A molecular phylogeny of the Sylvia cantillans complex: cryptic species within the Mediterranean basin

The subalpine warbler Sylvia cantillans is formally considered a polytypic species, with four subspecies, European S. c. cantillans, albistriata, moltonii (recently resumed name: subalpina) and North African S. c. inornata. They are very similar in external morphology but clearly differ in their vocalizations. We evaluated their uncertain taxonomic status reconstructing the phylogenetic and phylogeographic relationships among populations sampled across major biogeographical areas in the European species’ range, using nucleotide sequences of the mitochondrial cytochrome b gene (mtDNA cyt b). A variety of phylogenetic analyses concordantly led to identify four major groups, only partially corresponding to the three European nominal subspecies. Phylogenetic trees showed a monophyletic group including all moltonii individuals, well diverged from all other taxa. Populations taxonomically assigned to cantillans were polyphyletic being split into two distinct clades (western and southern cantillans), with monophyletic albistriata closely related to southern cantillans. Individuals of moltonii and southern cantillans sampled in sites of sympatry in central Italy were assigned to their respective groups, with perfect concordance between phenotypic and genetic identifications. All findings indicate that moltonii should be ranked as a distinct species. Former subspecies cantillans is polyphyletic, but additional data are needed to define the taxonomic status of its two clades. Albistriata is phylogenetically related to southern cantillans and should be provisionally kept as a subspecies of S. cantillans. The cantillans complex thus provides an interesting case-study illustrating geographical structuring across small geographical ranges, and it exemplifies speciation through differentiation in allopatry leading to reproductive isolation after a secondary contact.     

Extensive phenotypic diversification coexists with little genetic divergence and a lack of population structure in the White Wagtail subspecies complex (Motacilla alba)

Geographically clustered phenotypes often demonstrate consistent patterns in molecular markers, particularly mitochondrial DNA (mtDNA) traditionally used in phylogeographic studies. However, distinct evolutionary trajectories among traits and markers can lead to their discordance. First, geographic structure in phenotypic traits and nuclear molecular markers can be co‐aligned but inconsistent with mtDNA (mito‐nuclear discordance). Alternatively, phenotypic variation can have little to do with patterns in neither mtDNA nor nuclear markers. Disentangling between these distinct patterns can provide insight into the role of selection, demography and gene flow in population divergence. Here, we examined a previously reported case of strong inconsistency between geographic structure in mtDNA and plumage traits in a widespread polytypic bird species, the White Wagtail (Motacilla alba). We tested whether this pattern is due to mito‐nuclear discordance or discrepancy between morphological evolution and both nuclear and mtDNA markers. We analysed population differentiation and structure across six out of nine commonly recognized subspecies using 17 microsatellite loci and a combination of microsatellites and plumage indices in a comprehensively sampled region of a contact between two subspecies. We did not find support for the mito‐nuclear discordance hypothesis: nuclear markers indicated a subtle signal of genetic clustering only partially consistent with plumage groups, similar to previous findings that relied on mtDNA. We discuss evolutionary factors that could have shaped the intricate patterns of phenotypic diversification in the White wagtail and the role that repeated selection on plumage ‘hotspots’ and hybridization may have played.     

MITOCHONDRIAL DNA HOMOGENEITY IN THE PHENOTYPICALLY DIVERSE REDPOLL FINCH COMPLEX (AVES: CARDUELINAE: CARDUELIS FLAMMEA-HORNEMANNI)

Breeding redpoll finches (Aves: Carduelinae) show extensive plumage and size variability and, in many cases, a plumage polymorphism that is not related to age or sex. This has been ascribed to extreme phenotypic variation within a single taxon or to moderate variability within distinct taxa coupled with hybridization. The predominant view favors the recognition of two largely sympatric species: Carduelis flammea, comprised of four well-marked subspecies—flammea, cabaret, islandica, and rostrata; and C. hornemanni, comprised of two subspecies—hornemanni and exilipes. We studied representative samples of these putative subspecies (except islandica) for variation in mitochondrial DNA (mtDNA). Using 20 informative restriction enzymes that recognized 124 sites (642 base pairs [bp] of sequence or ≈ 3.7% of the molecule), we identified 17 RFLP haplotypes in the 31 individuals surveyed. The haplotypes formed a simple phylogenetic network with most clones diverging by a single site difference from a common haplotype found in almost half of the individuals. Within populations and taxa, levels of mtDNA diversity were similar to those observed in other avian species. The pattern of mtDNA divergence among populations was statistically unrelated to their geographic or traditional taxonomic relationships, and the estimated distance between the two traditionally recognized species was very small relative to those typically observed among avian sister species.     

