Chromosome evolution in a genus that deviates from the standard karyotype formula of Lacertidae

doi:10.21203/rs.3.rs-2917305/v1

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Chromosome evolution in a genus that deviates from the standard karyotype formula of Lacertidae

https://doi.org/10.21203/rs.3.rs-2917305/v1

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published 01 Sep, 2023

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This paper describes the preparation of flow-sorted chromosome paints from the Iberian Rock lizard I. monticola, and exemplifies their subsequent use in cross-species chromosome painting for carrying out comparative analyses of chromosome evolution in the congeneric species I. galani and I. bonnali, as well as in two other species of Lacertini, namely Lacerta schreiberi and Timon lepidus, whose sex chromosomes were also studied through comparative genomic hybridization. Most species of Lacertini possess a diplod number of 2n = 38, with 36 acrocentric macrochromosomes and 2 microchromosomes. However, the nine species included in the genus Iberolacerta do not possess microchromosomes, and additionally very conspicuous differences from the standard Lacertini karyotype are observed in the three Pyrenean species of this genus, including I. bonnali, with several biarmed metacentrics and a Z₁Z₂W multiple sex-chromosome system. All the species of the familiy Lacertidae, so far with the possible exception of L. schreiberi, appear to share homologous Z chromosomes, which date back to the last common ancestor of the whole group. We provide conclusive evidence that L. schreiberi should no longer be considered an exception to this rule, demonstrate that the loss of microchromosomes in Iberolacerta was produced by their fusion to a middle-sized chromosome, that the multiple sex-chromosome system of the Pyrenean species of Iberolacerta originated from the fusion of the ancestral W chromosome with one of the shortest autosomes, and provide additional evidence of the fast evolution of DNA sequences linked to the W chromosome in Lacertini.

Reptilia

ZOO-FISH

iCGH

sex-chromosomes

microchromosome loss

W-autosome fusion

satellite DNA

Evolution of chromosome number and chromosome form may seem of little significance in the current -omics era, with so many thousands of whole-genome sequencing projects (WGS) already fulfilled or underway. Actually, only a small fraction of these projects in Animals have reached the chromosome assembly level (1979 out of 10337 reports, NCBI Genome Database, last accessed 23/02/23). Paradoxically, in many respects, a similar situation was experienced in the first quarter of the 20th century, during what M.J.D. White called "the heyday of atomistic genetics", until the evolutionary importance of the physical basis of a species genetic system, its karyotype, was realized (White 1973).

Notwithstanding this apparent disregard for karyotypes, there is a special kind of chromosomes whose evolution has received continuing attention over the years, namely animal sex-chromosomes. This is particularly so for squamate reptiles, since this group exhibits an astonishing diversity of sex-determining systems, which range from environmental sex determination to genotypic sex determination, including male heterogamety (XX/XY), female heterogamety (ZZ/ZW) and multiple sex chromosomes (Sarre et al. 2004; Pokorná and Kratochvíl 2009; Ezaz et al. 2010; Nielsen et al. 2020; Rovatsos et al. 2022). So, the study of sex-chromosomes in reptiles during the last two decades has revealed that, contrary to previous claims of the overall high lability of their sex determination systems (Ezaz et al. 2010; Sarre et al. 2011; Matsubara 2019), and notwithstanding the independent origins of sex chromosomes in different families (Nielsen et al. 2020; Rovatsos et al. 2022), many reptile groups actually possess old, long-term stable sex-chromosomes (Rovatsos et al. 2014a, b, 2015; Augstenová et al. 2021; Thépot 2021), and that relatively recent turnovers of sex-chromosomes are restricted to just a few genera or families (Gamble et al. 2015; Patawang et al. 2017; Sidhom et al. 2020; Augstenová et al. 2021; Keating et al. 2021). The Old World Lizard family Lacertidae(Pyron et al. 2013) does not appear to be an exception to this rule (Rovatsos et al. 2016), although the portrait of the evolution of their sex-chromosomes is still unfinished. With 370 species in 45 genera (Uetz et al. 2022), it is the predominant lizard group in Europe and a substantial component of the squamate reptile diversity in Africa (Arnold et al. 2007; Hipsley et al. 2009). The family, whose diversification started roughly 87 Mya, in the Late Cretaceous Epoch (Garcia-Porta et al. 2019), contains two subfamilies, Gallotinae and Lacertinae, with the latter composed of two monophyletic clades or tribus, after Arnold et al. (2007), the mainly Paleartic Lacertini and the Eremiadni of Africa. Genetic analyses indicate a fast diversification and radiation of Lacertini in the late Eocene, roughly 37 Mya (Hipsley et al. 2009; Garcia-Porta et al. 2019). Most species of Lacertini (and Eremiadni, for that matter) possess a diploid number of 2n = 38, with 36 acrocentric macrochromosomes and 2 microchromosomes (Arnold et al. 2007; Mezzasalma et al. 2021), with the most conspicuous exceptions to this karyotypic formula being the three Pyrenean species of the genus Iberolacerta, I. bonnali, I. aranica and I. aurelioi, whose male karyotypes consist of 2n = 24 (bonnali) or 2n = 26 (aranica and aurelioi) chromosomes, and in two of which (bonnali and aurelioi) thre is a Z₁Z₂W multiple sex-chromosome system, very infrequent among Lacertidae (Odierna et al. 1996; Mezzasalma et al. 2021).

