Merging social hierarchies: Effects on dominance rank in male green swordtail fish (Xiphophorus helleri)

When the same set of individuals are placed in different social contexts, some groups members often experience a change in dominance status. We examined the context-dependence of social status using a group fusion protocol in male green swordtail fish (Xiphophorus helleri). Six individuals were matched for size and separated into two groups of three fish. Each triad established a stable hierarchy after which time the two subgroups were merged into one larger assemblage. The maintenance of within- and between-group rank relationships was examined. Relative rank was preserved within each subgroup across social contexts but we found no evidence that familiarity with dominant animals assists individuals of one subgroup in achieving higher rank (coat-tail effects). Dominant individuals from the pre-fusion groups were significantly likely to obtain high status in the merged group and vice versa for subordinate pre-fusion animals. These results demonstrate that social rank in swordtails is relatively impervious to changes in social context, but we address some deviations from this trend. Small differences in standard length were a significant predictor of the most dominant rank in the post-fusion hierarchy, with the largest animals tending to occupy the alpha position. We discuss our results in terms of the potential factors involved in within- and between-group rank maintenance, including individual recognition, winner and loser effects, or asymmetries in dominance-related characteristics.     

Distinguishing kinds of prior dominance and subordination experiences in males of green swordtail fish (Xiphophorus helleri)

In experiments, there are usually two general ways of obtaining dominants and subordinates to test for the effect of recent experience upon ulterior behavior and dominance. One is to ‘impose’ such an experience on the contestants by a priori deciding which individual of the pair will become the dominant and which will become the subordinate through the use of rigged contests. The second technique is to let contestants ‘self-select’ the winner and loser by waiting for the spontaneous outcome of dyadic encounters between two usually well matched opponents. These two techniques of obtaining dominants and subordinates probably represent extreme cases on a single continuum of investment made by animals to settle dominance. To test this, we compared dominants and subordinates obtained from these two techniques in Xiphophorus fish males. It was found that pairs obtained through rigged contest (R) were much more aggressive in subsequent encounters than pairs in which the dominant and subordinate could self-select (S). They recuperated more rapidly from handling, initiated contact earlier, took more time to assess each other, and fought for a longer period of time. Prior-winners and prior-losers of the R condition more frequently relied on aggressive behavior during contest than that of the S condition. As a consequence, prior-winners and prior-losers of the R condition won equally the subsequent contest. On the contrary, prior-winners of the S condition defeated their prior-loser opponent in a majority of cases. These results can be tentatively explained by the following principle, winning or losing against a well matched opponent would leave more ‘experience’ than winning over a much weaker opponent, or losing to a much stronger one. This reinforces the hypothesis that prior-experiences are not qualitative states but come in various degrees.     

Sperm cryopreservation of green swordtail Xiphophorus helleri, a fish with internal fertilization

Sperm cryopreservation for fishes with internal fertilization is essentially unexplored although many species of these fishes are valuable biomedical research models. To explore methods for sperm cryopreservation within the live-bearing genus Xiphophorus, this study used X. helleri to evaluate the effects of cryoprotectant, osmotic pressure, cooling rate, equilibration time, and sperm-to-extender ratio. Sperm motility and survival duration after thawing showed significant differences among different cryoprotectants with the highest motility at 10 min after thawing obtained with 14% glycerol. With subsequent use of 14% glycerol as the cryoprotectant, the highest motility after thawing was observed with Hanks' balanced salt solution (HBSS) at 300 mOsmol/kg. Samples cooled from 5 to -80 degrees C at 20 degrees C/min yielded the highest post-thaw motility although no significant difference was found in the first 4h after thawing for cooling rates across the range of 20-35 degrees C/min. Evaluation of equilibration time revealed no significant difference between 20 min and 2h, but the highest motility at 10 min after thawing was found with a 20-min equilibration. Dilution ratios of sperm-to-extender at 1:20, 1:60, and 1:120 showed no significant differences in motility and survival duration after thawing, but the dilution of sperm solutions with HBSS (320 mOsmol/kg) immediately after thawing reduced the decline of sperm motility, and significantly prolonged the survival duration. Based on these findings, the highest average sperm motility (77%) at 10 min after thawing was obtained when sperm were suspended in HBSS at 300 mOsmol/kg with 14% glycerol as cryoprotectant, diluted at a ratio of sperm to HBSS-glycerol of 1:20, equilibrated for 10 min, cooled at 20 degrees C/min from 5 to -80 degrees C before being plunged in liquid nitrogen, and thawed in a 40 degrees C water bath for 7s. If diluted immediately after thawing, sperm frozen by the protocol above retained continuous motility after thawing for more than 8 days when stored at 4 degrees C.     

