Boom-bust population dynamics drive rapid genetic change.

Increasing environmental threats and more extreme environmental perturbations place species at risk of population declines, with associated loss of genetic diversity and evolutionary potential. While theory shows that rapid population declines can cause loss of genetic diversity, populations in some environments, like Australia's arid zone, are repeatedly subject to major population fluctuations yet persist and appear able to maintain genetic diversity. Here, we use repeated population sampling over 13 y and genotype-by-sequencing of 1903 individuals to investigate the genetic consequences of repeated population fluctuations in two small mammals in the Australian arid zone. The sandy inland mouse (Pseudomys hermannsburgensis) experiences marked boom-bust population dynamics in response to the highly variable desert environment. We show that heterozygosity levels declined, and population differentiation (FST) increased, during bust periods when populations became small and isolated, but that heterozygosity was rapidly restored during episodic population booms. In contrast, the lesser hairy-footed dunnart (Sminthopsis youngsoni), a desert marsupial that maintains relatively stable population sizes, showed no linear declines in heterozygosity. These results reveal two contrasting ways in which genetic diversity is maintained in highly variable environments. In one species, diversity is conserved through the maintenance of stable population sizes across time. In the other species, diversity is conserved through rapid genetic mixing during population booms that restores heterozygosity lost during population busts.

Episodic population fragmentation and gene flow reveal a trade-off between heterozygosity and allelic richness.

In episodic environments like deserts, populations of some animal species exhibit irregular fluctuations such that populations are alternately large and connected or small and isolated. Such dynamics are typically driven by periodic resource pulses due, for example, to large but infrequent rainfall events. The repeated population bottlenecks resulting from fragmentation should lower genetic diversity over time, yet species undergoing these fluctuations appear to maintain high levels of genetic diversity. To resolve this apparent paradox, we simulated a metapopulation of constant size undergoing repeat episodes of fragmentation and change in gene flow to mimic outcomes experienced by mammals in an Australian desert. We show that episodic fragmentation and gene flow have contrasting effects on two measures of genetic diversity: heterozygosity and allelic richness. Specifically, fragmentation into many, small subpopulations, coupled with periods of infrequent gene flow, preserves allelic richness at the expense of heterozygosity. In contrast, fragmentation into a few, large subpopulations maintains heterozygosity at the expense of allelic richness. The strength of the trade-off between heterozygosity and allelic richness depends on the amount of gene flow and the frequency of gene flow events. Our results imply that the type of genetic diversity maintained among species living in strongly fluctuating environments will depend on the way populations fragment, with our results highlighting different mechanisms for maintaining allelic richness and heterozygosity in small, fragmented populations.

Metabolic consequences of sex reversal in two lizard species: a test of the like-genotype and like-phenotype hypotheses.

Vertebrate sex is typically determined genetically, but in many ectotherms sex can be determined by genes (genetic sex determination, GSD), temperature (temperature-dependent sex determination, TSD), or interactions between genes and temperature during development. TSD may involve GSD systems with either male or female heterogamety (XX/XY or ZZ/ZW) where temperature overrides chromosomal sex determination to cause a mismatch between genetic sex and phenotypic sex (sex reversal). In these temperature-sensitive lineages, phylogenetic investigations point to recurrent evolutionary shifts between genotypic and temperature-dependent sex determination. These evolutionary transitions in sex determination can occur rapidly if selection favours the reversed sex over the concordant phenotypic sex. To investigate the consequences of sex reversal on offspring phenotypes, we measured two energy-driven traits (metabolism and growth) and 6 month survival in two species of reptile with different patterns of temperature-induced sex reversal. Male sex reversal occurs in Bassiana duperreyi when chromosomal females (female XX) develop male phenotypes (maleSR XX), while female sex reversal occurs in Pogona vitticeps when chromosomal males (male ZZ) develop female phenotypes (femaleSR ZZ). We show metabolism in maleSR XX was like that of male XY; that is, reflective of phenotypic sex and lower than genotypic sex. In contrast, for Pogona vitticeps, femaleSR ZZ metabolism was intermediate between male ZZ and female ZW metabolic rate. For both species, our data indicate that differences in metabolism become more apparent as individuals become larger. Our findings provide some evidence for an energetic advantage from sex reversal in both species but do not exclude energetic processes as a constraint on the distribution of sex reversal in nature.

