All performed experiments were allowed by institutional Animal Care and Use Committee of the Charles University in Prague, and approved by Ethical Committee of Ministry of Education, Youth and Sports of the Czech Republic license no. 18147/203 and 24773/2008–10001. All animals from nature were purchased from a Czech company importing animals in the year 2002 and from private breeders. Eublepharis sp. does not belong to the species whose trade is limited by the CITES agreement or any other known regulations. According to the IUCN categorization it neither belongs to endangered species. After the study, geckos were used either for other behavioural experiments or for breeding purposes.

Experimental procedures

The breeding stocks of the parental species were 38 females and ten males of an E. macularius (the first generation of descendants of wild-caught animals imported from Pakistan) and only five females and three males of the rare E. angramainyu (wild-caught animals and their two daughters; a putative locality of origin: Choqa Zanbil, Khuzestan province, Iran, 32"00'N 48'31'E, for more details about the locality see [102]).

To obtain F 1 hybrids, 17 virgin females of the E. macularius were allowed to copulate with one breeding male of the E. angramainyu. The resulting F 1 hybrids were reared to sexual maturity and further bred to obtain F 2 hybrids and/or backcrosses with either E. macularius or with the same breeding male of the E. angramainyu (with their father). Fertility of some of the backcross hybrids was subsequently assessed by crossing with the parental species (for details see under the Results and Table 1). Because the geckos of the genus Eublepharis are able to store sperm for several months, each experimental female was allowed to copulate exclusively with a single male during a given mating season (lasting from January/February to July/August). In contrast, males were allowed to copulate with multiple females within a single breeding season. 15 F 1 hybrid females were experimentally crossed for more than one breeding season; this allowed us to test their fertility with two or three different males (first with F 1 male or one of the parental species and then with a male of the other parental species). As controls for the hybridization experiments, 16 females of the E. macularius and five E. angramainyu females were bred with conspecific unrelated males (with the exception of two E. angramainyu females, which were the daughters of the breeding male).

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larger image TIFF original image Download: Table 1. The incubation success of eggs (hatchability) and survival rates of hatchlings. The parental species (E. macularius—P M , E. angramainyu—P A ), their hybrids of the first (F 1 ) and second (F 2 ) filial generations, backcrosses of F 1 females to male of E. angramainyu (B 1A ; denoted as MAxA), the reciprocal backcrosses of F 1 males or females to E. macularius (B 1M ; the individuals with father F 1 hybrid are denoted as MxMA, while those with mother F 1 hybrid as MAxM), and two categories of higher order hybrids (crosses of MxMA females with males of either E. macularius or E. angramainyu). The above mentioned generations and/or crossings refer to the embryos and hatchlings. https://doi.org/10.1371/journal.pone.0143630.t001

The animals were housed individually in glass terrariums 60 x 30 x 20cm or 30 x 30 x 20cm in size. The ambient temperature in the breeding room was about 28°C with permanent presence of basking cables under every terrarium to maintain a temperature gradient. The floor of each cage was covered with bark substrate. Paper shelters, as well as feeding and drinking dishes, were provided. During the laying season, containers with adequately humid coconut substrate for egg deposition were added. The geckos had continuous access to water and were fed crickets and mealworms dusted with vitamins and minerals (Nutri Mix) weekly; AD 3 and E vitamins were provided once per 14 days. The hatchlings were housed singly in plastic boxes 20 x 20 x 15cm and were fed solely the vitamins dusted crickets up to the three months of their age.

We studied the following nine categories of the parental species and their hybrids that are further referred to as follows (the abbreviations are given in parentheses; on the first place there is always an abbreviation for a female, then cross (x) with a male on the second position; the number and the sexes of these specimens in Table 1):

