Disentangling the genetic effects of refugial isolation and range expansion in a trans-continentally distributed species.

In wide-ranging taxa with historically dynamic ranges, past allopatric isolation and range expansion can both influence the current structure of genetic diversity. Considering alternate historical scenarios involving expansion from either a single refugium or from multiple refugia can be useful in differentiating the effects of isolation and expansion. Here, we examined patterns of genetic variability in the trans-continentally distributed painted turtle (Chrysemys picta). We utilized an existing phylogeographic dataset for the mitochondrial control region and generated additional data from nine populations for the mitochondrial control region (n = 302) and for eleven nuclear microsatellite loci (n = 247). We created a present-day ecological niche model (ENM) for C. picta and hindcast this model to three reconstructions of historical climate to define three potential scenarios with one, two, or three refugia. Finally, we employed spatially-explicit coalescent simulations and an approximate Bayesian computation (ABC) framework to test which scenario best fit the observed genetic data. Simulations indicated that phylogeographic and multilocus population-level sampling both could differentiate among refugial scenarios, although inferences made using mitochondrial data were less accurate when a longer coalescence time was assumed. Furthermore, all empirical genetic datasets were most consistent with expansion from a single refugium based on ABC. Our results indicate a stronger role for post-glacial range expansion, rather than isolation in allopatric refugia followed by range expansion, in structuring diversity in this species. To distinguish among complex historical scenarios, we recommend explicitly modeling the effects of range expansion and evaluating alternate refugial scenarios for wide-ranging taxa.

Comparing and combining distance-based and character-based approaches for barcoding turtles.

Molecular barcoding can serve as a powerful tool in wildlife forensics and may prove to be a vital aid in conserving organisms that are threatened by illegal wildlife trade, such as turtles (Order Testudines). We produced cytochrome oxidase subunit one (COI) sequences (650 bp) for 174 turtle species and combined these with publicly available sequences for 50 species to produce a data set representative of the breadth of the order. Variability within the barcode region was assessed, and the utility of both distance-based and character-based methods for species identification was evaluated. For species in which genetic material from more than one individual was available (n = 69), intraspecific divergences were 1.3% on average, although divergences greater than the customary 2% barcode threshold occurred within 15 species. High intraspecific divergences could indicate species with a high degree of internal genetic structure or possibly even cryptic species, although introgression is also probable in some of these taxa. Divergences between species of the same genus were 6.4% on average; however, 49 species were <2% divergent from congeners. Low levels of interspecific divergence could be caused by recent evolutionary radiations coupled with the low rates of mtDNA evolution previously observed in turtles. Complementing distance-based barcoding with character-based methods for identifying diagnostic sets of nucleotides provided better resolution in several cases where distance-based methods failed to distinguish species. An online identification engine was created to provide character-based identifications. This study constitutes the first comprehensive barcoding effort for this seriously threatened order.

Adaptations to terrestrial overwintering of hatchling northern map turtles, Graptemys geographica.

We conducted a 3-year field and laboratory study of winter biology in hatchlings of the northern map turtle ( Graptemys geographica). At our study area in northern Indiana, hatchlings routinely overwintered in their natal nests, emerging after the weather warmed in spring. Winter survival was excellent despite the fact that hatchlings were exposed frequently to subfreezing temperatures (to -5.4 degrees C). In the laboratory, cold-acclimated hatchlings exhibited low rates of evaporative water loss (mean=2.0 mg g(-1) day(-1)), which would enable them to conserve body water during winter. Laboratory-reared hatchlings were intolerant of freezing at -2.5 degrees C for 24 h, conditions that are readily survived by freeze-tolerant species of turtles. Winter survival of hatchling G. geographica probably depended on their extensive capacity for supercooling (to -14.8 degrees C) and their well-developed resistance to inoculative freezing, which may occur when hatchlings contact ice and ice-nucleating agents present in nesting soil. Supercooled hatchlings survived a brief exposure to -8 degrees C. Others, held at -6 degrees C for 5 days, maintained ATP concentrations at control levels, although they did accumulate lactate and glucose, probably in response to tissue hypoxia. Therefore, anoxia tolerance, as evidenced by the viability of hatchlings exposed to N(2) gas for 8 days, may promote survival during exposure to subfreezing temperatures.

