MORPHOLOGICAL AND ALLOZYMIC EVIDENCE FOR SABATIA FORMOSA (GENTIANACEAE) IN THE SECTION CAMPESTRIA

An investigation of eleven populations of Sabatia, utilizing twelve morphological traits and fifteen allozymic loci, revealed the presence of three species in the section Campestria. Currently, the section has two recognized species, S. campestris Nutt. and S. arenicola Greenm. A stepwise discriminant analysis of the morphological data indicated the presence of the third previously described species, S. formosa Buckl. Eight of the loci were either monomorphic or there were no allozyme frequency differences among the three species. An analysis of the allozyme frequencies of the seven polymorphic loci substantiated the presence of S. formosa. Ecological and phenological phenomena further separate the three species. A distribution map of the populations analyzed, populations observed, and populations represented in herbarium specimens indicates that the ranges of the three species are separate although sympatry does occur. Thus, the morphological form described and designated S. formosa by Buckley in 1862 is a separate species.     

Geographical variation and diversification in the flightless leaf beetles of the Chrysolina angusticollis species complex (Chrysomelidae, Coleoptera) in northern Japan

Geographical variation and differentiation of flightless leaf beetles of the Chrysolina angusticollis species complex were analysed. Nine morphological groups were recognized on the basis of metric characters relating to body size, shape of hind wing and shape of male genitalia, and binary characters relating to females' elytral surface and larval pubescence. The nine groups were essentially parapatric or allopatric to each other. Five groups occupied a continuous geographical range each, whereas another four groups were disjunctively distributed. Morphometric distance and geographical distance between populations were significantly correlated. Analyses based on 13 allozyme loci demonstrated that six out of the nine morphological groups were further divided into more than one genetically distinct group each. Genetic similarity between populations was significantly correlated with morphometric similarity and geographical closeness. Disjunctively distributed populations of a single morphological group more often resembled geographically adjacent populations of different morphological groups than geographically remote populations of the same group. In a morphological group that occupied a wide continuous range, remote populations substantially differed genetically from each other. The results suggest that there exist at least 19 morphologically and/or genetically differentiated units in the C. angusticollis complex in northern Japan. The C. angusticollis complex is likely a superspecies composed of a number of semispecies. We postulate that the diversification of this species complex might have resulted from the repeated range contraction and expansion during the ice ages. Movement of tension zones that separate two adjacent forms might contribute to enhance their geographical differentiation.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 93 , 557–578.     

Floral and vegetative morphometrics of five Pleurothallis (Orchidaceae) species: correlation with taxonomy,phylogeny, genetic variability and pollination systems

Morphometric analyses of vegetative and floral characters were conducted in 21 populations of five Pleurothallis (Orchidaceae) species occurring in Brazilian 'campo rupestre' vegetation. A phylogenetic analysis of this species group was also carried out using nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2). Results of the ordination and cluster analyses agree with species' delimitation revealed by taxonomic and allozyme studies. The groups formed in ordination analysis correspond to the pollinator groups determined in a previous pollination study. Relationships among the species in the cluster analysis using only vegetative characters are similar to those found in a previous allozyme study, but those indicated by cluster analysis using only floral characters differ. These results support the hypothesis that floral similarities are due to convergence driven by similar pollination mechanisms, and therefore floral traits may not be good indicators of phylogenetic relationships in this group. The results of the phylogenetic analysis support this conclusion to some extent. There is no correlation between genetic (allozyme) and morphological variability in the populations nor in the way this variability is distributed among conspecific populations. We describe a new subspecies of Pleurothallis ochreata based on differences in vegetative and chemical characters as well as geographic distribution. Absence of differentiation in floral characters, attraction of the same pollinator species, interfertility and genetic similarity support the argument for subspecific rather than specific status.     

