Article

Cryptic diversity and molecular systematics of the Aegean Ophiomorus skinks (Reptilia: Squamata), with the description of a new species

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Abstract

In recent years, great attention has been devoted to the discovery and description of cryptic species, especially using DNA markers and new statistical approaches. Genetic data are useful for discovering new lineages that can then be treated as hypotheses to be tested using morphology. Here, we use multilocus genetic data and a thorough sampling to delimit species within the Greek legless skink. Phylogenetic analyses reveal high levels of genetic diversity, suggesting that Ophiomorus punctatissimus includes at least two species, East and West of the Aegean Sea. Moreover, species delimitation methods support at least two and up to five species within the western O. punctatissimus, and this result is shared among single-locus (ABGD, PTP, and GMYC) and multilocus coalescent (BPP) methods. We examined whether the two allopatric lineages exhibit morphological differentiation, but the different preservation methods used for the examined material led to inconclusive results. However, morphological conservatism in this semi-fossorial limbless skink possibly also played a key role. Finally, we formally recognize the East Aegean taxon as a separate species and we provide a differential diagnosis based on DNA diagnostic characters.

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... The most interesting finding is the isolated dentary attributed to Ophiomorus, because this represents the first fossil known of this scincid genus worldwide. Ophiomorus is a widespread genus in western Asia and present also in southeastern Europe, including 12 extant species (Kazemi et al., 2011;Kornilios et al., 2018) found in an area that goes from southeastern Europe to western India (Anderson and Leviton, 1966;Uetz et al., 2019). In Europe, it is present with a single species, Ophiomorus punctatissimus, which is the type species of the genus and only lives in the Peloponnese and in the Kythira Island Speybroeck et al., 2016;Kornilios et al., 2018; Fig. 1). ...
... Ophiomorus is a widespread genus in western Asia and present also in southeastern Europe, including 12 extant species (Kazemi et al., 2011;Kornilios et al., 2018) found in an area that goes from southeastern Europe to western India (Anderson and Leviton, 1966;Uetz et al., 2019). In Europe, it is present with a single species, Ophiomorus punctatissimus, which is the type species of the genus and only lives in the Peloponnese and in the Kythira Island Speybroeck et al., 2016;Kornilios et al., 2018; Fig. 1). Populations in southwestern Turkey and Kastellorizon Island in Greece, formerly assigned to O. punctatissimus, are now assigned to their own species, named Ophiomorus kardesi Kornilios et al., 2018 (Fig. 1). ...
... In Europe, it is present with a single species, Ophiomorus punctatissimus, which is the type species of the genus and only lives in the Peloponnese and in the Kythira Island Speybroeck et al., 2016;Kornilios et al., 2018; Fig. 1). Populations in southwestern Turkey and Kastellorizon Island in Greece, formerly assigned to O. punctatissimus, are now assigned to their own species, named Ophiomorus kardesi Kornilios et al., 2018 (Fig. 1). ...
Article
Together with the rest of the Balkan Peninsula, Greece acted as a refuge for thermophilic amphibians and reptiles during the Quaternary Glaciations. In this work we provide new herpetological data on the latest Early Pleistocene locality of Kaiafas, in western Peloponnese. The site yielded one indeterminate salamandrid urodelan (Salamandridae indet.), one tree frog (Hyla gr. H. arborea), one indeterminate testudinid (Testudinidae indet.), at least two lacertid lizards (Lacertidae indet. A and B), one scincid lizard (Ophiomorus sp.), one anguid lizard (Pseudopus sp.), and one indeterminate “colubrine” snake (“Colubrinae” indet.). This assemblage is indicative of a surrounding environment with water bodies, wooded areas, and possibly loose soils with rocks and stones. In spite of being based on few remains, the study of the amphibians and reptiles from Kaiafas resulted in the identification of the first fossil of Ophiomorus known worldwide. Moreover, the two lacertid taxa displayed very peculiar morphological features which are either previously unknown or rare in that clade.
... The scincid lizard genus Ophiomorus was first described by Dum eril & Bibron (1839) to accommodate the species Ophiomorus miliaris Dum eril & Bibron, 1839, intended as a 'junior objective synonym of Anguis punctatissimus' (Anderson & Leviton, 1966: 500, 525), which was subsequently renamed as Ophiomorus punctatissimus (Boulenger, 1887). The genus Ophiomorus currently comprises 12 extant species (Kazemi et al. 2011;Kornilios et al. 2018), ranging from southeastern Europe (mainland of Greece) to western India (Anderson & Leviton, 1966). Following the first revision of the genus by Boulenger (1887), in which six species were recognized (Ophiomorus blanfordi, Ophiomorus brevipes, Ophiomorus latastii, Ophiomorus persicus, Ophiomorus punctatissimus, Ophiomorus tridactylus), three more were added by Anderson & Leviton (1966): Ophiomorus chernovi, Ophiomorus raithmai and Ophiomorus streeti. ...
... The subsequent description of Ophiomorus nuchalis Nilson & Andr en, 1978 and Ophiomorus maranjabensis Kazemi et al. 2011 raised that number to 11. In 2018, a 12th species was described as Ophiomorus kardesi Kornilios et al. 2018, to accommodate the Anatolian populations previously attributed to O. punctatissimus (Kornilios et al. 2018). ...
... The subsequent description of Ophiomorus nuchalis Nilson & Andr en, 1978 and Ophiomorus maranjabensis Kazemi et al. 2011 raised that number to 11. In 2018, a 12th species was described as Ophiomorus kardesi Kornilios et al. 2018, to accommodate the Anatolian populations previously attributed to O. punctatissimus (Kornilios et al. 2018). ...
Article
The limbless skink Ophiomorus punctatissimus is a cryptozoic species found in the Peloponnese region of Greece and on the Greek island Kythira. To provide the first thorough description of the cranial and postcranial osteology of this species, both disarticulated specimens and X‐ray computed tomographies of wet‐preserved specimens were examined in detail. Resulting from this, an anatomical atlas of this species is provided. Two separate considerations, an evolutionary and an ecomorphological one, are made based on the observed adaptations related to limb loss in this skink. The structure of the girdles shows a particular pattern of reduction: whereas the pelvic girdle is mostly vestigial, the pectoral girdle is instead well developed, with all the elements typical of limbed lizards except for the actual limbs. This led us to hypothesize an asynchronous pattern of limb reduction during the evolution of this species, in which the hindlimbs regressed earlier than the forelimbs. Furthermore, considerations based on overall body morphology, osteology and the structure of the inner ear led to the recognition of this species as a burrowing ecomorph. In contrast to the morphology normally displayed in this ecomorph, O. punctatissimus is characterized by the retention of autotomic vertebrae in its tail. This is consistent with the habitats in which it lives, where active burrowing would be difficult because of the hard, rocky terrain. Instead, this skink hides among rocks on the surface and is, therefore, subject to greater predation risk.
... The Family Scincidae in Turkey was represented by ten species, including Asymblepharus bivittatus (Ménétries, 1832), Ablepharus budaki Göçmen, Kumlutaş and Tosunoğlu, 1996, A. chernovi Darevsky, 1953, A. kitaibelii (Bibron and Bory St-Vincent, 1833 Kornilios, Kumlutaş, Lymberakis andIlgaz, 2018 (Schmidtler, 1997a;Ilgaz et al., 2007;Kumlutaş et al., 2007;Poulakakis et al., 2008;Durmuş et al., 2011;Baran et al., 2012;Karin et al., 2016, Kornilios et al., 2018Bozkurt and Olgun, 2020). ...
... The Family Scincidae in Turkey was represented by ten species, including Asymblepharus bivittatus (Ménétries, 1832), Ablepharus budaki Göçmen, Kumlutaş and Tosunoğlu, 1996, A. chernovi Darevsky, 1953, A. kitaibelii (Bibron and Bory St-Vincent, 1833 Kornilios, Kumlutaş, Lymberakis andIlgaz, 2018 (Schmidtler, 1997a;Ilgaz et al., 2007;Kumlutaş et al., 2007;Poulakakis et al., 2008;Durmuş et al., 2011;Baran et al., 2012;Karin et al., 2016, Kornilios et al., 2018Bozkurt and Olgun, 2020). ...
... Also, Ophiomorus kardesi was known as O. punctatissimus (Bibron and Bory de Saint-Vincent, 1833) in Turkey until recently. According to a recent phylogenetic and morphological study, Ophiomorus populations distributed in Turkey were revealed to be a distinct species from O. punctatissimus (Kornilios et al., 2018). ...
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Turkey is biogeographically diverse and consequently has a rich herpetofauna. As a result of active herpetological research, the number of species has steadily increased in recent times. I here present a new checklist of amphibian and reptile species distributed in Turkey, revising the nomenclature to reflect the latest knowledge. In addition, information about the systematics of many species is also given. In total 35 (19.4%) amphibian and 145 (80.6%) reptile species comprise the Turkish herpetofauna. Among amphibians, 16 (45.7%) anurans and 19 urodelans (54.3%) are present. Among reptiles, 11 (7.6%) testudines, 71 (49%) saurians, 3 (2.1%) amphisbaenians and 60 (41.3%) ophidians are considered part of the fauna. The endemism rate in herptile species in Turkey is relatively high. A total of 34 amphibian (12 species – 34.3%) and reptile (22 species – 15.2%) species are endemic to Turkey, yielding a total herpetofsunal endemism of 18.9%. While 38 species have not been threat-assessed by the IUCN, 92 of the 180 Turkish herpetofaunal species are Least Concern (LC), 13 are Near Threatened (NT), 10 are Vulnerable (VU), 14 are Endangered (EN), and 7 are Critically Endangered (CR). In addition, 6 species are in the DD (Data Deficient) category.
... Finally, the morphological investigation of the samples we have at hand reveals that they do not differ with regard to all available meristic characters (scale counts) and body measurements, while the preservation of our samples in different mediums does not allow a safe comparison of scale measurements (e.g. length or width of certain head scales; see Kornilios et al., 2018 for a comparison of formalin-preserved to ethanol-preserved specimens). ...
... Many recent studies of east Mediterranean reptiles that used current molecular-taxonomy methods on multi-locus data, have uncovered new cryptic species, elevated subspecies to the species level or resurrected old ones (e.g. Sindaco et al., 2014;Kornilios et al., 2018;. These studies, including the present one, can provide a better assessment of biodiversity levels and a more stable taxonomic framework for the implementation of conservation actions. ...
Article
Scolecophidia (worm snakes) are a vertebrate group with high ecomorphological conservatism due to their burrowing lifestyle. The Eurasian or Greek blindsnake Xerotyphlops vermicularis is their only European representative, a species-complex with an old diversification history. However, its systematics and taxonomy has remained untouched. Here, we extend previous work that relied heavily on mitochondrial markers, following a multi-locus approach and applying several species-delimitation methods, including a Bayesian coalescence-based approach (STACEY). Four “species” delimitation analyses based on the mtDNA (ABGD, bGMYC, mPTP, parsimony networks) returned 14, 11, 9 and 10 clusters, respectively. By mitotyping twice as many specimens as before, we have a complete picture of each cluster’s distribution. With the exception of the highly-divergent Levantine lineage, the three independent nuclear markers did not help with phylogenetic resolution, as demonstrated in haplotype networks, concatenated and species-trees, a result of incomplete lineage sorting. The prevailing model from the coalescence-based species-delimitation identified two species: the lineage from the Levant and all others. We formally recognize them as distinct species and resurrect Xerotyphlops syriacus (Jan, 1864) to include the Levantine blindsnakes. Finally, X. vermicularis and X. syriacus may represent species-complexes themselves, since they include high levels of cryptic diversity.
... This date fits nicely with the opening of the mid-Aegean trench, estimated from geological information to have started around 12 Mya and being completed around 9 Mya. More recently, Kornilios et al. (2018) used mtDNA and nuclear DNA data (three nuclear genes) for single-locus and multilocus coalescent-based species delimitation. The established COI p-distance is substantial (12.7%). ...
... Based on the divergence time and level of genetic divergence between these two cryptic species, we accept the species status of O. kardesi. In our area, the new species occurs on the Greek island of Kastellorizo (Kornilios et al., 2018). ...
