Genetic Diversity of Neotropical Myotis (Chiroptera: Vespertilionidae) with an Emphasis on South American Species

Background

Cryptic morphological variation in the Chiropteran genus Myotis limits the understanding of species boundaries and species richness within the genus. Several authors have suggested that it is likely there are unrecognized species-level lineages of Myotis in the Neotropics. This study provides an assessment of the diversity in New World Myotis by analyzing cytochrome-b gene variation from an expansive sample ranging throughout North, Central, and South America. We provide baseline genetic data for researchers investigating phylogeographic and phylogenetic patterns of Myotis in these regions, with an emphasis on South America.

Methodology and Principal Findings

Cytochrome-b sequences were generated and phylogenetically analyzed from 215 specimens, providing DNA sequence data for the most species of New World Myotis to date. Based on genetic data in our sample, and on comparisons with available DNA sequence data from GenBank, we estimate the number of species-level genetic lineages in South America alone to be at least 18, rather than the 15 species currently recognized.

Conclusions

Our findings provide evidence that the perception of lower species richness in South American Myotis is largely due to a combination of cryptic morphological variation and insufficient sampling coverage in genetic-based systematic studies. A more accurate assessment of the level of diversity and species richness in New World Myotis is not only helpful for delimiting species boundaries, but also for understanding evolutionary processes within this globally distributed bat genus.     

Phylogeny of New World Paspalum (Poaceae,Panicoideae, Paspaleae) based on plastid and nuclear markers

Phylogenetic analyses of 131 terminals of Paspalum and related genera, based on both plastid and nuclear markers, were performed under maximum parsimony and Bayesian methods. The total evidence analyses generated a hypothesis showing that Paspalum would be monophyletic if Spheneria, Thrasyopsis and Reimarochloa are included within the genus. Paspalum inaequivalve and P. microstachyum, two species of the Inaequivalvia group were related to genus Anthaenantiopsis, excluded from Paspalum, or nested within it by plastid and nuclear markers, respectively. Subgenera Anachyris and Harpostachys were partially recovered as monophyletic assemblages, while subg. Ceresia and Paspalum resolved as polyphyletic. Within subgenus Paspalum, some informal groups were recovered as monophyletic, while others were resolved as paraphyletic or polyphyletic. Phylogenetic relationships among species of Paspalum were partially recovered possibly due to reticulation events among species, autopolyploidization and apomixis; all these processes being common in Paspalum, thus obscuring the infrageneric classification.     

Mitochondrial DNA based phylogeny of the woodpecker genera Colaptes and Piculus,and implications for the history of woodpecker diversification in South America

The woodpecker genus Colaptes (flickers) has its highest diversity in South America and the closely related genus Piculus is restricted to South and Central America. Two species of flickers occur in North America, and one species is endemic to Cuba. We conducted a Bayesian phylogenetic analysis of three mitochondrial encoded genes (cyt b, COI, 12S rRNA) and confirmed that the two genera are paraphyletic. Three species historically classified as Piculus are actually flickers. We found that the Cuban endemic C. fernandinae is the most basal species within the flickers and that the Northern Flicker is the next most basal species within the Colaptes lineage. The South American clade is most derived. The age of the South American diversification is estimated to be 3.6 MY, which is synchronous with the emergence of the Isthmus of Panama. The pattern of diversification of South American flickers is common among South American woodpeckers. Although woodpeckers have their greatest diversity in South America, we hypothesize that woodpeckers (Family Picidae) originated in Eurasia, dispersed to North America via the Bering land bridge, and multiple lineages entered South America as the Isthmus approached its final closing.     

Genetic relationship among Paspalum species of the subgenus Anachyris: Taxonomic and evolutionary implications