Dactylorhiza majalis s.l. (Orchidaceae) in acid habitats: variation patterns, taxonomy, and evolution

Morphometric and allozyme data were collected from 11 populations of Dactylorhiza majalis s.l. in the Ardennes (Belgium, France) and North Jutland (Denmark). All study populations were growing in obviously acid habitats. Genetic distances were calculated and illustrated by UPGMA dendrograms, while principal components analyses (PCAs) were used to summarize morphological variation patterns. Based on the PCAs, the probability of each character to distinguish correctly between the main groups of populations was estimated. The study populations from the Ardennes and from the Danish island of Læsø seem to agree with populations from Sweden previously studied by Hedrén—and to belong to D. majalis subsp. sphagnicola (comb. nov.). It should be noted that allozyme data suggest a polytopic origin of this taxon. The study populations from the Danish region of Thy differ morphologically from the others and exhibit much less genetic variation. They are described as a new subspecies, D. majalis subsp. calciñigiens. The evolution and biogeography of the two taxa are discussed, and brief taxonomic accounts are provided.     

Morphometric and allozyme variation in the African catfishes Clarias gariepinus and C. anguillaris

This study investigated morphological characters and electrophoretic polymorphism at 25 protein loci in nine wild populations of the African clariid catfish Clarias gariepinus and seven wild populations of C. anguillaris. Two other clariid species, Clarias albopunctatus and Heterobranchus longifilis , were used as outgroups in the allozyme study. Morphometric and allozyme data are congruent for the Nilo-Sudanian populations of C. gariepinus and C. anguillaris. Both approaches also distinguished two groups amongst the C. gariepinus populations, one containing Nilo-Sudanian populations and the other including Lake Victoria and southern African populations. However, allozyme data suggest that C. gariepinus is not a monophyletic group and show that C. albopunctatus is more divergent from C. gariepinus and C. anguillaris than it is from H. longifilis , stressing the need for a revision of clariid systematics. The variation observed in C. gariepinus is discussed in terms of palaeogeographical events and its use in aquaculture.     

Genetic relationships and origin of two geographic groups of the freshwater threespine stickleback, 'hariyo'

'Hariyo' comprise the southernmost freshwater populations of the threespine stickleback, Gasterosteus aculeatus species complex, distributed in central Honshu, Japan. Two geographic groups (Gifu and Shiga) of the 'hariyo' have been recognized as differing from each other in some morphological and ecological features. In order to elucidate the genetic characteristics and phylogenetic position of these populations, partial sequences of the mitochondrial cytochrome b gene were compared in 123 specimens from 10 'hariyo' populations, and eight other freshwater and anadromous populations of threespine stickleback in Japan. Phylogenetic analysis resolved 22 haplotypes in a single most-parsimonious tree. In contrast to previous allozyme analyses, the haplotype tree indicated that 'hariyo' populations are monophyletic. Each of two geographic groups of the 'hariyo' was also shown to be nearly monophyletic. The two geographic groups differed from each other in sequence by an average of 0.47% and from other populations by 0.70%. Based on a molecular clock, constructed from fossil records and mtDNA genetic distances of Gasterosteus and Pungitius, it was estimated that the two groups of 'hariyo' differentiated from the other threespine stickleback populations and also from each other in the middle Pleistocene. The results suggested that each of the two groups of 'hariyo' is a distinct evolutionarily significant unit with unique genetic features, as well as morphological and ecological characteristics.     

One species or several? Discordant patterns of geographic variation between allozymes and mtDNA sequences among spiders in the genus Metepeira (Araneae: Araneidae)

Paradoxically, an allozyme study of Metepeira "spinipes" (sensu lato) demonstrated extensive gene flow among four populations whose members are nevertheless morphologically and behaviorally distinct. Initially, the authors tentatively concluded that the populations exhibited panmixis and suggested that local environmental effects accounted for the apparent morphological and behavioral differences. However, they later concluded that such differences were too great to be accounted for by the environment alone and that the four populations actually represented three different species. To confirm that the allozyme results were, in fact, artifactual, we reexamined the relationships among these populations by sequencing a portion of the 12S mtDNA ribosomal subunit. In contrast to the allozyme result, our results demonstrate good agreement between patterns of genetic and morphological/behavioral variation. We suggest (1) that the allozyme allele frequencies are homogenized by balancing selection, not gene flow as was previously concluded, and therefore (2) that this study provides another instance in which inferences about population structures from allozyme data are misleading.     