Molecular and plumage analyses indicate the incomplete separation of two woodpeckers (Aves,Picidae)

We evaluated the relationship between Celeus undatus and Celeus grammicus, with the objective of clarifying their evolutionary history. We analysed fragments of the mitochondrial and nuclear genes of 57 specimens. For comparative purposes, we inspected the plumage patterns of 77 skins. Our findings highlight the absence of reciprocal monophyly between the two taxa, given their reduced genetic divergence, and the lack of any clear separation of the two forms in the haplotype networks. A similar situation was found in the STRUCTURE analysis, with reciprocal contributions from the two taxa to the respective clusters, indicating that C. grammicus and C. undatus cannot be differentiated using the molecular markers. Corroborating the genetic data, our plumage analyses also failed to find any clear diagnostic characters between the polytypic C. undatus and C. grammicus, as they are defined at present. The genetic profile is consistent with either extensive historical gene flow between the species or, alternatively, incomplete lineage sorting, rather than recent secondary contact. The lack of monophyly between the two taxa impeded subspecies‐level phylogeographic inferences, with the subspecific variation being interpreted as a probable artefact of the phenotypic plasticity of the two forms. These findings indicate clearly that the two taxa form a single evolutionary unit, in which the morphological differentiation used to diagnose the species, combined with their geographic distribution, is at odds with the incomplete separation of the taxa. This may reflect disparities in the rates of differentiation between molecular and phenotypic markers, which is possibly due to the variation in selection pressures along a humidity gradient in Amazonia.     

MOLECULAR PHYLOGENETICS AND EVOLUTION OF SEXUAL DICHROMATISM AMONG POPULATIONS OF THE YARROW'S SPINY LIZARD (SCELOPORUS JARROVII)

Understanding evolution of geographic variation in sexually dimorphic traits is critical for understanding the role that sexual selection may play in speciation. We performed a phylogenetic analysis of geographic variation in sexual dichromatism in the Yarrow's spiny lizard (Sceloporus jarrovii), a taxon that exhibits remarkable diversity in male coloration among populations (e.g., black, red, green, yellow, blue, brown). An mtDNA phylogeny based on approximately 880 bp from the 12S ribosomal RNA gene and 890 bp from the ND4 gene was reconstructed for 30 populations of S. jarrovii and eight other species of the torquatus species group using maximum-likelihood and parsimony methods. The phylogeny suggests that S. jarrovii consists of at least five evolutionary species, none of which are sister taxa. Although intraspecific diversity in male coloration is less than indicated by previous taxonomy, two species formerly referred to as S. jarrovii exhibit impressive geographic variation in sexual dichromatism. In one of these species, the phylogeny shows the independent evolution of a distinctive blue color morph in different parts of the species range. This pattern suggests that sexual selection may lead to striking phenotypic divergence among conspecific populations and striking convergence. Results also demonstrate the importance of a phylogenetic perspective in studies of evolutionary processes within nominal species and the problematic nature of “polytypic” species recognized under the biological species concept.     

