The Wall Lizards of the Balkan Peninsula: Tackling Questions at the Interphase of Phylogenomics and Population Genomics

[Abstract] Wall lizards of the genus Podarcis (Sauria, Lacertidae) are the predominant reptile group in southern Europe, including 24 recognized species. Mitochondrial DNA data have shown that, with the exception of P. muralis, the Podarcis species distributed in the Balkan peninsula form a species group that is further sub-divided into two subgroups: the one of “P. tauricus” consisting of P. tauricus, P. milensis, P. gaigeae, and P. melisellensis, and the other of “P. erhardii” comprising P. erhardii, P. levendis, P. cretensis, and P. peloponnesiacus. In an attempt to explore the Balkan Podarcis phylogenomic relationships, assess the levels of genetic structure and to re-evaluate the number of extant species, we employed phylogenomic and admixture approaches on ddRADseq (double digested Restriction site Associated DNA sequencing) genomic data. With this efficient Next Generation Sequencing approach, we were able to obtain a large number of genomic loci randomly distributed throughout the genome and use them to resolve the previously obscure phylogenetic relationships among the different Podarcis species distributed in the Balkans. The obtained phylogenomic relationships support the monophyly of both aforementioned subgroups and revealed several divergent lineages within each subgroup, stressing the need for taxonomic re-evaluation of Podarcis’ species in Balkans. The phylogenomic trees and the species delimitation analyses confirmed all recently recognized species (P. levendis, P. cretensis, and P. ionicus) and showed the presence of at least two more species, one in P. erhardii and the other in P. peloponnesiacus.This study was funded by NSFR 2007-2013 programme for development, European Social Fund, Operational Programme, Education and Lifelong Learning investing in knowledge society, Ministry of Education and Religious Affairs, Managing Authority, Co-financed by Greece and the European Union. Part of this work was funded by the Klaus Tschira Foundation, by the Ministry of Science and Innovation of Spain (PID2019-104184RB-I00 / AEI / 10.13039/501100011033), and by the Xunta de Galicia and FEDER funds of the EU under the Centro de Investigación de Galicia accreditation 2019-2022 (ED431G 2019/01)Xunta de Galicia; ED431G 2019/0

Behavioural mechanisms and adaptations of the European populations of Stellagama stellio

