DNA SEQUENCE DATA DEMONSTRATE THE POLYPHYLY OF THE GENUS CYSTOSEIRA AND OTHER SARGASSACEAE GENERA (PHAEOPHYCEAE)1

Phylogenetic relationships in the Sargassaceae were explored using three DNA markers, and the monophyly of its genera was challenged. Nineteen out of 24 currently recognized genera were sampled, representing 63 species. The variable mt23S‐tRNA Val intergenic spacer could only be aligned within genera and could not be used to infer intergeneric relationships. The partial mt23S was also useful to delineate genera and was alignable at the family level but provided few informative characters. Analysis of mt23S DNA sequences together with chloroplast‐encoded psbA sequences resulted in a better resolved phylogeny. Hormophysa was the first genus to branch off within the Sargassaceae, followed by Myriodesma; then the three genera Caulocystis, Carpoglossum, and Scaberia in unresolved order; and then Acrocarpia. The other taxa studied here were divided over three major clades, but there was no branch support for the monophyly of two of these. The genera Bifurcaria, Cystoseira, Halidrys, and Sargassum appeared polyphyletic. The following taxonomic changes are proposed: a new genus Brassicophycus for Bifurcaria brassicaeformis (Kützing) E. S. Barton; reinstatement of the genus Sargassopsis for Sargassum decurrens (R. Brown ex Turner) C. Agardh; reinstatement of the genus Sirophysalis for Indo‐Pacific Cystoseira trinodis (Forsskål) C. Agardh; reinstatement of the genus Polycladia for the western Indian Ocean species Cystoseira indica (Thivy et Doshi) Mairh, Cystoseira myrica (S. G. Gmelin) C. Agardh, and Acystis heinii Schiffner; and reinstatement of the genus Stephanocystis for the North Pacific Cystoseira species and Halidrys dioica N. L. Gardner. The European Cystoseira species should be split into three genera, but no name changes are proposed yet, because diagnostic characters were found only for the clade including the type species. Some evolutionary trends could be discerned from the mt23S + psbA phylogeny.     

High intra-individual sequence variation in the nuclear rDNA LSU-5S intergenic spacer in the Sargassaceae (Fucales, Phaeophyceae)

Of four species of Sargassaceae, representing the genera Carpophyllum, Cystoseira, Landsburgia, and Sargassum, the intergenic spacer of the ribosomal cistron was amplified using a forward primer annealing at the 3′-end of the large subunit (LSU) of the ribosomal cistron and a reverse primer annealing at the 5S rRNA gene. The PCR products were cloned and the DNA sequences of multiple clones were determined. Almost each clone showed a unique DNA sequence. Intra-individual variation of this LSU-5S intergenic spacer was extremely high and was characterized by great length variation and a high number of short tandem repeats. Sequences were unalignable and therefore it was concluded that the LSU-5S intergenic spacer is unsuitable for phylogenetic and phylogeographic studies of sargassacean taxa.     

Molecular Relationships of the New Guinean Bandicoot Genera Microperoryctes and Echymipera (Marsupialia: Peramelina)

The complete 12S rRNA gene was sequenced for multiple exemplars of the New Guinean bandicoot genera Microperoryctes and Echymipera representing many of the currently recognized subspecies. These two genera are resolved as monophyletic sister taxa but there was no genetic support for the family Peroryctidae proposed by Groves and Flannery (1990). Within Microperoryctes, M. papuensis was sister to M. longicauda. Although there was only weak support for recognition of the subspecies M. l. magnus, our results demonstrate the need for further genetic and morphological studies of variation among populations of Microperoryctes. Haplotype relationships within Echymipera did not reflect current species boundaries. Although E. clara was the most divergent species, no clear separation could be made between E. rufescens and E. kalubu (E. kalubu samples from New Britain were consistently more closely allied with the E. rufescens exemplars collected from north and east of the Central Cordillera than with their congeners). We suggest the need for a major morphological reassessment of New Guinean bandicoot relationships.     