Clinal variation or validation of a subspecies? A case study of the Graptemys nigrinoda complex (Testudines: Emydidae)

Widely distributed species often display intraspecific morphological variation due to the abiotic and biotic gradients experienced across their ranges. Historically, in many vertebrate taxa, such as birds and reptiles, these morphological differences within a species were used to delimit subspecies. Graptemys nigrinoda is an aquatic turtle species endemic to the Mobile Bay Basin. Colour pattern and morphological variability were used to describe a subspecies (G. n. delticola) from the lower reaches of the system, although it and the nominate subspecies also reportedly intergrade over a large portion of the range. Other researchers have suggested that these morphological differences merely reflect clinal variation. Our molecular data (mtDNA) did not support the existence of the subspecies, as the haplotypes were differentiated by only a few base pairs and one haplotype was shared between the putative subspecies. While there were significant morphological and pattern differences among putative specimens of G. n. nigrinoda, G. n. delticola and G. n. nigrinoda × delticola, these differences probably represent clinal variation as they were also related to environmental variables [i.e. cumulative drainage area and drainage (categorical)]. Specimens occupying slow‐current, high‐turbidity river reaches (e.g. the Tensaw River) exhibited greater relative carapace heights and more dark pigmentation, while specimens occupying fast‐current, clearer rivers (e.g. the upper Alabama, Cahaba and Tallapoosa rivers) exhibited lower carapace heights and more yellow pigmentation. Given the absence of clear molecular and morphological differences that are related to drainage characteristics, we suggest that there is not sufficient evidence for the recognition of G. n. delticola as a distinct subspecies. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 810–822.     

Relationships of morphological groups in the northern flicker superspecies complex (Colaptes auratus & C. chrysoides)

Many species complexes have diversified rapidly and recently, resulting in morphologically diverse populations; however, the rapid pace of diversification often prevents identification of clear phylogeographic structure. Recently, the use of large genomic and reduced-representation genomic datasets has improved resolution of the evolutionary histories in such species and allowed identification of lineages on distinct evolutionary trajectories. The northern flicker (Colaptes auratus) and gilded flicker (Colaptes chrysoides) form a polytypic superspecies group with a complex taxonomic history. The superspecies group includes up to 13 described subspecies, which represent slight geographic variation among five main morphological groups: red-shafted flickers of western North America (cafer group), yellow-shafted flickers of eastern North America (auratus group), Cuban flickers of the Caribbean (chrysocaulosus group), gilded flickers of the U.S. south-west and Mexican north-west (chrysoides group), and Guatemalan flickers of Central America (mexicanoides group). These groups are largely differentiable by variation in feather shaft colour, malar colour, throat colour, crown colour, and back barring. Here, using mitochondrial DNA (mtDNA) and hundreds of single nucleotide polymorphisms (SNPs), we characterized the genetic relationships and genomic distinctiveness of the five morphological groups. We found the mexicanoides group to be the most genetically distinct in both mtDNA (～1.4% sequence divergence) and large SNP panels. The chrysocaulosus group is differentiated by a single basepair mutation in a small mtDNA fragment. In both mtDNA and SNP panels, there is little genetic distinctiveness between auratus, cafer, and chrysoides morphological groups, with evidence of admixture and a lack of fixed differences.     

AFLP analysis shows high incongruence between genetic differentiation and morphology‐based taxonomy in a widely distributed tortoise

Morphology has traditionally been used to diagnose the taxa of various taxonomic ranks. However, there is growing evidence that morphology is not always able to reveal cryptic taxa, and that pronounced morphological variation could reflect phenotypic plasticity rather than evolutionary divergence. Spur‐thighed tortoises (the Testudo graeca complex), distributed in the western Palaearctic region, are characterized by high morphological variability and complicated taxonomy, which are under debate. Previous molecular studies using mainly mitochondrial DNA (mtDNA) sequences have revealed incongruence between genetic differentiation and morphology‐based taxonomy, suggesting that morphological variability is the result of phenotypic plasticity and stabilizing selection, which masks the true genealogies. In the present study, we used a range‐wide sampling and nuclear Amplified fragment length polymorphism (AFLP) markers to investigate genetic differentiation within the T. graeca complex. We found that spur‐thighed tortoises are differentiated into four geographically well‐defined AFLP groups: Balkans–Middle Eastern, western Mediterranean, Caucasian and central‐eastern Iranian. Compared with the distribution of mtDNA lineages, the groups are largely concordant, although the AFLP markers are less sensitive and distinguish fewer groups than do mtDNA sequences. The AFLP groups show an allopatric or parapatric distribution. The AFLP differentiation conflicts with the previously proposed morphology‐based taxonomy of the complex, suggesting that local adaptation to different environmental conditions may have led to the great extent of morphological variation within the same lineages. We propose a re‐evaluation of the taxa that were confirmed genetically using a thorough morphological analysis corrected for phenotypic plasticity. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ●● , ●●–●●.     

