Conservation of a flagship species: Health assessment of the pink land iguana, Conolophus marthae.

The pink land iguana, Conolophus marthae, is one of four species of iguanas (three terrestrial and one marine) in the Galápagos Islands, and the only one listed as critically endangered by the IUCN. The species can only be found on the north-west slopes of the highest volcano on Isabela Island and was first described to science in 2009. As part of a population telemetry study, a health assessment was authorized by the Galápagos National Park. Wild adult iguanas were captured on Wolf Volcano in September 2019 and April 2021 to record morphological and physiological parameters including body temperature, heart rate, intraocular pressures, tear formation, and infrared iris images. Blood samples were also collected and analyzed. An i-STAT portable blood analyzer was used to obtain values for base excess in the extracellular fluid compartment (BEecf), glucose (Glu), hematocrit (HctPCV), hemoglobin (Hb), ionized calcium (iCa), partial pressure of carbon dioxide (pCO2), partial pressure of oxygen (pO2), percent oxygen saturation (sO2%), pH, potassium (K), and sodium (Na). When possible, data were compared to previously published and available data for the other Galápagos iguanas. The results reported here provide baseline values that will be useful in detecting changes in health status among pink land iguanas affected by climate change, invasive species, anthropogenic threats, or natural disturbances. The collected data also provide an invaluable resource for conservation scientists planning to implement conservation strategies, like translocations, that may temporarily alter these baseline values.

Introduction

The Galápagos pink land iguana, Conolophus marthae, was first sighted on Wolf Volcano (WV hereafter), Isabela Island, Galápagos, in 1986, but was not described to science as a separate and unique species until 2009 [1]. Despite its recent description, this species rapidly became a conservation flagship, as it helped raising awareness about the value of biodiversity locally and internationally [2, 3]. The species is under the direct protection of Galápagos National Park Directorate (GNPD) and listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora [4]. It is classified as critically endangered by the IUCN Red List of Threatened Species [5]. Among the major threats for the species, we recognize: very small population size (200 < N < 300 adult individuals); small distribution area; lack of recruitment, with no hatchlings and very few juveniles observed since 2005; introduced alien species such as cats and rats. With such a small population, these animals are at risk of extinction, from both natural and anthropogenic events. Although information on the natural history and biology of Conolophus marthae have been accreting since its description [1, 3, 5–9], very little is known about the animal’s overall physiology and baseline medical parameters. This called for an urgent action aimed at providing data concerning the relative health status for individuals of this species.

Peripheral blood biochemical, blood gas, and hematology parameters are useful for assessing lizards’ health [10-14]. It is important to establish and publish species-specific baseline blood and other parameters for healthy individuals since disease, injury, pollutants, or starvation can result in blood value perturbations. As potential alteration of baseline values could also come from specific conservation actions, baseline data can be used to assess individuals’ health status after a conservation action has been implemented [15, 16]. Several reference intervals have been established for iguanids and include: Conolophus pallidus and C. subcristatus [13], Amblyrhynchus cristatus [14], Basiliscus plumifrons [17], Cyclura cychlura inornata [18], Cyclura ricordii [19], and Iguana iguana [20-24]. To date, hematological parameters and base line values have never been recorded in Conolophus marthae iguanas. As part of a radio-satellite tagging ecology and population study, a health assessment study for the pink iguanas was authorized by the GNPD. A detailed description of the satellite tracking study is beyond the scopes of this article, and we refer to Loreti and colleagues for further information [25, 26]. Nevertheless, we took advantage of opportunity provided by this tracking study and proceeded in two ways: i) we sampled wild pink iguanas on WV, Isabela Island, in September 2019 and April 2021 to analyze blood chemistry and hematological parameters and establish an intra-specific baseline report of such parameters; ii) we collected all published blood chemistry data available for other Galápagos iguanas to perform, when possible, an inter-specific comparison of such parameters. For wild samples collected in 2019 and 2021 a complete veterinary health examination was performed. The examination included measurement of body temperature, heart rate, length, body weight, intraocular pressure, ocular tear production, collection of blood samples, ectoparasites, and, in some cases, feces. In the current study, we report on the blood chemical analysis and status of clinically healthy wild adult Galápagos pink land iguanas.

Materials and methods

Ethic statement

This health assessment was authorized by the Galápagos National Park Directorate (Permit # PC-04-21 issued to G. Gentile). The techniques used during this health assessment were also approved by the University of Rome “Tor Vergata” ethics and animal handling protocol and the San Diego Zoo Wildlife Alliance IACUC. All procedures were performed by and carried out under the supervision of a licensed veterinarian and author of this study (GL).