Article
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The last species list of the European herpetofauna was published by Speybroeck, Beukema and Crochet (2010). In the meantime, ongoing research led to numerous taxonomic changes, including the discovery of new species-level lineages as well as reclassifications at genus level, requiring significant changes to this list. As of 2019, a new Taxonomic Committee was established as an official entity within the European Herpetological Society, Societas Europaea Herpetologica (SEH). Twelve members from nine European countries reviewed, discussed and voted on recent taxonomic research on a case-by-case basis. Accepted changes led to critical compilation of a new species list, which is hereby presented and discussed. According to our list, 301 species (95 amphibians, 15 chelonians, including six species of sea turtles, and 191 squamates) occur within our expanded geographical definition of Europe. The list includes 14 non-native species (three amphibians, one chelonian, and ten squamates).
... Ten years after the last integrated publication on the Greek herpetofauna , the list of newly described species on the Aegean archipelago has increased (e.g. Lymberakis et al., 2008;Stöck et al., 2012;Kornilios, Kumlutaş, Lymberakis, & Ilgaz, 2018), due to the rapid development of innovative molecular tools for studying biodiversity, which unveiled considerable cryptic diversity that led to the redefinition of the current taxonomy. Moreover, the involvement of more researchers in the study of the Aegean herpetofauna enriched our knowledge with numerous new distribution records (indicatively Spaneli & Lymberakis, 2014;Itescu, Slavenko, Schwarz, Meiri, & Pafilis, 2016;Mizerakis & Strachinis, 2017;Christopoulos, 2018). ...
Chapter
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Aegean Islands host a rich herpetofauna comprising a plethora of endemic taxa. Since the early 19th century, this unique diversity attracted numerous herpetologists that described the Greek species and provided records of their distribution. Interestingly, many new records on insular ranges are still added to the literature every year. During the last two decades, modern Systematics use powerful tools from the arsenal of molecular biology to unravel complicated phylogenies and shed light to hidden diversities. Current herpetological research focuses on the detection of cryptic species, yielding impressive findings. Another important research avenue includes the clarification of the particular adaptations that insular species adopted to survive under the demanding conditions of the Aegean Islands. Food scarcity, water dearth, high temperatures, relaxed predation and strong intraspecific competition are the main parameters shaping the ecophysiological profile of Aegean herptiles. However, the amphibians and reptiles of the Archipelago have to deal with serious threats. The most severe among them are habitat degradation and the loss of breeding-sites due to human activities and climate change. New phylogenetic data and well-justified gene trees together with the ongoing enhancement of our knowledge of species biology pave the way for the effective conservation of the matchless Aegean herpetofauna.
... More specifically, four individuals were found around the settlement and near the Acheres plain, while the other two were spotted basking by low stone walls on the island's plateau. (Fig. 4D).-A very recent genetic analysis (Kornilios et al., 2018) based on a multilocus DNA dataset, comprising of mitochondrial and nuclear genetic markers, described the Ophiomorus populations of Kastellorizo and SW Turkey as a new species, namely O. kardesi. The Anatolian Limbless Skink is mentioned by Valakos and Papapanagiotou (1985), who collected one specimen from the island's plateau. ...
Article
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Based on several recent herpetological surveys on the Kastellorizo archipelago in the Eastern Mediterranean, we present a review of the herpetofauna of the area. Active searching and visual encounter surveys on Kastellorizo confirmed the presence of two species of amphibians and thirteen species of reptiles, and in conjunction with the available literature data, brought the terrestrial herpetofauna of the island to a total of 17 species. In addition, during a one-day herpetological survey on Psomi islet, we encountered individuals of Anatololacerta sp. and Mediodactylus cf. danilewskii. Based on the analysis of the mitochondrial gene cytb, the recently discovered lizard population from the settlement of Megisti was identified as Anatololacerta pelasgiana (Mertens, 1959), while that of Psomi islet as Anatololacerta budaki (Eiselt and Schmidtler, 1986), a new addition to the herpetofauna of Greece. We reported on the extant herpetofauna of Kastellorizo and Psomi islet providing a species list that includes four new species records, namely A. pelasgiana, A. budaki, Platyceps najadum (Eichwald, 1831), and Telescopus fallax (Fleischmann, 1831). Our results expand our knowledge on the biogeography of the region and the insular distribution of the species. We also discuss the possible introduction ways of the non-native lizard A. pelasgiana, recent taxonomic revisions, and possible erroneous records.
... Five of the species sampled herein were represented by only a single sequence; in combination with variable effective sample sizes of these anoles, these samples might have contributed to the more liberal GMYC delimitations (Ahrens et al., 2016). This method recovered more clusters than distance-based analyses across our data, as well as in studies of Socotran reptiles (Vasconcelos et al., 2016), Madascincus lizards (Miralles and Vences, 2013), and Ophiomorus geckos (Korniolios et al., 2018). Interestingly, Blair and Bryson (2017) found it to be the most conservative method applied to their data. ...
Article
Full-text available
Single-locus molecular barcoding is a useful method for identifying overlooked and undescribed biodiversity, providing the groundwork for further systematic study and taxonomic investigation. A variety of methods for delimiting species from barcoding libraries have been developed and applied, allowing for rapid estimates of species diversity in a broad range of taxa. However, tree-based and distance-based analyses can infer different group assignments, potentially over- or underestimating the number of putative species groups. Here, we explore diversity of mainland species of anole lizards from the Chortís Block biogeographical province of northern Central America using a DNA barcoding approach, generating and analyzing cytochrome oxidase subunit I (COI) sequences for over 400 samples assignable to 33 of 38 (86.8%) native and one introduced mainland species. We subsequently tested the effects different models of nucleotide substitution, different species-delimitation algorithms, and reducing our dataset had on species delimitation estimates. We performed of two distance-based (ABGD, RESL) and three tree-based (bPTP, mPTP, GMYC) analyses on both the full dataset and a dataset consisting only of unique halotypes. From 34 nominal taxa, analyses of the full dataset recovered between 34 and 64 operational taxonomic units (OTUs), while analyses of the reduced dataset inferred between 36 and 59. Reassigning individuals to either mPTP-inferred or ABGD clustered (7.2% threshold) groups improved the detection of a barcoding gap across three different models of nucleotide substitution, removing overlap between intra- and interspecific distances. Our results highlight the underestimated diversity of mainland Chortís Block anoles, but the lack of congruence between analyses demonstrates the importance of considering multiple analytical methods when dealing with single-locus datasets. We recommend future studies consider the effects of different models of nucleotide substitution on proposed barcoding gaps, as well as the effect reducing a dataset to unique haplotypes may have on proposed diversity estimates.
... However, where several techniques are employed simultaneously, species delimitation results are often inconclusive (e.g. Blair and Bryson, 2017;Jacobs et al., 2018;Kornilios et al., 2018). Molecular delimitation analyses have been shown to be affected by the ratio of population sizes to species divergence times, gene flow, number of species involved, and number of sampling singletons and to a lesser degree sample size and number of loci (Luo et al., 2018;Esselstyn et al., 2012;Reid and Carstens, 2012;Fujisawa and Barraclough, 2013;Dellicour and Flot, 2015;Ahrens et al., 2016). ...
Article
We assess the phylogeographic structure and test several species delimitation methods in the forest-living Pondo flat-gecko, Afroedura pondolia, by sampling specimens from across the entire distribution in the Eastern Cape and KwaZulu-Natal provinces of South Africa. Phylogenetic results, using Bayesian inferences and maximum likelihood, from the combined mitochondrial and nuclear DNA sequence data derived from four partial loci (ND4, cyt b, PRLR and RAG1), suggest the presence of four geographically discrete clades, which represent candidate species. We applied four species-delimitation methods (ABGD, bGMYC, PTP and STACEY), the results of which were largely incongruent in the number of putative species nested within A. pondolia. Multivariate morphological analyses indicate statistically significant differences among the four candidate species, corroborating the presence of four cryptic species within A. pondolia. Divergence-time estimates suggest that cladogenesis was driven by forest fragmentation from the late Miocene to the Plio/Pleistocene with the onset of more pronounced xeric climatic conditions causing forest fragmentation.
... Systematics has largely benefitted from the ongoing development of molecular tools, in addition to clarifying the taxonomic classification of organisms and uncovering cryptic diversity (e.g., Kornilios et al. 2018;Tamar et al. 2018), the use of an integrative approach combining morphology and molecular phylogenetics helps reveal evolutionary relationships among taxa (Samadi and Barberousse 2009;Skoracka et al. 2015;Radenkovic et al. 2018), including mites belonging to the Eriophyoidea superfamily (Skoracka and Dabert 2010;Skoracka et al. 2012;Lewandowski et al. 2014;Cvrkovic et al. 2016). ...
Article
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Phytophagous mites belonging to the Eriophyoidea are extremely diverse and highly host-specific. Their accurate morphological identification is hampered by their reduced size and simplified bodies and by the existence of cryptic species complexes. Previous studies have demonstrated the urgency of applying multisource methods to accurate taxonomic identification of eriophyoid mites, especially species belonging to the genus Abacarus. This genus comprises 65 species, of which 37 are associated with grasses and four with sugarcane Saccharum (Poaceae). Recently, Abacarus specimens very similar to Abacarus sacchari were collected from the sugarcane crop in Brazil; however, their taxonomic placement was uncertain. In this study, we used an integrative approach to determine whether A. aff. sacchari specimens belong to A. sacchari or constitute a cryptic species. Morphological data were combined with molecular phylogeny based on the nucleotide sequences of three markers, one mitochondrial (COI) and two nuclear (D2 region of 28S and ITS). Morphological differences were observed between A. aff. sacchari, A. sacchari and A. doctus. The phylogenetic relationships among these three taxa and the genetic distances separating them revealed an interspecific divergence. The results of the morphological and molecular methods were congruent and supported the existence of a new species: Abacarus neosacchari n. sp. Duarte and Navia, herein described. This species belongs to the Abacarus cryptic species complex associated with sugarcane in the Americas. The results of this study, presenting the occurrence of multiple Abacarus species associated with sugarcane, contribute to the knowledge on plants and mites diversity by adding up one more clue highlighting that plant hybridization can be an important mechanism contributing to the speciation of plant-feeding arthropods.
... Cryptic reptile and amphibian lineages have continuously been uncovered in the circum Aegean region during the past decade (Dufresnes et al., 2018;Kornilios et al., 2014), and in many cases, new species have been described or elevated from subspecies (Kornilios, Kumlutaş, Lymberakis, & Ilgaz, 2018;Kotsakiozi et al., 2018;Psonis et al., 2017;Sindaco, Kornilios, Sacchi, & Lymberakis, 2014). This is not only the outcome of a better targeted and more thorough representation of populations, but also the extensive use of molecular and genetic tools applied in recent studies to detect divergence and infer phylogenetic relationships. ...
Article
Lacerta pamphylica and Lacerta trilineata are two currently recognized green lizard species with a historically problematic taxonomy. In cases of tangled phylogenies, next‐generation sequencing and double‐digest restriction‐site‐associated DNA protocols can provide a wealth of genomic data and resolve difficult taxonomic issues. Here, we generated genome‐wide SNPs and mitochondrial sequences, and applied molecular species delimitation approaches to provide a stable taxonomy for the Aegean green lizards. Mitochondrial gene trees, genetic cluster delimitation and population structure analyses converged into recognizing the populations of (a) L. pamphylica, (b) east Aegean islands, Anatolia and Thrace (diplochondrodes lineage), (c) central Aegean islands (citrovittata), and (d) remaining Balkan populations and islands (trilineata), as separate clusters. Phylogenomic analyses revealed a split into two major clades, east and west of the Aegean Barrier, unambiguously showing a sister–clade relationship between pamphylica and diplochondrodes, rendering L. trilineata paraphyletic. Species delimitation models were tested in a Bayesian framework using the genomic SNPs: lumping all populations into a single ‘species’ had the lowest likelihood but the current taxonomy was also outperformed by all other models. All lines of evidence support the Pamphylian green lizard as a valid species; thus, east Aegean L. trilineata should also be considered a distinct species under the name Lacerta diplochondrodes. Finally, evidence from the mitochondrial and nuclear genomes is overwhelmingly in favour of recognizing the morphologically distinct Cycladian green lizards as a distinct species. We propose their elevation to full species under the name Lacerta citrovittata. All remaining insular and continental populations of the Balkan Peninsula represent the species L. trilineata.