Phylogeny and evolution of dorsal pattern in the Mexican endemic lizard genus Barisia (Anguidae: Gerrhonotinae)

The phylogeny of the Mexican lizard genus Barisia was assessed using an 878 bp fragment of the mtDNA ND4 gene and a section of associated tRNA genes, as well as 16 external morphological characters. The terminal taxa comprised the currently recognized members of Barisia, including the four subspecies of the polytypic Barisia imbricata and individuals from different populations of the widespread B. i. imbricata and Barisia i. ciliaris, although for Barisia levicollis only morphology could be examined. The ‘step‐matrix frequency’ and the ‘step‐matrix gap‐weighting’ coding approaches were employed simultaneously for the morphological data set, and three different scaling methods were evaluated for the last approach. Maximum parsimony (MP) analyses were performed for the separate and combined data sets and Bayesian analysis was also performed for the mtDNA sequence data. The hypothesis obtained from the simultaneous MP analysis strongly supports the monophyly of Barisia, but the ‘exclusivity’ of B. imbricata as well as of B. i. imbricata and B. i. ciliaris were not recovered. Moreover, inclusion of the morphological data showed B. levicollis nested within a clade together with the taxa assigned to B. i. ciliaris. This, together with the genetic distances and geographic concordance among the haplotypes examined, confirms that B. imbricata represents several species, although the actual species limits in this composite taxon are still unclear. Applying previously published rates of molecular evolution to the mtDNA data gives ages of divergence similar to the times proposed for some Pleistocene–Miocene geological and climatic phenomena that occurred in the Mexican territory. Variation of the dorsal pattern within Barisia was mapped onto the simultaneous morphological and molecular phylogeny, indicating that the two main states present in the taxa assigned to B. imbricata, an adult dorsal pattern present in females and absent in males and the absence of any pattern in both sexes, occur each in separate lineages. This suggests a possible scenario, where sexual dichromatism within Barisia has been repeatedly lost in different lineages.     

Subgroups in theHordeum patagonicum complex (Poaceae)

Seeds of theHordeum patagonicum complex were collected from the field and grown in the greenhouse. The aim was to take a sample of members of the complex, and on the basis of the phenotypic similarities in some morphological and physiological characters, determine whether distinct groups exist. When cluster analyses, to generate hypotheses, and orthodox statistical procedures, for hypotheses obtained a priori, were applied to the reproductive morphology, germination and flowering patterns, onlyH. patagonicum subsp.magellanicum, out of the five recognized taxa, could be distinguished consistently. The remaining four taxa, which overlapped considerably, could be re-formed into three groups whose centroids were different but whose ranges of variation were not distinct from each other. We conclude that the highly cross-compatible members of theH. patagonicum complex, first defined as species and later redefined as subspecies are probably no more than biotypes.     

Evidence for Introgressive Hybridization of Captive Markhor (Capra falconeri) with Domestic Goat: Cautions for Reintroduction

Markhors (Capra falconeri) are among the most endangered mammal species, and several conservation measures, including ex situ breeding, are implemented to prevent their extinction. We studied sequence diversity and differentiation of the first hypervariable segment of the mitochondrial DNA control region among C. f. heptneri and C. f. megaceros kept in four zoos in relationship to lineages of other wild and domestic goats, to assess for the first time the level of molecular distinctness and variability among those subspecies, and to check for possible introgression by related Capra taxa, such as domestic goats. Levels of differentiation between some Capra falconeri lineages and modern domestic goats were similar to levels between other wild goat species (i.e., Capra aegagrus, Capra ibex) and domestic goats. Among pure markhor lineages, paraphyly was observed for C. f. heptneri, suggesting occurrence of shared ancestral polymorphism among markhor subspecies and/or ancient or recent gene exchange between subspecies. Interestingly, 35.7% of all studied markhors from three zoos are introgressed by the domestic goat. Furthermore, despite relatively small breeding group sizes, markhors have maintained a relatively high proportion of mtDNA variation within zoo groups. In any case, the existence of markhors introgressed with domestic goat DNA in zoos should be considered when selecting markhors for ex situ breeding programs with the aim of building up a stock for later reintroduction into the wild.     