Evolutionary stability inferred for a free ranging lizard with sex-reversal.

The sex of vertebrates is typically determined genetically, but reptile sex can also be determined by developmental temperature. In some reptiles, temperature interacts with genotype to reverse sex, potentially leading to transitions from a chromosomal to a temperature-dependent sex determining system. Transitions between such systems in nature are accelerated depending on the frequency and fitness of sex-reversed individuals. The Central Bearded Dragon, Pogona vitticeps, exhibits female heterogamety (ZZ/ZW) but can have its sex reversed from ZZ male to ZZ female by high incubation temperatures. The species exhibits sex-reversal in the wild and it has been suggested that climate change and fitness of sex-reversed individuals could be increasing the frequency of reversal within the species range. Transitions to temperature-dependent sex determination require low levels of dispersal and high (>50%) rates of sex-reversal. Here, we combine genotype-by-sequencing, identification of phenotypic and chromosomal sex, exhaustive field surveys, and radio telemetry to examine levels of genetic structure, rates of sex-reversal, movement, space use, and survival of P. vitticeps in a location previously identified as a hot spot for sex-reversal. We find that the species exhibits low levels of population structure (FST ~0.001) and a modest (~17%) rate of sex-reversal, and that sex-reversed and nonsex-reversed females have similar survival and behavioural characteristics to each other. Overall, our data indicate this system is evolutionary stable, although we do not rule out the prospect of a more gradual transition in sex-determining mechanisms in the future in a more fragmented landscape and as global temperatures increase.

Sex reversal triggers the rapid transition from genetic to temperature-dependent sex.

Sex determination in animals is amazingly plastic. Vertebrates display contrasting strategies ranging from complete genetic control of sex (genotypic sex determination) to environmentally determined sex (for example, temperature-dependent sex determination). Phylogenetic analyses suggest frequent evolutionary transitions between genotypic and temperature-dependent sex determination in environmentally sensitive lineages, including reptiles. These transitions are thought to involve a genotypic system becoming sensitive to temperature, with sex determined by gene-environment interactions. Most mechanistic models of transitions invoke a role for sex reversal. Sex reversal has not yet been demonstrated in nature for any amniote, although it occurs in fish and rarely in amphibians. Here we make the first report of reptile sex reversal in the wild, in the Australian bearded dragon (Pogona vitticeps), and use sex-reversed animals to experimentally induce a rapid transition from genotypic to temperature-dependent sex determination. Controlled mating of normal males to sex-reversed females produces viable and fertile offspring whose phenotypic sex is determined solely by temperature (temperature-dependent sex determination). The W sex chromosome is eliminated from this lineage in the first generation. The instantaneous creation of a lineage of ZZ temperature-sensitive animals reveals a novel, climate-induced pathway for the rapid transition between genetic and temperature-dependent sex determination, and adds to concern about adaptation to rapid global climate change.

Karyotype Characterisation of Two Australian Dragon Lizards (Squamata: Agamidae: Amphibolurinae) Reveals Subtle Chromosomal Rearrangements Between Related Species with Similar Karyotypes.

Agamid lizards (Squamata: Agamidae) are karyotypically heterogeneous. Among the 101 species currently described from Australia, all are from the subfamily Amphibolurinae. This group is, with some exceptions, karyotypically conserved, and all species involving heterogametic sex show female heterogamety. Here, we describe the chromosomes of 2 additional Australian agamid lizards, Tympanocryptis lineata and Rankinia diemensis. These species are phylogenetically and cytogenetically sisters to the well-characterised Pogona vitticeps, but their sex chromosomes and other chromosomal characteristics are unknown. In this study, we applied advanced molecular cytogenetic techniques, such as fluorescence in situ hybridisation (FISH) and cross-species gene mapping, to characterise chromosomes and to identify sex chromosomes in these species. Our data suggest that both species have a conserved karyotype with P. vitticeps but with subtle rearrangements in the chromosomal landscapes. We could identify that T. lineata possesses a female heterogametic system (ZZ/ZW) with a pair of sex microchromosomes, while R. diemensis may have heterogametic sex chromosomes, but this requires further investigations. Our study shows the pattern of chromosomal rearrangements between closely related species, explaining the speciation within Australian agamid lizards of similar karyotypes.

Climate change, sex reversal and lability of sex-determining systems.

