removed for histological study.

The remaining adult-sized hybird offspring (AMNH R-153157) appeared to be a male with paired testes, epididymides, and vasa deferentia. The vas deferens and epididymus were neither enlarged nor convoluted, so this individual did not appear to be reproductively functional at the time of preservation. There was no evidence macroscopically of any ovary, oviduct, or uterus. A testis and epididymus were removed for histological study.

Reproductive Histology

PATERNAL PARENT: An adult male of Aspidoscelis t. marmorata, AMNH R-153156 was the probable father of the laboratory hybrids discussed here. His testis appeared normal with well-defined seminiferous tubules (fig. 9A). The seminiferous tubules were thin-walled and appeared similar in structure (fig. 9B) to those studied earlier in other specimens of this taxon (Taylor et al., 2001: fig. 19D–F). The tubules of this specimen contained cellular and noncellular debris, few spermatocytes, and sperm only in a few peripheral tubules (figs. 9A, 10A, 10B). However, the vas deferens was packed with sperm (figs. 9C, 9D, 10C). The testis was adjacent to a normal-appearing adrenal gland with adrenal (chromaffin) cells and interrenal cells (fig. 10A), and the edge away from the testis contained both small and large mesonephric tubules (figs. 9A, 10C), representing the epididymus. This specimen was not undergoing additional spermatogenesis when preserved but was reproductive with a sperm-packed epididymus that had not been completely evacuated.

ADULT-SIZED, APPARENTLY FEMALE HYBRID (AMNH R-153158): The smaller tissue sample sectioned (AMNH R-153158B) consisted of 20 slides. The major structures in this sample included part of the mesonephros and adrenal gland (fig. 11A) and a small piece of liver. The adrenal gland was predominantly composed of interrenal cells with small, scattered clusters of chromaffin or adrenal cells (fig. 11B). The largest tubules visible were the mesonephric duct concentrated near the posteriomedial portion of the adrenal (fig. 11C). Mesonephric tubules formed a layer on the lateral and posteriolateral edge of the adrenal and consisted of larger tubules toward the outer surface and smaller tubules closer to the adrenal gland (fig. 11D). The physical relationship of the mesonephric tubules to the adrenal was consistent with that seen in other species of lizards studied previously (Hardy et al., 1989). There was no evidence of any gonadal tissue in this sample; however, the relationship of the mesonephros and adrenal gland was similar to that seen in male hybrids of A. tesselata × A. t. marmorata (Taylor et al., 2001: fig. 19G) and the mesonephros even resembled an epididymus in AMNH R-153158. This particular structure is more malelike than femalelike in this hybrid.

FIGURE 9.

Testis, adrenal gland, mesonephros, and vas deferens of A. t. marmorata, the father of the hybrids. A. Testis, adrenal gland, mesonephros, and vas deferens (AMNH R-153156, slide 8, row 1, section 2). B. Seminiferous tubule (AMNH R-153156, slide 8, row 1, section 2). C. Vas deferens containing mature spermatozoa (AMNH R-153156, slide 9, row 1, section 6). D. Vas deferens with mature spermatozoa (AMNH R-153156, slide 6, row 1, section 2, Mallory Triple, Pantin method). Scale bar: 0.1 mm except for A, 1.0 mm.

FIGURE 10.

Testis, adrenal gland, mesonephros, and vas deferens of A. t. marmorata, the father of the hybrids. A. Testis and adrenal gland (AMNH R-153156, slide 9, row 1, section 7). Area outlined by the black rectangle indicates an active seminiferous tubule adjacent to the adrenal gland and is enlarged in figure 10B. Note that the surrounding seminiferous tubules were empty. B. A productive seminiferous tubule (AMNH R-153156, slide 9, row 1, section 7). C. Adrenal gland, mesonephros, and vas deferens (AMNH R-153156, slide 7, row 1, section 6). Note the small and large tubules of the mesonephros and the vas deferens, all of which comprise the epididymus. Scale bars: 0.1 mm.