Sampling procedure

We captured, tagged, and sampled 15 and 27 adult pink iguana individuals during field expeditions conducted in September 2019 and April 2021, respectively. Individuals were collected from an area on WV comprising approximately 6 km2 at an altitude of 1600–1700 m (Fig 1). All iguanas were captured by hand or noose, either inside of or adjacent to their burrows. The animals were quickly transported to a field laboratory area (usually located within 10 m of the capture site) for blood drawing (usually within 5 min from capture). A blood sample of approximately 2.5 mL was obtained from the coccygeal hemal arch of each iguana using a heparinized 22-gauge 3.8 cm needle attached to a 10.0 mL syringe. This volume is considerably less than the safety threshold established for lizards (at 0.7 ml per 100 g [27]). The blood was divided into sub-samples and stored in a field cooler kept refrigerated using icepacks. Once the blood sample was secured the animal was examined, measured, weighed, and a custom designed GPS tracker was attached ([25, 26] for further details on the tracker devices).

Fig 1

Map of the Galápagos archipelago.

The archipelago is located ≈ 1000 km off mainland Ecuador. The inset shows a detail of the northwestern slopes of Wolf Volcano, the only site where pink iguanas are known to persist.

Cloacal temperature and heart rate were recorded shortly after the blood was taken, and usually within few minutes from capture. Heart rate was recorded via a Doppler ultrasound probe (Parks Medical Electronics, Inc., Aloha, Oregon, USA) over the heart. An EBRO® Compact J/K/T/E thermocouple thermometer (model EW-91219-40; Cole-Parmer, Vernon Hills, Illinois, USA 60061) with a T-PVC epoxy-tipped 24 GA probe was used to determine core body temperature. Snout-vent length (SVL) and tail length (TL) were recorded with a flexible measuring tape and used to determine the total length of each individual. Body mass was measured with a digital scale (± 0.1 kg). The sex of the iguanas was determined by the presence or absence of hemipenes or by visually inspecting secondary sexual characteristics (presence/absence of femoral pores, size of the individual, prominence of nuchal crest). Prior to release, ectoparasite load was determined by counting the number of ticks. For each animal, a sample of ticks was collected and preserved in 70% ethanol. Each animal was also scanned for the presence of a Passive-Integrated-Transponder (PIT) and checked for the presence of a brand. For never-before captured individuals a PIT was placed under the femoral skin of the right leg for long term identification and population monitoring.

A complete examination of the eyes was performed. Within 15 minutes of capture, internal ocular pressure (IOP) measurements were taken of the left (IOP_L) and right (IOP_R) eye using a rebound tonometer (TonoVet®, iCare, Tiolat, Helsinki, Finland). Intraocular pressures were measured on the Tonovet’s® rebound tonometer on undefined patient setting (p). Disposable probes were used and changed between every patient. The tonometer was held in position perpendicular to the patient eye and approximately 5 mm from the corneal surface. Tear production was measured using the endodontic absorbent point paper test (EPPT) in iguanas sampled in 2019 but not for samples collected in 2021. Size 30, 40, and 45 endodontic paper points (Parallax® Veterinary Absorbent Paper Points) were used. The tapered end of the clean points was placed into the fornix in both the left and right eye. The paper was removed after 60 s and the length of moisture that was wicked on the point was measured with a millimeter ruler. External infrared photographs of each eye (Panasonicⓒ Lumix DMC-ZS50 12.1MP) were obtained for each animal sampled in 2019 and reviewed for ocular abnormalities by a board-certified veterinary ophthalmologist (HDW). Before releasing the animal in the exact location where it was caught, a unique ID number was painted with non-toxic and washable paint to prevent recapture. The ID was painted on both flanks and the tip of the tail was also colored with non-toxic, washable white paint.

Hematology parameters

The blood samples were used for measuring various hematological parameters. Approximately one drop was used for lactate analysis; approx. one drop for glucose analysis; about 0.1 mL was loaded into an i-STAT Clinical Analyzer (Heska Corporation, Fort Collins, Colorado, USA) utilizing Chem8 cartridges (see later in text for details); about 0.05 mL was used for centrifugation with a portable microcentrifuge (Eppendorf North America, Inc., centrifuge model 5424) 5 min. at 14,000 G to determine packed cell volume (PCV) and total solids (TS). The PCV was determined by measuring the percentage of cellular material compared to plasma in the tubes. Two drops of plasma were placed on a refractometer (Ade Advanced Optics, Oregon City, Oregon 97045, USA) and the total solids values recorded. We additionally used ≈ one drop of blood for making blood films on clean glass microscope slides (samples not yet analyzed). All the blood not used for hematological analyses was stored in 2% SDS lysis buffer in the field until long term storage in laboratory freezers.