The present study focuses on the intraspecific divergence of the Roughtail Rock Agama (Stellagama stellio; Agamidae) in ecophysiology and evolutionary history. First, the effects of the environment on the digestive efficiency and the effectiveness of thermoregulation of insular and mainland populations were studied along a NW-SE cline. Apparent digestive efficiency (ADE) is not linked to phenotypic traits of populations nor to their phylogenetic relatedness but rather to their area of origin and its climatic features. Climate affects digestion indirectly, through food availability. Lizards from the northernmost ‘wet’ edge of the species’ distribution achieve the lowest ADEs for all nutrients. Insularity does not seem to affect digestion. Populations have adapted well to their current habitat conditions, having developed efficient physiological mechanisms, so that no single rule can account for all cases but particularities of each habitat lead to different patterns. Age also affects digestion, with adults being more capable to extract lipids and sugars, while juveniles to extract proteins. This can be due to effects of body size and sexual/reproductive behavior in adults and somatic growth in juveniles. Concerning thermoregulation, animals from Delos exhibit the highest effectiveness, while populations at the edges of the geographic cline exhibit the lowest. These differences are not related to phylogenetic relationships among populations but can be attributed to differences in the thermal regime of their habitats. Regarding selected temperatures, there is a significant difference between the Cypriot population and all the rest, with animals from Cyprus selecting lower temperatures. Thermoregulation in S. stellio is not a stable but rather a labile physiological function. The phylogeny of S. stellio based on five mitochondrial and three nuclear genes supports its monophyly and its divergence dates back to the Lower Miocene. All morphologically described subspecies represent distinct phylogenetic lineages, while seven more distinct lineages are found, four of which could be named as subspecies. The Cypriot subspecies branched off first at 3.99 MYA, and in combination with genetic distances, haplotype diversity and results of species-delimitation analysis, it should be revised and possibly elevated to species level. A series of vicarianistic events, geo- and/or overseas dispersal, as well as human-aided dispersal, combined with known biogeographic barriers contributed to the species’ diversification during the Pleistocene. The phylogenetic lineages revealed herein are congruent with the high morphological variation exhibited by populations along the species’ distribution. In conclusion, this research highlights the importance of studying physiological and evolutionary patterns and processes at population level for a better understanding of mechanisms and factors promoting differentiation even within monophyletic clades. The study organism seems to comprise a superspecies under intensive diversification promoted by a variety of natural and human-driven factors.Η παρούσα διατριβή εστιάζει στην ενδοειδική διαφοροποίηση της σαύρας Stellagama stellio (Agamidae) ως προς την οικοφυσιολογία και τη φυλογένεση. Αρχικά ερευνάται η επίδραση του περιβάλλοντος στην απόδοση της πέψης και στην αποτελεσματικότητα της θερμορρύθμισης σε νησιωτικούς και ηπειρωτικούς πληθυσμούς κατά μήκος γεωγραφικού ΒΔ-ΝΑ κλινούς. Αναδεικνύεται η επίδραση του κλίματος στην πεπτική αποδοτικότητα, η οποία ασκείται κυρίως έμμεσα. Οι πληθυσμοί του βόρειου, «υγρού» άκρου εμφάνισαν τη μικρότερη πεπτική αποδοτικότητα σε όλα τα θρεπτικά συστατικά. Η σύγκριση μεταξύ ηπειρωτικών και νησιωτικών πληθυσμών δεν ανέδειξε τον νησιωτισμό ως σημαντική παράμετρο για την πέψη. Οι διαφορετικοί πληθυσμοί έχουν προσαρμοστεί στις τοπικές συνθήκες του ενδιαιτήματος όπου ζουν με τις ιδιαιτερότητές του να οδηγούν σε διαφορετικά προσαρμοστικά πρότυπα. Η ηλικία επηρεάζει εξίσου την πέψη, οδηγώντας σε οντογενετική αλλαγή στην αποδοτικότητα των τριών βασικών θρεπτικών συστατικών. Τα ενήλικα είναι ικανότερα στην εξαγωγή λιπιδίων και σακχάρων ενώ τα ανήλικα στην εξαγωγή πρωτεϊνών, διαφορές που αποδίδονται στο σχετικό μέγεθος των ατόμων και στις ενεργοβόρες διεργασίες της αναπαραγωγής στα ενήλικα, και στις ανάγκες σωματικής αύξησης στα ανήλικα. Όσον αφορά τη θερμορρύθμιση, αποτελεσματικότερος εμφανίζεται ο πληθυσμός της Δήλου, ενώ εκείνοι στα δύο άκρα του γεωγραφικού κλινούς θερμορρυθμίζουν με μικρότερη αποτελεσματικότητα. Οι διαφορές αυτές δεν αντανακλούν τις φυλογενετικές συγγένειες των πληθυσμών και αποδίδονται στα θερμικά χαρακτηριστικά των ενδιαιτημάτων. Στις επιλεγόμενες θερμοκρασίες διαφοροποιείται σημαντικά ο κυπριακός πληθυσμός, επιλέγοντας χαμηλότερες θερμοκρασίες από τους υπόλοιπους. Στην περίπτωση του S. stellio, η θερμορρύθμιση δεν αποτελεί «στατική» αλλά «μεταβαλλόμενη» λειτουργία. Η μελέτη της εξελικτικής ιστορίας του είδους, στη βάση πέντε μιτοχονδριακών και τριών πυρηνικών γονιδίων, δείχνει ότι το S. stellio αποτελεί μονοφυλετικό κλάδο που απέκλινε περ. 18 εκ. χρ. πριν. Τα γνωστά υποείδη αντιστοιχούν σε ξεχωριστές γενεαλογικές γραμμές και υπάρχουν και επτά επιπλέον διακριτές γραμμές, τέσσερεις τουλάχιστον από τις οποίες θα μπορούσαν να αποτελούν νέα τάξα. Το κυπριακό υποείδος αντιπροσωπεύει την παλαιότερη γενεαλογική γραμμή (3,99 εκ. χρ. πριν) και, σε συνδυασμό με τις γενετικές αποστάσεις, τους απλοτύπους και την ανάλυση οριοθέτησης ειδών, φαίνεται αναγκαία η ταξινομική αναθεώρησή του και η αναβίβασή του στο επίπεδο του είδους. Συμβάντα βικαριανισμού, διασποράς και ανθρωπόχωρης μεταφοράς, σε συνδυασμό με γνωστά βιογεωγραφικά φράγματα, συνέβαλαν στην εξελικτική διαφοροποίηση του είδους κατά το μέσο και ανώτερο Πλειστόκαινο. Οι διαφορές μεταξύ των γενεαλογικών γραμμών υποστηρίζουν την υψηλή μορφολογική ποικιλότητα των διαφορετικών πληθυσμών του είδους σε όλο το εύρος εξάπλωσής του. Συνοπτικά, η παρούσα έρευνα αναδεικνύει τη σημασία της μελέτης των φυσιολογικών και εξελικτικών προτύπων και διεργασιών σε επίπεδο πληθυσμών για την καλύτερη κατανόηση των μηχανισμών και των παραγόντων που συμβάλλουν στη διαφοροποίηση ακόμα και εντός μονοφυλετικών ομάδων. Ο οργανισμός μελέτης φαίνεται να αποτελεί «υπερείδος» που διαφοροποιείται έντονα υπό τη δράση τόσο φυσικών όσο και ανθρωπογενών παραγόντων

