Category: New Research Page 10 of 66

A Case of Cryptic Back-Introduction

On August 21, 2018

Figure 1. Native and non-native ranges of Anolis sagrei. Map from Kolbe et al. (2017).In this study, Kolbe and collaborators (2017) surveyed A. sagrei populations across Cayman Brac. First, they looked for red-dewlapped lizards to determine whether invasive A. sagrei from Grand Cayman have invaded Cayman Brac. Second, they collected brown anole lizards on Grand Cayman and Little Cayman to determine the source of red-dewlapped A. sagrei. For all lizards captured, they quantified dewlap phenotypes (i.e., reflectance spectra) using spectrophotometric methods, measured structural habitat use (i.e., perch height and diameter) and body size (i.e., snout-vent length (SVL) and mass), and genotyped ten nuclear microsatellite loci. For lizards with intermediate multilocus genotypes or with a genotype that did not match their island, they sequenced mitochondrial DNA (mtDNA) haplotypes (ND2) to test for nuclear-mitochondrial mismatches. Genomic data was combined with previously published microsatellite genotypes (Kolbe et al. 2008) and mtDNA (ND2) sequences for the Cayman Islands (Kolbe et al. 2004, 2007). With these data, they evaluated whether invasive A. sagrei from Grand Cayman have been introduced to native populations on Cayman Brac, and if so, whether invasive lizards have interbred with native lizards.

Under current trends of globalization, human activities impact the distribution of species by facilitating dispersal of propagules. Human-mediated dispersal prevents geographic distance from being a barrier to the introduction and movement of many species. These long-distance colonization events can gather evolutionary distinct lineages that might have been separated for millions of years (e.g., Kolbe et al. 2004). Moreover, dispersal events can potentially reintroduce individuals from an invasive population back into their native range; either back into their original source population or to any part of their native range. This previously undocumented dimension of biological invasion was termed cryptic back-introduction by Guo (2005).

Anolis sagrei is an excellent colonist, judging by its geographical distribution. This species has reached many islands and mainland areas in the Caribbean by overwater dispersal (Williams 1969). About 2.5 million years ago, A. sagrei naturally colonized Cayman Brac and Little Cayman. These populations subsequently differentiated into the yellow-dewlapped endemic subspecies A. sagrei luteosignifer on Cayman Brac and the red-dewlapped A. s. sagrei on Little Cayman (Schwartz and Henderson 1991); the dewlap (i.e., an extendible flap of skin attached to the throat) is used for mate attraction, male-male and interspecific competition, and predator deterrence (Losos 2009). However, this species failed to naturally colonize the third of the Cayman Islands, Grand Cayman. In the early 1980s, through human-mediated dispersal, a red-dewlapped form of A. sagrei established on Grand Cayman. These populations resulted from the introduction of genetically admixed lizards from non-native populations in south Florida (Minton and Minton 1984; Kolbe et al. 2004, 2008; Figure 1). Since then, inter-island supply shipments by air and sea within the Caymans could have transported invasive and native brown anole lizards among the three islands. Kolbe et al. (2017) explored whether cryptic back-introduction is occurring in brown anole (A. sagrei) lizards and the implications of this type of invasion for native populations.

Figure 2. Results of PCA for dewlap reflectance (Kolbe et al. 2017).

Kolbe et al. (2017) found no differences among islands in structural habitat use. They conducted a principal component analysis (PCA) for dewlap reflectance data using the average wavelength of each lizard. PCA results show that there is strong differentiation in dewlap reflectance between yellow-dewlapped lizards on Cayman Brac and the red-dewlapped lizards on Little Cayman and Grand Cayman (Figure 2), which supports their field observations of red-dewlapped lizards occurring on Cayman Brac (Figure 3B). This suggests the introduction of brown anole lizards to Cayman Brac from either of the two other Cayman Islands.

Figure 3. Examples of Anolis sagrei dewlaps from the Cayman Islands (Kolbe et al. 2017).

Furthermore, this study reports strong population-genetic structure among the three Cayman Islands and evidence for non-equilibrium. They identified intermediate multilocus genotypes between Grand Cayman and Cayman Brac (Figure 4). Also, the authors found an intermediate microsatellite genotype in one individual from Cayman Brac. This lizard had a red dewlap and a mtDNA haplotype from Grand Cayman. This mismatch among genetic and phenotypic data suggests that A. sagrei lizards (with different colored dewlaps) from Grand Cayman and Cayman Brac are interbreeding.