Molecular phylogeny and species recognition in the mealybug genus Ferrisia Fullaway (Hemiptera: Pseudococcidae)

Previous allozyme and DNA nucleotide sequence studies of the mealybug genus Ferrisia Fullaway (Hemiptera: Pseudococcidae), although limited, have suggested greater species diversity than is recognized by the current morphology‐based taxonomy. Here we analyse nucleotide sequence data from one mitochondrial (cytochrome oxidase I) and two nuclear (EF‐1α and 28S D₂D₃) genes and recover ten well‐supported groups that allow us to reassess the taxonomic utility of morphological characters used for species recognition. We report on previously used morphological characters for which states are highly variable within genetic groups and identify new characters (of the wax‐exuding cuticular ducts and pores) with taxonomically informative states. The widespread pest species F. virgata (Cockerell), commonly called the striped mealybug, should be diagnosed more narrowly. From samples identified as F. virgata, we recover six clades that we equate with species and that can be distinguished with the newly identified morphological characters. We determine that five of the ‘electrophoretic species' identified informally by the late Uzi Nur based on electrophoretic mobility of 20 enzymes correlate with four of our genetic groups. This matching of Nur's putative species with ours was possible only because some of Nur's slide‐mounted voucher specimens were deposited in a museum and thus available for morphological study. Species confused with F. virgata are either new to science or were placed erroneously in synonymy with F. virgata by earlier authors: they will be described elsewhere. The most important characters of the adult female for distinguishing these species from F. virgata are the positions and characteristics of minute discoidal pore(s) associated both with the ventral oral‐collar tubular ducts and with the sclerotized area surrounding each dorsal enlarged tubular duct, and the number of sizes of the ventral oral‐collar tubular ducts. In addition, we determine that adult females of F. gilli Gullan from different populations on different host plants vary substantially in the number and size of clusters of small ventral oral‐collar ducts on the body margins – features previously suspected to indicate distinct species.     

Molecular phylogenetics and delimitation of species in Cortinarius section Calochroi (Basidiomycota, Agaricales) in Europe

Cortinarius is the most species rich genus of mushroom forming fungi with an estimated 2000 spp. worldwide. However, species delimitation within the genus is often controversial. This is particularly true in the section Calochroi (incl. section Fulvi), where the number of accepted taxa in Europe ranges between c.60 and c.170 according to different taxonomic schools. Here, we evaluated species delimitation within this taxonomically difficult group of species and estimated their phylogenetic relationships. Species were delimited by phylogenetic inference and by comparison of ITS sequence data in combination with morphological characters. A total of 421 ITS sequences were analyzed, including data from 53 type specimens. The phylogenetic relationships of the identified species were estimated by analyzing ITS data in combination with sequence data from the two largest subunits of RNA polymerase II (RPB1 and RPB2). Seventy-nine species were identified, which are believed to constitute the bulk of the diversity of this group in Europe. The delimitation of species based on ITS sequences is more consistent with a conservative morphological species concept for most groups. ITS sequence data from 30 of the 53 types were identical to other taxa, and most of these can be readily treated as synonyms. This emphasizes the importance of critical analysis of collections before describing new taxa. The phylogenetic separation of species was, in general, unambiguous and there is considerable potential for using ITS sequence data as a barcode for the group. A high level of homoplasy and phenotypic plasticity was observed for morphological and ecological characters. Whereas most species and several minor lineages can be recognized by morphological and ecological character states, these same states are poor indicators at higher levels.     