Female heterogamety is the only sex-chromosome system that has been found in Lacertidae (Mezzasalma et al. 2021), and all tested species, except for two discordant reports involving L. agilis (Srikulnath et al. 2014)d schreiberi (Rojo 2015), appear to share homologous Z chromosomes, which date back to the last common ancestor of the whole group, thus supporting the long-term stability of their ZZ/ZW chromosome systems (Rovatsos et al. 2019). The case for L. agilis has been strongly contested by Lisachov et al. (2020), while that for L. schreiberi must also be rejected on the basis of the evidence to be shown in this article. On the other hand, cytogenetic analyses, mainly accomplished through Giemsa staining, C-banding and G-banding (Olmo et al. 1986, 1987; Odierna et al. 1993; Rojo et al. 2014), revealed extensive variability in the morphology and degree of differentiation of the W chromosome across the family, spanning from W's completely euchromatic and homomorphic with the Z, to others strongly heterochromatic and morphologically distinct. These cytological observations were complemented by fluorescence in situ hybridization (FISH) studies, whereby it was concluded that the extent of heterochromatinization appears to be associated with independent, species-specific, extensive accumulation of microsatellite repeats, thus pointing to the W chromosome as one of the most dynamic parts of Lacertidae genomes (Pokorná et al. 2011; Giovannotti et al. 2018; Suwala et al. 2020).

When whole genome sequences are not available, chromosome homology among species is best determined through molecular cytogenetic analyses, that can reveal the evolutionary complexity hidden under morphologically similar (using standard banding and staining techniques) chromosome patterns (Matsubara et al. 2014). Here, we describe the preparation of flow-sorted chromosome paints from the Iberian Rock lizard I. monticola (BOULENGER, 1905), and exemplify their subsequent use in cross-species chromosome painting to carry out comparative analyses of chromosome evolution. There are currently nine recognized species in Iberolacerta, a genus almost entirely confined to small widely separated mountain areas in the Iberian Peninsula and in the Balkan Peninsula (Arribas et al. 2014). Cytogenetic surveys based on conventional staining and banding techniques showed that, except for the three Pyrenean species, with reduced diploid numbers and many biarmed chromosomes that probably evolved from the ancestral acrocentric complement through a series of Robertsonian fusions, Iberolacerta species possess a similar karyotypic macrostructure, with a diploid number of 2n = 36 and all chromosomes acrocentric (they thus depart from the standard Lacertini pattern by not posssessing microchromosomes), but different degrees of W chromosome differentiation (Odierna et al. 1996; Arribas and Odierna 2004; Arribas et al. 2006; Rojo et al. 2014). In this preliminary analysis, we applied the I. monticola chromosome paints to study the chromosome evolution in the following lacertid species: the congeneric I. galani ARRIBAS, CARRANZA & ODIERNA, 2006 (2n = 36), with ZW sex chromosomes (Arribas et al. 2006); I. bonnali (LANTZ, 1927) (2n = 24 in males, 2n = 23 in females), with 12 biarmed chromosomes and a multiple Z₁Z₂W chromosome system (Odierna et al. 1996); Lacerta schreiberi BEDRIAGA, 1878 (2n = 38), possessing the standard Lacertini karyotype (Mateo and Cano 1991); and Timon lepidus ibericus (LÓPEZ-SEOANE, 1885) (2n = 36), with a metacentric chromosome pair presumably produced by fusion of two large acrocentric chromosomes, a pair of microchromosomes, and a W sex microchromosome (de Smet 1981; Olmo et al. 1987; Mateo et al. 1999). Comparison of sex chromosomes at the molecular level was further extended through comparative genomic hybridization (CGH) between I. monticola, L. schreiberi and T. lepidus.

