Integrated analyses of chromosome, molecular and morphological variability in the Andean mice Eligmodontia puerulus and E. moreni (Rodentia, Cricetidae, Sigmodontinae)

Patterns of evolution and systematics of sigmodontine rodents are matters of continuous revision and debate. The silky mouse, Eligmodontia, is a phyllotine rodent adapted to arid environments. Chromosomal and molecular data have identified six species in this genus. Among these E. puerulus and E. moreni are sister taxa from the high Andean and lowland deserts, respectively, with large chromosomal differences and intermediate levels of molecular divergence. The purpose of our study was to quantify the degree of variability (morphological, cytogenetic, and molecular) and to analyze its evolutionary implications within, and between, these sister species in the Monte and Puna biomes of Argentina. Our results show a high variability at the chromosomal and molecular level, but low morphological differentiation among populations of E. puerulus. Diploid numbers vary from 31 to 37 due to a complex Robertsonian system, whereas cytochrome-b distances range from 0.15% to 5.75%. On the other hand, E. moreni shows high morphological differentiation between populations, but low intraspecific differentiation at the molecular (from 0.73% to 1.4%) and chromosomal level (2n = 52). Comparison of E. puerulus with E. moreni reveals high morphological and chromosomal distinction between them, but absence of molecular differentiation. Our results suggest that: (1) the high genetic variability of E. puerulus could be associated to its geographic distribution in the complex topography of the high Andean Puna; (2) the high morphological differentiation between E. moreni and E. puerulus could be the result of natural selection; and (3) molecular polyphyly between E. puerulus and E. moreni could be due to introgression or a recent split of these taxa. Finally, our results emphasize the need to integrate different datasets in the analysis of species variability and diversification, as tools for understanding their evolutionary histories.     

Genetic and morphological analyses reveal a complex biogeographic pattern in the endemic barbel populations of the southern Italian peninsula

The Italian peninsula is a biodiversity hotspot, with its freshwater fish fauna characterized by high levels of local endemism. Two endemic fluvio‐lacustrine fishes of the genus Barbus (barbel, family Cyprinidae) have allopatric distributions in the Tyrrhenian and Adriatic basins of Italy. Barbus plebejus inhabits the mid‐ to northern Adriatic basins, while B. tyberinus is widespread in all central‐northern basins draining into the Tyrrhenian Sea. For basins in Southern Italy draining into the southern parts of these seas, there remains a knowledge gap on their barbel populations due to no previous genetic and morphological studies, despite their apparent biogeographic isolation. Correspondingly, this study quantified the presence and distribution of barbels in the Adriatic and Tyrrhenian basins of Southern Italy through genetic and morphological analyses of 197 fish sampled across eight populations. Testing of how local isolation has influenced the evolution and persistence of these populations was completed by examining sequence variation at two mitochondrial loci (cytochrome b and D‐loop) and performing geometric morphometric analyses of body shape, plus measuring 11 morphometric and meristic characters. Phylogenetic and morphological analyses revealed the presence of two genetically distinct lineages that differed significantly from adjacent B. tyberinus and B. plebejus populations. These two new taxa, here described as SI1 and SI2 Barbus lineages, are highly structured and reflect a complex mosaic biogeographic pattern that is strongly associated with the underlying hydrographical scenarios of the basins. The geographic isolation of these basins thus has high evolutionary importance that has to be considered for maintaining endemism.     

Polyploid evolution and biogeography in Chelone (Scrophulariaceae): morphological and isozyme evidence

Chelone is a genus of perennial herbs comprising three diploid species (C. cuthbertii, C. glabra, and C. lyonii) and a fourth species (C. obliqua) that occurs as tetraploid and hexaploid races. To assess patterns of isozyme and morphological variation, and to test hypotheses of hybridization and allopolyploidy, we analyzed variation among 16 isozyme loci from 61 populations and 16 morphological characters from 33 populations representing all taxa and ploidy levels. Based on morphological analyses using clustering (unweighted pair group method using an arithmetic average) and ordination (principal components analysis and canonical variance analysis) methods, we recognize three diploid species without infraspecific taxa. Polyploids in the C. obliqua complex were most similar morphologically to diploid populations of C. glabra and C. lyonii. Patterns of isozyme variation among polyploids, which included fixed heterozygosity and recombinant profiles of alleles present in diploids, suggested polytopic origins of tetraploids and hexaploids. Our data indicate independent origins of polyploids in or near the southern Blue Ridge, Interior Highlands and Plains, and Atlantic Coastal Plain regions from progenitors most similar to C. glabra and C. lyonii. Extant tetraploids were not implicated in evolution of hexaploids, and plants similar to C. cuthbertii appeared unlikely as diploid progenitors for polyploids. We propose multiple differentiation and hybridization/polyploidization cycles in different geographic regions to explain the pattern of allopatry and inferred polytopic origins among polyploids.     