Sex reversal at high temperatures during embryonic development (e.g., ZZ females) provides the opportunity for new genotypic crosses (e.g., ZZ male × ZZ female). This raises the alarming possibility that climatic warming could lead to the loss of an entire chromosome-one member of the sex chromosome pair (the Y or W)-and the transition of populations to environmental sex determination (ESD). Here we examine the evolutionary dynamics of sex-determining systems exposed to climatic warming using theoretical models. We found that the loss of sex chromosomes is not an inevitable consequence of sex reversal. A large frequency of ZZ sex reversal (50% reversal from male to female) typically divides the outcome between loss of the ZW genotype and the stable persistence of ZZ males, ZW females and ZZ females. The amount of warming associated with sex chromosome loss depended on several features of wild populations-environmental fluctuation, immigration, heritable variation in temperature sensitivity and differential fecundity of sex-reversed individuals. Chromosome loss was partially or completely buffered when sex-reversed individuals suffered a reproductive fitness cost, when immigration occurred or when heritable variation for temperature sensitivity existed. Thus, under certain circumstances, sex chromosomes may persist cryptically in systems where the environment is the predominant influence on sex.

Amplification of microsatellite repeat motifs is associated with the evolutionary differentiation and heterochromatinization of sex chromosomes in Sauropsida.

The sex chromosomes in Sauropsida (reptiles and birds) have evolved independently many times. They show astonishing diversity in morphology ranging from cryptic to highly differentiated sex chromosomes with male (XX/XY) and female heterogamety (ZZ/ZW). Comparing such diverse sex chromosome systems thus provides unparalleled opportunities to capture evolution of morphologically differentiated sex chromosomes in action. Here, we describe chromosomal mapping of 18 microsatellite repeat motifs in eight species of Sauropsida. More than two microsatellite repeat motifs were amplified on the sex-specific chromosome, W or Y, in five species (Bassiana duperreyi, Aprasia parapulchella, Notechis scutatus, Chelodina longicollis, and Gallus gallus) of which the sex-specific chromosomes were heteromorphic and heterochromatic. Motifs (AAGG)n and (ATCC)n were amplified on the W chromosome of Pogona vitticeps and the Y chromosome of Emydura macquarii, respectively. By contrast, no motifs were amplified on the W chromosome of Christinus marmoratus, which is not much differentiated from the Z chromosome. Taken together with previously published studies, our results suggest that the amplification of microsatellite repeats is tightly associated with the differentiation and heterochromatinization of sex-specific chromosomes in sauropsids as well as in other taxa. Although some motifs were common between the sex-specific chromosomes of multiple species, no correlation was observed between this commonality and the species phylogeny. Furthermore, comparative analysis of sex chromosome homology and chromosomal distribution of microsatellite repeats between two closely related chelid turtles, C. longicollis and E. macquarii, identified different ancestry and differentiation history. These suggest multiple evolutions of sex chromosomes in the Sauropsida.

Identification of interleukin genes in Pogona vitticeps using a de novo transcriptome assembly from RNA-seq data.

Interleukins are a group of cytokines with complex immunomodulatory functions that are important for regulating immunity in vertebrate species. Reptiles and mammals last shared a common ancestor more than 350 million years ago, so it is not surprising that low sequence identity has prevented divergent interleukin genes from being identified in the central bearded dragon lizard, Pogona vitticeps, in its genome assembly. To determine the complete nucleotide sequences of key interleukin genes, we constructed full-length transcripts, using the Trinity platform, from short paired-end read RNA sequences from stimulated spleen cells. De novo transcript reconstruction and analysis allowed us to identify interleukin genes that are missing from the published P. vitticeps assembly. Identification of key cytokines in P. vitticeps will provide insight into the essential molecular mechanisms and evolution of interleukin gene families and allow for characterization of the immune response in a lizard for comparison with mammals.

Transitions between sex-determining systems in reptiles and amphibians.

