Squirrels

For each of the four species we constructed simulated datasets consisting of 100 replicates each of the following sample sizes: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75 and 100 individuals. Each replicate contained a random subset of individuals from the empirical dataset and replicates were created using a macro in excel, designed to assign each individual in the empirical dataset a random number (between 1 and 10,000), sort the dataset by the random numbers, then select the first 5 (or 10, 15, etc. depending on the sample size category) to a new worksheet, 100 times, resulting in 100 simulated ‘populations’ that are independent, random subsamples of the empirical dataset, at each sample size. Sampling was done without replacement, so no individual was present more than once in the same replicate (as in a real population genetic dataset), but as replicates were independent of each other, the same individual could be present in more than one replicate of the simulated dataset at each sample size. GenAlEx 6.2 [9] was then used to calculate allele frequencies, heterozygosity expected under Hardy-Weinberg Equilibrium (H_E) and pairwise F_ST between the simulated and empirical datasets for each replicate at each sample size. When we refer to the ‘empirical dataset’ we mean the real dataset of 547 ant, 107 squirrel, 616 albatross or 98 kakī individuals (see below for dataset details). Because the kakī dataset comprised fewer than 100 individuals, the largest sample size assessed for this species was 75 individuals. Throughout this paper, ‘individuals’ means diploid individuals.

Table 1 summarizes the MPXV isolates used in our phylogenetic analysis and provides references for the original description of the cases; they include (a) four isolates that correspond to MPXV obtained from outbreaks recorded in laboratories; (b) the five isolates included in the study by Likos et al.; (c) twelve isolates available from human case reports in Sub-Saharan Africa between 1970 and 2010; (d) one isolate obtained from a squirrel from Yambuku, DRC, which is the only MPXV isolate derived directly from wildlife included in this study; and (e) 23 isolates from human cases reported in Sankuru District, DRC between 2006 and 2007 [31 (link)]. In all, the 45 analyzed isolates cover the known range for MPX in Central and West Africa (Figure 1). Isolates form Nigeria, Cameroon, and Gabon are of particular interest for the present analysis since they are closer to one of the proposed biogeographic barriers (Cameroon Highlands) than the rest of the isolates from Western Africa or Congo Basin.
Genomes were sequenced using Sanger sequencing or Illumina^® (Illumina Inc., San Diego, CA USA) paired end sequencing. Whole genomes of MPXV isolates were aligned using MAFFT v7.017 [32 (link)]. Cowpox virus (Grisham 1990, X94355) and horsepox virus (Mongolia 1976, DQ792504) isolates were included as outgroup taxa. The original alignment was 241,258 bp in length. The first 25 kb and last 26 kb are highly variable between CPXV and other Orthopoxviruses since they contain a large number of indels; thus, they were trimmed. Subsequently, all columns containing indels were removed, resulting in an alignment of 173,804 bases from the central conserved region of the genome. A majority-rules consensus tree was estimated from the alignment of all genomes using MrBayes v3.2.2 [33 (link),34 (link)]. Settings included a general time reversible model (lset nst = 6) with estimated stationary state frequencies and substitution rates, and a model of gamma-shaped rate variation across sites (rates=gamma). The tree search was carried out over five million generations.
A patristic distances matrix was obtained from the consensus tree and separated into the groups representing the two recognized MPXV clades. We tested the distance values within each group for normality via the Shapiro-Wilk test, compared their variances via the F-test and compared the two groups using a Student’s t-test; all statistical test were performed in R 3.1.1 [35 ]. MEGA v6.06 [36 (link)] was used to calculate within group uncorrelated p-distances for a subset of samples from the Lomela Health Zone in DRC.

Animals were obtained from various sources such as abattoirs, zoos, veterinary practices and scientific establishments (see Acknowledgements). They had either been used for other scientific procedures, sacrificed for food production, died naturally or were put down owing to injury or illness. No animals were killed specifically for this project. Eyes were obtained either immediately following death, or soon thereafter, and were either used immediately or frozen dry for several days before thawing. Variable numbers of lenses were available for each species and in four species a range of lens sizes/ages were examined (see table 1 for details).
Table 1.

Summary of mammalian lenses examined ranked by the amount of UVA they transmit. ‘50%T’ is the wavelength at which the lens transmits 50% of the incident illumination. ‘%UVA transmitted’ is a measure of the proportion of light between 315 and 400 nm that is transmitted by the lens (see the electronic supplementary material, S2). For most species, lens transmission and axial diameter (pathlength) varied little between individuals and averages are shown. Where there were significant differences between individuals, ranges are given. Where the transmission of the lens varied with lens size/age, the % UVA on the retina was calculated using specific ages/lens sizes as described in footnotes.