One Species or Several? Discordant Patterns of Geographic Variation between Allozymes and mtDNA Sequences among Spiders in the Genus Metepeira (Araneae: Araneidae)

Paradoxically, an allozyme study of Metepeira “spinipes” (sensu lato) demonstrated extensive gene flow among four populations whose members are nevertheless morphologically and behaviorally distinct. Initially, the authors tentatively concluded that the populations exhibited panmixis and suggested that local environmental effects accounted for the apparent morphological and behavioral differences. However, they later concluded that such differences were too great to be accounted for by the environment alone and that the four populations actually represented three different species. To confirm that the allozyme results were, in fact, artifactual, we reexamined the relationships among these populations by sequencing a portion of the 12S mtDNA ribosomal subunit. In contrast to the allozyme result, our results demonstrate good agreement between patterns of genetic and morphological/behavioral variation. We suggest (1) that the allozyme allele frequencies are homogenized by balancing selection, not gene flow as was previously concluded, and therefore (2) that this study provides another instance in which inferences about population structures from allozyme data are misleading.     

Genetic differences among host-associated populations of water mites (Acari: Unionicolidae: Unionicola): allozyme variation supports morphological differentiation

Unionicola poundsi and U. lasallei are recognized as closely related, morphologically distinct species of water mites living in symbiotic association with the mussels Villosa villosa and Uniomerus declivus, respectively. However, results of a transplant experiment suggested that the morphological characters used to separate these species are plastic and are influenced by the host species in which these mites metamorphose. These results indicate that U. poundsi and U. lasallei are variants of the same species. To test the validity of these contrasting notions, the genetic structure of mite populations from Uniomerus declivus and V. villosa was compared. An examination of allozyme variation at 9 enzyme loci revealed a high degree of genetic differentiation between these host-associated populations, with mites from U. declivus and V. villosa being fixed for different alleles at 3 loci and exhibiting significant allele heterogeneity at 71% of their polymorphic loci. Coefficients of genetic similarity and genetic distance for mites from U. declivus and V. villosa were 0.36 and 0.95, respectively. The results of this study suggest that mite populations from U. declivus and V. villosa are genetically distinct and complement morphological data recognizing them as valid species.     

Falling apart and merging: diversification of slender salamanders (Plethodontidae: Batrachoseps) in the American West

The plethodontid genus Batrachoseps , the slender salamanders, is the most diverse clade of salamanders in western North America, but it has posed taxonomic difficulties because it contains many morphologically cryptic species. A segment of the mitochondrial DNA gene cytochrome b was studied for 278 individuals densely sampled from throughout the range of all 18 described species and several undescribed species. Phylogenetic analyses of the mtDNA data identify six major clades, one corresponding to the subgenus Plethopsis and five within a monophyletic subgenus Batrachoseps. All major clades and most species within these clades display strong phylogeographic structuring. Comparisons of mtDNA and allozyme data show that several allozymically cohesive groups are not monophyletic with respect to mtDNA. We suggest that this phenomenon results from fragmentation of populations, divergence in allopatry, and then recontact and gradual merging of units caused predominantly by male-mediated gene flow. The mtDNA offers evidence that populations were once more isolated than they are now, while the patterns of allozyme variation reflect recent and current interactions among populations. The complex patterns of morphological, allozymic and mtDNA variation associated with the constantly changing geological landscape give insight into the nature of processes responsible for species formation in Batrachoseps .  © 2002 The Linnean Society of London. Biological Journal of the Linnean Society , 2002, 76 , 361–391.     

Morphological and allozymic variation in Hynobius boulengeri and H. stejnegeri (Amphibia: Urodela: Hynobiidae)

We studied morphological and allozymic variation in populations of Japanese salamanders, Hynobius boulengeri and H. stejnegeri. Adult H. boulengeri showed sexual dimorphism, and juveniles differed greatly from adults in many morphological characters. From the results of multivariate analyses of morphological characters, the populations were divided into four groups: (I) H. boulengeri from Honshu, (II) H. boulengeri from Shikoku, (III) H. boulengeri from the Sobo-Katamuki Mountains of Kyushu and H. stejnegeri, and (IV) H. boulengeri from the Amakusa Islands and the Osumi Peninsula. Phenotypic relationships among the four groups were identical to relationships clarified by allozymic analyses, except for group IV, which was included in group III in the allozyme tree. Some morphometric characters were significantly correlated with environmental variables. We consider H. stejnegeri to be a valid species based on its unique color pattern, morphometric characters, and allelic composition, even though it was nested within group III of H. boulengeri by both morphological and allozymic analyses. We propose that group I from Honshu and group II from Shikoku should be treated as H. boulengeri sensu stricto and H. hirosei, respectively. Resolving the taxonomic status of the remaining populations of groups III and IV from Kyushu requires further study.     