Genetic Differentiation Among Annual Fishes of the Genus Cynolebias (Cyprinodontiformes, Rivuluidae) in a Biosphere Reserve Site from Uruguay

The annual killifishes of the genus Cynolebias include one well supported clade composed of three taxa: Cynolebias adloffi, from Porto Alegre (southern Brazil), C. cf. adloffi and Cynolebias viarius, parapatrically distributed in southeastern Uruguay. Cynolebias cf. adloffi has always been considered an intergradation complex of populations between C. adloffi and C. viarius showing high levels of morphological variability and its systematic affinities have been historically discussed. This study clarified the patterns of genetic differentiation of C. cf. adloffi by means of RAPD markers to define its taxonomic status as a possible separated taxon. Multidimensional scaling analysis reveals that C. cf. adloffi is composed of two highly structured genetic groups (northern and southern) with discrete geographical distributions. The estimate of gene diversity for RAPD markers could suggest a scenario in which the rainy season flood pool populations across large distances could make intrapopulation variation high but interpopulation variation low. Phylogenetic analyses (UPGMA and parsimony) show different and unstable patterns of relationships among the taxa analyzed. In the UPGMA tree, C. viarius and C. cf. adloffi appear as sister taxa, while parsimony analysis does not support a unique hypothesis of relationship. Our analyses are in agreement with the hypothesis that C. cf. adloffi may well be a case of ancient hybrid ancestry followed by its divergence associated with Pleistocene and post-Pleistocene environmental changes in this region.     

Taxonomic differentiation of Cupressus sempervirens and C. atlantica based on morphometric evidence

We investigated the interspecific morphological relationships of two related Mediterranean Cupressus taxa and the possible geographic differentiation of Eastern Mediterranean C. sempervirens using morphometric analysis. A total of 446 individuals, representing eight C. atlantica (Morocco) and 10 C. sempervirens (Greece, Turkey and Lebanon) populations were examined. The 17 morphological characters of cone, seed and shoot with leaves were measured and analysed statistically. Significant morphological differences between C. atlantica and C. sempervirens were detected for the majority of analysed characters. For C. sempervirens, significant differences were also detected and these were related to its three regions of origin, the Greek Islands, Taurus Mts and Lebanon Mts. Multivariate analyses did not indicate a clear geographic pattern of differentiation in C. sempervirens populations. A relatively low level of phenotypic differentiation was detected between populations originating from the Greek Islands and Taurus Mts. We also observed a moderate multivariate differentiation between the Lebanese and remaining C. sempervirens populations. Phenotypic differentiation detected in the Asian part of the C. sempervirens range reflects patterns of differentiation similar to those described for other taxa occurring in the Taurus and Lebanon Mts.     

Identifying hybridizing taxa within the <Emphasis Type="Italic">Daphnia longispina</Emphasis> species complex: a comparison of genetic methods and phenotypic approaches

Daphnia galeata Sars, D. longispina O. F. Müller and D. cucullata Sars (Crustacea: Cladocera) are closely related species which often produce interspecific hybrids in natural populations. Several marker systems are available for taxon determination in this hybridizing complex, but their performance and reliability has not been systematically assessed. We compared results from identifications by three molecular methods. More than 1,200 individuals from 10 localities in the Czech Republic were identified as parental species or hybrids by allozyme electrophoresis and the analysis of the restriction fragment length polymorphism of the internal transcribed spacer (ITS-RFLP); over 440 of them were additionally analyzed and identified by 12 microsatellite loci. Identification by microsatellite markers corresponded well with allozyme analyses. However, consistent discrepancies between ITS-RFLP and other markers were observed in two out of 10 studied localities. Although some marker discrepancies may have been caused by occasional recent introgression, consistent deviations between ITS-RFLP and other markers suggest a long-term maintenance of introgressed alleles. These results warn against its use as a sole identification method in field studies. Additionally, we quantitatively evaluated the discriminatory power of geometric morphometric (elliptic Fourier) analysis of body shapes based on photos of over 1,300 individuals pre-classified by allozyme markers. Furthermore, a randomly selected subset of 240 individuals was independently determined from photos by several experts. Despite a tendency for morphological divergence among parental Daphnia species, some taxa (especially D. galeata, D. longispina, and their hybrids) substantially overlapped in their body shapes. This was reflected in different determination success for particular species and hybrids in discriminant analysis based on shape data as well as from photographs.     