Figure 4. Results of a PCoA using multilocus genotypes from ten microsatellite loci (Kolbe et al. 2017).

This study reports the first evidence of cryptic back-introduction; however the frequency with which this phenomenon occurs is still unknown. By studying cryptic back-introductions we can eventually understand how lineages change though a brief period of isolation from its native range and determine if these are incompatible when brought together again. Likewise, future studies should address how phenotypic variation affects ecological interactions with native species and its consequences.

Article:

Kolbe, J. J., J. E. Wegener, Y. E. Stuart, U. Milstead, K. E. Boronow, A. S. Harrison, and J. B. Losos. 2017. An Incipient Invasion of Brown Anole Lizards (Anolis sagrei) Into Their Own Native Range in the Cayman Islands: A Case of Cryptic Back-introduction. Biological Invasions 19:1989–1998.

Cited Literature:

Guo, Q. 2005. Possible cryptic invasion through “back introduction”?

Kolbe, J. J., R. E. Glor, L. R. Schettino, A. C. Lara, A. Larson, and J. B. Losos. 2004. Genetic variation increases during biological invasion by a Cuban lizard. Nature 431:177–181.

Kolbe, J. J., A. Larson, and J. B. Losos. 2007. Differential admixture shapes morphological variation among invasive populations of the lizard Anolis sagrei. Molecular Ecology 16:1579–1591.

Kolbe, J. J., A. Larson, J. B. Losos, and K. de Queiroz. 2008. Admixture determines genetic diversity and population differentiation in the biological invasion of a lizard species. Biology letters 4:434–437.

Losos, J. B. 2009. Lizards in an Evolutionary Tree: Ecology and Adaptive Radiation of anoles. University of California Press.

Minton SA, Minton MR (1984) Anolis sagrei (brown anole). Herpetol Rev 15:77

Schwartz A, Henderson RW (1991) Amphibians and reptiles of the West Indies: descriptions, distributions, and natural history. University of Florida Press, Gainesville

Williams, E. E. 1969. The ecology of colonization as seen in the zoogeography of anoline lizards on small islands.

ESA 2018: The Consequences of Malarial Infection on the Puerto Rican Yellow-Chinned Anole in Post-Hurricane Conditions

By Johann D. Crespo

On August 19, 2018

In New Research, Research Methods

Reduced host fitness and impaired immune functions are some of the most well-known consequences of parasitic infections. However, some parasites play important ecological roles by influencing their host’s populations and community composition. In eastern Caribbean islands, the malaria parasite Plasmodium azurophilum has been suggested to mediate competition and determine distribution patterns on some anole species. In Puerto Rico, P. azurophilum is known to infect at least five Anolis species – its main host being the yellow-chinned anole (Anolis gundlachi).

David Clark, a master’s student at the University of Puerto Rico – Río Piedras Campus, along with his research mentor (Dr. Miguel A. Acevedo), assessed the negative ecological consequences of P. azurophilum infection on A. gundlachi within the Luquillo Experimental Forest in eastern Puerto Rico. They quantified this by measuring body condition, dewlap size and site fidelity, all of which were exclusively measured in male anoles, as these are the most often infected by P. azurophilum. Moreover, to determine if infected individuals perform worse after a major disturbance event, the body condition was measured again after Hurricane Maria. They used the residual index for body condition, which is calculated using the regression of the log weight and log size. Dewlap size was measured by taking photos of anoles with their dewlaps extended and calculating the area in ImageJ. To diagnose the presence of P. azurophilum infection, blood samples were collected and then examined using a light microscope under oil immersion. Finally, to examine movement patterns and quantify the site fidelity of male individuals, they conducted a mark-resight study within the forest. For statistical analysis they performed linear regression for body condition and dewlap area, and log-linear regression for distance moved.

Tagged male Anolis gundlachi (a) and Plasmodium under oil immersion (b, c & d) (Image by David Clark)

David and Miguel found that P. azurophilum infection did not influence the site fidelity of A. gundlachi males, and that infected individuals tend to exhibit slightly larger dewlaps. The presence of this malaria parasite did not seem to negatively influence body condition before Hurricane Maria. However, their results show that after this major disturbance, body condition was better for infected anoles, suggesting that these individuals are more tolerant to disturbance conditions than the uninfected ones. All in all, no evidence was found to suggest that P. azurophilum infection has negative consequences on the ecological factors assessed here on A. gundlachi. David and his team are currently performing experimental competition trials to assess intraspecific interactions between infected and uninfected yellow-chinned anoles, as well as immunological studies to determine immune responses to infection. Future studies could possibly bring light on the ecological consequences of interspecific interactions between Puerto Rican anoles infected with malaria parasites.