The larger tissue sample sectioned from the same lizard (AMNH R-153158A) consisted of 197 slides. The tissue had some clear organization; approximately the anterior half was composed of adrenal (fig. 12A–D) and the posterior half was a disorganized mass of stromalike material (fig. 12A, C–E). As the left adrenal gland is normally anterior to the left ovary and the right adrenal gland is normally posterior to the right ovary in other species of unisexual lizards (Hardy et al., 1989), the anterior position of the identifiable adrenal gland in this specimen suggests that it was the left adrenal gland. Consequently, the tissue posterior to the adrenal gland (figs. 12, 13) is identified here as the left ovary. The apparent division between the adrenal portion and the ovarian portion is evident macroscopically (arrows in fig. 8). The adrenal gland contained interrenal cells and scattered adrenal (chromaffin) cells (figs. 12B, 13B, 13C). Even though the anterior-posterior organization was still evident (fig. 13) deeper in the tissue, the whole structure was poorly organized. The adrenal was recognizable (fig. 13B, C) and the transition between the adrenal and the ovary could be seen (fig. 13D, E). The tissue of the ovary adjacent to the transition zone (fig. 13G) had the same composition as that seen more posteriorly (fig. 13F, H, I) and as that part of the ovary visible in the transition zone (fig. 13D, E). The ovary seemed to be composed of irregularly shaped stroma (fig. 13F–I) with spaces that were lined with an irregular cuboidal epithelium and which contained loose cells and amorphous material. The ends of individual trabeculae of the ovary sometimes resembled Bowman's capsules with vascularization; however, no glomeruli, Bowman's capsules, or any other indication of the mesonephros were present.

FIGURE 11.

The small tissue sample from the adult-sized, apparently female (but intersex) laboratory hybrid (AMNH R-153158). A. Mesonephros and adrenal gland (AMNH R-153158B, slide 6, row 2, section 1). B. Adrenal gland (AMNH R-153158B, slide 3, row 1, section 10). C. Mesonephros and adrenal gland (AMNH R-153158B, slide 5, row 1, section 6). D. Mesonephros and adrenal gland (AMNH R-153158B, slide 6, row 2, section 1). Scale bars: 0.1 mm.

FIGURE 12.

The large tissue sample from the adult-sized, apparently female (but intersex) laboratory hybrid (AMNH R-153158). A. An entire section (AMNH R-153158A, slide 1, section 3; scale bar: 1 mm). Rectangles identify enlarged views in B—E. B. Adrenal gland (AMNH R-153158A, slide 1, section 3). C and D. Adrenal gland and transition to adjacent ovary (AMNH R-153158A, slide 1, section 3). E. Ovary (AMNH R-153158A, slide 1, section 3). Scale bars for B—E: 0.1 mm.

The ovary contained irregular spaces (lacunae; fig. 13H–I). Small groups of yolk granules (fig. 13I) varied in diameter, stained yellowish or orange in Mallory Triple, and had the same appearance as the yolk granules in female hybrids of A. tesselata × A. t. marmorata illustrated in Taylor et al. (2001, their fig. 16F). A von Kossa test (Sheehan and Hrapchak, 1987) for calcium was negative, thus confirming that the yolk granules were not dystrophic calcium concretions. The ovarian stroma was composed of papillary structures that resembled a benign tumor; however, given the enlarged adrenal gland, we believe this pattern represented secondary hyperplasia produced in response to sex hormone production by the enlarged adrenal cortex rather than a neoplastic process. If the adrenal gland was actually enlarged, this could represent a case of adrenocorticohyperplasia. To test for this, measurements of the adrenal gland were made on several specimens of Aspidoscelis that were used in previous studies (table 5). The volume of the adrenal gland in this laboratory hybrid female was significantly larger (more than 50 times) than that of any of the other specimens studied, confirming adrenocorticohyperplasia, and thus would suggest other manifestations such as secondary masculinization. Additional corroboration that this lizard was an intersex came from the histology of the small tissue sample (AMNH R-153158B), which showed a malelike mesonephros with striking resemblance to an epididymus (see above).