Seasonal variation in the thermoregulation pattern of an insular agamid lizard

Abstract: Simple Summary The ability of animals to maintain their body temperature within an optimal range, known as thermoregulation, is essential for their survival, overall health, and daily activities. Ectotherms, including reptiles, rely on external energy resources to regulate their body temperature. How well they can achieve this, heavily depends on various environmental factors, such as the climate and its seasonal changes. Islands typically have a mild climate, which is expected to favor the thermoregulation of reptiles throughout the year. In this study, we investigate the effect of seasonality on the thermoregulation efficiency and behavior of a population of lizards found on Naxos Island, in the Cyclades, Greece. Our results reveal that seasonal fluctuations significantly influence how easily and precisely lizards can regulate their body temperature, with summer being the most favorable period, and autumn being the least favorable. Interestingly, lizards adjusted their thermal preferences and thermoregulation efficiency depending on the challenges imposed by each season and thus managed to maintain stable body temperatures. Whether these adjustments represent evolutionary adaptations or simply reversible shifts, awaits further research. Understanding how lizards adapt to their changing environment can provide valuable insights into their survival strategies and how they may cope with future environmental changes.Abstract Ectotherms, including lizards, rely on behavioral thermoregulation to maintain their body temperature within an optimal range. The benign climate of islands is expected to favor the thermoregulation efficiency of reptiles throughout their activity period. In this study, we investigated the seasonal variation in thermoregulation in an insular population of the roughtail rock agama (Laudakia stellio) on Naxos Island, Greece. We measured body, operative, and preferred temperatures across three seasons (spring, summer, and autumn), and we evaluated the effectiveness of thermoregulation, using the Hertz index (E). Our results revealed that the effectiveness of thermoregulation was significantly influenced by seasonality. E was quite high in summer (0.97) and spring (0.92), and lowest in autumn (0.81). Accordingly, the quality of the thermal environment was significantly low during autumn, and maximum during summer. However, despite the environmental temperature fluctuations, lizards exhibited remarkable stability in body temperatures. They also adjusted their preferred temperatures seasonally and doubled the thermal niche breadth they occupied during summer, thus enhancing thermoregulation efficiency. Whether or not these adjustments are plastic or fixed local adaptations remains to be explored in further research across multiple years and seasons, including additional insular populations