P M −the parental generation of the E. macularius, both parents belong to the E. macularius (M); P A −the parental generation of the E. angramainyu, both parents belong to the E. angramainyu (A); F 1 –the first generation hybrid, a mother of the E. macularius and a father of the E. angramainyu (MA); F 2 –the second generation hybrid, both parents are F 1 hybrids of the E. macularius and E. angramainyu (MAxMA); B 1A –the first generation backcross with the E. angramainyu, a mother is an F 1 hybrid and a father belongs to the E. angramainyu (MAxA); B 1M –the first generation backcross with the E. macularius, a mother is an F 1 hybrid and a father belongs to the E. macularius (MAxM); B 1M –the first generation backcross with the E. macularius (reciprocal to 6), a mother belongs to the E. macularius and a father is an F 1 hybrid (MxMA); B 1M x P A −a higher order hybrid, a mother is the B 1M hybrid (cf. 7) and a father belongs to the E. angramainyu ((MxMA)xA); B 2M –the second generation backcross with the E. macularius, a mother is the B 1M hybrid (cf. 7) and a father belongs to the E. macularius ((MxMA)xM).

Respective to the nocturnal activity pattern of the geckos and their thermal preferences [103], the mating attempts were conducted in the evening (after 7 p.m.) in a temperature-controlled breeding room (28°C) illuminated by a single red 25-W light bulb. Prior to the experiment, the females were weighed and were controlled for their receptivity by a visual inspection of the folicular growth through the abdomen wall [93]. We gently placed the male into the female’s terrarium for 30 min and we recorded the copulation behaviour using a night vision video camera. If mating did not occur within this interval, we repeated the trial the other day. The primary aim was to allow successful mating and to enable the production of fertilized eggs.

During the egg-laying season (since February to September), we controlled the egg-deposition containers for three times a week. The eggs were weighted and placed to the temperature-controlling incubator in plastic boxes, each containing a single clutch. We set the temperature to 28.5 ± 0.5°C, which is an optimal and preferred incubation temperature in the E. macularius [97, 104, 105]. Nevertheless, according to our previous experience with the E. angramainyu, the successful development of their embryos require slightly lower temperatures and longer incubation time. At 28°C incubation temperature (an upper limit for successful incubation), some hatchlings possessed a prolapsed yolk pouch. After consultation with other experienced colleagues at this field (e.g. Lukáš Kratochvíl, Charles University), we set the incubation temperature to 26 ± 0.5°C for the eggs laid by the E. angramainyu. The only feasible solution was to perform the experiments within the temperature range of 26–28°C, among which the incubation temperature overlaps in both species included in the experiment. Consequently, the eggs laid by the F 1 hybrid females were initially incubated either at 26°C or at 28°C to compare the hybrid hatchability at the optimum incubation temperature for both parent species (at 26°C in E. angramainyu and at 28°C in E. macularius). The temperature was selected at random for the first clutch and then regularly switched in successive ones (see Table 1). In additional backcrossing of the F 1 females with the E. macularius males in the breeding season 2013, which was aimed to prove their fertility, the incubation temperature was set to 28°C.

For every egg we took down the identity of the parents, the dates of laying and hatching, the weights of egg and hatchling and the incubation temperature. In order to perform formal tests of the hatchability, we used GLMs, in which the hatching of the incubated eggs of an individual clutch (number of hatchlings of one clutch and number of non-hatched eggs of the same clutch) was given as a dependent variable with binomial distribution and logit link function; the juvenile form, the incubation temperature and its interactions, and the clutch sequence were introduced as category explanatory variables. The calculations were performed in the R (R Development Core Team, Vienna, Austria).

Most eggs that have failed to hatch until the standard terms [106] were dissected to prove the presence and developmental stage of the embryos. Nevertheless, the content of many rotten eggs was entirely decayed, which precluded a reliable dissection. Thus, in many cases, we were unable to distinguish the fertilized eggs from those unfertilized.

The hatchlings were weighted and scanned (a ventral and a dorsal view of the body) in standardized positions. This procedure was repeated in adulthood at the age of 2–3 years. In order to provide a reference in the form additional fully grown individuals, the data set was supplemented with adult specimens of E. macularius from Pakistan and E. angramainyu from Iran (both wild-caught individuals and their descendants). In total, we collected 91 valid records for juveniles (E. angramainyu– 4 specimens, E. macularius– 32 spec., MA– 25 spec., MAxMA– 3 spec., MxMA– 11 spec., MAxM– 16 spec.) and 139 valid records for the animals older than two years (E. angramainyu– 10♀, 5♂, E. macularius– 55♀, 13♂), MA– 24♀, 3♂, MAxMA– 1♀, MxMA– 15♀, 2♂, MAxM– 7♀, 3♂, MMAxA– 1♀).