... Lizards constitute the most numerous reptile group in Greece containing 41 species of which 21 belong in lacertid family Valakos et al., 2008;Gvoždík et al., 2010;Psonis et al., 2017;Kalaentzis et al., 2018;Kornilios et al., 2018;Kotsakiozi et al., 2018;Strachinis et al., 2019). Mediterranean lacertid lizards consume almost all orders of Arthropoda and some Gastropoda, very small vertebrates and even some plant elements (Carretero, 2004), fruits (Brock et al., 2014;Mačát et al., 2015) or eggs (Brock et al., 2014;Žagar et al., 2016). ...
Article
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On 9th July 2019, during aquatic sampling in a backwater riparian wood of the Spercheios river valley (38.5350°N, 22.1859°E; 33 m a.s.l.) we observed the an event of saurophagy on the range-restricted anguid. The event was photographed by the lead author. The importance of this documented observation is reviewed.
... On the Aegean Islands, it is found only on Kythera and Kastellorizo. Recent genetic and morphological studies have highlighted the divergences between the western and the eastern populations of O. punctatissimus, which has led to the identification of the Anatolian population and the population of Kastellorizo, as a separate species: O. kardesi (Kornilios, et al., 2018). ...
Article
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In this paper, an update on the Dodecanese (Greece) herpetofauna is provided. In this Archipelago , 8 Amphibians (
... In this case, the PRLR topology suggests admixture between geographically proximate localities that can be explained by secondary contact during forest expansion events.In morphologically conservative reptiles where traditional taxonomy has proven to be problematic, molecular methods have widely been used to delineate species (e.g.Blair and Bryson, 2017;Kotsakiozi et al., 2018;Laver et al., 2018;Pinto et al., 2019;Šmíd et al., 2017;Travers et al., 2014). However, where several techniques are employed simultaneously, species delimitation results are often inconclusive (e.g.Blair and Bryson, 2017;Jacobs et al., 2018;Kornilios et al., 2018). Molecular delimitation analyses have been shown to be affected by the ratio of population sizes to species divergence times, gene flow, number of species involved, and number of sampling singletons and to a lesser degree sample size and number of loci(Luo et al. 2018;Esselstyn et al. 2012;Reid and Carstens 2012;Fujisawa and Barraclough, 2013;Dellicour and Flot, 2015;Ahrens et al. 2016). ...
Thesis
This study investigates the phylogeographic structure of three co-distributed forest-living reptile species, the Pondo flat gecko (Afroedura pondolia), the forest thread snake (Leptotyphlops sylvicolus) and the Natal black snake (Macrelaps microlepidotus), by sampling specimens from the Eastern Cape and KwaZulu-Natal provinces of South Africa. Phylogenetic results, using Bayesian inferences and maximum likelihood, from the combined mitochondrial sequence data (ND4 and cyt b), along with population genetic analyses suggest the presence of broadly congruent biogeographic breaks among the study taxa. Sequence divergence values suggest that A. pondolia and L. sylvicolus represent species complexes comprising several cryptic species while M. microlepidotus exhibits population level differentiation. Divergence-time estimates indicate that cladogenesis within the study taxa occurred during the late Miocene to the Plio/Pleistocene climatic shifts, suggesting that cladogenesis was driven by climatic oscillations and suitable habitat fragmentation. We further investigate the species level divergence within A. pondolia and L. sylvicolus by including two partial nuclear loci (PRLR and RAG1) and employing several species delimitation methods (ABGD, bGMYC, PTP and STACEY). The species delimitation results were generally incongruent, estimating between two and 14 species nested within A. pondolia and between ten and 12 species nested within L. sylvicolus. In both taxa, the species hypotheses retrieved by STACEY based on the total-evidence data were preferred and used to define groups in the morphological analyses. In A. pondolia the multivariate morphological analyses indicate statistically significant differences among the four putative species, corroborating the presence of four species. In L. sylvicolus the morphological analyses exhibit large overlap among the ten putative species but indicate differences between grassland and forest species. The narrow distributions of the putative species identified in the present study have further implications for the conservation status of A. pondolia and L. sylvicolus and suggest that the fragmented forest habitat along the east coast of South Africa may harbor significantly higher levels of diversity than currently recognized.
... Continuously and increasingly, researchers have incorporated nuclear loci into phylogenetic works in order to avoid the problems occurring when relying on mtDNA alone (e.g., selective sweeps, non-constant mutation rates, maternal inheritance) (Lemmon & Lemmon, 2013). Fortunately, this also happened in the phylogenetic studies on the Greek herpetofauna, with several works on several taxa in which the researchers used a combination of mitochondrial and nuclear DNA loci [e.g. the limbless skink, O. punctatissimus ( Kornilios et al., 2018), the Balkan Green Lizard Lacerta trilineata ( ), the snake-eyed skink, Ablepharus kitaibelii ( Skourtanioti et al., 2016), the Balkan Wall Lizard Podarcis tauricus ( Psonis et al., 2017), the Kotschy's Gecko M. kotschyi (Kot- sakiozi et al., 2018 ...
... The use of heavy-duty machinery should be prohibited in areas with high potential for habitat suitability as identified by scientific studies. Operations of this kind would not only affect salamanders but also the endemic fossorial Ophiomorus kardesi (Kornilios et al., 2018), as well as other amphibians and reptiles living in these areas. ...
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The Lycian salamanders consist of seven allopatric endangered and endemic species restricted to Mediterranean Turkey and some adjacent Aegean islands. Mega forest fires occurred consecutively over a prolonged period of time in the distribution areas of six of the species in 2021, leading to habitat fragmentation and habitat loss. In this study, we find that a total of 751.9 hectares of Lycian salamander habitats were lost due to the mega-fires that occurred in summer 2021. Our results suggest that L. atifi is the most vulnerable with a loss of 285.84 hectares of habitat area, followed by L. flavimembris with 242.54 hectares, and L. antalyana with 124.16 hectares. L. fazilae is the species which suffered the least habitat loss, at 25.83 hectares. L. billae and L. luschani suffered habitat losses of 30.83 and 43.40 hectares respectively. When the transformation of morphological classes was examined, a significant decrease of all species was observed in the core areas which ensure spatial connectedness, and the edge magnitude, which was taken as an indicator of fragmentation, increased. Bridges providing connectedness were observed to have increased for some species. This indicates that while existing connections in habitats were fragmented due to the forest fires, potential connections may be formed after the forest fires. When the fragmentation values were examined according to the results of pattern analysis, the most notable marginal increase in fragmentation before and after the fires was found to have occurred in the habitat of L. atifi. In addition, we discuss recommendations for the sustainability of species populations in habitat restoration and forest management.
... Species are increasingly being delimited with molecular genetic data, resulting in the recognition of new diversity that went overlooked by previous researchers who largely relied on phenotypic data (Bickford et al., 2007;Pfenninger & Schwenk, 2007;Scheffers et al., 2012). In many cases, these new species are not distinguished by known phenotypic traits or have boundaries that conflict with phenotypic variation (Leache & Fujita, 2010;Satler et al., 2013;Kealley et al., 2018;Kornilios et al., 2018). Such 'cryptic' species pose several challenges (Fišer et al., 2018;Singhal et al., 2018). ...
Article
Cryptic species – genetically distinct species that are morphologically difficult to distinguish – present challenges to systematists. Operationally, cryptic species are very difficult to identify and sole use of genetic data or morphological data can fail to recognize evolutionarily isolated lineages. We use morphometric data to test species boundaries hypothesized with genetic data in the North Caribbean bark anole (Anolis distichus), a suspected species complex. We use univariate and multivariate analyses to test if candidate species based on genetic data can be accurately diagnosed. We also test alternative species delimitation scenarios with a model fitting approach that evaluates normal mixture models capable of identifying morphological clusters. Our analyses reject the hypothesis that the candidate species are diagnosable. Neither uni- nor multivariate morphometric data distinguish candidate species. The best-supported model included two morphological clusters; however, these clusters were uneven and did not align with a plausible species divergence scenario. After removing two related traits driving this result, only one cluster was supported. Despite substantial differentiation revealed by genetic data, we recover no new evidence to delimit species and refrain from taxonomic revision. This study highlights the importance of considering other types of data along with molecular data when delimiting species.
... Application of molecular data might alleviate this difficulty . As an important complement to species identification , some common molecular species delimitations have been applied successfully in many vertebrates (Smith et al., 2013;Kornilios et al., 2018;Musher & Cracraft, 2018) and insects (Huang & Knowles, 2016;Jin et al., 2018;Yu et al., 2018). For example, the single-locus species delimitation method, automatic barcode gap discovery (ABGD; Puillandre et al., 2012), and the multilocus Bayesian coalescent species delimitation method, Bayesian phylogenetics and phylogeography (BP&P; Yang & Rannala, 2010Rannala & Yang, 2013), are most commonly applied. ...
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Phylogenetic relationships of the two largest species groups in the genus Amiota [the Amiota alboguttata group (29 known and six new species) and the Amiota basdeni group (17 known and three new species)] were reconstructed using two mitochondrial gene sequences (COI and ND2). The paraphyly of the A. alboguttata group was identified based on molecular evidence. The monophyletic A. basdeni group is found to be nested in the A. alboguttata group, rendering the latter paraphyletic. Automatic barcode gap discovery and Bayesian phylogenetics and phylogeography methods were used to assess species limits of the A. alboguttata and A. basdeni groups. The results indicate that most analysed species could be delimited clearly, including nine new species (Amiota beama sp. nov., Amiota cyclophylla sp. nov., Amiota flormontana sp. nov., Amiota obtusa sp. nov., Amiota planiceps sp. nov., Amiota scrobicula sp. nov., Amiota jianjuni sp. nov., Amiota tentacula sp. nov. and Amiota xinglaii sp. nov.), except for some closely related morphospecies. Furthermore, based on the highly biased distributions of these two groups, southwestern China is hypothesized to be a possible centre of origin and diversification for Amiota in East Asia.
... Of the species we did not detect, O. punctatissimus was only reported by Bedriaga (1883) and further reports (Bischoff 1981, Chondropoulos 1986) refer to Bedriaga's finding. According to recent research on its systematics, O. punctatissimus is limited to the Peloponnese peninsula south of Attica, two islands, and one mainland site (Iraion, Prefecture of Corinth) (Kornilios et al. 2018). It is dubious whether this species ever occurred in Attica. ...
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Although the insular distribution of the rich herpetofauna of Greece has been studied more extensively, the mainland one is still underexplored. The region of Attica in central Greece represents one of these “black spots” in the mainland. Thus, in 2019 we surveyed for the first time the herpetofauna of Attica in a systematic way. We collected 794 field records of 31 species (4 amphibians, 27 reptiles); Testudo graeca and Trachemys elegans were documented for the first time for Attica.
... The existence of highly divergent phylogenetic lineages with morphological similarity, known as cryptic species, is increasingly emerging in recent studies of reptiles, and in most cases their occurrence is mostly associated with the geoclimatic history of the study-region Cacciali et al., 2018;Jablonski et al., 2019;Kornilios et al., 2018;Kotsakiozi et al., 2018;Mendes et al., 2018;Montgelard et al., 2020;Psonis et al., 2017;Sim o-Riudalbas et al., 2017;Sindaco et al., 2014;Tamar et al., 2015Tamar et al., , 2019. As a result of our phylogenetic analyses for D. valentini, previously undiscovered genetically divergent lineages were newly detected (Figs. 2, 3). ...