The i-STAT Clinical Analyzer is a handheld, battery-powered, device that measures selected blood gas, biochemical, and hematology parameters using approx. 0.095 mL of non-coagulated whole blood. The following parameters were measured: base excess in the extracellular fluid compartment (BEecf), bicarbonate (HCO3-), glucose (Glu), hematocrit (HctPCV), hemoglobin (Hb), ionized calcium (iCa), partial pressure of carbon dioxide (pCO2), total carbon dioxide (tCO2), partial pressure of oxygen (pO2), pH, potassium (K), and sodium (Na). The i-STAT automatically produces temperature corrected values for pCO2, pH and pO2 once the animal’s body temperature is entered. Blood lactate (Lact.) was determined using a portable Lactate PlusTM analyzer (Nova Biomedical, Waltham, Massachusetts, 02454 USA). A glucometer (Accu-Check® Active, Roche) was sometimes used to obtain near instant glucose values in the field and compare them to the glucose values obtained by the i-STAT Clinical Analyzer. To compare the validity and consistency of i-Stat readings, glucose, calcium, sodium, potassium and hematocrit parameters were measured using two different i-Stat cartridge types (Chem8 and CG-8) in samples collected in 2019. Readings were compared using a Wilcoxon Rank Sum test.

Data compiling procedure

We reviewed available and published health and hematological data from other Galápagos iguanas. Data were sourced primarily from two publications by Lewbart and colleagues [13, 14] from individuals of C. pallidus, C. subcristatus, the two other species of Galápagos land iguanas, and A. cristatus, the Galápagos marine iguana. We were able to compile hematological and morphological data from 28 A. cristatus (four females and 24 males), 21 C. pallidus (12 females and nine males) and 30 C. subcristatus (19 females and 11 males). The protocols adopted while sampling individuals of these other species are the same as the ones adopted for this study, in fact they were performed by the same author (GL). For this reason, when possible, the data were used to perform inter-specific comparisons.

Data analysis

Unless otherwise stated, all statistical analyses and data manipulation were performed in R v4.0.5 [28]. We first built a series of boxplots to identify potential outlier values in our data. While it is hard to imagine a scenario where outliers exist when looking at morphological and physiological characteristics, it is possible that mistakes while transcribing data in the field have occurred. Also, considering the relatively small sample size we had at our hand, we wanted to avoid outlier values to potentially influence intra and inter-specific comparisons. Outliers in all measured parameters were identified by looking at values falling outside of the inter-quartile range of data distribution and removed from further analyses (S1 Fig).

We calculated summary statistics (mean, standard deviation, minimum and maximum values) for all recorded parameters in males and females. For C. marthae samples, we report on the differences between sexes in all measurements considered using a Wilcoxon Rank Sum test. In pink iguanas we also used linear morphological features to calculate an estimate of individual’s body condition as suggested by Peig and Green [29]. We first used the smatr R-package [30] to compute a scaling exponent (derived from a standardized major axis regression). This exponent was then used to calculate the Scaled Mass Index (SMI) for each individual using the following expression:

where Mi and Li are the body mass and the linear body measurement of individual i respectively; bSMA is the scaling exponent estimated by the SMA regression of M on L; L0 is an arbitrary value of L (in this case we used the SVL arithmetic mean value for the study population). We accounted for the significant difference between sexes by computing two separate scaling exponents for different sexes prior to calculate the SMI for each individual. The SMI is a better predictor of individuals’ health, as it should be an unbiased estimator of the overall body condition of the individual [29].

Considering the non-normal distribution of most measured variables and acknowledging for the reduced sample sizes, we used Wilcoxon Rank Sum test to compare all measured variables between species while also considering for sex differences. Significant differences were computed accounting for multiple comparisons using Bonferroni correction [citation needed]. The potential relation between morphological measurements (SVL, Mass, and SMI) and the other parameters measured in pink iguanas was investigated using Spearman rank correlation coefficient (rs). Linear regressions were calculated also between each measured biochemical parameter. Significance level was adjusted for multiple comparisons using Bonferroni correction.