Nine of the 11 taxa examined (table 5) had adrenal volumes less than 0.7 mm³ except A. inornata and the A. inornata × A. t. marmorata hybrid, which had 1.88 and 108 mm³, respectively (fig. 14). The A. sonorae × A. t. marmorata hybrid was closer to A. sonorae (fig. 14) and contained only one marmorata genome. The A. tesselata × A. t. marmorata hybrid was closer to A. t. marmorata (fig. 14) and contained two complements of marmorata in its ancestral genomes. The A. inornata × A. t. marmorata laboratory hybrid was closer to A. inornata (fig. 14) and contained only one marmorata genome. All the A. t. marmorata and the other hybrids involving marmorata were clustered below 0.7 mm³ (fig. 14). The A. inornata and the A. inornata × A. t. marmorata laboratory hybrid had large adrenals. Both A. velox and A. uniparens have two complements of A. inornata in their ancestral genomes plus a complement of the A. burti genome (Reeder et al., 2002).

FIGURE 13.

The large tissue sample from the adult-sized, apparently female (but intersex) laboratory hybrid (AMNH R-153158). A. An entire section (AMNH R-153158A, slide 23, section 1; scale bar: 1 mm). Rectangles identify enlarged views in B—I. B—C. Adrenal gland (AMNH R-153158A, slide 23, section 1). D—E. Adrenal gland and ovary (AMNH R-153158A, slide 23, section 1). F—I. Ovary (AMNH R-153158A, slide 23, section 1). H. An enlargement from slide 23, section 2 showing an atretic follicle in the ovary. I. An enlargement from slide 23, section 2, showing yolk granules. Scale bars for B—I: 0.1 mm.

TABLE 5.

Comparison of adrenal gland sizes in several species of Aspidoscelis. Museum number refers to the American Museum of Natural History (AMNH) or Museum of Life Sciences, Louisiana State University, Shreveport (LSUS). Heavy lines separate three hybrids above, seven specimens representing six unisexual species in the middle, then four specimens of bisexual species below, including the mother of the hybrids (A. i. arizonae).

One could speculate that two copies of the A. inornata genome alone or with a third genome from another species causes enlarged adrenal growth; however, the burti genome with two complements of the inornata genome in the triploid A. velox and A. uniparens has no effect on adrenal growth. Nor is there a problem in typical specimens of A. neomexicana, which also has one genome each from A. inornata and A. t. marmorata, as in this laboratory hybrid (but the reciprocal cross; Brown and Wright, 1979). Perhaps the species represented by the maternal genome makes a difference with respect to abnormal adrenal enlargement.

Some of the lacunae (fig. 13H) of the large, apparently female hybrid contained disorganized clumps of cells and debris. The cellular debris, including nuclei and cytoplasmic strands, was derived from the matrix (= granulosa?) of the trabeculae. Remnants of ooplasm had a stellate surface and contained clear vacuoles, often oblong in shape (fig. 13H). The degenerating ooplasm was stained more intensely than was the acellular material in other lacunae and appeared to be abnormal atretic material very similar to that illustrated by Betz (1963, his fig.9). The presence of several scattered yolk granules and isolated masses of degenerated ooplasm that resembled atretic follicle material suggests that the ovary contained some normal cells that possibly could have produced follicles, yolk, and atretic follicles. However, the disorganization of the ovary resulted in atresia in some areas and vitellogenesis in other isolated places rather than normal oogenesis, and this hybrid never laid eggs although it was more than four years old when sacrificed. Follicles had not developed but some vittelogenesis had occurred (i.e., the isolated yolk granules) and follicle cells that could not mature became atretic in the lacunae, which might represent the cavities of abnormal follicles. The adrenal gland might have hypertrophied owing to the lack of physiological feedback that would be coming from a normally developing ovary and the resulting ovulation. This large structure is a hybrid sex organ, containing few characteristics of a normal ovary or of a normal testis. This hybrid individual, in final analysis, appeared to be a sterile intersex.

FIGURE 14.

Volume of the adrenal gland compared to the snout-vent length (SVL) of the specimens listed in table 5. Hybrids are depicted by rectangles (i × m = A. i. arizonae × A. t. marmorata; s × m = A. sonorae × A. t. marmorata; t × m = A. tesselata × A. t. marmorata), parthenogens by squares (e = A. exsanguis; s = A. sonorae; n = A. neomexicana; v = A. velox; u = A. uniparens; t = A. tesselata), and bisexual species by circles (i = A. i. arizonae; m = A. t. marmorata).

FIGURE 15.