Initial studies on sperm cryopreservation of a live-bearing fish, the green swordtail Xiphophorus helleri

Swordtails and platyfish of the genus Xiphophorus are valuable models for biomedical research and are also commercially raised as ornamental fish valued by aquarists. While research use and commercial interest increases yearly in these fish, cryopreservation of sperm is unexplored in this genus. Xiphophorus are live-bearing fishes characterized by small body sizes, limited sperm volumes, and internal fertilization, an atypical reproductive mode for fish. These attributes make research involving cryopreservation of Xiphophorus germplasm challenging. To explore methods for sperm cryopreservation, this study evaluated the effect of different loading volumes of sperm suspension in 0.25-ml French straws, different dilution ratios of sperm to extender, an osmolality range of extender without cryoprotectant and with dimethyl sulfoxide (DMSO) as cryoprotectant, and short-term storage at room temperature and 4 degrees C after thawing. No significant difference in sperm motility due to straw loading volume was observed after thawing. Sperm motility was observed to decrease with increasing dilution. The osmolality of Hanks' balanced salt solution (HBSS) without cryoprotectant in which the highest sperm motility (67%) was observed was 320 +/- 3 mOsm/kg, which was also the osmolality of X. helleri blood plasma. When cryopreserved with 10% DMSO, however, the highest motilities within 10 min after thawing were observed with HBSS in the range of 240-300 mOsm/kg. Sperm suspended in HBSS at 320 mOsm/kg with a dilution factor of 100 maintained motility for 24h at room temperature, but persisted for 10 days when stored at 4 degrees C. These results provided the first evidence that cryopreservation may be applied to conservation of genetic resources in live-bearing fishes.     

Dominance hierarchies of male green swordtails (Xiphophorus helleri) in nature

Swordtail males formed dominance hierarchies at shallow sites in creeks or streams and invested much time in maintaining a high rank-order position (mean 11·1 chases per 5 min).     

Eavesdropping on visual cues in green swordtail (Xiphophorus helleri) fights: a case for networking

Aggressive contests probably occur in networking environments where information about fighting ability is conveyed both to an opponent and to individuals peripheral to the fight itself, the bystanders. Our primary aim was to investigate the relative influences of eavesdropping and prior social experience on the dynamics of aggressive contests in Xiphophorus helleri. A bystander's ability to witness an encounter was manipulated using clear, one-way mirror, and opaque partitions. After watching (or not watching) the initial contest, the bystander encountered either the winner or loser of the bout. Treatment comparisons of bystander-winner or bystander-loser contest dynamics indicated the presence or absence of winner, loser, or eavesdropping effects. Winner and loser effects had negligible influences on bystander contest dynamics. Eavesdropping significantly reduced the bystander's propensity to initiate aggression, escalate, and win against seen winners regardless of whether the watched bout had escalated or not. Though eavesdropping had relatively little effect on bystander-loser contest dynamics, bystanders were less prone to initiate aggression and win against losers that had escalated in the witnessed bout. Thus, bystanders appear to preferentially retain and utilize information gained about potentially dangerous opponents (winners or persistent losers). Our data lend clear support for the importance of eavesdropping in visually based aggressive signalling systems.     

Effects of social isolation on aggressiveness in fish with special reference to the swordtail (Xiphophorus helleri)