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Taxonomy of the lacertid genus Darevskia has been complex since the first morphology-based studies. Darevskia valentini, mostly distributed in Turkey, currently includes three subspecies, but its interspecific and intraspecific taxonomic status has so far been shaped only by morphological evaluations, without molecular phylogenetic assessments. In this study, samples from the entire distribution of this species and its closest relatives were evaluated with molecular phylogenetic analyses of a multi-marker dataset, comprised of maternally-inherited mitochondrial and biparentally-inherited nuclear markers. We discovered two previously unrecognized genetically highly divergent lineages. The distinction of these two lineages is supported by gene trees, haplotype networks, divergence time estimation and species-delimitation analyses. We discuss possible taxonomic resolutions given that the phylogenies do not seem to agree with currently accepted taxonomy. Our results advance the knowledge of the ancestry of parthenogenesis as a result of past hybridization events between parental species pairs, because the paternal origin of some parthenogenetic species lies within the new lineages discovered here.
... The use of heavy-duty machinery should be prohibited in areas with high potential for habitat suitability as identified by scientific studies. Operations of this kind would not only affect salamanders but also the endemic fossorial Ophiomorus kardesi (Kornilios et al., 2018), as well as other amphibians and reptiles living in these areas. ...
... The three tree-based methods employ a coalescent framework based on a single-locus or multi-locus datasets in order to independently identify evolving lineages without gene flow and separate the intraspecific population from interspecific divergence, therefore with each representing a putative species [20,57]. In contrast to ABGD's underestimation of species diversity, GMYC is known to oversplit species [58][59][60][61]. The results could be affected by higher substitution rates, uneven sampling, variation in population size among species, ongoing gene flow, or unresolved nodes [27,48,49,62,63]. ...
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Chrysomyxa rusts are fungal pathogens widely distributed in the Northern hemisphere, causing spruce needle and cone rust diseases, and they are responsible for significant economic losses in China. Taxonomic delimitation and precise species identification are difficult within this genus because some characters often overlap in several species. Adequate species delimitation, enhanced by the use of DNA-based methodologies, will help to establish well-supported species boundaries and enable the identification of cryptic species. Here, we explore the cryptic species diversity in the rust genus Chrysomyxa from China. Species delimitation analyses are conducted using a distance-based method (ABGD) and three tree-based methods (GMYC, bPTP, and mPTP) based on combined LSU and ITS sequences of over 60 specimens. Although there is some incongruence among species delimitation methods, two new species and three putative cryptic species are identified. The key to 20 Chrysomyxa species distributed in China is presented. These results suggest that a significant level of undiscovered cryptic diversity is likely to be found in Chrysomyxa from China. Future studies should consider multiple analytical methods when dealing with multi-locus datasets.
... These three monophyletic lineages were supported with high or moderate values of bootstrap and posterior probability on main nodes in any of our tree topologies (Figs. 3 and S1). The phenomenon called cryptic species, which means the existence of highly divergent phylogenetic lineages with morphological similarity, is of ubiquitous impact on the taxonomy for reptiles Jablonski et al., 2019;Kornilios et al., 2018;Psonis et al., 2017;Simó-Riudalbas et al., 2017;Sindaco et al., 2014;Tamar et al., 2015Tamar et al., , 2019. The genus Darevskia, particularly, is an extraordinary group due to containing both parthenogenetic and cryptic species, which put it on an eminent position among faunal species of the earth Candan et al., 2021;Freitas et al., 2016a). ...
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In this study, we re-examine the Darevskia parvula group comprehensively using morphology, osteology and mitochondrial phylogeny, and describe a new endemic species from Turkey: Darevskia tuniyevisp. nov. A total of 257 adult specimens were evaluated for external morphology (scalation and biometry) with univariate (descriptive statistics and ANOVA with post-hoc tests) and multivariate (Discriminant Analysis and ANOSIM) analyses. In parallel, osteological data and molecular analyses using three DNA markers (mitochondrial 16S rRNA and Cyt-b, nuclear Rag-1) were used to complete the description of the new taxon. The molecular phylogenetic analyses indicated that the D. parvula group is composed of three taxa as D. parvula, D. adjarica and D. tuniyevisp. nov., and showed that D. adjarica and D. tuniyevisp. nov. are reciprocal sister taxa. On the other hand, D. adjarica is morphologically very different from other two forms, while D. parvula is hardly distinguishable externally from D. tuniyevisp. nov. Therefore, we can consider that D. parvula and D. tuniyevisp. nov. are cryptic species. These two cryptic species retain their primitive morphology within the group, while D. adjarica has changed, perhaps due to different bioclimatic conditions in its Pleistocene refuge and current area.
... For this reason, a 'refugia-withinrefugia' pattern during the Pleistocene climatic oscillations has also been proposed for Anatolia, with several studies describing secondary contacts and hybridization events (Fritz et al., 2009;Guicking, Joger, & Wink, 2009;Kindler et al., 2013;Stöck et al., 2012;Vamberger et al., 2013). This high level of genetic diversity in Anatolia has also reported in several other organisms (Dubey et al., 2007;Korkmaz et al., 2014;Rokas et al., 2003) and reptile groups (Bellati et al., 2015;Kornilios et al., 2012Kornilios et al., , 2018Kornilios et al., , 2020Rato et al., 2021;Sindaco et al., 2013;Tamar et al., 2016). ...
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The smooth snake Coronella austriaca (Laurenti, 1768) is distributed across the western Palearctic throughout north‐central, southern Europe and western Asia. So far, only few phylogenetic studies, based on mitochondrial DNA, have been carried out on this species focusing mainly on the Iberian Peninsula and northern Europe, leaving unstudied some of important areas of its distribution. This study aims to cover this gap and to explore the phylogenetic relationships between the populations of in the Eastern Mediterranean and to detect possible historical refugia. With this objective, total genomic DNA was extracted from samples originated from the Balkans, Anatolia and a large part of Europe. Two mitochondrial (cyt b, 16S) and six nuclear (BDNF, NKTR, RAG1, RAG2, MC1R and PRLR) markers were combined for phylogenetic and chronophylogenetic analyses. The different analyses confirmed previous phylogeographic hypothesis showing the presence of several well‐supported clades distributed in Anatolia, Central Europe, Eastern Europe, Western Europe, the Balkans and the Iberian Peninsula and cryptic diversity within Anatolia and the Balkans. Moreover, dating analysis confirmed the diversification of the species during the Pleistocene, as well as the role of the Southern Balkans and Anatolia region as refugia during this period.
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Ablepharus Lichtenstein, 1823, which includes ten species, is distributed in eastern Europe and Asia. Four species are recorded in Turkey: A. kitaibelii, A. chernovi, A. bivittatus, and A. budaki. After molecular and morphological studies in Anatolia, the phylogenetic relationship of the genus is still very complicated. Here, we investigate the taxonomic status of Ablepharus in Anatolia using morphological and molecular methods. The genetic structure of Ablepharus populations in Anatolia was analyzed using both the nuclear (CMOS) and mitochondrial (cyt b and COI) gene regions. The maximum likelihood and Bayesian inference methods were applied for phylogenetic tree reconstruction, together with calculation of p-distances and reconstruction of haplotype networks. The morphological analysis was evaluated with SPSS by using 12 metric and 17 meristic characteristics. In total, 41 specimens and 279 specimens were used for the molecular and morphological analysis, respectively. A. bivittatus has highly different genetic and morphological characteristics compared to the others, and it shares similar morphological characteristics with the genus Asymblepharus, which are elliptical tympanum, hidden upper eye opening under 3 or 4 large shields, light and dark longitudinal stripes on the back, adpressed hind-limbs reaching to knees, and well-developed limbs with five toes. For this reason, A. bivittatus was placed in the genus Asymblepharus as Asymblepharus bivittatus comb. nov. A. budaki anatolicus was elevated to species level as A. anatolicus based on morphological and genetic data. In this study, Anatolian A. kitaibelii comprised two sister subclades, A. kitaibelii kitaibelii and A. kitaibelii stepaneki, and this research is the first record of A. kitaibelii stepaneki in Turkey. A. chernovi was divided into three subclades: A. chernovi chernovi, A. chernovi eiselti, and A. chernovi isaurensis.
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The Mediterranean basin is a major centre for land-snail diversity, with many localized endemics, but there are also species widely spread by humans. Both endemics and introductions can be found in the snail genus Helix, which comprises many large-bodied species used for human consumption in the past and present. The Mediterranean clade of Helix is currently distributed throughout this region, but the phylogenetic and biogeographic relationships among its forms from different parts of the basin remain enigmatic. The reasons include insufficient sampling, taxa with unclear taxonomy and a significant impact of human-assisted transport obscuring the natural distribution of phylogenetic lineages. We provide evidence that European and Anatolian populations of H. cincta and its relatives are not native to those regions, but originate from the northern Levant. These results have implications for taxonomy of the genus, but also for the understanding of its evolutionary history. We posit that the Mediterranean clade consists of four geographically separated groups, which diversified in Northern Africa, the Apennine Peninsula and Corsica, the Aegean and Greece, and the northern Levant. This geographic pattern has been subsequently blurred by multiple instances of human-assisted dispersal. However, revealing the founding populations with certainty requires thorough sampling in currently inaccessible countries.
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Knowledge on the spatial distribution of taxa is crucial for the decision-making processes in the conservation and management of biodiversity that rely on precise distribution data. We present an annotated list for a total of 37 amphibian (20 caudatans and 17 anurans) and 139 reptile species (11 chelonians, 68 lizards, 3 amphisbaenians and 57 snakes) in Turkey, using both available scientific literature up to December 2020 and our own fieldwork data from 1987 to 2020. We provide a comprehensive listing of taxonomy, names, distribution and conservation status of Turkish amphibians and reptiles. The herpetofauna list will be particularly useful for establishing national conservation priorities as well as for placing Turkish fauna into phylogenetic and biogeographic contexts. We compiled information published in books, journals and various web sources with our personal data. We projected the data in the WGS84 coordinate system and created an overlay grid with cells of 50x50 km2. The database comprises more than 500 grid cells and 11,912 records. As a result, the distribution of Turkish amphibians and reptiles has been extensively mapped with geographical information systems and a database has been created. The obtained data will be useful in planning future studies on taxonomy, ecology and conservation of Turkish amphibians and reptiles.
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Self-published taxon descriptions, bereft of a basis of evidence, are a long-standing problem in taxonomy. The problem derives in part from the Principle of Priority in the International Code of Zoological Nomenclature, which forces the use of the oldest available nomen irrespective of scientific merit. This provides a route to ‘immortality’ for unscrupulous individuals through the mass-naming of taxa without scientific basis, a phenomenon referred to as taxonomic vandalism. Following a flood of unscientific taxon namings, in 2013 a group of concerned herpetologists organized a widely supported, community-based campaign to treat these nomina as lying outside the permanent scientific record, and to ignore and overwrite them as appropriate. Here, we review the impact of these proposals over the past 8 years. We identified 59 instances of unscientific names being set aside and overwritten with science-based names (here termed aspidonyms), and 1087 uses of these aspidonyms, compared to one instance of preference for the overwritten names. This shows that when there is widespread consultation and agreement across affected research communities, setting aside certain provisions of the Code can constitute an effective last resort defence against taxonomic vandalism and enhance the universality and stability of the scientific nomenclature.
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Cenozoic sediments of the Taatsiin Tsagaan Nuur region in the Valley of Lakes are known for their exceptional fossil content of the Oligocene and early Miocene, whereas, until now, middle and late Miocene records are very poor. Here, we report the late Miocene vertebrate fauna of Builstyn Khudag. Wet screening of large samples from the upper part of section BUK-A (from BUK-A/12 +14) display a rich fossil collection, comprising three lizard and five snake taxa, and sixteen small mammal species in nine families: Ochotonidae, Erinaceidae, Soricidae, Sciuridae, Eomyidae, Cricetidae, Zapodidae, Dipodidae and Gerbillidae. Surface findings of bones and teeth of large mammal families, Rhinocerotidae and Gomphotheriidae, complete the collection. The structures and the included fossils assume floodplain environments with locally dense vegetation and laterally dry open areas. Stratigraphically, we correlate the Builstyn Khudag fauna with the early late Miocene, specifically with the early part of the Asian Bahean LMS/A and with the European Mammal Zone MN9 of the early Vallesian.