order	family	species	number of lenses	pathlength (mm)	50%T (nm)	%UVA transmitted
Rodentia	Muridae	mouse (Mus musculus)	29	1.9–2.8	313–337	81.4a
Rodentia	Muridae	black rat (Rattus rattus)	11	3.7–5.2	317–372	80.5b
Erinaceomorpha	Erinaceidae	hedgehog (Erinaceus europaeus)	4	3.0	326	65.5
Carnivora	Canidae	dog (Canis lupus familiaris) (labrador)	2	5.0	335	61.3
Chiroptera	Pteropodidae	Livingstone's fruit bat (Pteropus livingstonii)	4	5.0–6.0	332–422	60.8c
Carnivora	Felidae	cat (Felis catus)	6	7.0	345	58.9
Carnivora	Mustelidae	ferret (Mustela putorius furo)	4	3.9	344	56.1
Rodentia	Muridae	brown rat (Rattus norvegicus)	2	4.2	339	55.8
Artiodactyla	Giraffidae	okapi (Okapia johnstoni)	2	7.0	355	53.4
Artiodactyla	Suidae	pig (Sus scrofa)	5	5.5	375	43.6
Rodentia	Caviidae	guinea pig (Cavia porcellus)	11	3.7	377	34.6
Carnivora	Ailuridae	red panda (Ailurus fulgens)	1	5.8	386	30.2
Rodentia	Sciuridae	flying squirrel (Glaucomys volans)	2	4.9	423	29.3
Chiroptera	Pteropodidae	Rodrigues flying fox (Pteropus rodricensis)	1	4.8	388	28.1
Artiodactyla	Cervidae	reindeer (Rangifer tarandus)	5	10.1	384	26.5
Artiodactyla	Cervidae	pudú (Pudu puda)	2	7.0	386	25.0
Artiodactyla	Bovidae	cattle (Bos primigenius)	8	11.1	384	22.1
Artiodactyla	Bovidae	sheep (Ovis aries)	4	7.7	393	15.2
Rodentia	Dasyproctidae	agouti (Dasyprocta punctata)	1	6.1	406	15.0
Lagomorpha	Leporidae	rabbit (Oryctolagus cuniculus)	2	6.7	392	12.7
Artiodactyla	Tragulidae	java mouse deer (Tragulus javanicus)	2	9.0	403	12.4
Artiodactyla	Bovidae	Arabian oryx (Oryx leucoryx)	1	10.3	400	8.5
Artiodactyla	Camelidae	alpaca (Vicugna pacos)	5	10.2	405	6.0
Perissodactyla	Equidae	horse (Equus ferus caballus)	1	12.0	416	4.6
Primates	Cebidae	squirrel monkey (Saimiri sciureus sciureus)	2	4.6	420	2.8
Primates	Lemuridae	ring-tailed lémur (Lemur catta)	1	6.5	425	2.0
Carnivora	Herpestidae	meerkat (Suricata suricatta)	3	2.4–3.4	420–436	1.7d
Primates	Callitrichidae	marmoset (Callithrix jacchus)	1	3.0	427	0.9
Artiodactyla	Bovidae	lowland anoa (Bubalus depressicornis)	1	8.0	478	0.6
Rodentia	Sciuridae	ground squirrel (Urocitellus richardsonii)	2	3.1	462	0.6
Primates	Cercopithecidae	macaque (Macaca fascicularis)	5	3.3	424	0.5
Primates	Atelidae	red-faced spider monkey (Ateles paniscus)	1	3.8	438	0.4
Primates	Callitrichidae	golden lion tamarin (Leontopithecus rosalia)	1	3.0	441	0.4
Scandentia	Tupaiidae	Tree shrew (Tupaia glis)	1	3.2	435	0.3
Primates	Lemuridae	Alaotran gentle lemur (Hapalemur alaotrensis)	1	5.9	425	0.3
Rodentia	Sciuridae	grey squirrel (Sciurus carolinensis)	2	3.6	441	0
Rodentia	Sciuridae	prairie dog (Cynomys ludovicianus)	7	3.6	463	0
Primates	Cebidae	capuchin (Cebus apella)	1	3.9	426	0

^aAged 69–72 days with lens pathlength 2.2 mm.

^bPathlength 3.8 mm.

^cPathlength 5.0 mm.

^dPathlength 3.4 mm.

Lenses, and usually corneas, were removed from the eye, briefly rinsed in phosphate-buffered saline (PBS) and mounted in purpose-built holders in air in front of an integrating sphere within a Shimadzu 2101 UVPC spectrophotometer. Vitreous humour was also removed from the eyes of some animals with a syringe and placed in a standard quartz cuvette within the same apparatus. Transmission at 700 nm was set to 100% and ocular media scanned at 1 nm intervals from 300 to 700 nm.
To determine the effect of freezing on lens transmission, three fresh bovine lenses were scanned soon after death, frozen in air at −25°C for 4 days, thawed and rescanned.
The pigments responsible for lens pigmentation were also extracted and spectrally characterized for six species (see the electronic supplementary material, S4).