Characterization of polymorphic microsatellite loci in the tropical ant‐plant Leonardoxa africana (Fabaceae: Caesalpinioideae)

Ten microsatellite loci were isolated from the African ant‐plant Leonardoxa africana (Fabaceae: Caesalpinioideae). They differentiate the two most divergent subspecies of this polytypic complex, L. a. africana and L. a. gracilicaulis, showing promise for the study of gene flow. In each of these two subspecies, high levels of within‐population variation were observed, with a number of alleles ranging from one to 10 in ssp. africana and from one to 7 in ssp. gracilicaulis, and heterozygosity from 0 to 0.933 in ssp. africana and to 0.867 in ssp. gracilicaulis. All loci amplified successfully in the two other subspecies of L. africana.     

Evolutionary history of Trachylepis skinks in the Seychelles islands: introgressive hybridization,morphological evolution and geographic structure

The Seychelles is a remarkably interesting archipelago for evolutionary studies, but only recently have molecular markers been used to explore its biogeographic patterns. Here we used morphological and molecular data to examine diversity and phylogenetic relationships of two endemic skink sister‐species from this archipelago: Trachylepis sechellensis and Trachylepis wrightii. Mitochondrial DNA genealogy rendered a monophyletic T. wrightii nested within a paraphyletic T. sechellensis, whereas nuclear DNA sequences from five unlinked markers reflected the accepted taxonomy. Hybridization and massive mtDNA introgression leading to the complete replacement of the native mtDNA lineage of T. sechellensis in some of the islands were invoked to explain this result, and morphological variation also seemed to reflect this pattern of reticulation. A Mio‐Pliocene divergence between both species is suggested. Multilocus molecular data were used to uncover biogeographic patterns within the archipelago, which reflected shared patterns with other co‐distributed lizard taxa; specifically a north–south marked structure, a close relationship between populations from Fregate and the southern islands, and a detectable isolation within the southern group, between Mahé, and Silhouette and North Islands. Gene flow from these latter islands towards the northern group was also suggested. These results add to the growing body of evidence of the influence of geographic distance and sea‐level oscillations in shaping the genetic structure of Seychellois taxa and of the existence of common biogeographic patterns across the archipelago.     

Phylogeography of the Black-tailed Godwit Limosa limosa: substructuring revealed by mtDNA control region sequences

Black-tailed (Limosa limosa) and Hudsonian Godwits (L. haemastica) are sometimes described as a superspecies. The Black-tailed Godwit is further split into three subspecies on the basis of morphological differences (L. l. limosa, L. l. islandica and L. l. melanuroides). We studied variation in partial mtDNA control region sequences among Black-tailed and Hudsonian Godwits which showed 5% divergence. Black-tailed and Hudsonian Godwits were thus clearly differentiated and the separate species status for the two taxa is validated. All three subspecies described for the Black-tailed Godwit had unique haplotypes but the genetic distances were small (0.3–0.6%). Despite small genetic differences we could not detect any substantial gene flow between any of the subspecies as haplotypes were private to each subspecies. Thus, genetic variation within Black-tailed Godwits showed a clear geographic structure. We found a high proportion of rare private haplotypes in three fringe populations of the nominate subspecies of the Black-tailed Godwit (L. l. limosa) where godwits breed in low numbers, but no genetic variation at all in a sample from the Netherlands where godwits are abundant. This suggests that Dutch Godwits may have been affected by a founder effect.     