The oviduct (part) and ovary of laboratory hybrid AMNH R-148432, which was partly decomposed. A. Smooth muscle band and the distal oviduct (AMNH R-148432, slide 1, row 3, section 13). B. Ovary (AMNH R-148432, slide 5, row 2, section 3). Scale bars: 0.1 mm.

SMALLEST LABORATORY HYBRID (AMNH R-148432): There were very few histological characteristics about this specimen that could verify the sex. However, the distal oviduct and associated smooth muscle band seen in other species (Hardy and Cole 1981: fig. 10; Hardy et al., 1989: figs. 11, 12) were visible (fig. 15A). The ovary was tiny, poorly developed, and showed the lip and a few oogonia (fig. 15B). This specimen was nonreproductive and appeared to be an immature and infertile female.

ADULT-SIZED, APPARENTLY MALE HYBRID (AMNH R-153157): The well-vascularized testis of the male contained seminiferous tubules with possible spermatogonia in the tubule walls and cellular material in the lumena (fig. 16A). The material in the lumena consisted of cells and noncellular debris, but neither spermatozoa nor meiotic figures were seen (fig. 16B). The mesonephros (fig. 16C) contained large and small mesonephric tubules adjacent to the adrenal gland anteriorly and the testis posteriorly (the latter not visible in fig. 16C). The larger mesonephric tubules contained loosely organized or packed material (fig. 17A); however, the contents were debris and cells that were not spermatozoa, spermatids, or secondary spermatocytes (fig. 17B). A portion of the mesonephros contained large coiled tubules (the epididymus; fig. 17C) that contained packed material having the superficial appearance of packed sperm. However, the packed material consisted only of cells or cellular debris; neither spermatozoa nor spermatids were present (fig. 17D). This male was not reproductive at the time of sacrifice.

FIGURE 16.

The testis of laboratory hybrid AMNH R-153157. A. Testis with seminiferous tubules. B. Seminiferous tubules containing debris and some cellular material. C. Mesonephros with small and large mesonephric tubules. Scale bars: 0.1 mm.

FIGURE 17.

Mesonephros of laboratory hybrid AMNH R-153157. A. Small and large mesonephric tubules with loosely organized material. B. Detail of a large mesonephric tubule. C. Large coiled tubules of the mesonephros (= epididymus); note the lack of small tubules in this region. D. Detail of large tubules of the epididymus containing debris and cellular material but not spermatozoa.

The Hybrids

The following is a conservative estimate of the hybrid offspring that we think the lizards produced in the laboratory at the AMNH, listed chronologically with the reasons for including them on the list. Our uncertainty is based on the fact that we do not have genetic evidence bearing on all possible hybrid individuals, and many hatchlings (not included on this list) died after a short time without evidence that they were hybrids, even if their mother mated with a male of a different species; sometimes important characters of coloration do not emerge until after several months of life (Hardy and Cole, 1998).

There were several other lizards hatched in the laboratory that appeared as if they might be hybrids but for which we have no genetic evidence to confirm or reject their hybrid status. We decided not to list them because this might be misleading, as indicated by two pertinent experiences: (1) one morphologically aberrant specimen hatched in the laboratory from an egg deposited by a normal A. neomexicana appeared to be a hybrid with A. t. marmorata, but genetic data showed she was not; and (2) a similarly morphologically aberrant specimen was caught in the field and allowed to reproduce in the laboratory; she produced normal offspring and genetic data showed that she and her offspring were not hybrids. This was discussed and illustrated by Dessauer and Cole (1989: 50–51), who cautioned that such aberrations may be caused by environmental or regulatory phenomena, rather than hybridization. Although hybridization occurs more often among whiptail lizards than nearly all other lizards, we should exercise caution in deciding which individuals are hybrids and which are not.

Effort Expended to Obtain the Hybrids

How readily do species of Aspidoscelis hybridize? How readily do males of bisexual species mate with females of unisexual species? How readily are new clones of parthenogenetic species created (whether through interbreeding either between two bisexual species or between a male and parthenogenetic female of either a diploid or triploid species)? Definitive answers to these questions are difficult to get, but at least we can present some observations and discuss issues.