Phylogenetic and biogeographical relationships among some holarctic frog lung flukes (Digenea: Haematoloechidae).

A phylogenetic study of 8 North American and European species of frog lung flukes belonging to Haematoloechus was conducted using approximately 850 to 1,000 bases of the intemal transcribed spacer region (ITS 1 + 5.8S + ITS 2) and 1,250 bases of the large subunit (LSU) of the nuclear ribosomal DNA. Adequate phylogenetic resolution could not be obtained from 5.8S or ITS 2 data. Analysis of ITS 1 data produced 2 equally parsimonious trees that differed only in the position of Haematoloechus breviplexus relative to H. medioplexus and H. varioplexus. Single, identical trees were produced by analysis of both LSU sequence data and a data set comprised of all ITS and LSU data. All trees demonstrated 3 distinct evolutionary lineages within the Holarctic Haematoloechus examined. The results confirmed the taxonomic validity of H. abbreviatus and demonstrated that the presence or absence of extracecal uterine loops is not a character meaningful to the recognition of evolutionary lineages or differentiation of genera. Examination of ITS sequence data revealed almost no intraspecific variation within 5 species of Haematoloechus and demonstrated an approximately 150-base indel common to the North American H. longiplexus and the European H. asper. Two of 3 clades revealed by the phylogenetic analyses are comprised of both European and North American species, indicating that lineages of Haematoloechus arose before the breakup of Laurasia and radiated after Eurasia and North America split. Within each of 3 evolutionary lineages, members share similar patterns of arthropod host specificity distinct from patterns found in the other lineages. This suggests that second intermediate host specificity may be a trait that has been conserved through evolutionary time.     

Phylogenetic Reappraisal of the Genus Monomorphina (Euglenophyceae) Based on Molecular and Morphological Data

A morphological and molecular examination of the genus Monomorphina was conducted on 46 strains isolated mainly from Korea. The strains were divided into two types based on morphological data: Monomorphina aenigmatica and M. pyrum ‐ like species. Phylogenetic analysis based on a combined data set of nuclear SSU and LSU and plastid SSU and LSU rDNA showed that the strains could be divided into eight clades: Clade A of M. aenigmatica, Clade B of the isolates (M. pyropsis) from Michigan, USA, Clade C of M. pseudopyrum, Clade D of the isolates (M. pyroria) from Bremen, Germany, Clade E of M. soropyrum, Clade F of M. pyriformis, Clade G of M. parapyrum, and Clade H of M. pyrum. Six of these clades came from strains that would be considered M. pyrum sensu Kosmala et Zakry?, one of which could be recognized as a traditional species (M. pyrum) and five were designated as new species; each species had unique molecular signatures at nr SSU rDNA helix 17 and 17′ and spacer E23_14′‐E23_15. The species of Monomorphina had a wide range of genetic diversity with interspecies sequence similarity of 85.6%–97.1% and intraspecies similarity of 96.4%–99.9%. Our results suggested that genetic diversity found in the M. pyrum complex justifies the recognition of a minimum of eight species within this genus, based on specific molecular signatures and gene divergence of the nr SSU rDNA sequences.     

Species delimitation of three species within the Russula subgenus Compacta in Korea: R. eccentrica,R. nigricans,and R. subnigricans

Distinguishing individual Russula species can be very difficult due to extensive phenotypic plasticity and obscure morphological and anatomical discontinuities. In this study, we use the internal transcribed spacer (ITS) and 28S nuclear ribosomal large subunit (LSU) markers to identify and study the genetic diversity of species in the Russula subgenus Compacta in Korea. We focus on two morphologically similar species that are often misidentified for each other: R. nigricans and R. subnigricans. Based on molecular phylogenetic analyses, we identify three subgroups of R. nigricans, with two from Asia and one from Europe/North America. Surprisingly, we find Korean R. subnigricans are more closely related to R. eccentrica from North America than the type specimen of R. subnigricans from Japan. These molecular data, along with habitat data, reveal that Korean R. subnigricans had previously been misclassified and should now be recognized as R. eccentrica. Both ITS and LSU exhibit high interspecific and low intraspecific variation for R. eccentrica, R. nigricans, and R. subnigricans. These markers provide enough resolutional power to differentiate these species and uncover phylogeographic structure, and will be powerful tools for future ecological studies of Russula.     