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Aim To understand the patterns of genetic, geographical and morphological diversification in the Arabian gecko subspecies Pristurus rupestris rupestris, and to discuss the various processes that generate and shape diversity in arid mountain ranges. Location Hajar Mountains, southeastern Arabian Peninsula. Methods We sampled 466 individuals across the entire range of P. r. rupestris in the Hajar Mountains and sequenced one mitochondrial gene for all the samples, and one extra mitochondrial and four nuclear genes for a subset of 75 specimens. A total of 155 individuals were characterized morphologically. Multilocus coalescent-based methods were used for species delimitation, for phylogenetic inference of species trees and to investigate the diversification dynamics. Multivariate statistics were used to analyse morphological data. Results Our molecular methods revealed 14 candidate species hidden within the subspecies P. r. rupestris. Diversification started c. 15 Ma, but most of the lineages have evolved in the last 7 Ma of the mountains' orogeny, producing a mosaic of allopatric taxa. Secondary interchange has occurred between high and low elevation lineages, and along the mountain chain between unrelated lineages formerly separated by mountain blocks. Morphological variation between the 14 candidate species is slight, but a high elevation robust morpho-type is identified. Main conclusions Our results support the notion that mountain ranges constitute important centres of diversification in arid regions, and also act as 'spe-cies pumps' into surrounding areas. We also demonstrate that arid mountains may constitute important reservoirs of cryptic diversity, even in common and widespread species. This, and previous evidence, suggest that the Hajar Mountains are a biological hotspot and an important reservoir of diversity. We therefore suggest that these mountains should be a priority focal point for conservation in Arabia.
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Motivation: In recent years, molecular species delimitation has become a routine approach for quantifying and classifying biodiversity. Barcoding methods are of particular importance in large-scale surveys as they promote fast species discovery and biodiversity estimates. Among those, distance-based methods are the most common choice as they scale well with large datasets; however, they are sensitive to similarity threshold parameters and they ignore evolutionary relationships. The recently introduced "Poisson Tree Processes" (PTP) method is a phylogeny-aware approach that does not rely on such thresholds. Yet, two weaknesses of PTP impact its accuracy and practicality when applied to large datasets; it does not account for divergent intraspecific variation and is slow for a large number of sequences. Results: We introduce the multi-rate PTP (mPTP), an improved method that alleviates the theoretical and technical shortcomings of PTP. It incorporates different levels of intraspecific genetic diversity deriving from differences in either the evolutionary history or sampling of each species. Results on empirical data suggest that mPTP is superior to PTP and popular distance-based methods as it, consistently yields more accurate delimitations with respect to the taxonomy (i.e., identifies more taxonomic species, infers species numbers closer to the taxonomy). Moreover, mPTP does not require any similarity threshold as input. The novel dynamic programming algorithm attains a speedup of at least five orders of magnitude compared to PTP, allowing it to delimit species in large (meta-) barcoding data. In addition, Markov Chain Monte Carlo sampling provides a comprehensive evaluation of the inferred delimitation in just a few seconds for millions of steps, independently of tree size. Availability and implementation: mPTP is implemented in C and is available for download at http://github.com/Pas-Kapli/mptp under the GNU Affero 3 license. A web-service is available at http://mptp.h-its.org . Contact: : paschalia.kapli@h-its.org or alexandros.stamatakis@h-its.org or tomas.flouri@h-its.org. Supplementary information: Supplementary data are available at Bioinformatics online.
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Using nearly range-wide sampling, we analyze up to 1848 bp of mitochondrial DNA of 183 helmeted terrapins and identify a minimum of 12 deeply divergent species-level clades. Uncorrected p distances of these clades equal or clearly exceed those between the currently recognized species of Pelusios, the genus most closely related to Pelomedusa. We correlate genetic discontinuities of Pelomedusa with data on morphology and endoparasites and describe six new Pelomedusa species. Moreover, we restrict the name Pelomedusa subrufa (Bonnaterre, 1789) to one genetic lineage and resurrect three further species from its synonymy, namely P. galeata (Schoepff, 1792), P. gehafie (Rüppell, 1835), and P. olivacea (Schweigger, 1812). In addition to these ten Pelomedusa species, we identify two further clades from Cameroon and Sudan with similar levels of genetic divergence that remain unnamed candidate species. We also note that some problematical terrapins from South Africa and Somalia may represent two additional candidate species. Some of the Pelomedusa species are morphologically distinctive, whilst others can only be identified by molecular markers and are therefore morphologically cryptic taxa.
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Background High rates of species discovery and loss have led to the urgent need for more rapid assessment of species diversity in the herpetofauna. DNA barcoding allows for the preliminary identification of species based on sequence divergence. Prior DNA barcoding work on reptiles and amphibians has revealed higher biodiversity counts than previously estimated due to cases of cryptic and undiscovered species. Past studies have provided DNA barcodes for just 14% of the North American herpetofauna, revealing the need for expanded coverage. Methodology/Principal Findings This study extends the DNA barcode reference library for North American herpetofauna, assesses the utility of this approach in aiding species delimitation, and examines the correspondence between current species boundaries and sequence clusters designated by the BIN system. Sequences were obtained from 730 specimens, representing 274 species (43%) from the North American herpetofauna. Mean intraspecific divergences were 1% and 3%, while average congeneric sequence divergences were 16% and 14% in amphibians and reptiles, respectively. BIN assignments corresponded with current species boundaries in 79% of amphibians, 100% of turtles, and 60% of squamates. Deep divergences (>2%) were noted in 35% of squamate and 16% of amphibian species, and low divergences (<2%) occurred in 12% of reptiles and 23% of amphibians, patterns reflected in BIN assignments. Sequence recovery declined with specimen age, and variation in recovery success was noted among collections. Within collections, barcodes effectively flagged seven mislabeled tissues, and barcode fragments were recovered from five formalin-fixed specimens. Conclusions/Significance This study demonstrates that DNA barcodes can effectively flag errors in museum collections, while BIN splits and merges reveal taxa belonging to deeply diverged or hybridizing lineages. This study is the first effort to compile a reference library of DNA barcodes for herpetofauna on a continental scale.
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This article presents W-IQ-TREE, an intuitive and user-friendly web interface and server for IQ-TREE, an efficient phylogenetic software for maximum likelihood analysis. W-IQ-TREE supports multiple sequence types (DNA, protein, codon, binary and morphology) in common alignment formats and a wide range of evolutionary models including mixture and partition models. W-IQ-TREE performs fast model selection, partition scheme finding, efficient tree reconstruction, ultrafast bootstrapping, branch tests, and tree topology tests. All computations are conducted on a dedicated computer cluster and the users receive the results via URL or email. W-IQ-TREE is available at http://iqtree.cibiv.univie.ac.at. It is free and open to all users and there is no login requirement.
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A total of 282 specimens belonging to 13 lizard species from the Western Taurus Range were investigated. Morphological and biological data as well as missing pieces of information about the species were sought to be completed with the addition of a large number of new localities. Moreover, the data obtained were compared with those in the relevant literature in an attempt to shed light on the taxonomical status of the species.
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In the last decade, taxonomic studies have drastically increased the number of species known to inhabit the Arabian deserts. While ongoing phylogenetic studies continue to identify new species and high levels of intraspecific genetic diversity, few studies have yet explored the biogeographic patterns in this arid region using an integrative approach. In the present work, we apply different phylogenetic methods to infer relationships within the Palearctic naked-toed geckos. We specifically address for the first time the taxonomy and biogeography of Bunopus spatalurus Anderson, 1901, from Arabia using multilocus concatenated and species tree phylogenies, haplotype networks and morphology. We also use species distribution modelling and phylogeographic interpolation to explore the phylogeographic structure of Bunopus spatalurus hajarensis in the Hajar Mountains and the roles of climatic stability and possible biogeographic barriers on lineage occurrence and contact zones in this arid mountain endemism hot spot. According to the inferred topology recovered using concatenated and species tree methods, the genus ‘Bunopus’ is polyphyletic. Bunopus tuberculatus and B. blanfordii form a highly supported clade closely related to Crossobamon orientalis, while the two subspecies of ‘Bunopus’ spatalurus branch together as an independent highly supported clade that diverged during the Miocene according to our estimations. Within B. s. hajarensis, three geographically structured clades can be recognized that according to our estimations diverged during the Late Miocene to Pliocene. The paleodistribution models indicate climatic stability during the Late Pleistocene and the lineage occurrence, and predicted contact zones obtained from phylogeographic interpolation therefore probably result from the older splits of the groups when these lineages originated in allopatry. As demonstrated by the results of the multilocusmolecular phylogenetic analyses and the topological test carried out in this study, the genus ‘Bunopus’ is not monophyletic. To resolve this, we resurrect the genus Trachydactylus Haas and Battersby, 1959; for the species formerly referred to as Bunopus spatalurus. Considering the morphological differences, the high level of genetic differentiation in the 12S mitochondrial gene and the results of the phylogenetic and the cmos haplotype network analysis, we elevate Trachydactylus spatalurus hajarensis to the species level Trachydactylus hajarensis (Arnold, 1980).
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Burrowing is an important form of locomotion in reptiles, but no study has examined the energetic cost of burrowing for reptiles. This is significant because burrowing is the most energetically expensive mode of locomotion undertaken by animals and many burrowing species therefore show specialisations for their subterranean lifestyle. We examined the effect of temperature and substrate characteristics (coarse sand or fine sand) on the net energetic cost of burrowing (NCOB) and burrowing rate in two species of the Egernia group of skinks (Liopholis striata and Liopholis inornata) compared with other burrowing animals. We further tested for morphological specialisations among burrowing species by comparing the relationship between body shape and retreat preference in Egernia group skinks. For L. striata and L. inornata, NCOB is 350 times more expensive than the predicted cost of pedestrian terrestrial locomotion. Temperature had a positive effect on burrowing rate for both species, and a negative effect on NCOB for L. striata but not L. inornata. Both NCOB and burrowing rate were independent of substrate type. Burrows constructed by skinks had a smaller cross-sectional area than those constructed by mammals of comparable mass, and NCOB of skinks was lower than that of mammals of similar mass. After accounting for body size, retreat preference was significantly correlated with body shape in Egernia group skinks. Species of Egernia group skinks that use burrows for retreats have narrower bodies and shorter front limbs than other species. We conclude that the morphological specialisations of burrowing skinks allow them to construct relatively narrow burrows, thereby reducing NCOB and the total cost of constructing their burrow retreats. © 2015. Published by The Company of Biologists Ltd.
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The limb-reduced, cryptozoic scincid lizard genus Ophiomorus (10 species) is diagnosed vis-à-vis the primitive genus Eumeces. The species O. latastii is unusual in showing truncation in both the snout and neck. O. persicus is unusual in having the distal ends of the sacral pleurapophyses widely separated despite retaining a rear limb with two digits. Sexual dimorphism is evident in that females are generally larger and have more transverse scale rows than males. Strong interspecific correlations are: the number of digits on the front and rear limbs; the number of digits and the number of longitudinal scale rows at midbody (an index of attenuation?), and the number of presacral vertebrae and the number of transverse scale rows on the body. Cladistic analysis of 26 characters resulted in a single most parsimonious tree that only partly supported previous views of species relationships. The cladistic analysis suggests that changes in limb morphology may have involved reversals and saltations and that the external ear opening was lost twice. However, a tree one step longer suggests that limb reduction could have occurred without reversals, although it also suggests that the external ear opening may have been reacquired.
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In phylogenomics the analysis of concatenated gene alignments, the so-called supermatrix, is commonly accompanied by the assumption of partition models. Under such models each gene, or more generally partition, is allowed to evolve under its own evolutionary model. Though partition models provide a more comprehensive analysis of supermatrices, missing data may hamper the tree search algorithms due to the existence of phylogenetic (partial) terraces. Here we introduce the phylogenetic terrace aware (PTA) data structure for the efficient analysis under partition models. In the presence of missing data PTA exploits (partial) terraces and induced partition trees to save computation time. We show that an implementation of PTA in IQ-TREE leads to a substantial speedup of up to 4.5 and 8 times compared with the standard IQ-TREE and RAxML implementations, respectively. PTA is generally applicable to all types of partition models and common topological rearrangements thus can be employed by all phylogenomic inference software.