A multivariate analysis of geographic variation in morphology inPersoonia mollis (Proteaceae)

This study highlights the utility and limitations of multivariate procedures for the analysis and taxonomic interpretation of geographic variation within a complex species. Morphological data from herbarium specimens ofPersoonia mollis (Proteceae) were analyzed by two ordination procedures, multidimensional scaling (MDS) and canonical variate analysis (CVA). Support for the earlier recognition of nine infraspecific taxa was found. However, plots of canonical variate scores against latitute or longitude across parapatric subspecies boundaries revealed unexpected phenetic homogeneity within subspecies with sharp transitions between subspecies. The geographic variation in morphology withinP. mollis can be best described as a mosaic of nine recognisably distinct and internally homogeneous allopatric or parapatric taxa, where neighbouring taxa share surprisingly narrow zones of morphological transition at ecotones. The utility of these procedures was highlighted by the recovery of unexpected patterns of variation. However, questions of rank remain unresolved by phenetic analysis alone.     

Phenetic studies in Nuphar Sm. (Nymphaeaceae):variation in sect. Nuphar

Previous taxonomic assessments of the hydrophytic genus Nuphar Sm. (Nymphaeaceae) have inadequately considered the range of morphological variation existing in the genus. A comprehensive statistical analysis of morphological features among the recognized taxa of Nuphar is presented here, first using 10 fruit characters to delimit the separation of two infrageneric groups of taxa, and secondly using 31 characters to quantify the morphological variation found within sect. Nuphar. Results from combined phenetic analyses provided empirical support for recognition of two infrageneric groups of species in the genus (largely by fruit, stigmatic disk margin, neck, and stigmatic disk size) and for separation of taxa within sect. Nuphar (by leaf size, and fruit disk and neck size). Statistical analyses demonstrate that extensive variation in ranges of character states exists among sect. Nuphar taxa. However, a suite of qualitative and quantitative characters can be used to recognize five species and three subspecies within this predominantly Old World assemblage.     

Molecular phylogeny of JapaneseAmanita species based on nucleotide sequences of the internal transcribed spacer region of nuclear ribosomal DNA

The molecular phylogeny of 36 specimens of JapaneseAmanita species was studied based on nucleotide sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. The phylogenetic tree obtained supported the traditional classification systems of Bas (1969) and Singer (1986), which are based on morphological characters, in the division of the genusAmanita is divided into subgeneraAmanita andLepidella by the amyloidity of basidiospores. However, at section-level, we suggest that subgenusAmanita should be divided into three sections (Amanita, Vaginatae, andCaesareae). Our results also showed the necessity to modify the taxonomic treatments at section-level in the subgenusLepidella. It appears that the establishment ofA. muscaria andA. pantherina from a common ancestral species might be a very recent event, or these might be lower taxa of same species. As for three subspecies ofA. hemibapha and three varieties ofA. vaginata, it is necessary to grade up their taxonomical ranks from subspecies/variety to species. A new combination,A. javanica, is proposed forA. hemibapha subsp.javanica.     

Molecular Systematics of the Malagasy Babblers (Passeriformes: Timaliidae) and Warblers (Passeriformes: Sylviidae), Based on Cytochrome b and 16S rRNA Sequences

The phylogenetic relationships of the Timaliidae (babblers) and Sylviidae (warblers) have long challenged ornithologists. We focus here on three Malagasy genera currently assigned to the Timaliidae, Mystacornis, Oxylabes, and Neomixis, and on their relationships with other babblers and warblers using the sequences of two mitochondrial genes (cytochrome b and 16S rRNA). Maximum parsimony analyses show that the Malagasy “babblers” are not related to any of the other African and Asian babblers. The genus Mystacornis is neither a babbler nor a warbler. The other Malagasy “babblers” are members of warbler groups (the monophyly of the Sylviidae is not demonstrated). Oxylabes madagascariensis and Hartertula flavoviridis (we recognize Hartertula as a genus for the species flavoviridis, previously Neomixis flavoviridis) constitute, with two presumed sylviine taxa, Thamnornis chloropetoides and Cryptosylvicola randrianasoloi, a warbler radiation endemic to the island of Madagascar. The other Neomixis species (tenella, striatigula, and viridis) belong to another warbler group comprising cisticoline taxa. These results show that the Timaliidae did not disperse to Madagascar. Rather, the island has been colonized, independently, by at least two clades of warblers, probably originating from Africa, where the Sylviidae radiation has been the most extensive.