The coloration pattern analysis of the E. angramainyu (29 spec.), E. macularius (29 spec.), F 1 (28 spec.) and the B 1M (27 spec.) hybrids we conducted on a dorsal view of the head. For this purpose, we examined the scans of the animals older than one year with fully developed adult coloration pattern (Fig 1, also in [97]) First, the scans were set to black and white colors (converted to Grayscale mode, then to Bitmap mode by 50% Threshold method in Adobe Photoshop CS2; Adobe Systems Incorporated, USA). The total number of dark (melanistic) spots and the length of the longest continuous spot were performed by UTHSCSA Image Tool (San Antonio, Texas). The area of the largest continuous dark spot was measured in ImageJ program (National Institutes of Health, USA) (Fig 2). All measurements were calibrated using a squared paper present in each scan.

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larger image TIFF original image Download: Fig 1. The external appearance and coloration. E. macularius (P M ), E. angramainyu (P A ), their hybrid of the first (F 1 ) and second filial generations (F 2 ), backcrosses of the F 1 with male or female E. macularius (B 1M : MAxM and B 1M : MxMA, respectively), and a cross between a female of the latter backcross and a male of the E. angramainyu (B 1M xP A ). The scale bar used was 10mm. https://doi.org/10.1371/journal.pone.0143630.g001

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larger image TIFF original image Download: Fig 2. Measurements of the body and the head. SVL: snout-vent length; DEX1: from the margin of the front leg to the cloacal lips; DEX2: from the margin of collar to the cloacal lips; TW: tail width; UFL: upper fore-limb length; CFW: chest and upper fore-limb width; LFL: lower fore-limb length; FL: finger length; HHW: hip upper hind-limb width; KHL: knee to heel length; HL: head length; HW: head width; EEL: length between eyes; REL: rostrum to eye length; the largest spot: length and area was measured; the number of spots was computed. https://doi.org/10.1371/journal.pone.0143630.g002

To test the effect of species/hybrid category on the adult coloration pattern on the head, we analyzed the Number of spots (square-root transformed), Spot size (area of the largest spot scaled to the head size and natural log-transformed) and Spot length (length of the largest spot scaled to the head length and natural log-transformed) using linear models with the form of the animal (P M , P A , F 1 , B 1M ) as a factor. Post hoc Tukey tests were adopted to compare the factor levels. The calculations were performed using STATISTICA, version 6.0 (StatSoft Inc., Tulsa, USA).

For morphometric analyses we adopted and/or modified standard measurements from Kratochvíl et al. (2003) [107] and Frýdlová et al. (2011) [108]. We used the following 14 measurements that were measured by UTHSCSA Image Tool from digital images: (1) SVL–snout-vent length; (2) DEX1 –distance between the extremities (from the posterior margin of the front leg to the cloacal lips); (3) DEX2 –from the posterior margin of collar to the cloacal lips; (4) TW–tail width (the largest width of the tail); (5) UFL–upper fore-limb length; (6) CFW–chest and upper fore-limb width; (7) LFL–lower fore-limb length (without hand); (8) FL–middle finger length without the claw; (9) HHW–hip upper hind-limb width; (10) KHL–knee to heel length; (11) HL–head length (from rostrum to the posterior margin of collar); (12) HW–head width, the largest width of the head; (13) EEL–distance between anterior corners of eyes; (14) REL–rostrum to eye length, from tip of the snout to the anterior corner of eye. In case of juveniles we measured only SVL. For the definition of these measurements, see Fig 2.

In order to separate a shape component of the morphometric variation, we performed the size-adjustment of the original variables. For this purpose, we used the method published by Somers (1986, 1989) [109, 110] as implemented in the Size analysis v02 [111–113]. This software computes not only generalized (multivariate) isometric size of the original untransformed measurements, but also partial isometric size-adjusted measurements. These size-free data were further analyzed by a multivariate exploratory statistics as implemented in the discriminant function analysis (DFA) subroutine of STATISTICA, version 6.0. The data were checked for normality prior to the statistical analyses. Deviations from normality were small, and most distributions were both unimodal and symmetrical as required for the used multivariate procedures.