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Large phylogenomics data sets require fast tree inference methods, especially for maximum-likelihood (ML) phylogenies. Fast programs exist, but due to inherent heuristics to find optimal trees, it is not clear whether the best tree is found. Thus, there is need for additional approaches that employ different search strategies to find ML trees and that are at the same time as fast as currently available ML programs. We show that a combination of hill-climbing approaches and a stochastic perturbation method can be time-efficiently implemented. If we allow the same CPU time as RAxML and PhyML, then our software IQ-TREE found higher likelihoods between 62.2% and 87.1% of the studied alignments, thus efficiently exploring the tree-space. If we use the IQ-TREE stopping rule, RAxML and PhyML are faster in 75.7% and 47.1% of the DNA alignments and 42.2% and 100% of the protein alignments, respectively. However, the range of obtaining higher likelihoods with IQ-TREE improves to 73.3–97.1%. IQ-TREE is freely available at http://www.cibiv.at/software/iqtree.
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A method was developed for simultaneous Bayesian inference of species delimitation and species phylogeny using the multi species coalescent model. The method eliminates the need for a user-specified guide tree in species delimitation and incorporates phylogenetic uncertainty in a Bayesian framework. The Nearest-Neighbor Interchange (NNI) algorithm was adapted to proposes changes to the species tree, with the gene trees for multiple loci altered in the proposal to avoid conflicts with the newly proposed species tree. We also modify our previous scheme for specifying priors for species delimitation models to construct joint priors for models of species delimitation and species phylogeny. As in our earlier method, the modified algorithm integrates over gene trees, taking account of the uncertainty of gene tree topology and branch lengths given the sequence data. We conducted simulation study to examine the statistical properties of the method using 6 populations (2 sequences each) and a true number of 3 species, with values of divergence times and ancestral populations sizes that are realistic for recently diverged species. The results suggest that the method tends to be conservative with high posterior probabilities being a confident indicator of species status. Simulation results also indicate that the power of the method to delimit species increases with an increase of the divergence times in the species tree, and with an increased number of gene loci. Re-analyses of two datasets of cavefish and coast horned lizards suggest considerable phylogenetic uncertainty even though the data are informative about species delimitation. We discuss the impact of the prior on models of species delimitation and species phylogeny and the prior on population size parameters (θ) on Bayesian species delimitation.
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We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements our RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www.megasoftware.net free of charge.
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Molecular methods of species delineation are rapidly developing and widely considered as fast and efficient means to discover species and face the 'taxonomic impediment' in times of biodiversity crisis. So far, however, this form of DNA taxonomy frequently remains incomplete, lacking the final step of formal species description, thus enhancing rather than reducing impediments in taxonomy. DNA sequence information contributes valuable diagnostic characters and --at least for cryptic species -- could even serve as the backbone of a taxonomic description. To this end solutions for a number of practical problems must be found, including a way in which molecular data can be presented to fulfill the formal requirements every description must meet. Multi-gene barcoding and a combined molecular species delineation approach recently revealed a radiation of at least 12 more or less cryptic species in the marine meiofaunal slug genus Pontohedyle (Acochlidia, Heterobranchia). All identified candidate species are well delimited by a consensus across different methods based on mitochondrial and nuclear markers. The detailed microanatomical redescription of Pontohedyle verrucosa provided in the present paper does not reveal reliable characters for diagnosing even the two major clades identified within the genus on molecular data. We thus characterize three previously valid Pontohedyle species based on four genetic markers (mitochondrial cytochrome c oxidase subunit I, 16S rRNA, nuclear 28S and 18S rRNA) and formally describe nine cryptic new species (P. kepii sp. nov., P. joni sp. nov., P. neridae sp. nov., P. liliae sp. nov., P. wiggi sp. nov., P. wenzli sp. nov., P. peteryalli sp. nov., P. martynovi sp. nov., P. yurihookeri sp. nov.) applying molecular taxonomy, based on diagnostic nucleotides in DNA sequences of the four markers. Due to the minute size of the animals, entire specimens were used for extraction, consequently the holotype is a voucher of extracted DNA ('DNA-type'). We used the Character Attribute Organization System (CAOS) to determine diagnostic nucleotides, explore the dependence on input data and data processing, and aim for maximum traceability in our diagnoses for future research. Challenges, pitfalls and necessary considerations for applied DNA taxonomy are critically evaluated. To describe cryptic species traditional lines of evidence in taxonomy need to be modified. DNA sequence information, for example, could even serve as the backbone of a taxonomic description. The present contribution demonstrates that few adaptations are needed to integrate into traditional taxonomy novel diagnoses based on molecular data. The taxonomic community is encouraged to join the discussion and develop a quality standard for molecular taxonomy, ideally in the form of an automated final step in molecular species delineation procedures.
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Sequence-based methods to delimit species are central to DNA taxonomy, microbial community surveys, and DNA meta-barcoding studies. Current approaches either rely on simple sequence similarity thresholds (OTU-picking) or on complex and compute-intensive evolutionary models. OTU-picking methods scale well on large data sets, but the results are highly sensitive to the similarity threshold. Coalescent-based species delimitation approaches often rely on Bayesian statistics and MCMC sampling, and can therefore only be applied to small data sets. We introduce the Poisson Tree Processes (PTP) model to infer putative species boundaries on a given phylogenetic input tree. We also integrate PTP with our Evolutionary Placement Algorithm (EPA-PTP) to count the number of species in phylogenetic placements. We compare our approaches to popular OTU-picking methods and the General Mixed Yule Coalescent (GMYC) model. For de novo species delimitation, the stand-alone PTP model generally outperforms GMYC as well as OTU-picking methods when evolutionary distances between species are small. PTP neither requires an ultrametric input tree, nor a sequence similarity threshold as input. In the open reference species delimitation approach, EPA-PTP yields more accurate results than de novo species delimitation methods. Finally, EPA-PTP scales on large datasets because it relies on the parallel implementations of the EPA and RAxML, thereby allowing to delimit species in high-throughput sequencing data. The code is freely available at www.exelixis-lab.org/software.html. Alexandros.Stamatakis@h-its.org SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics on-line.
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Estimating genealogical relationships among genes at the population level presents a number of difficulties to traditional methods of phylogeny reconstruction. These traditional methods such as parsimony, neighbour-joining, and maximum-likelihood make assumptions that are invalid at the population level. In this note, we announce the availability of a new software package, TCS, to estimate genealogical relationships among sequences using the method of Templeton et al. (1992) .
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DNA barcoding-type studies assemble single-locus data from large samples of individuals and species, and have provided new kinds of data for evolutionary surveys of diversity. An important goal of many such studies is to delimit evolutionarily significant species units, especially in biodiversity surveys from environmental DNA samples. The Generalized Mixed Yule Coalescent (GMYC) method is a likelihood method for delimiting species by fitting within- and between-species branching models to reconstructed gene trees. Although the method has been widely used, it has not previously been described in detail or evaluated fully against simulations of alternative scenarios of true patterns of population variation and divergence between species. Here, we present important reformulations to the GMYC method as originally specified, and demonstrate its robustness to a range of departures from its simplifying assumptions. The main factor affecting the accuracy of delimitation is the mean population size of species relative to divergence times between them. Other departures from the model assumptions, such as varying population sizes among species, alternative scenarios for speciation and extinction, and population growth or subdivision within species, have relatively smaller effects. Our simulations demonstrate that support measures derived from the likelihood function provide a robust indication of when the model performs well and when it leads to inaccurate delimitations. Finally, the so-called single threshold version of the method outperforms the multiple threshold version of the method on simulated data: we argue that this might represent a fundamental limit due to the nature of evidence used to delimit species in this approach. Together with other studies comparing its performance relative to other methods, our findings support the robustness of GMYC as a tool for delimiting species when only single-locus information is available.
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Nonparametric bootstrap has been a widely used tool in phylogenetic analysis to assess the clade support of phylogenetic trees. However, with the rapidly growing amount of data, this task remains a computational bottleneck. Recently, approximation methods such as the RAxML rapid bootstrap (RBS) and the Shimodaira–Hasegawa-like approximate likelihood ratio test have been introduced to speed up the bootstrap. Here, we suggest an ultrafast bootstrap approximation approach (UFBoot) to compute the support of phylogenetic groups in maximum likelihood (ML) based trees. To achieve this, we combine the resampling estimated log-likelihood method with a simple but effective collection scheme of candidate trees. We also propose a stopping rule that assesses the convergence of branch support values to automatically determine when to stop collecting candidate trees. UFBoot achieves a median speed up of 3.1 (range: 0.66–33.3) to 10.2 (range: 1.32–41.4) compared with RAxML RBS for real DNA and amino acid alignments, respectively. Moreover, our extensive simulations show that UFBoot is robust against moderate model violations and the support values obtained appear to be relatively unbiased compared with the conservative standard bootstrap. This provides a more direct interpretation of the bootstrap support. We offer an efficient and easy-to-use software (available at http://www.cibiv.at/software/iqtree) to perform the UFBoot analysis with ML tree inference.
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Squamate reptiles (lizards and snakes) are one of the most diverse groups of terrestrial vertebrates. Recent molecular analyses have suggested a very different squamate phylogeny relative to morphological hypotheses, but many aspects remain uncertain from molecular data. Here, we analyse higher-level squamate phylogeny with a molecular dataset of unprecedented size, including 161 squamate species for up to 44 nuclear genes each (33 717 base pairs), using both concatenated and species-tree methods for the first time. Our results strongly resolve most squamate relationships and reveal some surprising results. In contrast to most other recent studies, we find that dibamids and gekkotans are together the sister group to all other squamates. Remarkably, we find that the distinctive scolecophidians (blind snakes) are paraphyletic with respect to other snakes, suggesting that snakes were primitively burrowers and subsequently re-invaded surface habitats. Finally, we find that some clades remain poorly supported, despite our extensive data. Our analyses show that weakly supported clades are associated with relatively short branches for which individual genes often show conflicting relationships. These latter results have important implications for all studies that attempt to resolve phylogenies with large-scale phylogenomic datasets.
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The statistical rigor of species delimitation has increased dramatically over the past decade. Coalescent theory provides powerful models for population genetic inference, and is now increasingly important in phylogenetics and speciation research. By applying probabilistic models, coalescent-based species delimitation provides clear and objective testing of alternative hypotheses of evolutionary independence. As acquisition of multilocus data becomes increasingly automated, coalescent-based species delimitation will improve the discovery, resolution, consistency, and stability of the taxonomy of species. Along with other tools and data types, coalescent-based species delimitation will play an important role in an integrative taxonomy that emphasizes the identification of species limits and the processes that have promoted lineage diversification.
Conference Paper
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Understanding the evolutionary history of living organisms is a central problem in biology. Until recently the ability to infer evolutionary relationships was limited by the amount of DNA sequence data available, but new DNA sequencing technologies have largely removed this limitation. As a result, DNA sequence data are readily available or obtainable for a wide spectrum of organisms, thus creating an unprecedented opportunity to explore evolutionary relationships broadly and deeply across the Tree of Life. Unfortunately, the algorithms used to infer evolutionary relationships are NP-hard, so the dramatic increase in available DNA sequence data has created a commensurate increase in the need for access to powerful computational resources. Local laptop or desktop machines are no longer viable for analysis of the larger data sets available today, and progress in the field relies upon access to large, scalable high-performance computing resources. This paper describes development of the CIPRES Science Gateway, a web portal designed to provide researchers with transparent access to the fastest available community codes for inference of phylogenetic relationships, and implementation of these codes on scalable computational resources. Meeting the needs of the community has included developing infrastructure to provide access, working with the community to improve existing community codes, developing infrastructure to insure the portal is scalable to the entire systematics community, and adopting strategies that make the project sustainable by the community. The CIPRES Science Gateway has allowed more than 1800 unique users to run jobs that required 2.5 million Service Units since its release in December 2009. (A Service Unit is a CPU-hour at unit priority).