Important technological advances in genomics are driving a new understanding of the evolution of sex determination in vertebrates. In particular, comparative chromosome mapping in reptiles has shown an intriguing distribution of homology in sex chromosomes across reptile groups. When this new understanding is combined with the widespread distribution of genetic and temperature-dependent sex-determination mechanisms among reptiles, it is apparent that transitions between modes have occurred many times, as they have for amphibians (particularly between male and female heterogamety). It is also likely that thermosensitivity in sex determination is a key factor in those transitions in reptiles, and possibly in amphibians too. New models of sex determination involving temperature thresholds are providing the framework for the investigation of transitions and making possible key predictions about the homologies and sex-determination patterns expected among taxa in these groups. Molecular cytogenetics and other genomic approaches are essential to providing the fundamental material necessary to make advances in this field.

Non-homologous sex chromosomes in two geckos (Gekkonidae: Gekkota) with female heterogamety.

Evaluating homology between the sex chromosomes of different species is an important first step in deducing the origins and evolution of sex-determining mechanisms in a clade. Here, we describe the preparation of Z and W chromosome paints via chromosome microdissection from the Australian marbled gecko (Christinus marmoratus) and their subsequent use in evaluating sex chromosome homology with the ZW chromosomes of the Kwangsi gecko (Gekko hokouensis) from eastern Asia. We show that the ZW sex chromosomes of C. marmoratus and G. hokouensis are not homologous and represent independent origins of female heterogamety within the Gekkonidae. We also show that the C. marmoratus Z and W chromosomes are genetically similar to each other as revealed by C-banding, comparative genomic hybridization, and the reciprocal painting of Z and W chromosome probes. This implies that sex chromosomes in C. marmoratus are at an early stage of differentiation, suggesting a recent origin.

Creating new evolutionary pathways through bioinvasion: the population genetics of brushtail possums in New Zealand.

Rapid increases in global trade and human movement have created novel mixtures of organisms bringing with them the potential to rapidly accelerate the evolution of new forms. The common brushtail possum (Trichosurus vulpecula), introduced into New Zealand from Australia in the 19th century, is one such species having been sourced from multiple populations in its native range. Here, we combine microsatellite DNA- and GIS-based spatial data to show that T. vulpecula originating from at least two different Australian locations exhibit a population structure that is commensurate with their introduction history and which cannot be explained by landscape features alone. Most importantly, we identify a hybrid zone between the two subspecies which appears to function as a barrier to dispersal. When combined with previous genetic, morphological and captive studies, our data suggest that assortative mating between the two subspecies may operate at a behavioural or species recognition level rather than through fertilization, genetic incompatibility or developmental inhibition. Nevertheless, hybridization between the two subspecies of possum clearly occurs, creating the opportunity for novel genetic combinations that would not occur in their natural ranges and which is especially likely given that multiple contact zones occur in New Zealand. This discovery has implications for wildlife management in New Zealand because multiple contact zones are likely to influence the dispersal patterns of possums and because differential susceptibility to baiting with sodium fluoroacetate between possums of different origins may promote novel genetic forms.

Molecular evolution of Dmrt1 accompanies change of sex-determining mechanisms in reptilia.

In reptiles, sex-determining mechanisms have evolved repeatedly and reversibly between genotypic and temperature-dependent sex determination. The gene Dmrt1 directs male determination in chicken (and presumably other birds), and regulates sex differentiation in animals as distantly related as fruit flies, nematodes and humans. Here, we show a consistent molecular difference in Dmrt1 between reptiles with genotypic and temperature-dependent sex determination. Among 34 non-avian reptiles, a convergently evolved pair of amino acids encoded by sequence within exon 2 near the DM-binding domain of Dmrt1 distinguishes species with either type of sex determination. We suggest that this amino acid shift accompanied the evolution of genotypic sex determination from an ancestral condition of temperature-dependent sex determination at least three times among reptiles, as evident in turtles, birds and squamates. This novel hypothesis describes the evolution of sex-determining mechanisms as turnover events accompanied by one or two small mutations.

The genetic signature left by the range expansion of red foxes in Australia is detectable after more than 80 years of population stability.