Phylogenetic analyses (Reeder et al., 2002: 26) coupled with analyses of mitochondrial DNA (= mtDNA, which identifies the female in hybridization events; Brown and Wright, 1979; Densmore et al., 1989a, 1989b; Moritz et al., 1989) provided a low-end number of at least six hybridization events between two ancestral bisexual species to explain the origins of the diploid parthenogenetic species of Aspidoscelis. At least five additional hybridization events between males and diploid parthenogenetic females would be necessary to produce the founders of the triploid clones, for a total of at least 11 founder hybridization events over hundreds or thousands of years. Owing to the very low level of mtDNA variation in parthenogens, it appeared as if their hybrid origins were very rare as well as recent (Densmore et al., 1989a, 1989b; Moritz et al., 1989).

Alternatively, considerably more frequent hybrid origins of clonal diversity were suggested by relatively high levels of variation in allozymes and external morphology within some parthenogenetic species, especially A. tesselata (e.g., Parker and Selander, 1976; Parker, 1979; Dessauer and Cole, 1989). This alternative is not supported by the most recent data. The various forms of A. tesselata are now known to be histocompatible (i.e., they do not reject skin transplants from each other), suggesting that they all originated from a single F₁ hybrid individual (Cordes and Walker, 2006), and that the diverse clones recognized in morphology and allozymes are probably results of postformational mutations (Taylor et al., 2003). Another recent study showed that although there is frequent hybridization between the bisexual A. tigris marmorata and unisexual A. tesselata in the vicinity of Roswell, Chaves County, New Mexico, none of the triploid hybrids studied to date were able to clone themselves or reproduce by backcrossing (Taylor et al., 2001). It now appears as if reproductively competent hybrids appear to be rare, except when hybridization involves close relatives (e.g., A. t. marmorata × A. t. punctilinealis; Dessauer et al., 2000), and these hybrids are bisexual, not parthenogens.

Our few successes in obtaining hybrids among various species of Aspidoscelis in captivity is consistent with the above, particularly considering that apparently none was fertile. For more than 29 years, from 18 June 1973–5 August 2002 (349 months), we had at least one male and one female of different species caged together in hopes that they would reproduce. Throughout this period of time, we had a total of 74 males of four species caged with 156 females of nine species (table 6) and obtained only the five genetically confirmed hybrid lizards from three clutches of eggs (see above). In most instances, we caged only one or two males per cage with one to four females. Before discussing the results in table 6, however, it is appropriate to discuss the following general caveats:

With the above caveats in mind, we now discuss the results shown in table 6. These include attempts to have hybridization between two bisexual species (inornata, gularis, sexlineata, and tigris), between a bisexual species and diploid parthenogen (laredoensis, neomexicana, and tesselata), and between a bisexual species and triploid parthenogen (exsanguis and sonorae [which included a few individuals that some herpetologists would refer to A. flagellicauda]).

Our greatest efforts (table 6) were to recreate the hybrid origins of A. neomexicana (A. t. marmorata × A. inornata and the reciprocal cross) and A. laredoensis (A. gularis × A. sexlineata [see Wright et al., 1983] and the reciprocal cross). Only the reciprocal of the first of these crosses produced laboratory hybrids, and all were from a single clutch of eggs.

TABLE 6.

Combinations of lizards^a caged together for possible hybridization^b

When a total sample of 10 or more females was involved, the highest frequency of mating or attempted mating (90.0%) involved males of inornata and females of sexlineata, the latter of which included individuals from population samples ranging from New Mexico to Georgia. Considering the bisexual species included in the phylogenetic analysis (Reeder et al., 2002), which included all of these species, inornata and sexlineata are among the closest relatives analyzed. Nevertheless, only a single clutch of eggs resulted in hatchlings from these couplings (see above), and the hatchlings all died within a matter of weeks.

When a sample of 10 or more females was involved, the second highest frequency of mating or attempted mating (70.0%) involved males of sexlineata and females of neomexicana, and only one hybrid was produced from these couplings (see above). In case it is pertinent, inornata was one of the ancestors of neomexicana, and as mentioned above, sexlineata is a close relative. Nevertheless, captive males in most cases attempted to mate with their female cagemates, regardless of distance of relationship. Captive males also frequently mated with males of their own species.

Despite the considerable caveats, we now conclude that hybrid origins of unisexual species of whiptails are rather rare events, although the evidence indicates that all unisexual whiptails had a hybrid origin (with postformational mutations creating clonal diversity).