Animal samples

Two adult females and one adult male of I. monticola were collected from the population of the fluvial valley of the river Eume (A Coruña, Spain). The tail tips from one adult female of L. schreiberi and another one of T. lepidus were collected at the Natural Park Montes do Invernadeiro (Ourense, Spain); additionally, one adult female of L. schreiberi was collected at Aranga (A Coruña, Spain). Finally, two adult females of I. galani and the tail tip of one adult female of I. bonnali were collected at the localities of A Ponte, Pena Trevinca (Ourense, Spain) and Pico de Urdiceto, Pirineos (Huesca, Spain), respectively. The sex of each animal was determined by examination of sexually dimorphic external morphology. All these samples were used to make metaphase chromosomes. Permissions for fieldwork and ethics approval of experimental procedures were issued by the competent authorities (Xunta de Galicia, Junta de Castilla-León and Gobierno de Aragón, in Spain), in accordance with the Spanish legislation (Royal Decree 1201/2005 and Law 32/2007, on the protection of animals used for experimentation and other scientific purposes). All the animal samples used in this study were generously supplied by Pedro Galán.

Metaphase chromosomes preparation

The tail tip collected from each specimen (approximately 10 mm) was pre-treated before setting up the cell cultures as described in Ezaz et al. (2008), with slight modifications. Briefly, the surfaces of the tail tips were sterilized by wiping with gauze soaked in 70% ethanol, clipped and incubated at 30ºC for 24 h in Collection Medium [RPMI 1640 Medium containing 25 mM HEPES (Sigma) with 1 mg/mL kanamycin (Sigma) and 1% antibiotic-antimycotic (Life Technologies-Gibco)].

Fibroblast cell lines and metaphase chromosome spreads were prepared as described in Rojo et al. (2014). Cultures for flow-sorting were split up to 4 passages before the chromosomes were harvested.

Probe preparation

Chromosome paints from a female I. monticola were prepared from chromosomes sorted with a dual laser cell sorter (Mo-Flo, Dako) at the Cambridge Resource Centre for Comparative Genomics, Department of Veterinary Medicine, University of Cambridge, Cambridge, UK, as previously described (Yang et al. 1995). Sorted chromosomes were used as templates for DNA amplification by DOP-PCR (Telenius et al. 1992). Primary DOP-PCR products were used as templates in a secondary DOP-PCR to incorporate biotin-16-dUTP (Roche).

TaqI sat DNA species-specific probes were prepared as described in Rojo et al. (2015).

Karyotyping and C-banding

For karyotypying, the slides were stained with DAPI (1.5 µg/mL) in anti-fade medium Vectashield (Vector Laboratories). Sequential C-banding + CMA₃ + DAPI staining was performed as described in Rojo et al. (2014).

Fluorescence in situ hybridization and signal detection

The chromosome content and purity of flow-sorted fractions was first determined by FISH onto metaphase spreads of female I. monticola. Unidirectional chromosome painting with the probe containing the W sex chromosome of I. monticola was performed on I. galani, I. bonnali, L. schreiberi and T. lepidus. Three additional probes were applied for the characterization of I. bonnali, whereas the full set of chromosome-specific probes of I. monticola was used in cross-species hybridization to metaphase spreads of L. schreiberi and T. lepidus.