Reconstructing biological invasions from historical sources can provide insights into how they occur but are difficult to do when invasions are poorly documented. Genetic signatures left by invaders can also offer insights into invasion routes, points of origin and general biology but often present conclusions that are contradictory to expectations. Here, we test the ability of continental-wide microsatellite genotype data from 29 loci and 3122 samples to reconstruct the well-documented invasion of red foxes Vulpes vulpes from the United Kingdom into Australia over 150 years ago, an invasion that has led to the extinction of many native species. Our analysis reveals several key signals of invasion evident in Australian foxes. They display lower levels of diversity than foxes sampled from the UK, exhibit clines in diversity from the point of introduction (south-east Australia) to the edge of their range, and show strong evidence of allele surfing in westerly and north-easterly directions. These characteristics are consistent with a single point of origin followed by rapid expansion in westerly and north-easterly directions as suggested by historical records. We also find little genetic structure in foxes across Australia with only the vast Nullarbor Plains and Great Victoria Desert region presenting a detectable barrier to their dispersal. As such, no mainland region within the current range of foxes can be considered genetically isolated and therefore appropriate for localised eradication efforts. Overall, our analyses demonstrate the ability of comprehensive population genetic studies to reconstruct invasion histories even after more than 80 years since colonisation was stabilised.

slimr: An R package for tailor-made integrations of data in population genomic simulations over space and time.

Software for realistically simulating complex population genomic processes is revolutionizing our understanding of evolutionary processes, and providing novel opportunities for integrating empirical data with simulations. However, the integration between standalone simulation software and R is currently not well developed. Here, we present slimr, an R package designed to create a seamless link between standalone software SLiM >3.0, one of the most powerful population genomic simulation frameworks, and the R development environment, with its powerful data manipulation and analysis tools. We show how slimr facilitates smooth integration between genetic data, ecological data and simulation in a single environment. The package enables pipelines that begin with data reading, cleaning and manipulation, proceed to constructing empirically based parameters and initial conditions for simulations, then to running numerical simulations and finally to retrieving simulation results in a format suitable for comparisons with empirical data - aided by advanced analysis and visualization tools provided by R. We demonstrate the use of slimr with an example from our own work on the landscape population genomics of desert mammals, highlighting the advantage of having a single integrated tool for both data analysis and simulation. slimr makes the powerful simulation ability of SLiM directly accessible to R users, allowing integrated simulation projects that incorporate empirical data without the need to switch between software environments. This should provide more opportunities for evolutionary biologists and ecologists to use realistic simulations to better understand the interplay between ecological and evolutionary processes.

Sequence and gene content of a large fragment of a lizard sex chromosome and evaluation of candidate sex differentiating gene R-spondin 1.

BACKGROUND: Scant genomic information from non-avian reptile sex chromosomes is available, and for only a few lizards, several snakes and one turtle species, and it represents only a small fraction of the total sex chromosome sequences in these species. RESULTS: We report a 352 kb of contiguous sequence from the sex chromosome of a squamate reptile, Pogona vitticeps, with a ZZ/ZW sex microchromosome system. This contig contains five protein coding genes (oprd1, rcc1, znf91, znf131, znf180), and major families of repetitive sequences with a high number of copies of LTR and non-LTR retrotransposons, including the CR1 and Bov-B LINEs. The two genes, oprd1 and rcc1 are part of a homologous syntenic block, which is conserved among amniotes. While oprd1 and rcc1 have no known function in sex determination or differentiation in amniotes, this homologous syntenic block in mammals and chicken also contains R-spondin 1 (rspo1), the ovarian differentiating gene in mammals. In order to explore the probability that rspo1 is sex determining in dragon lizards, genomic BAC and cDNA clones were mapped using fluorescence in situ hybridisation. Their location on an autosomal microchromosome pair, not on the ZW sex microchromosomes, eliminates rspo1 as a candidate sex determining gene in P. vitticeps. CONCLUSION: Our study has characterized the largest contiguous stretch of physically mapped sex chromosome sequence (352 kb) from a ZZ/ZW lizard species. Although this region represents only a small fraction of the sex chromosomes of P. vitticeps, it has revealed several features typically associated with sex chromosomes including the accumulation of large blocks of repetitive sequences.

Are some chromosomes particularly good at sex? Insights from amniotes.

Several recent studies have produced comparative maps of genes on amniote sex chromosomes, revealing homology of gene content and arrangement across lineages as divergent as mammals and lizards. For example, the chicken Z chromosome, which shares homology with the sex chromosomes of all birds, monotremes, and a gecko, is a striking example of stability of genome organization and retention, or independent acquisition, of function in sex determination. In other lineages, such as snakes and therian mammals, well conserved but independently evolved sex chromosome systems have arisen. Among lizards, novel sex chromosomes appear frequently, even in congeneric species. Here, we review recent gene mapping data, examine the evolutionary relationships of amniote sex chromosomes and argue that gene content can predispose some chromosomes to a specialized role in sex determination.