The attack readiness of socially isolated male swordtails ($\text{Xiphophorus helleri}$) was measured in two different aggression tests: (a) In the standard–opponent test the aggressive responses to a small, rather passive opponent swimming in a small adjacent compartment of the test aquarium were counted. (b) In the mirror test the subjects were confronted with their own mirror image. Social isolation for 4 weeks resulted in a decrease of attack readiness in both test situations. It is concluded that -- similar to cichlids -- external stimuli from conspecifics are essential for maintaining the level of aggression readiness characteristic for socially living males. There are good reasons to propose that the effect of external stimuli is mediated by the pituitary-gonadal axis. On the other hand, it is well known from previous studies that, in escalated encounters between freely swimming males, isolated males fight longer and at higher intensities for rank-order position than non-isolated males. The hypothesis is proposed that the high fighting intensity of previously isolated males could be due to an experience levelling effect of social isolation, i.e. the influence of different agonistic experience is reduced by isolation.A critical review of the literature on isolation-induced decremental and incremental effects on aggression in teleosts is presented. Often it is extremely difficult to compare the results of different authors because the methods of behavioural measurement differ to a large extent. However, it is a striking fact that social isolation of immature fish in all known cases has resulted in an increase of aggressiveness. Probably in mature males, depending on species and circumstances, either an incremental or a decremental effect of social isolation may be adaptive. On the one hand, a spontaneous increase of attack readiness may be advantageous for defence of a territory or a position in the social hierarchy and improves the ability of a single individual to join a group of strange conspecifics. On the other hand, it may be economic to lower the level of androgenic hormones to some extent in the absence of conspecifics, and this could lead to a reduction of the aggression level.     

Integrating personality research and animal contest theory: aggressiveness in the green swordtail Xiphophorus helleri

Aggression occurs when individuals compete over limiting resources. While theoretical studies have long placed a strong emphasis on context-specificity of aggression, there is increasing recognition that consistent behavioural differences exist among individuals, and that aggressiveness may be an important component of individual personality. Though empirical studies tend to focus on one aspect or the other, we suggest there is merit in modelling both within- and among-individual variation in agonistic behaviour simultaneously. Here, we demonstrate how this can be achieved using multivariate linear mixed effect models. Using data from repeated mirror trials and dyadic interactions of male green swordtails, Xiphophorus helleri, we show repeatable components of (co)variation in a suite of agonistic behaviour that is broadly consistent with a major axis of variation in aggressiveness. We also show that observed focal behaviour is dependent on opponent effects, which can themselves be repeatable but were more generally found to be context specific. In particular, our models show that within-individual variation in agonistic behaviour is explained, at least in part, by the relative size of a live opponent as predicted by contest theory. Finally, we suggest several additional applications of the multivariate models demonstrated here. These include testing the recently queried functional equivalence of alternative experimental approaches, (e.g., mirror trials, dyadic interaction tests) for assaying individual aggressiveness.     

Plasma membrane structure in spermatogenic cells of the swordtail (teleostei,Xiphophorus helleri)

The plasma membrane of spermatogenic cells of the teleost Xiphophorus helleri was examined ultrastructurally and cytochemically in order to characterize the temporal development of the membrane specializations characteristic of the mature spermatozoon. Mature sperm display a mosaic distribution of Concanavalin A and Ricinus comrnunis I binding sites; the anterior region of the head displays an intense binding that is not seen in other surface regions. This asymmetric binding is evident in early spermatids and the area of lectin binding appears associated with the plasma membrane overlying the nucleus. Transmission electron microscopy reveals that the plasma membrane over the anterior region of the head is characterized by an ordered glycocalyx and a tight adherence to the underlying nucleus. Additional membrane differentiations were revealed both in the midpiece region where a “submitochondrial net” is attached to the plasma membrane and at the base of the axoneme where the plasma membrane possesses a “collar-like” arrangement of circumferential rings. The possible functions of these differentiations, as well as their correlation to differentiations seen in sperm of other animal groups, are discussed.     

The role of individual differences and patterns of resolution in the formation of dominance orders in domestic hen triads

This research compares the role of initial individual characteristics to that of patterns of resolution in which successive dominance relationships are established during the formation of triads in the domestic hen. Combining weight and comb size with prior victory or defeat in the site of encounter, we created three levels of asymmetries of characteristics for triads of hens. The effects of these asymmetries were then examined on the resultant hierarchies and on the order of conflict resolution within triads under two different conditions of assembly. In one condition (simultaneous triad), the three hens were simultaneously introduced to each other and could thus freely choose their opponent. In the other condition (step-assembled triad), the hen predicted to occupy the highest rank was left on standby and introduced once the other two hens had settled dominance. This condition disrupts the normal process of hierarchy formation by imposing the first sequence of dominance settlement. We found that the structure of triadic hierarchies can be predicted from individual characteristics existing prior to hierarchy formation. No difference in the resultant structures were found between conditions of introduction, though different paths of conflict resolution were followed indicating that individual differences had a more determining role on the resultant hierarchies than patterns of resolution. In addition to demonstrating that individual differences determine resultant triadic structures, the present results also show that the same end structures can be reached by following resolution paths that are not necessarily of the Double Dominance and Double Subordinance types as prescribed by Chase's model. It is also found that in the simultaneous condition hens select each other to form pairs. Therefore, individuals do not meet at random but choose each other as opponents. The two hens predicted from individual differences to occupy the highest ranks first settle dominance, followed by settlement between the winner of the previous encounter and the bystander.     