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Computational evolutionary biology, statistical phylogenetics and coalescent-based population genetics are becoming increasingly central to the analysis and understanding of molecular sequence data. We present the Bayesian Evolutionary Analysis by Sampling Trees (BEAST) software package version 1.7, which implements a family of Markov chain Monte Carlo (MCMC) algorithms for Bayesian phylogenetic inference, divergence time dating, coalescent analysis, phylogeography and related molecular evolutionary analyses. This package includes an enhanced graphical user interface program called Bayesian Evolutionary Analysis Utility (BEAUti) that enables access to advanced models for molecular sequence and phenotypic trait evolution that were previously available to developers only. The package also provides new tools for visualizing and summarizing multispecies coalescent and phylogeographic analyses. BEAUti and BEAST 1.7 are open source under the GNU lesser general public license and available at http://beast-mcmc.googlecode.com and http://beast.bio.ed.ac.uk
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Since its introduction in 2001, MrBayes has grown in popularity as a software package for Bayesian phylogenetic inference using Markov chain Monte Carlo (MCMC) methods. With this note, we announce the release of version 3.2, a major upgrade to the latest official release presented in 2003. The new version provides convergence diagnostics and allows multiple analyses to be run in parallel with convergence progress monitored on the fly. The introduction of new proposals and automatic optimization of tuning parameters has improved convergence for many problems. The new version also sports significantly faster likelihood calculations through streaming single-instruction-multiple-data extensions (SSE) and support of the BEAGLE library, allowing likelihood calculations to be delegated to graphics processing units (GPUs) on compatible hardware. Speedup factors range from around 2 with SSE code to more than 50 with BEAGLE for codon problems. Checkpointing across all models allows long runs to be completed even when an analysis is prematurely terminated. New models include relaxed clocks, dating, model averaging across time-reversible substitution models, and support for hard, negative, and partial (backbone) tree constraints. Inference of species trees from gene trees is supported by full incorporation of the Bayesian estimation of species trees (BEST) algorithms. Marginal model likelihoods for Bayes factor tests can be estimated accurately across the entire model space using the stepping stone method. The new version provides more output options than previously, including samples of ancestral states, site rates, site d(N)/d(S) rations, branch rates, and node dates. A wide range of statistics on tree parameters can also be output for visualization in FigTree and compatible software.
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Shark fisheries worldwide are mostly unmanaged, but the burgeoning shark fin industry in the last few decades has made monitoring catch and trade of these animals critical. As a tool for molecular species identification, DNA barcoding offers significant potential. However, the genetic distance-based approach towards species identification employed by the Barcode of Life Data Systems may oftentimes lack the specificity needed for regulatory or legal applications that require unambiguous identification results. This is because such specificity is not typically realized by anything less than a 100% match of the query sequence to an entry in the reference database using genetic distance. Although various divergence thresholds have been proposed to define acceptable levels of intraspecific variation, enough exceptions exist to cast reasonable doubt on many less than exact matches using a distance-based approach for the identification of unknowns. An alternative approach relies on the identification of discrete molecular characters that can be used to unambiguously diagnose species. The objective of this study was to assess the performance differences between these competing approaches by examining more than 1000 DNA barcodes representing nearly 20% of all known elasmobranch species. Our results demonstrate that a character-based, nucleotide diagnostic (ND) approach to barcode identification is feasible and also provides novel insights into the structure of haplotype diversity among closely related species of sharks. Considerations for the use of NDs in applied fields are also explored.
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PhyML is a phylogeny software based on the maximum-likelihood principle. Early PhyML versions used a fast algorithm performing nearest neighbor interchanges to improve a reasonable starting tree topology. Since the original publication (Guindon S., Gascuel O. 2003. A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52:696-704), PhyML has been widely used (>2500 citations in ISI Web of Science) because of its simplicity and a fair compromise between accuracy and speed. In the meantime, research around PhyML has continued, and this article describes the new algorithms and methods implemented in the program. First, we introduce a new algorithm to search the tree space with user-defined intensity using subtree pruning and regrafting topological moves. The parsimony criterion is used here to filter out the least promising topology modifications with respect to the likelihood function. The analysis of a large collection of real nucleotide and amino acid data sets of various sizes demonstrates the good performance of this method. Second, we describe a new test to assess the support of the data for internal branches of a phylogeny. This approach extends the recently proposed approximate likelihood-ratio test and relies on a nonparametric, Shimodaira-Hasegawa-like procedure. A detailed analysis of real alignments sheds light on the links between this new approach and the more classical nonparametric bootstrap method. Overall, our tests show that the last version (3.0) of PhyML is fast, accurate, stable, and ready to use. A Web server and binary files are available from http://www.atgc-montpellier.fr/phyml/.
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Morphological and DNA data support that the East Mediterranean snake-eyed skink Ablepharus kitaibelii represents a species complex that includes four species A. kitaibelii, A. budaki, Α. chernovi, and A. rueppellii, highlighting the need of its taxonomic reevaluation. Here, we used Bayesian and Maximum Likelihood methods to estimate the phylogenetic relationships of all members of the complex based on two mitochondrial (cyt b, 16S rRNA) and two nuclear markers (MC1R, and NKTR) and using Chalcides, Eumeces, and Eutropis as outgroups. The biogeographic history of the complex was also investigated through the application of several phylogeographic (BEAST) and biogeographic (BBM) analyses. Paleogeographic and paleoclimatic data were used to support the inferred phylogeographic patterns. The A. kitaibelli species complex exhibits high genetic diversity, revealing cases of hidden diversity and cases of non-monophyletic species such as A. kitaibelii and A. budaki. Our results indicate that A. pannonicus branches off first and a group that comprises specimens of A. kitaibelli and A. budaki from Kastelorizo Island group (southeast Greece) and southwest Turkey, respectively is differentiated from the rest A. kitaibelli and A. budaki populations and may represent a new species. The estimated divergence times place the origin of the complex in the Middle Miocene (∼16 Mya) and the divergence of most currently recognised species in the Late Miocene. The inferred ancestral distribution suggests that the complex originated in Anatolia, supposing that several vicariance and dispersal events that are related with the formation of Mid-Aegean Trench, Anatolian Diagonal and the orogenesis of the mountain chains in southern and eastern Anatolia have led to current distribution pattern of A. kitaibelii species complex in the Balkans and Middle East.
Article
We describe Vipera walser, a new viper species from the north-western Italian Alps. Despite an overall morphological resemblance with Vipera berus, the new species is remarkably distinct genetically from both V. berus and other vipers occurring in western Europe and shows closer affinities to species occurring only in the Caucasus. Morphologically, the new species appear to be more similar to V. berus than to its closest relatives occurring in the Caucasus, but can be readily distinguished in most cases by a combination of meristic features as confirmed by discriminant analysis. The extant population shows a very low genetic variability measured with mitochondrial markers, suggesting that the taxon has suffered a serious population reduction/bottleneck in the past. The species is extremely range-restricted (less than 500 km2) and occurs only in two disjunct sites within the high rainfall valleys of the Alps north of Biella. This new species should be classified as globally ‘endangered’ due to its small and fragmented range, and an inferred population decline. The main near-future threats to the species are habitat changes associated with reduced grazing, along with persecution and collecting.
Article
Descriptions and diagnoses are alternative choices in all Codes of Nomenclature because Linnaeus relied on diagnoses, not descriptions, to name ca. 13400 animals, plants, and fungi. A diagnosis names characters in which a new taxon differs from the most similar known taxon; a description mixes taxonomically informative and uninformative features, usually without indicating which is which. The first formal diagnoses of new taxa that included DNA-based characters came out in 2001, and by November 2015, at least 98 names of species of mites, acoels, lichens, angiosperms, annelids, alveolates, arachnids, centipedes, turtles, fishes, butterflies, mollusks, nematodes, and pathogenic fungi have been published based on diagnostic mitochondrial, plastid, or nuclear DNA substitutions, indels, or rarely genetic distances, with or without additional morphological features. Authors have found diverse ways to specify the diagnostic traits (all published studies are here tabulated). While descriptions try to 'cover' within-species variation, a goal rarely accomplished because of (i) the stochastic nature of specimen availability (thousands of species are known from single collections) and (ii) the subjective circumscription of species, the purpose of diagnoses was and is speedy identification. Linnaeus tried to achieve this by citing images, geographic occurrence, and previous literature. The renewed attention to sharp diagnosis now coincides with worldwide barcoding efforts, may speed up formal naming, and matches the increasing reliance on DNA for both classification and identification. I argue for DNA-based diagnoses of new species becoming a recommendation in all Codes, not just the bacterial code.
Article
The Aegean region, located in the Eastern Mediterranean, is an area of rich biodiversity and endemism. Its position, geographical configuration and complex geological history have shaped the diversification history of many animal taxa. Mygalomorph spiders have drawn the attention of researchers, as excellent model systems for phylogeographical investigations. However, phylogeographic studies of spiders in the Aegean region are scarce. In this study, we focused on the phylogeography of the endemic ctenizid trap-door spider CyrtocarenumAusserer, 1871. The genus includes two morphologically described species: C. grajum (C. L. Koch, 1836) and C. cunicularium (Olivier, 1811). We sampled 60 specimens from the distributions of both species and analyzed four mitochondrial and two nuclear markers. Cyrtocarenum served as an example to demonstrate the importance of natural history traits in the inference of phylogeographic scenarios. The mtDNA substitution rates inferred for the genus are profoundly higher compared to araneomorph spiders and other arthropods, which seems tightly associated with their biology. We evaluate published mtDNA substitution rates followed in the literature for mygalomorph spiders and discuss potential pitfalls. Following gene tree (maximum likelihood, Bayesian inference) and species tree approaches (∗BEAST), we reconstructed a time-calibrated phylogeny of the genus. These results, combined with a biogeographical ancestral-area analysis, helped build a biogeographic scenario that describes how the major palaeogeographic and palaeoclimatic events of the Aegean may have affected the distribution of Cyrtocarenum lineages. The diversification of the genus seems to have begun in the Middle Miocene in the present west Aegean area, while major phylogenetic events occurred at the Miocene-Pliocene boundary for C. cunicularium, probably related to the Messinian Salinity Crisis. Our results also demonstrate the clear molecular distinction of the two morphologically described species, but possible cryptic lineages may exist within C. cunicularium.
Article
Two slow worm species are distributed at the southernmost part of the Balkan Peninsula: Anguis cephallonica, an endemic of the Peloponnese and the islands Zakynthos, Ithaki and Kephallonia, and A. graeca. Here, we investigate the intraspecific genetic diversity of A. cephallonica from the Peloponnese and Kephallonia and analyse A. graeca, from the northern Peloponnese, where it is found in sympatry with A. cephallonica. MtDNA and nDNA phylogenetic analyses confirm the genetic similarity of Peloponnesian and Kephallonian populations of A. cephallonica and reveal significant mtDNA genetic variation within it, probably related to the occurrence of multiple subrefugia in the Peloponnese. Peloponnesian A. graeca populations are genetically similar to non-Peloponnesian conspecifics implying recent dispersal to the Peloponnese. In contrast to the genetic markers, morphological characteristics (such as the number of mid-body scale-rows) failed to distinguish between Peloponnesian A. cephallonica and A. graeca. Although the former species is believed to be well-differentiated from its congeneric taxa, a thorough morphological study is needed.
Article
The study of mitochondrial and nuclear DNA sequences reveals that the polytypic Blanus strauchi is a species complex including three well-defined allopatric clades, one of which consists of two sub-clades. Only the two sub-clades of the Western clade are morphologically diagnosable in the field, whereas obvious characters to distinguish the Central and the Eastern clades are lacking. However, all four clades show significant statistical differentiation on meristic traits, as well as in morphometric characters of the head when compared by means of the geometric morphometrics. The genetic distance between the three major mitochondrial clades is comparable to the p-distances for the same markers observed between Blanus species-pairs from Morocco and the Iberian Peninsula, respectively. The nuclear marker confirms the mitochondrial clades, and shows that the three major clades do not share any haplotypes, as an indication of restricted gene flow among them. On the basis of this evidence, the taxonomy of Blanus strauchi is re-assessed: the Western clade corresponds to B. strauchi, with two subspecies: B. s. strauchi and B. s. bedriagae. The Central clade corresponds to B. aporus, here elevated at the species rank. For the eastern clade there are no available names, and therefore it is described here as Blanus alexandri sp. nov.