Results

All pink iguanas sampled were deemed clinically healthy based upon physical examination. We found no significant differences in measurements obtained using different i-Stat cartridges (S1 Table). Almost all iguanas (95%) collected between 2019 and 2021 had ticks (between 9 and 90). Ticks were collected and stored in 70% ethanol but remain in Galápagos for identification at a later time. No significant ocular abnormalities were noted on initial examination or on review of the infrared images. A representative image showing the detail captured in the photographs is presented in Fig 2. Overall, the intraocular pressure values for right (R) and left (L) eyes were similar and not significantly different (WIOP = 734.5, p-value = 0.42). The comparison between ocular pressure in left eyes of males and females produced a slightly significant result (WIOP_L_m/f = 276, p-value = 0.03; Table 1). Very similar and not significantly different values were also recorded for overall tear production (WEPPT = 99.5, p-value = 0.69). As for IOP, we found a slightly significant difference in eye tear production between sexes in the left eye (WEPPT_L_m/f = 36.5, p-value = 0.01; Table 1 and S4 Fig). Overall, morphological characteristics of adult pink iguanas analyzed here are in lines with results from other studies (see for example [1, 3]), with males significantly larger and heavier than females (WSVL = 316.5, p-value << 0.001; WMass = 356.5, p-value << 0.001). Scaled mass index ranged from 4.40–6.24 in males and 2.21–4.65 in females and no outlier values were identified, suggesting all adults are relatively healthy (Fig 3). A detail of all biochemical parameters recorded in pink iguanas is reported in Table 1. We found a significant difference between males and females only in ionized calcium values (WiCa = 113, p-value = 0.02), sodium (WNa = 324.5, p-value << 0.01), and total solids (WTS = 52, p-value << 0.01). In C. marthae, heart rate was positively correlated with body temperature (rs = 0.61, p-value << 0.001), while respiratory rate was positively correlated with lactate values (rs = 0.487, p-value = 0.001). Values of HctPCV were very strongly correlated with hemoglobin (rs = 0.98, p-value << 0.001). An overview of other significant correlations is presented in S5 Fig.

Fig 2

Detail of infrared eye photography.

This image shows a detail of the infrared images collected and analyzed.

Table 1

Conolophus marthae intra-specific comparisons.

	Males				Females				P
	Mean	Sd	Min/Max	n	Mean	Sd	Min/Max	n
Svl (cm)	46.36	3.65	37.40/53.00	17	41.13	4.03	33.60/50.20	22	<<0.01
Mass (Kg)	5.36	0.64	4.40/6.50	16	3.76	0.82	2.20/4.87	23	<<0.01
HR (bpm)	69.83	18.34	32.00/92.00	18	77.83	14.81	60.00/108.00	23	0.32
RR (brpm)	23.06	7.88	12.00/36.00	18	21.33	7.09	10.00/36.00	21	0.58
T ˚C	31.43	5.67	21.30/39.20	18	31.77	5.35	22.40/40.10	21	0.95
IOP_R	7.18	2.44	2.40/11.00	18	6.94	2.43	3.20/12.00	23	0.60
IOP_L	8.21	2.24	5.00/11.20	18	6.63	1.31	4.80/10.00	22	0.03
EPPT_R	15.13	6.12	4.50/22.50	8	13.08	3.97	7.50/17.00	6	0.39
EPPT_L	15.06	2.70	10.50/18.50	8	11.10	1.29	10.00/12.50	5	0.01
BEecf	-2.71	1.50	-4.00/0.00	7	-7.14	5.90	-17.00/-1.00	7	0.15
Glu (mmol/L)	154.47	22.41	110.00/192.00	17	155.22	26.19	110.00/209.00	23	0.90
Hb (g/L)	10.89	1.68	7.50/14.30	18	10.51	1.34	8.50/12.90	22	0.52
HCO3- (mmol/L)	22.95	2.83	19.30/27.80	8	19.19	4.32	12.00/24.00	7	0.08
HctPCV (%)	32.12	5.01	22.00/42.00	17	30.95	4.10	23.00/38.00	22	0.58
iCa (mmol/L)	1.33	0.16	1.00/1.62	17	1.45	0.15	1.13/1.77	23	0.02
K (mmol/L)	3.64	0.85	2.00/4.40	7	3.13	1.20	2.00/5.30	7	0.30
Lactate (mmol/L)	9.19	4.46	3.40/18.10	18	10.25	4.35	1.90/17.10	23	0.39
Na (mmol/L)	160.82	3.94	154.00/168.00	17	155.09	3.32	150.00/161.00	22	<<0.01
pCO2 (mmHg)	27.58	7.48	13.10/35.60	8	29.88	1.33	28.30/31.80	5	1.00
pH 37 ˚C	7.40	0.11	7.23/7.52	8	7.31	0.14	7.14/7.47	7	0.23
pO2 (mmHg)	45.25	18.95	20.00/78.00	8	43.00	14.12	26.00/57.00	7	0.86
tCO2 (mmHg)	23.88	3.79	16.00/29.00	17	22.91	3.99	16.00/29.00	23	0.39
TS (g/L)	7.63	1.16	5.40/9.50	18	10.22	1.65	7.10/12.50	23	<<0.01

Mean, standard deviation (Sd), minimum and maximum, number of individuals analyzed (n) of morphological features (Svl and Mass), general health parameters (Hearth rate–HR, Respiratory rate–RR, Temperature–T, Internal Ocular Pressure–IOP for right and left eyes, and tears production–EEPT for right and left eyes), and blood biochemical parameters (Base excess in extracellular fluid compartment–BEECF; Glucose–Glu; Hemoglobin–Hb; Bicarbonate–HCO3-; Hematocrit–HCT; ionized calcium–iCa; Potassium–K; Lactate; Sodium–Na; Partial pressure of carbon dioxide–pCO2; pH measured at standardized temperature–pH[37˚C]; Partial pressure of oxygen–pO2; Total carbon dioxide–tCO2; Total protein solid–TS) measured in C. marthae samples and separated according to sex. The last column in this table (P) reports the P-value from a Wilcoxon Rank Sum test comparison.