Variability in natural populations of Anopheles sacharovi (Diptera: Culicidae) from southeast Anatolia, revealed by morphometric and allozymic analyses.

Four populations of Anopheles sacharovi Favre occurring in different ecological subregions at altitudes between 353-1,126 m in the Sanliurfa Province of southeast Turkey were compared using morphometric and allozyme analyses. Four allozyme loci were assessed for genetic differentiation among samples from four localities. The similarity phenogram obtained from the allozyme data showed that populations at Birecik and Sandi branched as a separate group from the Pamuklu and Gedik populations. The Gedik population at the highest altitude (1,126 m) was clustered as a separate branch when linear measurements of 63 morphological characteristics were examined. The UPGMA phenogram also showed that Pamuklu and Sandi formed a cluster while Birecik and Gedik formed separate groups.     

Genetic variation and evolutionary history of holly oak: a circum-Mediterranean species-complex [Quercus coccifera L./Q. calliprinos (Webb) Holmboe, Fagaceae]

Holly oak is the only evergreen oak possessing a circum-Mediterranean range; it has two predominant morphological forms, calliprinos and coccifera, described in the eastern and western Mediterranean Basin respectively. The concordance of allozyme and morphotype variation was analysed in the whole holly oak range, and the most plausible historical events responsible for the current geographic pattern of genetic variation were investigated. Individuals from 24 populations were scored for allozyme variation at seven polymorphic loci. Multi-locus genotypes were analysed by using a correspondence analysis (CA) and a Bayesian clustering approach. The relative positions of the populations were obtained from multi-dimensional scaling coupled with UPGMA treatment. A continuous genotype distribution was observed in the CA, and two groups were identified using the Bayesian approach. With a 0.95 threshold, 66 and 69% of the individuals showing the calliprinos and the coccifera morphotypes respectively were assigned to the corresponding genetic groups, which differed by private alleles. As compared to coccifera, the genetic group calliprinos was characterized by higher allelic richness and a strong geographical genetic structure. In agreement with fossil records, the most parsimonious explanation for lack of geographical structure in coccifera is a substantial regression of holly oak in the western Mediterranean Basin during the glaciations and a fast westward post-glacial expansion of coccifera populations, probably from Greece. Two population groups were obtained from the scaling/UPGMA treatment. One included all of the calliprinos populations and a Greek coccifera population, suggesting that the two morphotypes are closely related genetically and constitute two components of the same species.     

Cryptic species and morphological plasticity in long-lived bivalves (Unionoida: Hyriidae) from inland Australia

Molecular (mitochondrial DNA, isozyme) and morphological diversity of freshwater mussels (Family Hyriidae) was examined at 21 sites encompassing four large river systems, across southwest Queensland, Australia. Evidence was found for two major morphological groups. One group, which occurred in every river system, closely matched a recognized species (Velesunio ambiguus) both morphologically and in a well-supported lineage within a mitochondrial phylogeny generated from partial cytochrome c oxidase subunit I (COI) sequences. The second group most closely matched Velesunio wilsonii in shell morphology but formed three deeply divergent mitochondrial DNA lineages. All four lineages occurred sympatrically in some areas and displayed corresponding fixed differences at nuclear allozyme loci, which suggests an absence of recent hybridization and the presence of separate species.     