FISH was performed using the protocols described in Yang et al. (Yang et al. 1995) and Rens et al. (2006), with several modifications. Briefly, slides were dehydrated through ethanol series; aged at 65°C for 1 h; denatured in 70% formamide/2x saline-sodium citrate (SSC) at 70°C for 1 up to 3 min (time depending on species and metaphase preparation) and dehydrated again. One microlitre of biotinylated probe was made up to 12 µL with hybridization buffer (50% deionized formamide (v/v), 10% dextran sulfate, 2x SSC, 0.05 M phosphate buffer, pH 7.3). This mixture was denatured at 75°C for 10 min, preannealed at 37°C for 30 min and applied to each slide. Hybridization was carried out at 37°C overnight, for the same species, and over 48h and 72h, for congeneric and more distantly related species, respectively. Posthybridization washes were performed in 50% formamide/2x SSC twice for 5 min each, followed by 2x SSC twice for 5 min each and 4x SSC with 0.05% Tween-20 (4xT) once for 4 min. Washes were carried out at 42°C. Probe detection was performed using 200 µL of diluted (1:500) Cy3-Streptavidin antibody (Amersham) per slide at 37°C for 30 min. After detection, slides were washed in 4xT three times for 3 min each at 42°C and mounted in with anti-fade medium Vectashield (Vector Laboratories) containing 1.5 µg/mL DAPI.

Interspecies comparative genomic hybridization (iCGH)

Total genomic DNA was extracted from ethanol preserved tissues of I. monticola, L. schreiberi and T. lepidus females using a commercial kit (RealPure Genomic DNA Extraction Kit, Durviz), following the manufacturer's instructions. Total genomic DNA was labeled by random priming with the Prime-It Random Priming Labeling Kit (Agilent Technologies), according to the manufacturer's specifications. Genomic DNAs of I. monticola, L. schreiberi and T. lepidus were labeled, respectively, with TRITC-dUTP, FITC-dUTP, and both TRITC-dUTP and FITC-dUTP. iCGH was performed as described in Rojo et al. (2014). Reciprocal iCGH experiments were done between each pair of species. For each slide that was made, 250 ng of TRITC-labeled and 250 ng of FITC-labeled DNA were ethanol-precipitated with 20 µg of glycogen and 4 µg of unlabeled, sheared genomic DNA (as competitor) derived from a male of the same species as the target metaphases.

Microscopy and data analyses

At least 20 metaphase spreads were examined after each hybridization. Images were captured using the epifluorescence microscopes Leica DMRXA and Nikon Microphot-FXA, equipped with cooled CCD cameras [Photometrics Sensys and DS-Qi1Mc (Nikon Instruments), respectively]. The Leica CW4000 FISH and the NIS-Elements D 3.10 (Nikon Instruments) softwares were used to capture 16-bit grey-scale images of DAPI, Cy3/TRITC and FITC signals, which were then normalized and merged to a 24-bit colour image. For karyotyping, the DAPI images were displayed in contrast-adjusted reversed greyscale images. The final composition of the images was performed with Adobe Photoshop CS4 11.0.1 (Adobe Systems Inc.).

Karyotyping and C-banding

DAPI-stained karyotypes of all the analyzed species are shown in Fig. 1. The karyotypes of I. monticola and I. galani (2n = 36) consisted exlusively of acrocentric chromosomes of gradually decreasing size. A similar heteromorphic sex chromosome pair was found in female specimens of both species, in which the W chromosome is distinctly smaller than the Z counterpart, and showed an intense fluorescent signal after DAPI staining.

The diplod chromosome number in female I. bonnali was 2n = 23, and the karyotype comprised 13 biarmed and 10 acrocentric chromosomes. In this species, the W chromosome is a metacentric element, and its homologues—Z₁ and Z₂—are two smaller acrocentric elements. A bright DAPI-positive region was observed in the q arm of the W chromosome.