Fig 3

Scaled mass index comparison.

Boxplots showing the distribution of SMI values in males and females of analyzed pink iguanas.

For a graphical comparison of all measured parameters across species see supplementary materials (S1–S4 Figs). Based on our pairwise Wilcox test we found two variables, ionized calcium and potassium, that did not differ in any of the considered groups (Fig 4). Values of sodium were found significantly differing between A. cristatus (both males and females) and all other groups considered (Fig 4). Similarly, the respiratory rate of A. cristatus male individuals was different from every other group but from that of A. cristatus females and C. subcristatus males (Fig 4).

Fig 4

Pairwise comparisons.

This figure shows the results Wilcoxon Rank Sum test pairwise comparisons between samples grouped based on species and sex. Darker shades of blue color indicate highly significant differences. Level of significance in pairwise tests has been adjusted using Bonferroni correction.

Discussion

The pink iguanas we examined were alert, robust, and clinically healthy. Despite relatively high tick and hemogregarine burdens physical examination and diagnostic test results support our good health assessment. Hemogregarines are known to affect this species [31], and does not appear to produce any clinical signs, such as lethargy, open mouth breathing, weight loss, or dehydration that may be observed in immunocompromised animals [32]. In general, even though the C. subcristatus from Wolf volcano did show significant alteration in some measures of immune function, significant correlation between corticosteroid levels (or body condition index) and the number of ticks or parasitemia were not found in C. marthae [31]. This supports the hypothesis that, in C. marthae, ecto- and hemoparasites can be sufficiently tolerated and may have coevolved with their host, as is appears to be the case in other iguana species [33].

Most of the blood values we recorded for the pink land iguanas were similar to those reported in other iguanids. For example, the average PCV in green iguanas, Iguana iguana, [22] was 36.7%, slightly higher than the average found in pink iguanas (31.75%). This value, indeed, is more similar to values reported for the basilisk lizard (Basiliscus plumifrons, 31.4%), and much closer to that of C. subcristatus [13, 17]. Despite this, for reptile species PCV values less than 18–20% would be considered anemic, and potentially associated with blood loss, chronic infections, malnutrition and exposure to toxins [27]. PCV values recorded in C. marthae, therefore, suggest that individuals are healthy. The blood sodium levels for green iguanas and basilisk lizards are 160 and 153.5 mmol/L respectively [17, 22], very similar and comparable to the average sodium levels for C. marthae, C. subcristatus, and C. pallidus are calculated at 156.9, 154.0, and 153.7 mmol/L respectively. Another general and easy way to determine health status and low stress is blood glucose. Basilisk lizards (that were held in cloth bags overnight and sampled 12 hours post-capture) had a fairly high level (203 mg/dL [17]) while C. subcristatus and C. pallidus had much lower, levels (126 and 135 mg/dL respectively [13]). The pink iguana glucose values, based on the i-STAT, average to 154.9 mg/dL, similar to that of captive green iguanas (166 mg/dL for males and 180 mg/dL for females [22]). Wild Allen Cays rock iguanas (Cyclura cychlura inornata) had a mean glucose of 189 mg/dL [18]. Glucose values are indeed affected by intrinsic as well as extrinsic factors and unless a large sample is available, it is hard to measure real hypo- or hyper-glycemia in reptiles [27]. Despite this, and after comparing the measured values with what is known from other species of iguanas, the authors consider the recorded values in pink iguanas clinically healthy.

Point-of-care analyzers like the i-STAT may require caution in interpretation, as published studies have found that some blood gas and hematocrit values are not always accurate or reliable with certain non-mammalian species. In rainbow trout (Onchyrhynchus mykiss) results varied with temperature and only pH was a uniformly reliable value [34]. A study in sandbar sharks (Carcharhinus plumbeus) determined the i-STAT is not reliable for accurately measuring blood gases [35]. The i-STAT did not produce valid sO2 or hemoglobin values in the bar-headed goose (Anser indicus [36]). After a comparison between the four different species of iguanas we noticed that the blood gas values and pH recorded were fairly consistent. The only notable exception is represented by the much higher sodium levels in marine iguanas [14]. This is most likely a result of the dietary and habitat difference between the terrestrial and marine iguanas. The marine iguanas also had higher potassium levels than all three land iguanas. The calcium values were comparable between all four species. Moreover, our comparison of parameters calculated using different i-Stat cartridges did not produce a significant difference (S1 Table) indicating that the values are indeed comparable.