Cryptic diversity within and amongst spring‐associated Stygobromus amphipods (Amphipoda: Crangonyctidae)

Multiple species of troglomorphic, spring‐associated Stygobromus amphipods, including the endangered, narrow‐range endemic Stygobromus pecki, occupy sites in the Edwards Plateau region of North America. Given the prevalence of cryptic diversity observed in disparate subterranean, animal taxa, we evaluated geographical genetic variation and tested whether Stygobromus contained undetected biodiversity. Nominal Stygobromus taxa were treated as hypotheses and tested with mitochondrial sequence cytochrome oxidase C subunit 1, nuclear sequence (internal transcribed spacer region 1), and AFLP data. Stygobromus pecki population structure and diversity was characterized and compared with congeners. For several Stygobromus species, the nominal taxonomy conflicted with molecular genetic data and there was strong evidence of significant cryptic diversity. Whereas S. pecki genetic diversity was similar to that of congeners, mitochondrial data identified two significantly diverged but sympatric clades. AFLP data for S. pecki indicated relatively recent and ongoing gene flow in the nuclear genome. These data for S. pecki suggest either a substantial history of isolation followed by current sympatry and ongoing admixture, or a protracted period of extremely large effective population size. This study demonstrates that Edwards Plateau Stygobromus are a complex, genetically diverse group with substantially more diversity than currently recognized. © 2013 The Linnean Society of London     

Molecular phylogenetics of tribe Synandreae, a North American lineage of lamioid mints (Lamiaceae)

The five mint genera Brazoria, Macbridea, Physostegia, Synandra and Warnockia (Lamioideae: Lamiaceae) are all North American endemics. Together with the monotypic European genus Melittis and the Asian genus Chelonopsis, these taxa have been classified as subtribe Melittidinae. Previous morphological studies have failed to uncover synapomorphic characters for this group. We sequenced the plastid trnL‐trnF region and trnS‐trnG spacer and the nuclear ribosomal 5S non‐transcribed spacer (5S‐NTS) to assess phylogenetic relationships within Melittidinae. Standard parsimony and direct optimization (POY) analyses show Melittis, the type genus of the subtribe, as sister to Stachys. Thus, the monophyly of subtribe Melittidinae is not supported either by molecular or morphological data. However, the North American endemics form a monophyletic group that can be recognized as the recircumscribed tribe Synandreae. The molecular relationships among these genera are corroborated by both morphological and cytological data. The expected close relationship between the south‐central endemics Warnockia and Brazoria and their sister relationship to the widespread genus Physostegia is confirmed. Nevertheless, most of the North American endemics are restricted to the south‐east of the continent. Dispersal westwards and northwards is correlated with an increase in chromosome numbers. No specific Eurasian origin (i.e., transatlantic or transpacific) can be determined, but Synandreae are clearly distinct from the large Stachys clade, and therefore represent a separate migration into North America. © The Willi Hennig Society 2007.     