Climate warming drives a temperate-zone lizard to its upper thermal limits, restricting activity, and increasing energetic costs.

Lizards are considered vulnerable to climate change because many operate near their thermal maxima. Exposure to higher temperatures could reduce activity of these animals by forcing them to shelter in thermal refugia for prolonged periods to avoid exceeding lethal limits. While rising temperatures should reduce activity in tropical species, the situation is less clear for temperate-zone species where activity can be constrained by both low and high temperatures. Here, we measure the effects of natural variation in environmental temperatures on activity in a temperate grassland lizard and show that it is operating near its upper thermal limit in summer even when sheltering in thermal refuges. As air temperatures increased above 32 °C, lizard activity declined markedly as individuals sought refuge in cool microhabitats while still incurring substantial metabolic costs. We estimate that warming over the last two decades has required these lizards to increase their energy intake up to 40% to offset metabolic losses caused by rising temperatures. Our results show that recent increases in temperature are sufficient to exceed the thermal and metabolic limits of temperate-zone grassland lizards. Extended periods of high temperatures could place natural populations of ectotherms under significantly increased environmental stress and contribute to population declines and extinction.

Securing the demographic and genetic future of tuatara through assisted colonization.

Climate change poses a particular threat to species with fragmented distributions and little or no capacity to migrate. Assisted colonization, moving species into regions where they have not previously occurred, aims to establish populations where they are expected to survive as climatic envelopes shift. However, adaptation to the source environment may affect whether species successfully establish in new regions. Assisted colonization has spurred debate among conservation biologists and ecologists over whether the potential benefits to the threatened species outweigh the potential disruption to recipient communities. In our opinion, the debate has been distracted by controversial examples, rather than cases where assisted colonization may be a viable strategy. We present a strategic plan for the assisted migration of tuatara (Sphenodon punctatus), an endemic New Zealand reptile. The plan includes use of extant populations as reference points for comparisons with assisted-colonization populations with respect to demography, phenotypic plasticity, and phenology; optimization of genetic variation; research to fill knowledge gaps; consideration of host and recipient communities; and inclusion of stakeholders in the planning stage. When strategically planned and monitored, assisted colonization could meet conservation and research goals and ultimately result in the establishment of long-term sustainable populations capable of persisting during rapid changes in climate.

Experimental habitat fragmentation disrupts host-parasite interaction over decades via life-cycle bottlenecks.

Habitat loss and fragmentation are likely to seriously impact parasites, a less studied but critical component of ecosystems, yet we lack long-term experimental evidence. Parasites structure communities, increase connectivity in food webs, and account for a large proportion of an ecosystem's total biomass. Food web models predict that parasites with multiple obligate hosts are at greater risk of extinction because the local extinction, or reduction in abundance, of any host will result in a life-cycle bottleneck for the parasite. We examine the response of a parasite and its multiple hosts to forest fragmentation over 26 years in the Wog Wog Habitat Fragmentation Experiment in southeastern Australia. The parasite is the nematode Hedruris wogwogensis, its intermediate host is the amphipod, Arcitalitrus sylvaticus, and its definitive host is the skink, Lampropholis guichenoti. In the first decade after fragmentation, nematodes completely disappeared from the matrix (plantation forestry) and all but disappeared from their definitive host (skinks) in fragments, and by the third decade after fragmentation had not appreciably recovered anywhere in the fragmented landscape compared to continuous forest. The low prevalence of the nematode in the fragmented landscape was associated with the low abundance of one or the other host in different decades: low abundance of the intermediate host (amphipod) in the first decade and low abundance of the definitive host (skink) in the third decade. In turn, the low abundance of each host was associated with changes to the abiotic environment over time due largely to the dynamically changing matrix as the plantation trees grew. Our study provides rare long-term experimental evidence of how disturbance can cause local extinction in parasites with life cycles dependent on more than one host species through population bottlenecks at any life stage. Mismatches in the abundance of multiple hosts over time are likely to be common following disturbance, thus causing parasites with complex life cycles to be particularly susceptible to habitat fragmentation and other disturbances. The integrity of food webs, communities, and ecosystems in fragmented landscapes may be more compromised than presently appreciated due to the sensitivity of parasites to habitat fragmentation.