In this study we document, for the first time, the base-line blood gas, biochemistry, and hematology values of the critically endangered and elusive C. marthae. The measured parameters, provide evidence indicating that the analyzed animals are healthy. Although we recognize that our sample size is not large enough to provide accurate reference intervals [10], our results represent a useful resource for veterinary scientists and other researchers especially considering that conservation strategies recently developed for this species call for actions, like translocation or/and head-start, that may temporarily alter these baseline parameters [37]. Moreover, although C. marthae individuals seem to be in good health at present, we recommend that the only existing population of this species is regularly monitored in order to be able to immediately track possible changes in health status that may be induced by the harsh and changing environment where the species lives.

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10 Nov 2021

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Additional Editor Comments:

1. Introduction - paragraph 1 last sentence. The authors state that the pink land iguana is a "flagship" species. What is meant by this? How can a species that was only recently discovered by science and is little known to the broader world be a "flagship"?

2. Methods - the authors drew a blood sample of 2.5 ml, but don't mention that this is less than a threshold for safety based on % of total blood volume for the species. In addition, the authors seemed to use very little of this 2.5 ml for their analyses. What was the rationale for taking 2.5 ml?

3. Methods - why is tear production an important health parameter to measure, and why is it important to compare right and left eyes?

4. Methods - in the "Intra-Specific" section the authors mention removing outliers, but give no rationale for doing so, or the procedure for identifying outliers. In the case of SVL and mass, it is hard to imagine a scenario where true outliers can even exist. More information is required here.

5. Table 1 - I find the information content in this small table to be very low. It could likely be removed and the values summarized in the text.

6. Some might question the ethics of capturing and sampling blood from 40% of the individuals in a species that exists nowhere else in the world. Did any of the animals show signs of stress or capture injury? Is baseline data of the kind collected really worth it? Some additional text in the Discussion addressing this point would be useful.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a well-written manuscript outlining field-collected health data from apparently healthy individuals of an endangered species of iguana from the Galapagos islands. The health data collected is thorough and well presented.

There are too many figures with multiple figures demonstrating the same morphometric and clinical data. The number of figures could be reduced to 3 or 4 and the rest of the material submitted as supplemental information. The gross images should be collated into a single image.

In the results section, it is written that "one sample escaped", but the authors likely mean that the iguana escaped and an incomplete set of samples was taken.

More information could be added to the discussion that there are limitations in interpreting clinical data from a small sample of animals as reference intervals could not be obtained from only 15 individuals. The difficulties in interpretation from an iSTAT field analyzer can be explored with paired analyses from an in-lab analyzer, but that was not pursued in this case and this could be addressed by the authors.

Although there is a small sample size and there could be issues with interpretation of the results of a field analyzer, the information is still valuable as baseline data for use in potential captive breeding and reintroduction as well as captive collections (are there individuals of this species kept in zoological collections at the moment?). The information regarding the presence of hemogregarine parasites without an overall negative impact on health is an important take-home from this manuscript and could be highlighted more in the discussion and abstract as it it likely associated with co-evolution.

Reviewer #2: Really important work for an endangered species. Technical language, and easy to understand for the reader. Results presented in a kind graphical way. The authors developed a vigorous statistic analysis.

1) Abstract should highlight the main results and/or important points of discussion.

2) Should consider to include some extra results of descriptive/summary statistics for hematological/biochemistry results (range, min, max, maybe quartiles)

3) For results tables: important to clarify if all the variables have the same number of individuals analyzed, or if not, include an "n" data column in the tables.

4) Could define if there are "local seasons" that externally influence the variables or if it's not taken into account because there're no significant weather variations or according to other analytic factors

5) Did the authors measure the total proteins on in-house equipment to verify or compare the field refractometry results of total solids (TS)?

6) "Data compiling procedure" section could be included in the first methodology heading and so diminish the amount of text.

7) Data of re-sampled individuals is included as separated data (2 samples per individual)? any chance the authors developed a statistical comparison of intraindividual variations between the two samplings?

8) The "heavy mite-infested" individual was included in the general "n" for data presentation?

9) Maybe include a table to summarize the opthalmological results, so it does not get lost in the text.

10) Finally, did the authors make statistical correlations between the grade of parasitism (external ("tick charge") or hemoparasites) and hematological/biochemical analytes? beyond the previous report's explanation made on the discussion...