Genetic patterns in the Lathyrus pannonicus complex (Fabaceae) reflect ecological differentiation rather than biogeography and traditional subspecific division

The endangered European relict species complex Lathyrus pannonicus shows distinct morphological variation, reflected by the number of subspecies recognized, and complicated patterns of genetic variation. The traditionally recognized subspecies appear to possess different ecological preferences and disjunct distributions, particularly in the western range of the species. In this study, L. pannonicus was investigated by the correlation of distance matrices based on phytosociological, ecological, molecular and morphological data. Ecological characteristics of selected stands of L. pannonicus throughout Europe were assessed using ‘Ellenberg values’ of all the constituent taxa in phytosociological relevés. Genetic distances were calculated using recently developed methods to analyse high degrees of intra‐individual nuclear‐encoded internal transcribed spacer variability. We found that the remarkable genetic (and morphological) diversity in L. pannonicus could not be explained solely by the fragmentation of the distributional range. Instead, patterns of morphological and genetic differentiation were a reflection of the moisture regime in the sampled stands. Two major lineages could be identified: (1) a lineage adapted to dry conditions (Ellenberg indicator F‐value ≤ 3.5) and (2) a lineage preferring moist conditions (F‐value ≥ 4.5). Although both lineages occurred in close proximity in the Pannonian area, they appeared to be reproductively isolated in general. Further data are needed to determine whether these genetically and ecologically defined lineages, or ecospecies, within the L. pannonicus species complex can be formalized as (Linnaean) species or subspecies. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 165 , 402–421.     

The genus Pseudo-nitzschia (Bacillariophyceae) in a temperate estuary with description of two new species: Pseudo-nitzschia plurisecta sp. nov. and Pseudo-nitzschia abrensis sp. nov.

The genus Pseudo‐nitzschia contains potentially toxic species of problematic taxonomy, making it one of the most intensively studied diatom genera. The study of 35 clonal strains isolated from the Bilbao estuary, an area that experiences recurrent blooms of Pseudo‐nitzschia, revealed the presence of two new species, P. abrensis and P. plurisecta, differing from their congeners in both morphology and gene sequence. The morphological features were analyzed by LM and EM, whereas molecular analyses were based on the internal transcribed spacer (ITS) and large subunit (LSU) regions of the rDNA. P. plurisecta appears closely related to P. cuspidata/P. pseudodelicatissima in the phylogenetic tree, whereas P. abrensis forms a moderately supported clade with P. heimii/P. subpacifica and P. caciantha/P. circumpora. Comparison of the secondary structure of ITS2 regions reveals marked differences in the most highly conserved regions among related taxa. Morphologically, the new species differ from their closest congeners in the arrangement of the poroid sectors and the density of valve striae and fibulae. The two species share similar pigment composition, and belong to the group of Pseudo‐nitzschia species containing only chlorophyll c₂ and c₃.     

Cryptic Speciation in the Genus Cryptoglena (Euglenaceae) Revealed by Nuclear and Plastid SSU and LSU rRNA Gene

The photosynthetic euglenoid genus Cryptoglena is differentiated from other euglenoid genera by having a longitudinal sulcus, one chloroplast, two large trough‐shaped paramylon plates positioned between the chloroplast and pellicle, and lack of metaboly. The genus contains only two species. To understand genetic diversity and taxonomy of Cryptoglena species, we analyzed molecular and morphological data from 25 strains. A combined data set of nuclear SSU and LSU and plastid SSU and LSU rRNA genes was analyzed using Bayesian, maximum likelihood, maximum parsimony, and distance (neighbor joining) methods. Although morphological data of all strains showed no significant species‐specific pattern, molecular data segregated the taxa into five clades, two of which represented previously known species: C. skujae and C. pigra, and three of which were designated as the new species, C. soropigra, C. similis, and C. longisulca. Each species had unique molecular signatures that could be found in the plastid SSU rRNA Helix P23_1 and LSU rRNA H2 domain. The genetic similarity of intraspecies based on nr SSU rDNA ranged from 97.8% to 100% and interspecies ranged from 95.3% to 98.9%. Therefore, we propose three new species based on specific molecular signatures and gene divergence of the nr SSU rDNA sequences.     