The karyotype of L. schreiberi (2n = 38) was composed of 36 acrocentric chromosomes, gradually decreasing in size, and a pair of microchromosomes. The female specimen analyzed in the first instance for this study, from the population of Invernadeiro, showed a markedly heteromorphic pair formed by a very small, DAPI-positive element, and a medium-sized counterpart, tentatively identified as the Z in this species.

The karyotype of female T. lepidus (2n = 36) contained one large metacentric chromosome pair, 32 acrocentric chromosomes and two microchromosomes. The smallest acrocentric chromosome, barely larger than the microchromome pair, was distinctively stained by DAPI, and it is most likely to be the W sex chromosome, while the putative Z was identified as a medium-sized acrocentric element.

C-banding revealed similarities in the abundance and distribution of constitutive heterochromatin in the karyotypes of these species, such as the presence of DAPI- and CMA₃-positive centromeric and interstitial/pericentromeric blocks, and the occurrence of GC-rich, faint telomeric C-bands in at least the largest chromosomes of the karyotypes (Fig. 2). Differences in the C-banding patterns of these species were mainly associated to the sex chromosomes. The W chromosomes of I. monticola and I. galani are almost completely heterochromatic, with only a small euchromatic region located in an interstitial position (Figs. 2a–c). There is considerable heterogeneity in size and overall appearance of this chromosome among different metaphases, apparently brought about by differences in the extent of DNA denaturation and loss produced by the C-banding pretreatment of the preparations (see also Arribas et al. 2006; Rojo et al. 2014). The submetacentric W chromosome of I. bonnali shows a prominent C-band in the distal region of the q-arm (Fig. 2d). In L. schreiberi, the smallest chromosome of the heteromorphic pair (the putative W chromosome) is also easily recognizable after C-banding by bearing a prominent heterochromatin block in interstitial position (Fig. 2e). This same pattern is found in the W chromosome of T. lepidus, which, despite its small size, seems to be only partially heterochromatic, with an interstitial C-positive region surrounded by proximal and distal euchromatic areas (Fig. 2f). In all the cases, the heterochromatin of the W chromosomes resulted intensely stained after both DAPI and CMA₃ staining. On the other hand, the Z chromosome of I. monticola and I. galani could be distinguished from the autosomes by bearing a brighter, CMA₃-positive telomeric C-band, which is most clearly shown before DAPI staining (Figs. 2a–c).

Flow sorting of I. monticola chromosomes and characterization of painting probes

The 36 chromosomes of the karyotype of I. monticola were differentiated into 14 separate flow peaks (Fig. 3). Painting probes (pp) from each peak were hybridized onto I. monticola metaphase chromosomes to determine the chromosome content of these flow peaks (Fig. S1, Supplementary Information). Nine chromosome pairs could be resolved separately, and chromosome-specific painting probes were obtained from them (pp1–pp3, pp6–pp10, and pp17), two peaks contained two chromosomes each (pp4,5 and pp5,7), and three peaks contained three chromosomes each (pp11,12,Z, pp13,14,W, and pp14,15,16). The presence of the same chromosome in adjacent flow peaks, as it is the case with chromosomes 5, 7 and 14 (see again Fig. 3), could be an indication of the two homologues differing in their repetitive DNA content, but the close similarity to the sizes and DAPI banding patterns of other chromosomes of the karyotype preclude our exploration of this interesting possibility with the available data.

Cross-species chromosome painting

The study of chromosome synteny with the whole set of I. monticola probes on L. schreiberi and T. lepidus revealed a high degree of karyotype conservation between the three species (see Figs. S2 and S3, Supplementary Information, for the complete results of chromosome painting on these species). Most I. monticola chromosomes were completely preserved —both in DNA content and morphology— in the other lacertids. One of the few rearrangements detected involved I. monticola chromosomes 2 and 4, which correspond, respectively, to the q and p-arms of the metacentric chromosome 1 of T. lepidus (Figs. 4a, c), while being homologous to acrocentric chromosomes 2 and 4 of L. schreiberi (Figs. 4b, d). Arrows in Fig. 4c point to the p-arm of T. lepidus chromosome 1, which was painted by the pp4,5 probe, but not by pp5,7 (Fig. 4e).