Effect of osmotic immobilization on refrigerated storage and cryopreservation of sperm from a viviparous fish, the green swordtail Xiphophorus helleri

In this study, refrigerated storage and cryopreservation of sperm from the green swordtail Xiphophorus helleri were investigated. Previous cryopreservation research in this species utilized motile sperm because unlike in most fish species, Xiphophorus sperm can remain continuously motile after collection for a week with refrigerated storage. However, this species reproduces by internal fertilization, and given the significant requirements for motility within the female reproductive tract and potential limitations on sperm energetic capacities, immobilization of sperm prior to insemination could be used to improve fertilization success. Thus, the goal in this study was to use osmotic pressure to inhibit the motility of sperm after collection from X. helleri, and to test the effect of immobilization on refrigerated storage and cryopreservation. The objectives were to: (1) estimate the motility of sperm at different osmotic pressures, and determine an osmotic pressure suitable for immobilization; (2) cryopreserve the immobilized sperm, and estimate the motility after thawing with or without dilution, and (3) compare motility of non-immobilized and immobilized sperm after thawing, centrifugation, and washing to remove cryoprotectant. Motility was determined when sperm were suspended in 11 different osmotic pressures (24-500 mOsmol/kg) of Hanks' balanced salt solution (HBSS). Motility was observed between 116 and 425 mOsmol/kg. Sperm were not motile when the osmolality was lower than 116 or higher than 425 mOsmol/kg. Motility of the immobilized (non-motile) sperm could be activated by changing the osmotic pressure to 291-316 mOsmol/kg, and motility of immobilized sperm from hypertonic HBSS (425 mOsmol/kg) was significantly higher than that from hypotonic HBSS (145 mOsmol/kg) after 48 h of storage. At an osmolality of 500 mOsmol/kg, HBSS was used as extender to maintain immobilized sperm during cryopreservation with glycerol as the cryoprotectant. High motility (approximately 55%) was obtained in sperm after thawing when cryopreserved with 10-15% glycerol, and dilution of thawed sperm in fresh HBSS (1:4; V:V) was found to decrease the motility significantly. No difference was found in the motility of thawed sperm cryopreserved with 14% glycerol and extended in 310 and 500 mOsmol/kg HBSS. Washing by centrifugation prolonged the motility of thawed sperm from 24 to 72 h in HBSS at 310 and 500 mOsmol/kg. This study showed that sperm from X. helleri could be immobilized by use of specific osmotic pressures, and that the immobilization did not affect sperm motility after thawing. The immobilization of sperm by osmotic pressure could minimize reduction of the energetic capacities necessary for insemination, traversal, and residence within the female reproductive tract, and fertilization.     

The function of aggression in the swordtail, Xiphophorus helleri: resource defence

Competition for mating opportunities may involve exclusion of intrasexual competitors (direct) or defending resources necessary to attract mates (indirect). Male swordtails (Xiphophorus helleri) engage in direct competition. Moreover in natural populations they defend a home range. This study aimed to test whether this home range defence is a form of food resource defence, which may therefore have a female attraction function. Male swordtails did defend a food resource and showed increased aggression in the presence of both food and females. However, food resource defence decreased when females were present, suggesting that both food and females are treated as defendable resources.     