Article
While traditionally species recognition has been based solely on morphological differences either typological or quantitative, several newly developed methods can be used for a more objective and integrative approach on species delimitation. This may be especially relevant when dealing with cryptic species or species complexes, where high overall resemblance between species is coupled with comparatively high morphological variation within populations. Rock lizards, genus Darevskia, are such an example, as many of its members offer few diagnostic morphological features. Herein, we use a combination of genetic, morphological and ecological criteria to delimit cryptic species within two species complexes, D. chlorogaster and D. defilippii, both distributed in northern Iran. Our analyses are based on molecular information from two nuclear and two mitochondrial genes, morphological data (15 morphometric, 16 meristic and four categorical characters) and eleven newly calculated spatial environmental predictors. The phylogeny inferred for Darevskia confirmed monophyly of each species complex, with each of them comprising several highly divergent clades, especially when compared to other congeners. We identified seven candidate species within each complex, of which three and four species were supported by Bayesian species delimitation within D. chlorogaster and D. defilippii, respectively. Trained with genetically determined clades, Ecological Niche Modeling provided additional support for these cryptic species. Especially those within the D. defilippii-complex exhibit well-differentiated niches. Due to overall morphological resemblance, in a first approach PCA with mixed variables only showed the separation between the two complexes. However, MANCOVA and subsequent Discriminant Analysis performed separately for both complexes allowed for distinction of the species when sample size was large enough, namely within the D. chlorogaster-complex. In conclusion, the results support four new species, which are described herein.
Article
Species delimitation is the act of identifying species-level biological diversity. In recent years, the field has witnessed a dramatic increase in the number of methods available for delimiting species. However, most recent investigations only utilize a handful (i.e. 2-3) of the available methods, often for unstated reasons. Because the parameter space that is potentially relevant to species delimitation far exceeds the parameterization of any existing method, a given method necessarily makes a number of simplifying assumptions, any one of which could be violated in a particular system. We suggest that researchers should apply a wide range of species delimitation analyses to their data and place their trust in delimitations that are congruent across methods. Incongruence across the results from different methods is evidence of either a difference in the power to detect cryptic lineages across one or more of the approaches used to delimit species and could indicate that assumptions of one or more of the methods have been violated. In either case, the inferences drawn from species delimitation studies should be conservative, for in most contexts it is better to fail to delimit species than it is to falsely delimit entities that do not represent actual evolutionary lineages.
Article
The millions of conserved biological specimens that are stored upon the shelves of Natural History Museums across the world constitute a capital of biological information that is becoming increasingly accessible to students of various disciplines. Most students have taken measures of body size and shape of preserved museum specimens to test various elements of ecological and evolutionary theory. One possible hazard of using morphological measurements of museum specimens is that fixation and preservation may deform bodies or body parts, but most researchers implicitely assume that the magnitude of conservation-induced distortions are insufficient to jeopardize their analyses. However, no study to our knowledge has clearly quantified those possible distortions. In this study, we have measured 17 morphological variables on a set of 65 green iguanas (Iguana iguana), starting shortly after their death and then repeatedly over a two month period, a period during which they were fixated and preserved. Our aims were (1) to quantify and compare the deformation in different morphometrics frequently used in evolutionary studies; (2) to determine the amount of temporal variation that can be attributed to reader variability; and (3) to build conversion equations that should improve the reliability of morphological comparisons of life and conserved specimens. Conserved lizards revealed major reduction in snout vent length and body weight. Changes in other measured traits are more subtle, but persistent. These facts disturb analyses when using relative measurements, especially when comparing (often small) intraspecific differences or even morphological differences within populations in a temporal frame. We urge caution in using museum specimens as direct proxies for living organisms in ecological and taxonomic studies.
Article
The Ozark madtom, Noturus albater, is distributed in upland streams in the upper White, Black, and St. Francis rivers of Missouri and Arkansas, USA. Fixed chromosomal and biochemical differences between populations in the upper White River and the Black and St. Francis rivers led earlier researchers to suggest that the Ozark madtom comprised two distinct species. The current study reviewed previous data, and examined osteology, pigmentation, morphometrics and DNA sequences from the mitochondrial cytochrome b gene and the nuclear Recombination Activating Gene 2. Sequence data were analysed using parsimony and Bayesian methods to construct a phylogenetic hypothesis of relationships within Noturus and examine relationships of populations of Ozark madtoms. Results of the molecular analyses were consistent with earlier chromosomal and biochemical studies. Morphology could not distinguish between populations from the upper White River and populations from the Black and St. Francis rivers. However, karyotypes, allozyme variation, and DNA sequences all provide diagnostic characters demonstrating the presence of two species consistent with the phylogenetic species concept. We describe the populations from the Black and St. Francis rivers as Noturus maydeni, a new cryptic species of madtom catfish.
Article
Aim Australian scincid lizards represent three distinct groups within the cosmopolitan clade Lygosominae, the Egernia, Eugongylus and Sphenomorphus groups. This paper presents a time-calibrated phylogeny for Lygosominae that provides the necessary temporal framework for assessing the contributions of immigration from Asia and of Gondwanan inheritance in the derivation of the Australian scincid fauna. Location Australasia, Asia, Africa. Methods Phylogenetic relationships and divergence times were inferred from novel BDNF, c-mos and PTPN12 sequences (2408 aligned sites). Results Lygosomine monophyly is well supported, and there is strong support for monophyly of the Egernia, Eugongylus and Sphenomorphus groups. A sister-group relationship of Tribolonotus (distributed in Melanesia and the Papuan Region) and the Egernia group is strongly supported in both Bayesian and maximum likelihood analyses. Australian representatives of the Sphenomorphus group compose a significantly supported clade estimated to have originated c. 25 Ma. An age of c. 18 Ma is inferred for a strongly supported clade comprising Australian representatives of the Egernia group; this clade diverged from Corucia zebrata (confined to the Solomon Islands) c. 25 Ma and from Tribolonotus c. 54 Ma. A well-supported clade including all Australian Eugongylus group taxa sampled is estimated to have arisen c. 20 Ma. Main conclusions The Australian Sphenomorphus group is nested within the more inclusive Sphenomorphus group (distributed primarily in Asia and Australasia), suggesting comparatively recent descent from a colonizing Asian ancestor; the divergence times inferred here indicate that colonization occurred during the mid Cenozoic, subsequent to the rifting of Australia from Antarctica. An Oligocene origin of the extant Eugongylus group fauna of Australasia (the basal members of which are distributed in the Southwest Pacific) indicates that Eugongylus group lygosomines also dispersed to Australia relatively recently. The Egernia group diverged from Tribolonotus in the Early Eocene; however, extant Egernia group lygosomines originated only during the Late Oligocene, implying extensive pruning of stem taxa (i.e. extinction). As a result, inferences of the timing of dispersal into Australia are associated with substantial uncertainty, although independent palaeontological evidence suggests that the Egernia group entered Australia prior to the Oligocene, immediately after (or perhaps before) the separation of Australia and Antarctica.
Article
Museum fish specimens are invaluable resources for genetic studies, but extraction of high quality DNA is often problematic. In this study, hairtail fishes of the genera Trichiurus and Lepturacanthus (family: Trichiuridae) representing a wide range of preservation histories and three different methods of preservation were analyzed for mitochondrial DNA (mtDNA) extraction, amplification and sequencing of marker genes. A total of six protocols, including a commercially available kit, were compared in this study. Amplification of conserved genes such as16S rRNA and 12S rRNA were done using polymerase chain reaction with sequence analyses using automated capillary sequencing techniques. The results show that mtDNA extraction, amplification and sequencing of conserved genes could be obtained successfully from frozen (−20°C) preserved specimens (1–5 years) and also from ethanol (95%) fixed specimens (2–5 years) but not from any of the formalin (10%) fixed specimens (3–4 years). However, specimens that have been fixed for only 7 days in buffered formalin (10% formalin with phosphate buffer containing 173 mm salt) and ethanol (95%) could yield successful mtDNA extraction, amplification and sequence information of both 16S rRNA and 12S rRNA.
Article
The literature about species concepts might be larger than that about any other subject in evolutionary biology, but the issue of empirically testing species boundaries has been given little attention relative to seemingly endless debates over what species are. The practical issue of delimiting species boundaries is nevertheless of central importance to many areas of evolutionary biology. The number of recently described methods for delimiting species suggests renewed interest in the topic, and some methods are explicitly quantitative. Here, we review nine of these methods by summarizing the relevant biological properties of species amenable to empirical evaluation, the classes of data required and some of the strengths and limitations of each.
Article
Within uncharacterized groups, DNA barcodes, short DNA sequences that are present in a wide range of species, can be used to assign organisms into species. We propose an automatic procedure that sorts the sequences into hypothetical species based on the barcode gap, which can be observed whenever the divergence among organisms belonging to the same species is smaller than divergence among organisms from different species. We use a range of prior intraspecific divergence to infer from the data a model-based one-sided confidence limit for intraspecific divergence. The method, called Automatic Barcode Gap Discovery (ABGD), then detects the barcode gap as the first significant gap beyond this limit and uses it to partition the data. Inference of the limit and gap detection are then recursively applied to previously obtained groups to get finer partitions until there is no further partitioning. Using six published data sets of metazoans, we show that ABGD is computationally efficient and performs well for standard prior maximum intraspecific divergences (a few per cent of divergence for the five data sets), except for one data set where less than three sequences per species were sampled. We further explore the theoretical limitations of ABGD through simulation of explicit speciation and population genetics scenarios. Our results emphasize in particular the sensitivity of the method to the presence of recent speciation events, via (unrealistically) high rates of speciation or large numbers of species. In conclusion, ABGD is fast, simple method to split a sequence alignment data set into candidate species that should be complemented with other evidence in an integrative taxonomic approach.
Article
Lineages that exhibit little morphological change over time provide a unique opportunity to explore whether nonadaptive or adaptive processes explain the conservation of morphology over evolutionary time scales. We provide the most comprehensive evaluation to date of the evolutionary processes leading to morphological similarity among species in a cryptic species complex, incorporating two agamid lizard species (Diporiphora magna and D. bilineata). Phylogenetic analysis of mitochondrial (ND2) and nuclear (RAG-1) gene regions revealed the existence of eight deeply divergent clades. Analysis of morphological data confirmed the presence of cryptic species among these clades. Alternative evolutionary hypotheses for the morphological similarity of species were tested using a combination of phylogenetic, morphological, and ecological data. Likelihood model testing of morphological data suggested a history of constrained phenotypic evolution where lineages have a tendency to return to their medial state, whereas ecological data showed support for both Brownian motion and constrained evolution. Thus, there was an overriding signature of constrained evolution influencing morphological divergence between clades. Our study illustrates the utility of using a combination of phylogenetic, morphological, and ecological data to investigate evolutionary mechanisms maintaining cryptic species.
Article
Lizards have been model organisms for ecological and evolutionary studies from individual to community levels at multiple spatial and temporal scales. Here we highlight lizards as models for phylogeographic studies, review the published population genetics/phylogeography literature to summarize general patterns and trends and describe some studies that have contributed to conceptual advances. Our review includes 426 references and 452 case studies: this literature reflects a general trend of exponential growth associated with the theoretical and empirical expansions of the discipline. We describe recent lizard studies that have contributed to advances in understanding of several aspects of phylogeography, emphasize some linkages between phylogeography and speciation and suggest ways to expand phylogeographic studies to test alternative pattern-based modes of speciation. Allopatric speciation patterns can be tested by phylogeographic approaches if these are designed to discriminate among four alternatives based on the role of selection in driving divergence between populations, including: (i) passive divergence by genetic drift; (ii) adaptive divergence by natural selection (niche conservatism or ecological speciation); and (iii) socially-mediated speciation. Here we propose an expanded approach to compare patterns of variation in phylogeographic data sets that, when coupled with morphological and environmental data, can be used to to discriminate among these alternative speciation patterns. [Correction made after online publication (28/07/2010): (minor deletion in the last line of the abstract)].