COMPARISON OF THREE COMMON MOLECULAR TOOLS FOR DISTINGUISHING AMONG GEOGRAPHICALLY SEPARATED CLONES OF THE DIATOM SKELETONEMA MARINOI SARNO ET ZINGONE (BACILLARIOPHYCEAE)1

Skeletonema marinoi Sarno et Zingone is a planktonic marine diatom with a widespread geographic distribution. Different populations of this species may show distinct genetic signatures. We have evaluated the utility of three common molecular methods for distinguishing clones of S. marinoi from different geographic regions. Clonal cultures were isolated from the Canadian west coast, south west Portugal, and the east and west coasts of Sweden. All strains originated from resting stages in sediment. More than 90% of the individually isolated chains grew to densities suitable for DNA extraction. Genetic signatures of clones from each sample location were assessed by sequencing variable domains (D1–D3) of the nuclear large subunit (LSU) rRNA gene and internal transcriber spacer (ITS) (ITS‐1, 5.8S and ITS‐2) regions, and also by analysis of randomly amplified polymorphic DNA patterns. Analysis of molecular variance showed that strains from the four geographic areas were significantly separated by all three methods but that differences among European samples were best resolved by ITS 2 sequences.     

<Emphasis Type="Italic">Rhodocollybia</Emphasis> in neotropical montane forests

Traditional concepts of Collybia (Agaricales) have included a variety of morphologically disparate taxa. Reapplication of some characters used in circumscribing Collybia s.l. suggests realignment of well-known North American and European species into three genera, viz. Collybia s.s, Rhodocollybia, and Gymnopus. Rhodocollybia is recognized here at generic rank based on morphological characteristics and phylogenetic signaling. Seven new species epithets under Rhodocollybia, and a new combination under Gymnopus, are proposed. A key to species of Rhodocollybia found in neotropical montane forests is provided.     

我国北方岩溶泉域刚毛藻的系统发育及形态学研究

脆弱刚毛藻(Cladophora fracta)是一种大型丝状绿藻,生境分布广泛。然而,对于岩溶泉域分布的刚毛藻研究较少,它们的遗传多样性、生物地理亲缘性和生理特性都有待于深入研究。该研究对我国北方地区五个典型岩溶泉域的50个脆弱刚毛藻样本进行了形态学和分子系统学描述。主要研究目标:(1)对我国北方地区五个典型岩溶泉的刚毛藻生境进行描述;(2)根据形态学特征和分子序列对藻体进行鉴定;(3)探究生境对藻体生理特性的影响。结果表明:基于SSU和LSU序列的结果,发现所分析的50株刚毛藻个体为同一种,同时还发现了13个不同的核糖体基因型。基于SSU和LSU的系统发育树,刚毛藻属均未能形成单系分支,分布在三个不同的分支上。13个样本基因型在SSU和LSU树中的位置相似,与Cladophora vagabunda有很高的序列同源性,但是形态特征却差异很大。从显微结构结果来看,五个岩溶泉域采集到的刚毛藻在细胞直径上无显著差异,藻体的形态特征与脆弱刚毛藻相一致。但是,岩溶泉域采集的藻体细胞直径比文献报道中在湖泊和河流中采集的脆弱刚毛藻直径要大。另外,仅在两个地点(XA和ST)采集的标本中发现有假根状分枝。因此,基于形态学和分子序列的结果,将这五个泉域的刚毛藻鉴定为脆弱刚毛藻(Cladophora fracta)。     

CHROMOSOME NUMBERS OF HAWAIIAN FLOWERING PLANTS AND THE SIGNIFICANCE OF CYTOLOGY IN SELECTED TAXA

Chromosome numbers are reported for 128 species of flowering plants indigenous or endemic to Hawaii, including first reports for 13 genera and 82 species. The special significance of reports for Ilex, Tribulus, Keysseria, Pisonia, Boerhavia, Jacquemontia, Claoxylon, Lipochaeta, Railliardia, and Dubautia are discussed. The cytological and morphological variation in Railliardia and Dubautia is considered and their treatment as congeners is advocated. The cytogeogruphic pattern in Dubautia and Railliardia and other factors suggest that the ancestral chromosome number of the Hawaiian tarweeds is n = 14. Their derivation from a western North American progenitor similar to Adenothamnus is considered plausible if not indeed likely.     