Sperm acrosome antigen-1. Specific tissue distribution of immunoreactivity in a teleost fish,the swordtail ( Xiphophorus helleri)

The acrosome reaction is a fundamental requirement for mammalian fertilization. Its exact molecular mechanisms and cellular elements are still poorly understood. We have detected an acrosomal sperm antigen, SAA-1, by monoclonal antibodies directed against SAA-1, that appears to be critically involved in the regulation of the acrosome reaction. SAA-1 is conserved within a broad range of mammalian species, emphasizing its important role in mammalian reproduction. Here we demonstrate that SAA-1 is immunohistochemically detectable in a nonmammalian vertebrate whose sperm do not possess an acrosome. In the swordtail, a live-bearing teleost with special reproductive tactics, we were able to demonstrate immunoreactivity of sperm heads of spermatids and mature sperm in the testis using monoclonal antibodies against SAA-1. Due to the cystic spermatogenesis with synchronous sperm maturation, immunoreactive maturational stages could be clearly identified. Stored immunoreactive sperm were also identified in spermathecal tissue of the female genital tract. Interestingly, immunoreactivity was furthermore detected in defined cells of the compounded endocrine organs pituitary and endocrine pancreas. All these different cell systems are involved in paracrine regulation and exhibit exocytotic properties. The possible nature of SAA-1 is discussed. Additionally, some new aspects of the morphologic composition of the swordtail pituitary are described.     

A theoretically estimated optimal cooling rate for the cryopreservation of sperm cells from a live-bearing fish, the green swordtail Xiphophorus helleri

Sperm cryopreservation of live-bearing fishes, such as those of the genus Xiphophorus is only beginning to be studied, although these fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. To explore optimization of techniques for sperm cryopreservation of these fishes, this study measured the volumetric shrinkage response during freezing of sperm cells of Xiphophorus helleri by use of a shape-independent differential scanning calorimeter (DSC) technique. Volumetric shrinkage during freezing of X. helleri sperm cell suspensions was obtained in the presence of extracellular ice at a cooling rate of 20 degrees C/min in three different media: (1) Hanks' balanced salt solution (HBSS) without cryoprotective agents (CPAs); (2) HBSS with 14% (v/v) glycerol; and (3) HBSS with 10% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder of 33.3 microm in length and 0.59 microm in diameter with an osmotically inactive cell volume (V(b)) of 0.6V(o), where V(o) is the isotonic or initial cell volume. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best-fit membrane permeability parameters (reference membrane permeability to water, L(pg) or L(pg)[cpa] and the activation energy, E(Lp) or E(Lp)[cpa]) of the Xiphophorus helleri sperm cell membrane were determined. The best-fit membrane permeability parameters at 20 degrees C/min in the absence of CPAs were: L(pg)=0.776 x 10(-15)m3/Ns (0.0046 microm/min atm), and E(Lp)=50.1 kJ/mol (11.97 kcal/mol) (R2=0.997). The corresponding parameters in the presence of 14% glycerol were L(pg)[cpa]=1.063 x 10(-15)m3/Ns (0.0063 microm/min atm), and E(Lp)[cpa]=83.81 kJ/mol (20.04 kcal/mol) (R2=0.997). The parameters in the presence of 10% DMSO were L(pg)[cpa]=1.4 x 10(-15)m3/Ns (0.0083 microm/min atm), and E(Lp)[cpa]=90.96 kJ/mol (21.75 kcal/mol) (R2=0.996). Parameters obtained in this study suggested that the optimal rate of cooling for X. helleri sperm cells in the presence of CPAs ranged from 20 to 35 degrees C/min and were in close agreement with recently published, empirically determined optimal cooling rates.     

Covariation between predation risk, body size and fin elaboration in the green swordtail, Xiphophorus helleri

Natural and sexual selection can have either opposing or synergistic effects on the evolution of traits. In the green swordtail Xiphophorus helleri , sexual selection arising from female choice is known to favour larger males and males with longer swords. We examined variation in male and female size and fin morphology among 15 populations that varied in their predation environments. Males and females from populations in which piscivorous fishes were present had longer and deeper bodies than did males and females from populations in which piscivorous fishes were absent. Controlling for a positive effect of body size on sword length, males from populations in which piscivores were present had relatively shorter swords than did males from populations in which piscivores were absent. The associations between morphology and predation environment may be due to direct effects of predation, indirect effects of predation, other sources of selection that covary with predator presence, or other environmental effects on trait expression. These results suggest that while sexual selection favours longer swords, natural selection may have an opposing effect on sword length in populations with predators. Natural selection on body size, however, may act synergistically with sexual selection in populations with predators; both may favour the evolution of larger body size. The body size results for X. helleri contrast with related taxa that have become model systems for the study of life history evolution.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 83 , 87–100.     