The probe pp11,12,Z painted an odd number of medium-sized chromosomes in I. monticola (Fig. 4f). The unpaired chromosome —which, according to its size, could be the 11th largest chromosome— is presumably the Z sex chromosome. Chromosome painting with this probe on male I. monticola metaphases labeled an even number of chromosomes, thus confirming that this flow peak contains the Z chromosome (Fig. 4g). Similarly, pp11,12,Z hybridized to five medium-sized acrocentric chromosomes on female T. lepidus and L. schreiberi (two populations) metaphases, strongly suggesting the structural conservation of the Z, which could be, respectively, the tenth and ninth largest element of the karyotype (Fig. 1, Figs. 6h–j). In addition, this probe clearly marked the microchromosome pair in both species (asterisks in Figs. 6h–j), thus indicating that these elements were most likely fused to either chromosome 11, 12 or Z in the last common ancestor of Iberolacerta species.

The probe pp13,14,W, containing the I. monticola W sex chromosome together with autosomes 13 and 14, hybridized to the euchromatin of the W chromosome in I. galani (Fig. 4k), and to the euchromatin at the end of the q-arm of the submetacentric W chromosome in I. bonnali (Fig. 4l). It also painted two small, acrocentric chromosome pairs in both species. A screening with the remaining flow-sorted fractions of I. monticola showed that the p-arm of the W chromosome of I. bonnali only was marked by the probe pp14,15,16 (Fig. 4o), thus indicating that it must be homologous to either autosome 15 or 16 of I. monticola. When the probe pp13,14,W was used on T. lepidus and L. schreiberi, it painted a pair of small acrocentric chromosomes in each species (12 and 13 in T. lepidus; 14 and 15 in L. schreiberi), but no signal was detected on the W chromosome of either of them (Figs. 4m, n).

In the screening of metaphase plates to determine the hybridisation results described above, some metaphases with chromosomal mutations, such as trisomies (Figs. 4p, q) or segmental duplications (Figs. 4r-t), were detected, apparently produced during the culture of the fibroblast cell lines.

Interspecies comparative genomic hybridization (iCGH) and FISH with a satDNA probe

Absence of hybridization signal with the pp13,14,W probe on the W chromosomes of L. schreiberi and T. lepidus led us to further investigate the differentation of W chromosomes between the three species by carrying out iCGH. Reciprocal iCGH experiments highlighted the accumulation of species-specific sequences in the chromosomes previously identified as the W of each species (Fig. 5). For instance, the W chromosome of I. monticola was predominantly labeled by I. monticola genomic DNA when co-hybridized with genomic DNA of either L. schreiberi or T. lepidus (Figs. 5a, b). The same pattern was observed in metaphases of L. schreiberi and T. lepidus (Figs. 5c, d and 5e, f, respectively). Due to the bright signals produced by the repetitive content of the W chromosomes, it was not possible to elucidate if the molecular composition of sex chromosomes differed only at the heterochromatic or also at the euchromatic regions. Additional evidence on the nucleotide divergence of W chromosomes was obtained after FISH with a satellite DNA probe, TaqI, which showed that although this satellite family is dispersed over several or most chromosomes of the karyotype of these three species, only the W chromosome of L. schreiberi harbors repeats of it (Fig. 6).

Four main conclusions of evolutionary relevance can be drawn from the results of this work. The first concerns the characteristic microchromosomes of birds and reptiles. The genus Iberolacerta, by not possessing microchromosomes, may appear somewhat atypical among reptiles, whose karyotypes, with the exception of crocodiles, most frequently show several pairs of microchromosomes, which might represent an ancestral karyological character of amniotes (Olmo 2008; Uno et al. 2012; Deakin and Ezaz 2019; Waters et al. 2021). Truth be told, squamates show a marked tendency towards reduction in the number of microchromosomes, through lineage-specific fusions to different macrochromosomes and a concomitant increase in G-banding patterns, which may have a direct influence on recombination levels and chromosome mutation rates (Olmo 2008; Srikulnath et al. 2021). This trend is particularly enhanced in lacertid lizards, with most species showing just one pair of microchromosomes, or even their complete absence in some of them (e.g., Zootoca vivipara and Atlantolacerta andreanskyi) (Olmo et al. 1991; Olmo and Signorino 2005). We have determined that in the I. monticola karyotype the ancestral microchromosomes are most likely fused to either the Z sex chromosome or to one of the autosomes labelled as 11 and 12. The possibility of the hybridization signals on the L. schreiberi and T. lepidus microchromosomes being due to repetitive centromeric DNA shared by any of the I. monticola chromosomes in the pp11,12,Z is quite improbable. In previous works we found that the HindIII satDNA repeats overlap the centromeric heterochromatic blocks of all the chromosomes of the I. monticola karyotype (Giovannotti et al. 2014), so that if the hybridization signal from the microchromosomes were due to centromeric repeats, we should have obtained it with all our probes.