Three gene phylogeny of the Thoreales (Rhodophyta) reveals high species diversity

The freshwater red algal order Thoreales has triphasic life history composed of a diminutive diploid “Chantransia” stage, a distinctive macroscopic gametophyte with multi‐axial growth and carposporophytes that develop on the gametophyte thallus. This order is comprised of two genera, Thorea and Nemalionopsis. Thorea has been widely reported with numerous species, whereas Nemalionopsis has been more rarely observed with only a few species described. DNA sequences from three loci (rbcL, cox1, and LSU) were used to examine the phylogenetic affinity of specimens collected from geographically distant locations including North America, South America, Europe, Pacific Islands, Southeast Asia, China, and India. Sixteen species of Thorea and two species of Nemalionopsis were recognized. Morphological observations confirmed the distinctness of the two genera and also provided some characters to distinguish species. However, many of the collections were in “Chantransia” stage rather than gametophyte stage, meaning that key diagnostic morphological characters were unavailable. Three new species are proposed primarily based on the DNA sequence data generated in this study, Thorea kokosinga‐pueschelii, T. mauitukitukii, and T. quisqueyana. In addition to these newly described species, one DNA sequence from GenBank was not closely associated with other Thorea clades and may represent further diversity in the genus. Two species in Nemalionopsis are recognized, N. shawii and N. parkeri nom. et stat. nov. Thorea harbors more diversity than had been recognized by morphological data alone. Distribution data indicated that Nemalionopsis is common in the Pacific region, whereas Thorea is more globally distributed. Most species of Thorea have a regional distribution, but Thorea hispida appears to be cosmopolitan.     

Morphological and isozyme divergence in Korean Hepatica sensu stricto (Ranunculaceae)

 Morphological and electrophoretic data were studied to examine species delimitation, patterns of morphological and genetic variation in three Korean Hepatica including two endemics, H.␣maxima and H. insularis. Based on a phenogram using 15 morphological characters, taxa were distinct; it was consistent with the phenogram based on genetic distance. In the enzyme electrophoresis study, the genetic identities suggested that three taxa were genetically divergent enough to be recognized as different species, falling within the range expected␣for congeners. The genetic identity between H.␣asiatica and H. insularis was higher than the values between these two taxa and H.␣maxima, a restricted endemic of Ulleung Island. The least genetic variation was found in H. maxima and the greatest in widespread H. asiatica. These data are consistent with theoretical expectations that small populations are more likely to be genetically depauperate. Received November 13, 2001; accepted May 10, 2002 Published online: December 11, 2002     

Generic recircumscriptions of Oncidiinae (Orchidaceae: Cymbidieae) based on maximum likelihood analysis of combined DNA datasets

Phylogenetic relationships within the orchid subtribe Oncidiinae sensu Chase were inferred using maximum likelihood analyses of single and multilocus DNA sequence data sets. Analyses included both nuclear ribosomal internal transcribed spacer DNA and plastid regions (matK exon, trnH‐psbA intergenic spacer and two portions of ycf1 exon) for 736 individuals representing approximately 590 species plus seven outgroup taxa. Based on the well resolved and highly supported results, we recognize 61 genera in Oncidiinae. Mimicry of oil‐secreting Malpighiaceae and other floral syndromes evolved in parallel across the subtribe, and many clades exhibit extensive variation in pollination‐related traits. Because previous classifications heavily emphasized these floral features, many genera recognized were not monophyletic. Our classification based on monophyly will facilitate focused monographs and clarifies the evolution of morphological and biochemical traits of interest within this highly diverse subtribe. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 117–146.     