The Luquillo Experimental Forest after Hurricane Maria (Image by Miguel A. Acevedo)

Interspecific Differences in Genetic Divergence among Populations of Anolis Lizards in Cuba

By Masakado Kawata

On August 17, 2018

In New Research

Anolis allisoni. Photo by Masakado Kawata

Cuba is a fascinating country and the largest island in the Caribbean. Cuba has the highest diversity of Anolis lizards, including more than 60 species (see my Instagram page for photos of Cuban anoles and landscapes). Antonio Cádiz, Luis M. Díaz (National Museum of Natural History of Cuba) and a member of my lab published a paper comparing genetic divergence of Anolis species within Cuba (Cádiz et al. 2018, Zoological Letters, 4:21). The study was conducted when Tony was a PhD student at Tohoku University and lecturer at Havana University.

We constructed a phylogeny using nuclear and mitochondrial genes of 303 individuals from 33 Cuban Anolis species (Fig.2) . The phylogeny presented in this study follows the most comprehensive sampling of Cuban Anolis species to date. We added five species which had not been sequenced previously. We also estimated another phylogeny using mitochondrial genes of 51 Cuban and 47 non-Cuban Anolis species for estimated relative species ages (Fig.S3).

Map of Cuba showing our sampling locations.

Then, we tried to estimate factors affecting interspecific (or interclade) differences in genetic divergence among populations of Cuban Anolis species. We considered species age, environmental heterogeneity within species ranges, and ecomorph type as putative factors. For this purpose, we examined genetic divergence within species by using 177 populations of 26 species.The sampling locations of these species were selected for the best feasible coverage of known geographic ranges of each species. Phylogenetic Generalized Least Squares (PGLS) analyses showed that species age was positively correlated with species’ average genetic divergence among populations.

Previous studies have indicated deep interpopulation genetic divergence found in several Anolis species. Our results showed that relavie differences in genetic divergence was largely affected by species age and geographic distances within species (Fig. 3). This indicates that older species could have more divergent populations within species.

Phylogeny of Cuban anoles.

Cádiz, A., N. Nagata, L. Díaz, Y. Suzuki-Ohno, L.Echenique-Díaz, H. Akashi, T. Makino and M. Kawata. (2018) Factors affecting interspecific differences in genetic divergence among populations of Anolis lizards in Cuba. Zoological Letters 4:21 [Open Access] https://doi.org/10.1186/s40851-018-0107-x

Perch Use by Anolis polylepis Peters, 1874 (Polychrotidae) in a Tropical Humid Forest at the Piro Biological Station, Costa Rica

By Javier Cortes Suarez

On August 14, 2018

In Natural History Observations, New Research, Notes from the Field, Research Methods

Morazán Fernández, F., Gutiérrez Sanabria D. R., Coello-Toro H. L., Arévalo-Huezo, E. Ioli, A. G., Díaz Gutiérrez, N., Guerra, L. F, Burbano, D., Guevara, C., Lobos, L., Rico-Urones, A., Cortés-Suárez, J. E, Jiménez, R., Reinke, H., Narváez, V., Aranda, J.M. 2013. Relación entre la fauna silvestre y las plantaciones de palma africana (elaeis guineensis) y su efecto en la producción de pequeños y medianos productores en la península de osa, Costa Rica. Instituto Internacional de Conservación y Manejo de Vida Silvestre, Universidad Nacional, Costa Rica. Pp 104.

This image was taken as part of the integrated course developed by the XXIII promotion of the Masters in Conservation and Wildlife Management of the National University of Costa Rica.

Individuals of a species use habitats on different ways for refuge, feeding, reproduction, or perching. We studied the variation on perch use between sex and age classes of Anolis polylepis at the Piro Biological Station, Costa Rica. Our results point to a similar perch use pattern between sex, but different between age classes, considering only the lowest and
highest perches. Adult females and males use herbaceous and shrubby vegetation and avoid leaf litter. Juveniles use herbaceous vegetation and leaf litter, but avoid shrubby vegetation. We suggest that adult males use higher perches to defend territory.
Conversely, juveniles use lower perches to avoid predators and foraging. Adult females use middle and high perches. This result is in contrast with previous studies on this species.