Cold-hardiness and evaporative water loss in hatchling turtles.

North American turtles hatch in late summer and spend their first winter either on land or underwater. Adaptations for terrestrial overwintering of hatchlings in northern regions, where winter thermal and hydric regimes are harsh, have not been systematically investigated in many species. We measured intrinsic supercooling capacity, resistance to inoculative freezing, and desiccation resistance in hatchlings of terrestrial and aquatic turtles collected from northern (Terrapene ornata, Chrysemys picta bellii, Kinosternon flavescens, Chelydra serpentina) and southern (Chrysemys picta dorsalis, Trachemys scripta, Sternotherus odoratus, Sternotherus carinatus) locales. Supercooling capacity was estimated from the crystallization temperature of turtles cooled in the absence of external ice nuclei. Mean values ranged from -8.1 degrees to -15.5 degrees C and tended to be lower in terrestrial hibernators. Inoculation resistance was estimated from the crystallization temperature of turtles cooled in a matrix of frozen soil. These values (range of means: -0.8 degrees to -13.6 degrees C) also tended to be lower in the terrestrial hibernators, especially C. picta bellii. Mean rates of evaporative water loss varied markedly among the species (0.9-11.4 mg g(-1) d(-1)) and were lowest in the terrestrial hibernators. Most species tolerated the loss of a modest amount of body water, although half of the sample of S. carinatus died from desiccation. In general, turtles did not regain lost body water from wet soil, and immersion in free water was required for rehydration. Therefore, desiccation resistance may be an important adaptation to terrestrial hibernation. Resistances to inoculative freezing and desiccation were directly correlated, perhaps because they are governed by the same morphological characteristics.

Molecular phylogeny and biogeography of Kinosternon flavescens based on complete mitochondrial control region sequences.

Nucleotide sequences for the complete mitochondrial control region (1158 bp) were used to determine the phylogenetic relationships among populations of the yellow mud turtle, Kinosternon flavescens (Kinosternidae). Phylogenetic analysis of the mtDNA sequences reveals a polyphyletic K. flavescens with three distinct clades: (1) K. flavescens of the Central Plains, including isolated populations of Illinois and Iowa, (2) K. arizonense in the Sonoran Desert of Arizona and Mexico, and (3) K. durangoense in the Chihuahuan Desert of Durango, Coahuila, and Chihuahua, Mexico. Sequence divergence and nucleotide diversity calculations support a hypothesis of Great Plains K. flavescens dispersal and subsequent isolation of populations in the Midwest related to climatic change during the Pleistocene.

Seasonal changes in physiology and development of cold hardiness in the hatchling painted turtle Chrysemys picta.

Hatchling painted turtles (Chrysemys picta) commonly hibernate in shallow, natal nests where winter temperatures may fall below -10 degrees C. Although hatchlings are moderately freeze-tolerant, they apparently rely on supercooling to survive exposure to severe cold. We investigated seasonal changes in physiology and in the development of supercooling capacity and resistance to inoculative freezing in hatchling Chrysemys picta exposed in the laboratory to temperatures that decreased from 22 to 4 degrees C over a 5.5 month period. For comparison, we also studied hatchling snapping turtles (Chelydra serpentina), a less cold-hardy species that usually overwinters under water. Although Chrysemys picta and Chelydra serpentina differed in some physiological responses, both species lost dry mass, catabolized lipid and tended to gain body water during the acclimation regimen. Recently hatched, 22 degrees C-acclimated Chrysemys picta supercooled only modestly (mean temperature of crystallization -6.3+/-0.2 degrees C; N=6) and were susceptible to inoculation by ice nuclei in a frozen substratum (mean temperature of crystallization -1.1+/-0.1 degrees C; N=6) (means +/- s.e.m.). In contrast, cold-acclimated turtles exhibited pronounced capacities for supercooling and resistance to inoculative freezing. The development of cold hardiness reflected the elimination or deactivation of potent endogenous ice nuclei and an elevation of blood osmolality that was due primarily to the retention of urea, but was not associated with accumulation of the polyols, sugars or amino acids commonly found in the cryoprotection systems of other animals. Also, Chrysemys picta (and Chelydra serpentina) lacked both antifreeze proteins and ice-nucleating proteins, which are used by some animals to promote supercooling and to initiate freezing at the high temperatures conducive to freezing survival, respectively.