Glinal variation in morphology among eastern populations of the waterstrider, Aquarius remigis Say (Hemiptera: Gerridae)

We examined morphological variation among populations of the stream-dwelling waterstrider, Aquarius remigis. A previous analysis of allozyme variation along a transect from southern Quebec to southern North Carolina revealed two distinct 'types' of A. remigis, north and south of a transition zone in southern New York and northern Pennsylvania. To assess the concordance of morphological and allozyme differentiation, adults were sampled from 15 populations along the original transect, as well as 11 additional populations in New York, Connecticut and New Jersey. Eight measurements of leg and body size were taken of preserved individuals. Univariate and multivariate analyses (multiple regression, PCA, discriminant analysis) reveal significant differentiation between northern and southern populations with a transition zone between latitudes 39^oand 42^onorth. Strong clines within each region and a clear transition between regions support previous suggestions that there are two distinct 'types' of this species in eastern North America. The transition zone, in morphology coincides with that revealed by the allozyme variation, but extends further south. The covariance of allozyme frequency and morphometric traits suggests that differentiation has occurred through vicariance and secondary contact, and that in this system, cladistic and adaptive differentiation are coupled.     

Allozyme variation and racial differentiation in Swedish Carex lepidocarpa s.l. (Cyperaceae)

Two morphological races have previously been recognized within the sedge, Carex lepidocarpa , in Sweden. These largely allopatric races are accorded specific status, as C. lepidocarpa s.s. and C. jemtlandica , in Scandinavian floras. A study of allozyme variation in populations from 80 Swedish sites supports the morphological evidence for racial differentiation within C. lepidocarpa. The two races differ from each other in terms of allele frequencies at polymorphic loci and also show different levels of within-population genetic diversity. Material that is morphologically referable to C. lepidocarpa s.s. is characterized by relatively high levels of allozyme variation, both within and between populations. Carex lepidocarpa s.s. is widespread in southern Sweden. In contrast, material that is morphologically assignable to C. jemtlandica shows low levels of within-population genetic diversity, and there is little differentiation between the geographically separated isolates of C. jemtlandica in northern Sweden and on the Baltic island of Gotland. The high degree of morphological similarity and moderate levels of genetic differentiation between the two races within C. lepidocarpa indicate that it is more appropriate to recognize the races as subspecies than as species. The low levels of genetic variation in C. jemtlandica , both within and between populations, suggest that C. jemtlandica may have arisen from C. lepidocarpa (or a near ancestor of C. lepidocarpa) as a result of population fragmentation and isolation in glacial refugia, or during the process of post-glacial colonization of Scandinavia. Lack of allozyme evidence for extensive hybridization between the two races of C. lepidocarpa , despite their ability to hybridize freely where their ranges overlap at present, supports the suggestion that the two races have had separate post-glacial histories.     

Systematics of Andean gladiator frogs of the Hypsiboas pulchellus species group (Anura,Hylidae)

Köhler, J., Koscinski, D., Padial, J. M., Chaparro, J. C., Handford, P., Lougheed, S. C. & De la Riva, I. (2010). Systematics of Andean gladiator frogs of the Hypsiboas pulchellus species group (Anura, Hylidae). —Zoologica Scripta, 39, 572–590. We revisit the taxonomic status of Andean species and populations of frogs of the Hypsiboas pulchellus group using multiple lines of evidence potentially indicative of evolutionary lineage divergence in anurans: differences in qualitative morphological or bioacoustic character states, no overlap in quantitative characters of advertisement calls, and monophyly of gene genealogies. We found qualitative and quantitative morphological characters to be extremely variable among species and populations of the group and thus of very limited use in assessing lineage divergence. In contrast, phylogenetic analyses based on 16S rRNA and cytochrome b sequences resolved highly supported clades that are in concordance with bioacoustic differences. The results support the specific distinctness of most nominal species recognized in the group, including the Bolivian Hypsiboas balzani and Hypsiboas callipleura, two species that were considered to be synonymous, and revealed the presence of an undescribed species from southern Peru, which is here described as Hypsiboas gladiator sp. n. In contrast, Hypsiboas andinus and Hypsiboas riojanus were mutually paraphyletic, and showed no differences in morphology and acoustic characters. Consequently, we regard the former as a junior synonym of the latter. However, we discovered that populations of H. riojanus from central Bolivia exhibit some degree of genetic differentiation and advertisement call differences with respect to Argentine populations, but sampling of these Bolivian populations is too sparse to draw taxonomic conclusions. Our phylogenetic results support the hypothesis that ancestral lineages of the Andean H. pulchellus group experienced successive splitting events along a latitudinal gradient from north to south.