Evolutionary Variability of W-Linked Repetitive Content in Lacertid Lizards

Lacertid lizards are a widely radiated group of squamate reptiles with long-term stable ZZ/ZW sex chromosomes. Despite their family-wide homology of Z-specific gene content, previous cytogenetic studies revealed significant variability in the size, morphology, and heterochromatin distribution of their W chromosome. However, there is little evidence about the accumulation and distribution of repetitive content on lacertid chromosomes, especially on their W chromosome. In order to expand our knowledge of the evolution of sex chromosome repetitive content, we examined the topology of telomeric and microsatellite motifs that tend to often accumulate on the sex chromosomes of reptiles in the karyotypes of 15 species of lacertids by fluorescence in situ hybridization (FISH). The topology of the above-mentioned motifs was compared to the pattern of heterochromatin distribution, as revealed by C-banding. Our results show that the topologies of the examined motifs on the W chromosome do not seem to follow a strong phylogenetic signal, indicating independent and species-specific accumulations. In addition, the degeneration of the W chromosome can also affect the Z chromosome and potentially also other parts of the genome. Our study provides solid evidence that the repetitive content of the degenerated sex chromosomes is one of the most evolutionary dynamic parts of the genome

The story of a rock-star: multilocus phylogeny and species delimitation in the starred or roughtail rock agama, Laudakia stellio (Reptilia: Agamidae)

Situated at the junction of three continents, Europe, Asia and Africa, the Eastern Mediterranean is an ideal region to study the effects of palaeogeography, ecology and long human presence on animal evolution. Laudakia stellio (Squamata: Agamidae) is found across this region and offers an excellent opportunity for such studies. The high morphological variation across their range suggests that these lizards might represent a species complex. This is the first study exploring their evolutionary history, using molecular markers and individuals from all described subspecies. We employed the latest phylogenetic and species-delimitation methods to identify all distinct evolutionary lineages, their genetic variation and divergence times. The phenotypical diversity of L. stellio matches its genetic differentiation: almost all subspecies correspond to well-supported retrieved subclades and additional distinct lineages representing intermediate morphs have been retrieved. 'Laudakia stellio' represents three distinct evolutionary entities that diverged during the Plio-Pleistocene transition, which we propose as distinct species. One includes Greek and Turkish populations, as well as cryptic Anatolian lineages. The second comprises all other Near East populations and the third is endemic to Cyprus. Our results indicate a role of humans in shaping present distribution patterns, and highlight the importance of the Aegean, Anatolia and the Levant as glacial refugia and diversity hotspots

Resolving complex phylogeographic patterns in the Balkan Peninsula using closely related wall-lizard species as a model system

The Balkan Peninsula constitutes a biodiversity hotspot with high levels of species richness and endemism. The complex geological history of the Balkans in conjunction with the climate evolution are hypothesized as the main drivers generating this biodiversity. We investigated the phylogeography, historical demography, and population structure of closely related wall-lizard species from the Balkan Peninsula and southeastern Europe to better understand diversification processes of species with limited dispersal ability, from Late Miocene to the Holocene. We used several analytical methods integrating genome-wide SNPs (ddRADseq), microsatellites, mitochondrial and nuclear DNA data, as well as species distribution modelling. Phylogenomic analysis resulted in a completely resolved species level phylogeny, population level analyses confirmed the existence of at least two cryptic evolutionary lineages and extensive within species genetic structuring. Divergence time estimations indicated that the Messinian Salinity Crisis played a key role in shaping patterns of species divergence, whereas intraspecific genetic structuring was mainly driven by Pliocene tectonic events and Quaternary climatic oscillations. The present work highlights the effectiveness of utilizing multiple methods and data types coupled with extensive geographic sampling to uncover the evolutionary processes that shaped the species over space and time