Samples were collected from three different areas, including two natural sites and one anthropized site located in Tuscany, Central Italy (Figure 1).
The anthropized site (Area 1) is located in Pisa Province within the municipalities of Crespina Lorenzana and Casciana Terme Lari (10.56815° N–43.56796° E) and includes 18 towns, with an average human density of 134.08 people/km². The area is characterized by a highly anthropized fragmented agroecosystem in which small woody areas are interspersed with agricultural and urban zones [9 (link)]. A wide variety of wild mammals live in this area, such as crested porcupines (Hystrix cristata), wild boars (Sus scrofa), roe deer (Capreolus capreolus), pine martens (Martes martes), stone martens (Martes foina), skunks (Mustela putorius), badgers (Meles meles), hares (Lepus europeus), eastern cottontails (Sylvilagus floridanus), wild rabbits (Oryctolagus cuniculus), red foxes (Vulpes vulpes), wolves (Canis lupus), and the introduced invasive coypu (Myocastor coypus) [10 (link)].
The natural area includes two different sites: the Monterufoli Caselli Nature Reserve of (Val di Cecina, Pisa, Central Italy, 43°15′6.48″ N, 10°45′26.64″ E) (Area 2) and the Foreste Casentinesi National Park (43°50′36″ N 11°47′28″ E) (Area 3). The Monterufoli Caselli Nature Reserve covers an area of 4.978 ha and is characterized by wide woody areas of Mediterranean scrub and oaks (Quercus ilex). Hares; wild ungulates (mainly wild boars, fallow deer (Dama dama), and mouflons (Ovis musimon)); and carnivores such as red foxes, wolves, pine martens, weasels (Mustela nivalis), badgers, stone martens, and wild cats (Felis silvestris silvestris) are the most representative wild mammals in the area [11 ]. The Foreste Casentinesi National Park (43°50′36″ N 11°47′28″ E), which extends along the Tuscan-Romagna Apennine Ridge [12 ], is characterized by a great richness and variety of wild fauna. The most common mammals are the red deer (Cervus elaphus), the fallow deer, the roe deer, wild boars, mouflons, and the wolf, the largest predator present in the park. At least 21 species of micro- and meso-mammals have been observed in the park territory, among which the most common are the fox, the wild cat, the hare, the European mole (Talpa europaea), the blind mole (Talpa caeca), the red squirrel (Sciurus vulgaris), the crested porcupine, and the raccoon (Procyon lotor), as well as several mustelid species, such as badgers, weasels, stone martens, skunks, and martens.

Rectal temperature (T_re) was measured using a thermistor (Mallinckrodt Medical Inc., St. Louis, MO, USA), which participants inserted to a depth of ~10 cm. Area-weighted mean skin temperature (T_skin) was calculated from four right-side sites; chest, the centre of the bicep, the centre of the thigh, and the widest part of the calf [35 (link)]. Temperatures were logged at 30-s intervals (Grant 1200 series Squirrel Data Logger, Grant Instruments, Cambridge, UK). Respiratory gases were sampled at 1, 2, 3% ∆BM for 4 min during passive trials and for 8 min (exercise and recovery) during active trials (S-3A Oxygen Analyser and CD-3A Carbon Dioxide Analyser; AEI Technologies, Inc., Bastrop, TX, USA). The RER values > 1.00 or <0.70 were excluded from metabolic analyses. Urine was collected at baseline, 1, 2, 3% ∆BM and 1, 2, and 24 h of recovery, from which urine colour was determined using a urine colour chart (1–8; printed from the internet), and U_SG was determined using a hand-held refractometer (Uricon-N, Urine Specific Gravity Refractometer, Atago Co., Tokyo, Japan). Heart rate (HR) was recorded from baseline to 3% ΔBM, and at 1 and 2 h recovery using telemetry from a chest band receiver and stored at 15-s intervals (Polar S810i Heart Rate monitor, Polar Electro Inc., Port Washington, NY, USA). The blood pressure was measured manually and in triplicate with a sphygmomanometer. Thirst was measured on a validated 9-point Likert thirst scale (1: “not thirsty”—9: “very thirsty”) [36 (link)], and oral sensations were assessed using the thirst sensation scale (TSS), which contains 6 graded oral sensations associated with thirst [37 (link)]. Capillary blood samples were collected into three heparinised 100 μL tubes at baseline, 1, 2, 3% ∆BM and 1, 2 h. Haemoglobin concentration [Hb] (Model OSM3, Radiometer, Copenhagen, Denmark) and hematocrit (Hct) were measured in triplicate. Haematocrit was determined using a custom-made vernier calliper (University of Otago, Dunedin, New Zealand) after being spun for 10 min at 1520 G (Thermo IEC MicroCL 17, radius 8.5 cm). The remaining plasma was stored at −80 °C for later analysis of P_osm using a 3-point calibrated (100, 290, and 1000 mOsmol/kg) vapour pressure osmometer (5520 Wescor Vapro, Austin, TX, USA).