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Steven Barajas-Valero

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

8 Feb 2022

Response To Reviewers

6 December, 2021

PONE-D-21-27173

Dear Dr. Lewbart,

Thank you for submitting your manuscript to PLOS ONE. Your work has been evaluated by 2 subject experts and the Academic Editor. All 3 view the work to be meritorious, but after careful consideration, it does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

REQUIRED CHANGES

1. Remove redundant data displays. Several data displays are redundant to one another. For example, the data in Figures 1 and 3 are also presented in Tables 2 and 3. The authors need to decide on the best format for presenting data and only display the information once. Given that this paper is a reference collection for future research, the tables are likely better than the figures because they list specific values clearly.

Answer: Thank you. We have removed Figure 1 but kept Figure 2 as it shows values (like IOP and EPP) not currently present in tables. For a detailed reference of the values, which we agree is very important, we decided to maintain the tables as well, but move them to the Supplementary online materials.

2. Add a study site figure. PLOS ONE has an international readership and it important to provide the geographic context for the work. A study site figure showing the location of the Galapagos Islands, Isabela, and the portion of the island studied, is required.

Answer: Thank you. We have now added a map of the Galápagos Islands with an inset focusing on Wolf Volcano, where the only population of pink iguanas is located. It is now the new Figure 1.

3. Allocate some data displays to supplementary online material (SOM). The authors present large collections of data, for example the correlation matrix in Figure 7, but dedicate almost no text in the manuscript to interpretation. If these datasets are not central to the main manuscript, they should be allocated to SOM. The authors need to examine carefully which figures belong in the main manuscript, and which ones should be removed and placed in SOM.

Answer: Thank you. We agree that there were too many graphics. We have now moved some of them (like figures previously labelled as 1 and 7) to the SOM. We also made sure to discuss the content of each graphic a little bit more, even for the ones moved to the SOM.

4. Discussion should be reorganized. The Discussion begins with a short summary of the findings that does not provide any insight into the importance of, or context for the work. This paragraph contains no citations. The first paragraph needs to be re-written to lead the Discussion with a targeted assessment of the key finding(s) of the work that is appropriately referenced. The second paragraph of the Discussion is a cautionary one about the blood chemistry methods used. This should come later after the key biological findings have been discussed.

Answer: Thank you. We have now reorganized the Discussion to according to the editor and reviewer suggestions.

5. The authors should carefully consider the specific comments provided by the referees.

Answer: All comments have been considered carefully and addressed using the track change feature in word.

6. Data accessibility requirement is not met. The raw data for all aspects of the study need to be made publicly available. It is stated by the authors that it is all in the manuscript, but there are no raw data sets provided.

Answer: We have prepared an Excel spreadsheet to share the raw data analyzed in this manuscript.

Please submit your revised manuscript by Dec 25 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Christopher M. Somers

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at:

• https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf

• https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Answer: Thank you. We have now updated all the information and made sure that the manuscript meets PLOS ONE's style requirements.

2. In your Methods section, please provide additional location information about your study area, including geographic coordinates for the data set if available.

Answer: Thank you. We have added a map to give a better idea of the study location.

3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

Answer: Thank you. We have now updated the information and provided details re the funds used for this study.

4. Thank you for stating the following in the Acknowledgments Section of your manuscript:

This work is part of a long-term institutional agreement between the University Tor Vergata and the Galápagos National Park Directorate, aimed at the conservation of Galápagos iguanas. G.C. was supported by a Post-Doctoral Research Fellowship from the San Diego Zoo Wildlife Alliance funded by a donation from the Kenneth and Anne Griffin Foundation. G.G. was supported by grants from the International Iguana Foundation and from Friends of Galápagos, Switzerland. GAL thanks Diego Páez-Rosas, Juan Pablo Muñoz-Pérez, Carlos Mena, Stephen Walsh, and the Galápagos Science Center for their assistance and support.

We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Answer: Thank you. We have now changed the information in the manuscript and we will make sure to provide the correct funding statement during the resubmission process.

5. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

Answer: Thank you. We have submitted the dataset to Dryad servers and we have DOI that is provided in the supplementary on line material for full access to the data used in this article.

6. Please amend the manuscript submission data (via Edit Submission) to include author Hans Westermeyer.

Answer: Thank you. We have now added the author.

Additional Editor Comments:

1. Introduction - paragraph 1 last sentence. The authors state that the pink land iguana is a "flagship" species. What is meant by this? How can a species that was only recently discovered by science and is little known to the broader world be a "flagship"?

Answer: We have clarified this issue by modifying the sentence as follow: “Despite its recent description, this species rapidly became a flagship species, as it helped raising awareness about the value of biodiversity locally and internationally [2,3].”

2. Methods - the authors drew a blood sample of 2.5 ml, but don't mention that this is less than a threshold for safety based on % of total blood volume for the species. In addition, the authors seemed to use very little of this 2.5 ml for their analyses. What was the rationale for taking 2.5 ml?