From Asia to Europe across Siberia: phylogeography of the Siberian spined loach (Teleostei,Cobitidae)

We assessed the genetic variability of the Siberian spined loach Cobitis melanoleuca across its unusually broad distribution that encompasses areas greatly affected by Pleistocene glaciations. Due to extensive morphological variation among their populations, the taxonomic status of C. melanoleuca is complicated. It is unclear whether C. melanoleuca represents a single taxonomic unit or contains several species or subspecies. Our analyses showed low genetic variability in all populations without any phylogenetic structure. The absence of molecular distinctiveness indicates the conspecificity of all C. melanoleuca populations. Only a few common haplotypes shared by East Asian, Siberian and European populations were found at high frequency in the nuclear genes analysed. At the mitochondrial level, Siberian populations shared haplotypes with populations located at both extremes of the species’ range suggesting central populations as a source of current mitochondrial variability. Unimodal mismatch distributions and significant values from neutrality tests support a recent expansion of C. melanoleuca. Our time estimates suggest a postglacial colonisation of European waters around 1.0 MYA, indicating that C. melanoleuca may represent the last cobitid immigrant in Europe that used the northern route across Siberia to expand its range.     

Genetic analysis reveals multiple cryptic invasive species of the hydrozoan genus Cordylophora

Understanding the patterns and dynamics of biological invasions is a crucial prerequisite to predicting and mitigating their potential ecological and economic impacts. Unfortunately, in many cases such understanding is limited not only by ignorance of invasion history, but also by uncertainty surrounding the ecology, physiology, and even systematics of the invasive taxa themselves. The invasive, colonial euryhaline hydroid Cordylophora has invaded multiple regions outside of its native Ponto-Caspian range. However, extensive morphological and ecological plasticity has prevented consensus on both species-level classification within the genus and the environmental conditions conducive to establishment. The goal of this research was to explore the invasive history and species composition of the genus Cordylophora through molecular analyses. We addressed both issues using DNA sequence data from two mitochondrial loci [the small subunit 16S rRNA and cytochrome c oxidase subunit I (COI)] and one nuclear locus (28S large nuclear rRNA), generated from 27 invasive Cordylophora populations collected throughout the global range of the taxon. Phylogenetic analysis and comparisons of genetic distances between populations suggest the presence of multiple cryptic species within the genus. This conclusion is further supported by the observation of significantly different habitat preferences between invasive lineages. Geographic distribution of lineages is consistent with the introduction of multiple lineages to some non-native regions, indicating that repeated introductions may contribute to the current global distribution of Cordylophora. Applying molecular and morphological analyses to additional populations of Cordylophora is likely to assist in clarifying the taxonomy of this genus and in providing a better understanding of the invasive history of this hydroid.     

Systematics and evolution of the Jacchus group of marmosets (Platyrrhini)

Interspecific differentiation and geographic variation patterns in 39 skull traits of eastern Brazilian marmosets are analyzed. Eastern Callithrix taxa are distinct morphologically, and no evidence of intergradation among taxa is observed. Instead, there are sharp, stepped morphological boundaries among taxa, consistent with species‐level distinction. The morphological similarity cluster diagram obtained from Mahalanobis distances is different from available molecular trees, and the general picture emerging is that the eastern Callithrix taxa should be considered as good species arising recently in South American history. In particular, C. kuhlii is morphologically distinct from other marmoset taxa, including C. geoffroyi and C. penicillata, which were previously hypothesized to be the parental populations that formed C. kuhlii by hybridization. Furthermore, C. kuhlii populations from southeastern Bahia do not overlap morphologically with any C. penicillata population, including the upper São Francisco River populations that display skin colors and pelage patterns to some extent similar to true Kuhli's marmosets. There is a negative, though insignificant, correlation between the morphological distance matrix and a Mahalanobis distance matrix estimated from nine climatic variables, a pattern opposite to that expected under a parapatric speciation model. This result, together with the lack of clinal variation in skull traits, suggests that an allopatric model of speciation might best explain eastern marmoset diversification. Am J Phys Anthropol 2003. © 2002 Wiley‐Liss, Inc.     