Given the exceptionality of the absence of microchromosomes in reptile karyotypes, and since all the species of the genus Iberolacerta share this trait, it seems logical to assume that the fusion observed in I. monticola most likely constitutes a synapomorphy for Iberolacerta within the Lacertidae, i.e., a shared derived trait that should have been already present in the last common ancestor of this small species group. Phylogenetic reconstructions indicate that Iberolacerta originated 22–30 mya, together with many other genera of Lacertini (Hipsley et al. 2009, additional file 1; Garcia-Porta et al. 2019, supplementary Fig. 11), well before the Pleistocene glacial oscillations, with its now-recognisable species having been preserved in glacial refugia in Southern European mountains, thanks to its complex topography and history, that allowed their allopatric divergence (Carranza et al. 2004; Crochet et al. 2004; Arribas et al. 2014). Population fragmentation was probably enhanced during the Messinian Salinity Crisis (5.9–5.3 Mya), when the nearly complete desiccation of the Mediterranean Sea produced an extreme increase in aridity, forcing the mesic species, such as Iberolacerta spp., to retreat to the moister Atlantic-influenced areas and to the mountainous regions around the Mediterranean Basin. The same climatic events were experienced in much the same way by another one of the lacertid species showing independent microchromosome loss, namely Atlantolacerta andreanskyi (Werner, 1929), which is a member of the subtribe Eremiadini and endemic to the High Atlas Mountains in northern Africa, where their highly fragmented populations in different mountaintops could actually harbor at least six well differentiated species (Barata et al. 2012). The same as in Iberolacerta and Atlantolacerta, microchromosomes have been independently lost in Zootoca vivipara (Lichtenstein, 1823), the terrestrial reptile with the largest geographical and highest latitudinal distribution, inhabiting quite different biogeographic regions in the Northern Hemisphere (IUCN 2022), but exhibiting responses to glaciations as do temperate zone species (Horreo et al. 2018). Perhaps the most important ecological characteristic that these three taxa have in common is their independent conquest of cold environments (Garcia-Porta et al. 2019). Taking into consideration the increasing evolutionary importance attributed to microchromosomes (Uno et al. 2012; Srikulnath et al. 2021; Waters et al. 2021), and the apparently direct involvement of some of their gene contents in differential adaptations of other sqamate reptiles (Bentley et al. 2023), it can not be discarded that their loss has been due to selection, in a still unspecified way.