Soil hydric characteristics and environmental ice nuclei influence supercooling capacity of hatchling painted turtles Chrysemys picta.

Hatchling painted turtles (Chrysemys picta) hibernate in their shallow natal nests where temperatures occasionally fall below -10 C during cold winters. Because the thermal limit of freeze tolerance in this species is approximately -4 C, hatchlings rely on supercooling to survive exposure to extreme cold. We investigated the influence of environmental ice nuclei on susceptibility to inoculative freezing in hatchling C. picta indigenous to the Sandhills of west-central Nebraska. In the absence of external ice nuclei, hatchlings cooled to -14.6 1.9 C (mean s.e.m.; N=5) before spontaneously freezing. Supercooling capacity varied markedly among turtles cooled in physical contact with sandy soil collected from nesting locales or samples of the native soil to which water-binding agents (clay or peat) had been added, despite the fact that all substrata contained the same amount of moisture (7.5 % moisture, w/w). The temperature of crystallization (Tc) of turtles exposed to frozen native soil was -1.6 0.4 C (N=5), whereas turtles exposed to frozen soil/clay and soil/peat mixtures supercooled extensively (mean Tc values approximately -13 C). Hatchlings cooled in contact with drier (less than or equal to 4 % moisture) native soil also supercooled extensively. Thus, inoculative freezing is promoted by exposure to sandy soils containing abundant moisture and little clay or organic matter. Soil collected at turtle nesting locales in mid and late winter contained variable amounts of moisture (4-15 % w/w) and organic matter (1-3 % w/w). In addition to ice, the soil at turtle nesting locales may harbor inorganic and organic ice nuclei that may also seed the freezing of hatchlings. Bulk samples of native soil, which were autoclaved to destroy any organic nuclei, nucleated aqueous solutions at approximately -7 C (Tc range -6.1 to -8.2 C). Non-autoclaved samples contained water-extractable, presumably organic, ice nuclei (Tc range -4.4 to -5.3 C). Ice nuclei of both classes varied in potency among turtle nesting locales. Interaction with ice nuclei in the winter microenvironment determines whether hatchling C. picta remain supercooled or freeze and may ultimately account for differential mortality in nests at a given locale and for variation in winter survival rates among populations.

Character congruence and phylogenetic signal in molecular and morphological data sets: a case study in the living Iguanas (Squamata, Iguanidae).

The lizard family Iguanidae comprises eight living genera distributed throughout the New and Old World, and includes several island endemics. We reconstruct phylogenetic relationships among these genera using 90 previously published morphological characters, to which we add a molecular (mtDNA sequence) data set that includes 742 nucleotides of the ND4 gene and the complete sequences of the histidine, serine, and leucine tRNAs (217 nucleotides). Trees were initially constructed separately from these three data sets, and then tested for significant conflict in topologies that would suggest the influence of different evolutionary processes. The three data sets were then combined, and a single tree was obtained from the total evidence that permitted identification of potential sources of character incongruence. Several additional analyses of the combined data sets were repeated with sequential deletion of successive classes of homoplastic characters, and we show that the same single tree topology is recovered in most cases. However, part of the tree structure collapses when the matrix of combined characters is completely purged of all homoplastic characters. We argue that this extreme results in an unacceptable loss of phylogenetic information, and we present a single phylogenetic hypothesis for all living genera of iguanas. We show that this hypothesis is significantly more parsimonious than either of two previously published trees, and we discuss the evolution and biogeography of the Iguanidae based on the preferred hypothesis.