DNA barcoding the genus Chara: molecular evidence recovers fewer taxa than the classical morphological approach

Charophytes (Charales) are benthic algae with a complex morphology. They are vulnerable to ecosystem changes, such as eutrophication, and are red‐listed in many countries. Accurate identification of Chara species is critical for understanding their diversity and for documenting changes in species distribution. Species delineation is, however, complicated, because of high phenotypic plasticity. We used barcodes of the ITS2, matK and rbcL regions to test if the distribution of barcode haplotypes among individuals is consistent with species boundaries as they are currently understood. The study included freshly collected and herbarium material of 91 specimens from 10 European countries, Canada and Argentina. Results showed that herbarium specimens are useful as a source of material for genetic analyses for aquatic plants like Chara. rbcL and matK had highest sequence recoverability, but rbcL had a somewhat lower discriminatory power than ITS2 and matK. The tree resulting from the concatenated data matrix grouped the samples into six main groups contrary to a traditional morphological approach that consisted of 14 different taxa. A large unresolved group consisted of C. intermedia, C. hispida, C. horrida, C. baltica, C. polyacantha, C. rudis, C. aculeolata, and C. corfuensis. A second unresolved group consisted of C. virgata and C. strigosa. The taxa within each of the unresolved groups shared identical barcode sequences on the 977 positions of the concatenated data matrix. The morphological differences of taxa within both unresolved groups include the number and length of spine cells, stipulodes, and bract cells. We suggest that these morphological traits have less taxonomic relevance than hitherto assumed.     

Genetic structure of the rare and endangered Hieracium wiesbaurianum group (Asteraceae) in Bavaria

The rare and endangered Hieracium wiesbaurianum species group shows a scattered relictual distribution in Bavaria. Recently, a couple of populations were discovered which clearly differ from all other populations. If these must be considered as taxonomically independent units, they would be of crucial conservation interest, because of the sole responsibility that Bavaria has for these worldwide endemics. We therefore analysed the genetic structure of H. wiesbaurianum in a comparative approach. Our analysis comprised 37 populations of 13 taxa of H. wiesbaurianum, H. bifidum and H. laevigatum, including three potentially new taxa. We applied amplified fragment length polymorphism (AFLP) analysis and observed only limited genetic variation within populations and taxa. Nevertheless, each studied individual exhibited a unique genotype. An analysis of molecular variance revealed high levels of genetic variation between taxa, but populations were genetically less different. The clear genetic differentiation between the studied taxa was supported by neighbor‐joining cluster analyses and principal coordinate analyses in which every individual was clearly assigned to its respective taxon. The three potentially new taxa were genetically as well differentiated as the other taxa included in our study. This supports the assumption that they should be treated as taxonomically independent units of high conservation interest. Therefore, the genetic analysis confirmed the morphologically based classification of the studied Hieracium taxa. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 112–123.     

Characterisation and inheritance of nuclear microsatellite loci for use in population studies of the allotetraploid <Emphasis Type="Italic">Salix alba</Emphasis>–<Emphasis Type="Italic">Salix fragilis</Emphasis> complex