Answer: The safety threshold considered for lizards is 0.7 mL of blood per 100 g of mass. The amount of blood sampled for our study is considerably lower. We have now clarified this in the manuscript as well, also providing the appropriate reference.

3. Methods - why is tear production an important health parameter to measure, and why is it important to compare right and left eyes?

Answer: The overall rationale to collect baseline data is to have a spectrum of useful parameters. While tear production per se may not be directly correlated with the health status of an individual, it nevertheless represents a parameter that can contribute to the health assessment. For example, should an individual be found with an extremely high tear production it would be interesting to investigate the causes of such alteration, which in turn could lead to the discovery of a threatening condition.

4. Methods - in the "Intra-Specific" section the authors mention removing outliers, but give no rationale for doing so, or the procedure for identifying outliers. In the case of SVL and mass, it is hard to imagine a scenario where true outliers can even exist. More information is required here.

Answer: We have now clarified the issue and explained how and why we looked for outlier values in our dataset.

5. Table 1 - I find the information content in this small table to be very low. It could likely be removed, and the values summarized in the text.

Answer: We have removed Table 1 and summarized its content in the text.

6. Some might question the ethics of capturing and sampling blood from 40% of the individuals in a species that exists nowhere else in the world. Did any of the animals show signs of stress or capture injury? Is baseline data of the kind collected really worth it? Some additional text in the Discussion addressing this point would be useful.

Answer: Thank you for this comment. We indeed considered the implication of this kind of approach. All the procedures adopted in the field to collect individuals and take specific samples have been approved by all parties involved in this research (GNPD, SDZW, Tor Vergata, NCSU). We have now expanded the discussion to also address this issue.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a well-written manuscript outlining field-collected health data from apparently healthy individuals of an endangered species of iguana from the Galapagos islands. The health data collected is thorough and well presented.

There are too many figures with multiple figures demonstrating the same morphometric and clinical data. The number of figures could be reduced to 3 or 4 and the rest of the material submitted as supplemental information. The gross images should be collated into a single image.

In the results section, it is written that "one sample escaped", but the authors likely mean that the iguana escaped and an incomplete set of samples was taken.

More information could be added to the discussion that there are limitations in interpreting clinical data from a small sample of animals as reference intervals could not be obtained from only 15 individuals. The difficulties in interpretation from an iSTAT field analyzer can be explored with paired analyses from an in-lab analyzer, but that was not pursued in this case and this could be addressed by the authors.

Although there is a small sample size and there could be issues with interpretation of the results of a field analyzer, the information is still valuable as baseline data for use in potential captive breeding and reintroduction as well as captive collections (are there individuals of this species kept in zoological collections at the moment?). The information regarding the presence of hemogregarine parasites without an overall negative impact on health is an important take-home from this manuscript and could be highlighted more in the discussion and abstract as it it likely associated with co-evolution.

Reviewer #2: Really important work for an endangered species. Technical language, and easy to understand for the reader. Results presented in a kind graphical way. The authors developed a vigorous statistic analysis.

1) Abstract should highlight the main results and/or important points of discussion.

2) Should consider to include some extra results of descriptive/summary statistics for hematological/biochemistry results (range, min, max, maybe quartiles)

3) For results tables: important to clarify if all the variables have the same number of individuals analyzed, or if not, include an "n" data column in the tables.

4) Could define if there are "local seasons" that externally influence the variables or if it's not taken into account because there're no significant weather variations or according to other analytic factors

5) Did the authors measure the total proteins on in-house equipment to verify or compare the field refractometry results of total solids (TS)?

6) "Data compiling procedure" section could be included in the first methodology heading and so diminish the amount of text.

7) Data of re-sampled individuals is included as separated data (2 samples per individual)? any chance the authors developed a statistical comparison of intraindividual variations between the two samplings?

8) The "heavy mite-infested" individual was included in the general "n" for data presentation?

9) Maybe include a table to summarize the opthalmological results, so it does not get lost in the text.

10) Finally, did the authors make statistical correlations between the grade of parasitism (external ("tick charge") or hemoparasites) and hematological/biochemical analytes? beyond the previous report's explanation made on the discussion...

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Steven Barajas-Valero

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Response To Reviewers D-21-27173.docx

Click here for additional data file.

22 Feb 2022

Health assessment of the pink land iguana, Conolophus marthae.

PONE-D-21-27173R1

Dear Dr. Lewbart,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Christopher M. Somers

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

11 Mar 2022

PONE-D-21-27173R1

Conservation of a flagship species: Health assessment of the pink land iguana, Conolophus marthae

Dear Dr. Lewbart:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Christopher M. Somers

Academic Editor

PLOS ONE