Cortés-Suárez, J. E. and N. Díaz-Gutiérrez. 2013. Perch use by Anolis polylepis Peters, 1874 (Polychrotidae) in a tropical humid forest at the Piro Biological Station, Costa Rica. Herpetology Notes 6: 219–222.

Halt the Bustle of City Life: Thermal Spikes from the Urban Heat Island Slow Development of Anole Embryos

By Joshua Hall

On July 26, 2018

In Introduced Anoles, New Research

A brown anole emerging from the egg.

There is much talk these days about how human land use (e.g. urbanization) impacts wildlife. Although anoles have often taken center stage in this discussion (Winchell et al. 2016; Tyler et al. 2016; Chejanovski et al. 2017; Lapiedra et al. 2017; Winchell et al. 2018), most of this work has focused on measuring phenotypes of adult males. Very little work has been done to understand how massive habitat alteration impacts early life stages even though we know that these stages are extremely sensitive to environmental disturbance and have the potential to impact population dynamics (Carlo et al 2018). Embryos are particularly sensitive to changes in the environment because they lack the ability to respond to unfavorable conditions by adjusting their behavior (i.e., they can’t run away). Since the 1980’s, we’ve known that egg mortality can have massive effects on population densities and even determine how these densities cycle from year to year (Andrews 1982; Andrews 1988; Chalcraft and Andrews 1999). Still, comparatively little attention is given to embryo development and egg survival when considering how habitat alteration impacts species.

In a newly published paper (Hall & Warner 2018), we sought to understand how extreme ground temperatures in cities and suburbs (i.e., the urban heat island effect) influence patterns of embryo development. Due to a lack of canopy cover (i.e., trees) and an abundance of heat-absorbing surfaces (e.g., concrete), cities and suburbs tend to be much warmer than adjacent forested areas, and this means nest temperatures are higher in urban and suburban areas compared to adjacent forested sites (Tiatragul et al. 2017). Warm temperatures often have positive effects on embryo development; however, extremely warm temperatures can cause mortality and even slow developmental rates (Sanger et al. 2018).

Figure 1. An overview of our experimental design to understand how urban incubation regimes impact embryo development and survival. Eggs from both forest and city populations were factorially distributed into forest and city incubation treatments. At approximately a quarter of the way through development, some eggs were exposed to a spike in temperature measured from the field (either 39 or 43 °C peak). Eggs completed development at their assigned incubation profile (city vs. forest) and hatchling growth and survival were monitored in the lab for three months.

JMIH 2018: The Curious Case of Bark Anoles

By Jessica Pita Aquino

On July 20, 2018

In New Research

The Bark Anole (Anolis distichus ignigularis) from the Río Recodo. Photo from Richard Glor’s Flickr.

The Bark Anole (Anolis distichus) is a highly polymorphic lizard widely distributed in Hispaniola. Anolis distichus is divided into 16 subspecies with dewlap colors ranging from deep wine red to pale yellow (Glor and Laport 2012). In the early days scientists, such as Albert Schwartz, argued that A. distichus is divisible into multiple subspecies according to an analysis of variation in body color and dewlap pigmentation. But, are they really subspecies?

During the 2018 Joint Meeting of Ichthyologists and Herpetologists (JMIH), Richard Glor shared his lab’s advances on the curious case of Bark Anoles. Anolis distichus populations have ecological, phenotypic and genetic differences. Previous studies show a correlation between dewlap phenotype and environmental variation; in drier habitats, lizards have smaller, brighter, yellow dewlaps, while those in wetter habitats have larger, less bright, orange dewlaps (Ng et al. 2012).

Previously, the Glor Lab found strong support for the hypothesis that A. distichus is comprised of numerous genomically distinct populations (MacGuigan et al. 2016). Genetic divergence was associated with a biogeographic barrier, but not with dewlap color. Also, they found evidence for hybridization in contact zones with limited gene flow and intrinsic reproductive isolation between subspecies (MacGuigan et al. 2016; Ng et al. 2016). Overall, these studies suggest that geographic isolation, as well as ecological specialization, contribute to speciation.