MOLECULAR DELINEATION OF SPECIES AND SPECIES RELATIONSHIPS IN THE RED ALGAL AGAROPHYTES GRACILARIOPSIS AND GRACILARIA (GRACILARIALES)1

Delineation of species in the economically important agarophyte genera Gracilaria and Gracilariopsis has proven extremely difficult using available morphological characteristics. In this study, we examine the usefulness of two transcribed spacers for molecular systematic studies of these genera. The polymerase chain reaction was used to amplify the internal transcribed spacers (ITSs) and the intervening 5.8S ribosomal DNA of the nuclear ribosomal repeat region. In addition, a plastid spacer region and flanking regions of coding genes were amplified from the RUBISCO operon. Both regions were sequenced for individuals and populations of Gracilariopsis lemaneiformis (Bory) Dawson, Acleto, et Foldvik to determine the usefulness of these spacers in delimiting populations. These studies reveal that there is as much variation among individuals of a population as there is between individuals of geographically separate populations. In addition, the ITS spacer regions were compared between different species of Gracilariopsis and Gracilaria. The nuclear ITS spacer region is conserved at a species level in both genera and provides phylogenetically informative characters that can be used to examine species interrelationships among relatively closely related taxa. However, because of the difficulties of aligning this entire region among species from the two genera, the ITS region is not useful for examining intergenera relationships. ITS interspecies sequence comparisons indicate that Gracilariopsis lemaneiformis from California is significantly different from G. lemaneiformis from China and that a species of Gracilariopsis from Peru is more closely related to G. lemaneiformis from North Carolina than it is to the other Gracilariopsis species examined. In addition, these studies indicate that Gracilaria chilensis Bird, McLachlan, et Oliveira from New Zealand and Gracilaria tenuistipitata Chang et Xia from southeast Asia are as closely related as are Gracilaria verrucosa (Hudson) Papenfuss, G. pacifica Abbott, and Gracilaria robusta Kylin. Phylogenetic analysis of aligned plastid spacer sequences from Gracilaria and Gracilariopsis taxa provide similar conclusions about species relationships.     

The use of geostatistics and GIS for evolutionary history studies: the case of the nose‐horned viper (Vipera ammodytes) in the Balkan Peninsula

Geostatistics and geographical information system (GIS) procedures are novel techniques helpful for the identification of environmental correlates sustaining contact zones among subspecies or closely related species. In this paper, we tried to infer evolutionary scenarios for Vipera ammodytes across the European part of its distribution area using geostatistics and ecological niche‐based models, hence trying to solve several biogeographical questions that remained unclear after the application of classical morphological tools and genetic analyses. Eleven morphological traits from 871 vipers were analysed with geostatistics and ecological niche‐based modelling. Interpolation by kriging was used to generate surfaces of morphological variation, which were combined with spatial principal components analysis (SPCA). SPCA maps were used to test putative morphological differentiated groups with discriminant function analysis (DFA). Maximum entropy modelling and seven environmental variables were used to identify factors limiting the distribution of groups and areas for the potential occurrence of such groups. Three patterns of morphological variation were observed: a north‐west/south‐west cline, transition zones with steep clines of variation in a west–east arc, and particular character traits that disturbed the general cline. SPCA identified between three and nine putative population groups, of which three were supported by DFA. Areas of potential occurrence of these groups were coherent with the range of the three subspecies of V. ammodytes currently recognized. The distribution of all subspecies was mostly related to precipitation in the driest month. Areas of probable sympatry between subspecies are generally small and restricted. The main patterns of geographic variation of morphological characters for V. ammodytes were similar to the patterns obtained for Vipera latastei and Vipera monticola; the same environmental factors limit the distribution of differentiated groups of vipers in the Balkans and the Iberian Peninsula. The influence of humidity on the variation of morphological traits in spatially separated viper taxa from the two European peninsulas coincides with their phylogenetic relatedness. Geostatistics and GIS procedures were successful in the identification of environmental correlates sustaining contact zones among V. ammodytes subspecies in the Balkans. The same techniques should be applied for studying other parapatric forms and refugia regions. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 651–666.