We present nine polymorphic di- and tri-nucleotide repeat nuclear microsatellite markers selected specifically for their use in high throughput studies concerning the dioecious allotetraploid Salix alba–Salix fragilis willow complex. These taxa and their hybrids are difficult to discriminate using morphological characters. Thus, multiplex reactions were developed for these microsatellite loci and their effectiveness to distinguish individuals, especially hybrids, and their inheritance patterns in controlled crosses were determined. All loci displayed disomic–monogenic inheritance which allowed for the genotype data to be analysed as for a diploid organism. The nine loci produced a total of 67 alleles (mean, 7.4 alleles per locus; range, 3–11 alleles) in a reference panel of 57 individuals from two germplasm collections and natural populations. Gene diversity values (as measured by the expected heterozygosity) ranged from 0.000–0.820. A total of 53 distinct multilocus genotypes were observed, and ordination analysis revealed three separate clusters corresponding to S. alba, S. fragilis and hybrids. The microsatellite loci described here will be used in population genetic studies to investigate genetic variation, gene flow, levels of hybridisation and the extent of introgression in natural populations of the S. alba–S. fragilis complex. They are also useful for clonal identification, conservation and sustainable management of germplasm collections, genetic mapping and the selection of individuals and/or certification of controlled crosses for breeding programmes.     

Taxonomy,skull diversity and evolution in a species complex of Myotis (Chiroptera: Vespertilionidae): a geometric morphometric appraisal

Phylogenetic relationships between taxa are not necessarily reflected by morphological data due to widespread homoplasy and convergence. However, combining morphological and molecular data provides insights into the evolution of biological forms and into the potential factors involved. Here we focus on a complex of three taxa of bats with unclear taxonomic affinities: Myotis myotis, Myotis blythii and Myotis punicus. Traditional morphometric methods failed to separate them, whereas recent molecular‐based studies suggested that they constitute separate biological species. In the present study, landmark‐based geometric morphometrics methods have been used to analyse the skull variability of 218 specimens belonging to this species complex. Patterns of size and shape delimitate three morphological groups that are congruent with the proposed taxonomic assignments, and therefore support species rank for all three major groups. These morphometrics results, however, suggest that M. myotis and M. punicus share shape characteristics in the rostrum and in the posterior part of the skull that differ from M. blythii. Because previous molecular phylogenetic analyses suggested that M. myotis and M. blythii are sister species, we interpret the similitude in skull morphology between M. myotis and M. punicus as a convergence probably related to their similar feeding habits. Within the taxon M. punicus, the skull of Corsican and Sardinian populations significantly differs from that of Maghrebian ones, suggesting the existence of further cryptic taxonomic diversity. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 529–538.     

Integrative species delimitation reveals cryptic diversity in the southern Appalachian Antrodiaetus unicolor (Araneae: Antrodiaetidae) species complex

Although species delimitation can be highly contentious, the development of reliable methods to accurately ascertain species boundaries is an imperative step in cataloguing and describing Earth's quickly disappearing biodiversity. Spider species delimitation remains largely based on morphological characters; however, many mygalomorph spider populations are morphologically indistinguishable from each other yet have considerable molecular divergence. The focus of our study, the Antrodiaetus unicolor species complex containing two sympatric species, exhibits this pattern of relative morphological stasis with considerable genetic divergence across its distribution. A past study using two molecular markers, COI and 28S, revealed that A. unicolor is paraphyletic with respect to A. microunicolor. To better investigate species boundaries in the complex, we implement the cohesion species concept and use multiple lines of evidence for testing genetic exchangeability and ecological interchangeability. Our integrative approach includes extensively sampling homologous loci across the genome using a RADseq approach (3RAD), assessing population structure across their geographic range using multiple genetic clustering analyses that include structure , principal components analysis and a recently developed unsupervised machine learning approach (Variational Autoencoder). We evaluate ecological similarity by using large‐scale ecological data for niche‐based distribution modelling. Based on our analyses, we conclude that this complex has at least one additional species as well as confirm species delimitations based on previous less comprehensive approaches. Our study demonstrates the efficacy of genomic‐scale data for recognizing cryptic species, suggesting that species delimitation with one data type, whether one mitochondrial gene or morphology, may underestimate true species diversity in morphologically homogenous taxa with low vagility.     

DNA barcoding to analyse taxonomically complex groups in plants: the case of Thymus (Lamiaceae)

We evaluated the utility of the core barcode regions (matK and rbcL) and the plastid intergenic spacer trnH‐psbA to distinguish between Thymus spp. This is a taxonomically complex group that has been investigated so far mainly using morphological approaches. Thirty‐six samples representing nine different morphospecies were collected and used for molecular analysis. The three markers showed clear amplification and sequencing. However, the genetic variation and the resulting haplotype networks showed that only Thymus capitatus forms a well‐defined ‘barcoding gap’ compared with the other taxa. The identification problems observed in the other Thymus spp. may be related to reduced gene flow among populations, resulting in high intraspecific and low interspecific genetic variation. This situation does not permit the definition of species‐specific barcodes. A second hypothesis suggests that morphological traits used for the delimitation of Thymus spp. do not reflect real biological and molecular species boundaries. If this is the case, the taxonomy of Thymus should be revised through extensive sampling and analyses with different tools (i.e. molecular variability, morphology, geographical distribution, etc.) to define the natural units at the species level. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013 , 171 , 687–699.     