The Glor Lab continues putting together the pieces of the puzzle. Most recently, they sequenced and assembled whole genome sequence data for A. distichus to identify the genomic basis for species differences and speciation.

JMIH 2018: How Does Artificial Light at Night Affect Anoles?

By Jessica Pita Aquino

On July 17, 2018

In Introduced Anoles, New Research

Crested Anole (Anolis cristatellus) under a leaf. Photo by Chris Thawley.

Conservation biologists have long been concerned about the effects of human development on species and environments. Urban habitats can significantly change lighting patterns for animals by increasing nocturnal ambient illumination. Artificial light at night (ALAN) has the potential to disrupt an organism’s physiology, behavior, and ecology. However, light pollution remains poorly studied and is a concern for urban herpetofauna.

Anolis lizards in Miami, Florida are a great system to study the effects of ALAN on behavior, health, reproduction, and survival. Anoles are diurnal and are adapted to a distinct photic habitat appropriate to their sun/shade preferences. However, many anole species have been observed active at night where artificial lights are prevalent. So, what are the effects of ALAN on anole fitness?

Chris Thawley, a postdoctoral researcher in the Kolbe Lab at the University of Rhode Island, is interested in whether ALAN imposes selection on anoles and how they might adapt to these pressures. Chris conducted a field experiment introducing landscape lightning into a previously unlighted habitat within an urban matrix. For over two months, he assessed whether Brown Anoles (Anolis sagrei) and Crested Anoles (A. cristatellus) experienced higher levels of ALAN at their sleeping perches and if these lizards behaviorally avoided exposure to artificial light. Also, lizards were marked and followed to determine if light exposure impacted survival, growth, body condition, and physiology.

Chris found that A. sagrei and A. cristatellus lizards are not behaviorally avoiding ALAN at night. Anoles that were more exposed to artificial light had lower glucose levels compared to those that were less exposed. Also, there were no dramatic changes in reproduction, but ALAN reduced follicle size. Egg mass showed a positive relationship with snout-vent length (SVL) in lizards exposed to ALAN, which suggests that ALAN increases egg mass in larger lizards. Chris continues analyzing growth and survival data and aims to explore if there is a correlation between levels of corticosterone (CORT), melatonin, and glucose.

JMIH 2018: Brown Anoles Have Broader Diets Where They Co-occur with Other Anoles

By Chris Thawley

On July 17, 2018

In Introduced Anoles, New Research

A brown anole (Anolis sagrei) surveys its domain.

Trophic ecology deals with questions about the ways in which organisms acquire energy and how that process interacts with the communities and ecosystems surrounding them. Anole-focused research has played a strong role in our understanding of trophic ecology and ideas abut how communities come together and evolve, particularly in papers by Schoener, Roughgarden, and Lister. However, many trophic ecology studies have focused on specific communities or locations and haven’t dealt with how the ecology of one focal species varies across space and as a function of the presence of other close competitors.

Sean Giery, a post-doc at the University of Connecticut, in collaboration with James Stroud, a post-doc at Washington University in St. Louis, worked to address this gap in our knowledge by studying how the trophic ecology of the brown anole, Anolis sagrei, varies across its range. Brown anoles are voracious predators of insects, known to chow down on a diverse range of arthropods, including some of surprising size. Since the brown anole is also a prodigious invader, it occupies habitats with a variety of potential competitors, including locations with few competitors. Sean and James leveraged this situation to their advantage by compiling stomach content data from previously published papers (including a follow-up on Lister’s paper above). They also added their own sampling, including in Southern Florida, the Bahamas, and Hawaii…tough work! Sean and James then used the articles themselves, field guides, and citizen science sources like iNaturalist to determine the presence of other species which might compete with the brown anole, including other anoles and diurnal, insectivorous lizards.

Sean and James assembled an impressive database of the diet of A. sagrei.

They found that as community richness increases, the dietary niche of A. sagrei actually becomes broader, the opposite of the direction predicted by theories of ecological release. Additionally, average niche overlap between individual anoles declines as community richness increases. When only brown anoles are present in a community, individuals are highly similar in the types and proportions of what they eat, another finding which runs counter to models of how niche breadth should vary when a species is released from interspecific competition. Sean concluded his talk by suggesting that interference competition may be more important than generally recognized and soliciting suggestions for ways to continue looking at this impressive dataset. We’ll look forward to reading the paper!

JMIH 2018: Do Ecomorphs and Parasites Coevolve?