The second a priori evolutionary relevant result reported in this paper refers to the origin of the Z₁Z₂W multiple sex-chromosome system in two of the three Pyrenean species of Iberolacerta, I. bonnali and I. aurelioi. DNA phylogenies have not been able to resolve the splitting order of these species, probably because the time interval between their speciation events was too short (Mayer and Arribas 2003; Arribas et al. 2014), but allozymes (Mayer and Arribas 1996), and both karyological(Odierna et al. 1996) and osteological data(Arribas 1998) indicate that I. aranica is most likely to have separated first, before the rearrangement that gave rise to the multiple sex chromosome system, that would be a derived character shared by I. bonnali and I. aurelioi. According to our results, that rearrangement was a fusion between the ancestral W and one of the autosomes belonging to either pair 15 or 16, which gave rise to a biarmed neo-W, whereas the homologous chromosome in that pair became the Z₂. This is interesting, first because W-autosome fusions or translocations are not the only possible ways to produce a Z₁Z₂W sex chromosome system. In insects, for example, It is far more common for such complex sex chromosomes to appear due to Z fissions than to W fusions with an autosome (Blackmon et al. 2016). Then, it is also interesting because the evolutionary outcome of a W-autosome fusion could be analogous to other rearrangements that restrict recombination on the sex specific chromosome, always assuming that the fused autosome contains sexually antagonistic variation (Charlesworth and Charlesworth 1980). However, in contrast to many families of Iguania, where multiple sex chromosomes are extraordinarily frequent, in Lacertidae they appear to be evolutionary dead ends, since only two other species are known to present them, besides I. bonnali and I. aurelioi, namely Podarcis tauricus and Z. vivipara (Mezzasalma et al. 2021). That these species of Iberolacerta and Z. vivipara share two such extraordinary traits as multiple sex chromosomes and loss of microchromosomes may be a simple coincidence, but it is something to keep in mind, just in case.

The third conclusion of our research is that the previously reported independent origin of the Z chromosome of L. schreiberi (Rojo 2015) is not correct. Quite on the contrary, the results of chromosome painting on this species, as well as on all the others studied in this paper, bespeak of the conservation of Z chromosome homology, in agreement with the papers reporting the long term stability of the Z chromosome in lacertid lizards (Rovatsos et al. 2019; Lisachov et al. 2020). We must conclude now that the previous observations apparently supporting that claim were artifacts produced by chromosomal aberrations arisen spontaneously during fibroblast culture. In addition to the Z chromosome, the autosomes show an overall conservation of syntenic relationships among Iberolacerta spp., T. lepidus and L. schreiberi, with centric fusions between acrocentrics giving rise to the new biarmed metacentric chromosomes, and no insertional translocations having been ever observed.

Finally, as regards the evolution of the nucleotide sequences in the ancestral W chromosome of Iberolacerta, it is clear that their divergence among I. monticola, I. galani and I. bonnali is not as large as to preclude their hybridization with the I. monticola probe. On the contrary, since this probe did not paint the W chromosomes of either T. lepidus or L. schreiberi, we must conclude that this is due to the high divergence among their nucleotide sequences. The results from iCGH and FISH with a satDNA probe confirm that it is the W chromosome which evolves the fastest in the comparison among these species, and that its evolution is at least in part due to satellite DNA turnovers, as it has been repeatedly observed in other comparative analyses of lacertid lizards (Matsubara et al. 2015; Giovannotti et al. 2018; Suwala et al. 2020).

Acknowledgements

This work was partially funded by grant PGIDIT06RFO10301PR (Xunta de Galicia, Spain) awarded to Horacio Naveira. Verónica Rojo was supported by a Formación de Personal Investigador contract from the Ministerio de Educación, Cultura y Deporte (Spain), and by a Research Internship Grant from the Universidade da Coruña, for a short stay at the Cambridge Resource Centre for Comparative Genomics. Special thanks are given to Pedro Galán for providing the animal samples used in this study.

Ethical standards

Permissions for field work and experimental procedures were issued by the competent spanish authorities: Xunta de Galicia (for I. monticola, I. galani, L. schreiberi and T. lepidus), and Gobierno de Aragón (for I. bonnali). All institutional and national guidelines for the care and use of laboratory animals were followed.

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Competing interest reported. Non-financial interests: Horacio Naveira has been Co-Editor-in-Chief of Genetica since 01/01/2020.

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Chromosome evolution in a genus that deviates from the standard karyotype formula of Lacertidae

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Abstract

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Introduction

Material and Methods

Animal samples

Metaphase chromosomes preparation

Probe preparation

Karyotyping and C-banding

Fluorescence in situ hybridization and signal detection

Interspecies comparative genomic hybridization (iCGH)

Microscopy and data analyses

Results

Karyotyping and C-banding

Flow sorting of I. monticola chromosomes and characterization of painting probes

Cross-species chromosome painting

Interspecies comparative genomic hybridization (iCGH) and FISH with a satDNA probe

Discussion

Declarations

References

Additional Declarations

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