A taxonomic revision of western Eupholidoptera bush crickets (Orthoptera: Tettigoniidae): testing the discrimination power of DNA barcode

The genus Eupholidoptera includes 46 Mediterranean species distributed from Turkey to Greece, Italy and southern France. In the eastern part of its range, Eupholidoptera has been considered to consist of several distinct species, while in the Balkans and Italian peninsula only E. chabrieri has been recognized. However, the status of some Italian populations, confined to particular geographic areas, remains uncertain. To investigate the delimitation of the Italian taxa of Eupholidoptera, we performed both morphological and molecular analyses. Morphological analysis was carried out by considering diagnostic characters usually used to distinguish different taxa, such as the shape of titillators in males and the subgenital plate in females. Molecular analysis was performed by sequencing three mitochondrial genes: 12S rRNA, 16S rRNA, partially sequenced and the entire gene of cox1. Molecular markers were used to infer phylogenetic relationships among the Italian Eupholidoptera species and to reconstruct the historical processes that shaped their current geographic distribution. Results from both morphological and molecular analyses were used to revise the taxonomic arrangement of species. On the whole we were able to distinguish nine lineages of Italian Eupholidoptera, of which E. tyrrhenica sp.n. from Corsica is described as a new species. This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:EBD181A0-5263-4880-AC80-66F624506E3A .     

Contemporary factors influencing genetic diversity in the Alaska humpback whitefish Coregonus clupeaformis complex

Thirteen microsatellite loci were used to address three hypotheses regarding genetic diversity in the humpback whitefish Coregonus clupeaformis complex in Alaska. The test results provided further insight into the factors influencing C. clupeaformis complex population structure and level of genetic variation. First, the microsatellite data did not provide evidence of two spatially distinct Beringian and Eurasian refugial groups as revealed in previous phylogeographic analyses of mitochondrial DNA variation. Rather, the population structure inferred from the microsatellite variation appears to reveal the influence of factors on a more recent time scale, including gene flow among the refugial groups and isolation of some anadromous and freshwater resident populations. Second, anadromous C. clupeaformis complex collections exhibited higher intra‐population genetic diversity than freshwater resident collections. This outcome is consistent with previous meta analyses suggesting that freshwater resident populations probably have smaller historical effective population sizes and less conspecific gene flow because the habitat tends to be smaller and supports fewer and smaller populations. Finally, the analysis of contemporary immigration rates was consistent with, but did not provide statistical support for, the hypothesis that gene flow among anadromous C. clupeaformis complex populations along coastal Alaska is influenced by the Alaska Coastal Current. Further studies are needed to evaluate gene flow among coastal Alaska C. clupeaformis complex populations.     

Cardamine apennina: a new endemic diploid species of the C. pratensis group (Brassicaceae) from Italy

Italian representatives of the Cardamine pratensis group were investigated using morphometric, karyological, and molecular (AFLP-fingerprinting) analyses. Focus was on resolving the taxonomic and evolutionary position of C. granulosa from Piemonte (NW Italy) and of populations from central Italy, previously included in C. granulosa or C. pratensis s. str. Results show that C. granulosa is a morphologically and genetically well defined diploid species, but is extremely rare and restricted to a small area in Piemonte. Central Italian populations differ from both C. granulosa and C. pratensis s. str., and, although the morphological differentiation is not strongly pronounced, they show marked molecular divergence, suggesting recognition as a new species, Cardamine apennina. Because of their restricted distribution and diploid chromosomal level, C. apennina and C. granulosa may represent basal and quite isolated lineages, whereas reticulation on diploid and polyploid level has affected remaining lineages of the C. pratensis group. Both Italian endemics are endangered due to rarity and habitat loss. A key for identification of the Italian taxa of the C. pratensis group is also provided.