Production of F1 interspecies hybrid offspring with cryopreserved sperm from a live-bearing fish, the swordtail Xiphophorus helleri

Despite study of sperm cryopreservation in more than 200 fish species, production of broods from cryopreserved sperm in live-bearing fish has not been demonstrated. This has not been due to a lack of effort, but instead is a result of the unique morphology, biology, and biochemistry of reproduction in viviparous fishes. For example, sperm of Xiphophorus helleri have a cylindrical nucleus, can swim for days after being activated, have glycolytic capabilities, and can reside in the female reproduction tract for months before fertilization. These traits are not found in fishes with external fertilization. The long-standing research use of the genus Xiphophorus has led to development of over 60 pedigreed lines among the 26 species maintained around the world. These species and lines serve as contemporary models in medical research, although they must be maintained as live populations. Previous attempts at establishing sperm cryopreservation protocols for Xiphophorus have not produced live young. To address this we have been studying the parameters surrounding cryobiology of Xiphophorus sperm and applying this information to an improved understanding of internal fertilization and reproduction. Here we report the first successful fertilization and offspring production by cryopreserved sperm in any live-bearing fish. This claim is supported by our use of artificial insemination between two species that yield distinct hybrid offspring to verify paternity via cryopreserved sperm. We provide a practical approach for preservation of valuable genetic resources from live-bearing fish species, a group that is rapidly being lost due to destruction of native habitats.     

Some properties of an established fish cell line fromXiphophorus helleri (red swordtail)

Summary A cell line (SWT) was established from embryonic tissue of the red swordtailXiphophorus helleri. The SWT cells grew optimally, at 26°C to 30°C in Eagle's basal medium plus 10% fetal calf serum but failed to grow at 16°C and 37°C. After 50 subcultivations, the cells remained contact-inhibited and were pseudodiploid with a chromosomal modal number of 46. Virological studies demonstrated that SWT cells supported replication of the following viruses at the indicated temperatures: IPN virus (22°C), FV-3 (30°C), and VSV (33°C). The following mammalian, viruses failed to replicate at 33°C: vaccinia, poliovirus 2, herpes simplex, and reovirus 2. Although not replicating, reovirus induced interferon in there cells. This work was supported in part by a grant from the University Research Council. A part of these results was presented at the 22nd Annual Meeting of the Tissue Culture Association, Lake Placid, N. Y. 1971.     

Genetic variation and phylogeography of the swordtail fish Xiphophorus cortezi (Cyprinodontiformes, Poeciliidae)

Swordtail fish have been studied extensively in relation to diverse aspects of biology; however, little attention has been paid to the patterns of genetic variation within and among populations of swordtails. In this study, we sequenced the mtDNA control region from 65 individuals and 10 populations of Xiphophorus cortezi to investigate the genetic variation within and among populations, including tests for correlations between genetic and geographic distances and tests for species monophyly. We found low gene and nucleotide diversity within populations and high degrees of genetic differentiation among populations. Significant and positive correlations between genetic distance and both river and straight-line geographic distance indicate that genetic differentiation among X. cortezi populations can be explained, to some extent, by an isolation-by-distance model and provide evidence of stream capture. Phylogenetic analyses suggest that X. cortezi is paraphyletic relative to X. malinche, raising questions concerning the status of these taxa as separate species.     

Phylogeography and monophyly of the swordtail fish species Xiphophorus birchmanni (Cyprinodontiformes, Poeciliidae)

We used sequences of the mitochondria control region to assess the distribution of genetic variation within and among populations of the poeciliid fish species Xiphophorus birchmanni . We collected 122 X. birchmanni samples from 11 sites in three drainage systems comprising the distribution of the species. We found low levels of polymorphism among aligned sequences and low levels of genetic variation within populations but high levels of genetic differentiation among populations. Haplotypes are exclusive to three river drainages (Los Hules, Calabozo and San Pedro). Mantel tests revealed correlations between geographical (both straight-line and river distances) and genetic distance, consistent with an isolation by distance scenario, while nested clade analysis suggested allopatric fragmentation between haplotypes from two of the major drainages, and isolation by distance with restricted gene flow within those drainages. Finally, monophyly of X. birchmanni is strongly supported while the previous hypothesis of the evolutionary origin of this species from X. malinche is not.