By Jessica Pita Aquino

On July 15, 2018

In New Research

Spencer Asperilla presenting his poster, “A Biodiversity Survey of Parasites from Anolis Lizards on Andros Island, Bahamas”, at the 2018 Joint Meeting of Ichthyologists and Herpetologists (JMIH).

Anolis species inhabiting the Caribbean provide a great example of adaptive radiation and convergent evolution in ecology, morphology, and behavior. Adaptation, diversification, and specialization to a particular microhabitat and dietary resource, created a great diversity among anoles. But what about their parasite assemblages? Andros Island in the Bahamas is the fifth largest island in the Caribbean Archipelago. However, it is still unclear if the parasite fauna hosted by Anolis lizards show similar evolutionary pathways.

In 2016, after an amazing experience studying abroad at ForFar Field Station on Andros Island, Spencer Asperilla and Katie Brittain joined the Langford Lab at Florida Southern College. Spencer and Katie were interested in documenting parasite species present in Bahamian Anolis lizards to determine if these are specialists or generalists among ecomorphs and identify if parasite populations vary seasonally. They conducted parasite biodiversity surveys on three sites on Andros Island, which involved capturing lizards and collecting blood smears and fecal matter. Specimens and samples were transported to Florida Southern College where they were processed and analyzed for parasites.

Spencer and collaborators found that parasitic infection rate was highest during the Summer (66.66%), and lowest during the Spring (60.56%); however these differences were not significant. Climate variables, such as mean daily temperature and precipitation, were evaluated, but no seasonal pattern could be determined for parasite infections in Bahamian lizards. As for parasite diversity, Brown Anole (Anolis sagrei; trunk-ground ecomorph) lizards had most species of parasites present, while A. angusticeps (twig ecomorph), A. distichus (Bark Anole; trunk ecomorph), and A. smaragdinus (Green Anole; trunk-crown ecomorph) had lower species diversity. The authors suggest these differences are related to the biology of the different ecomorphs. Trunk-ground anoles, such as A. sagrei, might be more susceptible to parasite infection by descending to the ground to capture prey or interact with a conspecific, whereas the other ecomorphs remain higher up in the tree. Ground-dwelling insects may serve as intermediate hosts for parasites found in trunk-ground anoles. Spencer and collaborators propose that habitat use, as well as dietary composition, serve as an ecological explanation for parasite distribution among ecomorphs.

The big question remains unanswered: have parasite species coevolved with specific lizard hosts? The Langford Lab continues identifying parasites species to assess the diversity, host-specificity and infection patterns of Bahamian Anolis lizards. Spencer wants to resume this project as part of his master’s thesis and he looks forward to traveling back to Andros Island to collect additional samples.

JMIH 2018: How Can We Measure Immune Function in Anoles?

By Chris Thawley

On July 14, 2018

In New Research, Research Methods

Measuring the swelling induced in anole feet during the PHA assay may result in swelling in one’s own fingers.

The immune system is critical to the survival of animals, including anoles, which are faced with an environment full of potential pathogens and toxins. Ecoimmunologists have developed a myriad of assays to measure various aspects of the immune system and its function in a variety of species, but these assays are often applied to organisms without fully validating them. This issue can prevent a full and accurate interpretation of the results obtained. The PHA skin test is widely used in lizards, including anoles, to test immune function, but has exactly this problem: it has only been validated in cane toads and a crocodile…a large oversight!

Caty Tylan, a PhD candidate and DVM at Penn State University in Tracy Langkilde’s lab, set about rectifying this situation by validating the PHA assay in our favorite squamate lab “rat,” the green anole, Anolis carolinensis. To conduct the PHA test, Caty injected two different types of phytohemagglutinin (PHA-L and PHA-P) into the footpads of green anoles and compared the swelling produced to that of control injections. She also measured types of white blood cells in the blood and foot tissue at regular intervals after injections. Caty found that both types of PHA work well and induce similar levels of swelling with a standard assay protocol in green anoles, but that they induce different types of immune responses. PHA-P elicits a broader response with different types of immune function that varies with time after injection, meaning that the outcomes of this test may be harder to interpret. PHA-L on the other hand, induces higher concentrations of T-lymphocytes, a specific type of white blood cell. As a result, using PHA-L for PHA assays may lead to a test that is more interpretable, especially in studies looking at how the stress response affects immune function.