Paspalum is one of the most important genera of the Poaceae family due to its large number of species and diversity. The subgenus Anachyris comprises six species mainly from South America grouped together by sharing rare spikelet characteristics. A genetic analysis using ISSR markers, compared with the morphological and phenotypic variation observed in each one species, was used to establish genetic relationships among 40 accessions with several ploidy levels, belonging to 5 species of the subgenus Anachyris. Fourteen accessions of Paspalum malacophyllum (2x and 4x), 12 of P. simplex (2x, 3x, 4x and 6x), 4 of P. procurrens (2x and 4x), 4 of P. usterii (4x) and 6 of P. volcanensis (4x) were analysed. A total of 227 ISSR loci (98.7% polymorphic) were detected among all accessions, with variable loci number and percentages of polymorphism according to species delimitations. Six main groups were identified by cluster analysis based on Jaccard's genetic distance and UPGMA, four of which matched all the respective accessions of P. simplex, P. procurrens, P. usterii and P. volcanensis, while the other two were consistent with two different groups of accessions of P. malacophyllum, one involving most tetraploid accessions, and the other one grouping together a tetraploid and two diploid accessions. The distinctive morphological characteristics and the separate clustering of these tetraploid and diploid cytotypes suggest to consider a new multiploid species complex inside the subgenus Anachyris. Both cytotypes of P. procurrens, and the four co-specific cytotypes of P. simplex consistently clustered together forming two specific groups for the two multiploid taxons. This is in agreement with the existence of high phenotypic similarities between diploid and tetraploid cytotypes of P. procurrens, and among diploid, triploid, tetraploid and hexaploid cytotypes of P. simplex. Since the polyploid cytotypes of these species are reproduced by apomixis, the specific genetic clustering by ISSR markers and morphological and cytological results support the hypothesis that the two multiploid species were originated by autopolyploidy. Our results confirm previous studies suggesting a monophyletic origin for the subgenus Anachyris and are concordant with previous data regarding genomic homologies and phylogenetic analyses in the genus.     

Out of South America: phylogeny of non‐biting midges in the genus Labrundinia suggests multiple dispersal events to Central and North America

Non‐biting midges of the genus Labrundinia (Chironomidae: Tanypodinae) are minute dipterans with immature stages living in a variety of unpolluted water bodies, from small streams and ponds to lakes and bays. Extensively recorded in ecological studies, the genus comprises 39 species, all except one described from areas outside the Palearctic region. Internal structure among Labrundinia species was postulated by S. S. Roback, who recognized four species groups based on morphological characters of immature stages. We examined phylogenetic relationships among known Labrundinia species using partial DNA sequences of the nuclear protein‐coding gene CAD and morphological characters. Both analyses with Bayesian inference and parsimony methods recovered the monophyly of Labrundinia, strongly supported by five morphological synapomorphies. Internal relationships within the genus partly supported Roback's species groups with the addition of later described species. Biogeographical inferences were obtained by applying Bayesian binary MCMC (BBM) analysis and favoured a scenario where Labrundinia had its initial diversification in the Neotropical region and that current presence in the Nearctic region and southern South America is due to subsequent dispersal.     

Two new species of yellow-shouldered bats,genus Sturnira Gray, 1842 (Chiroptera,Phyllostomidae) from Costa Rica,Panama and western Ecuador

Two new species of yellow-shouldered bats Sturnira Gray, 1842 (Chiroptera, Phyllostomidae) from Central America and western South America are described using molecular and morphological data. The two new species, which occur in Costa Rica and Panama and in western Ecuador, were previously confused with S. ludovici, and S. lilium and S. luisi, respectively. Sturnira now includes 22 described species, making it the most speciose genus in the Neotropical family Phyllostomidae.     

Systematic Studies of the Genus A<Emphasis Type="Italic">egialomys</Emphasis> Weksler et al., 2006 (Rodentia: Cricetidae: Sigmodontinae): Geographic Variation,Species Delimitation,and Biogeography

Aegialomys occurs in open habitats west of the Ecuadorean and Peruvian Andes, including the Galapagos Archipelago. This genus currently includes two species, A. galapagoensis and A. xanthaeolus. We studied patterns of geographic variation to characterize the morphologic and morphometric variation and recognize diagnosable clusters of samples. Employing this evidence, within a phylogenetic framework employing morphological, molecular, and concatenated matrices, we diagnose monophyletic lineages and assign the appropriate names to species–group taxa. Qualitatively, we noted geographic variation in some characters, and quantitatively there is a pronounced increase in cranial dimensions along the north–south distribution axis, revealing the existence of four distinct clusters: North, South, Extreme South, and Galapagos. These results, along with the phylogenetic relationships, allowed us to hypothesize that Aegialomys exhibits four monophyletic species that we call: Aegialomys galapagoensis, restricted to the Galapagos Archipelago; Aegialomys xanthaeolus, distributed from Ecuador to northern Peru; Aegialomys baroni, ocurring in Central Perú; and Aegialomys ica, distributed in southern Peru. Our distributional data suggest that species discontinuities are associated with some well-known barriers in the western portion of South America. Through the Andes and trans–Andean area, there are some geographic features or areas, the Huancabamba Depression, that historically played a key role as barriers to plant and animal dispersion or as a boundaries to species distribution.     

Phylogeny of the tribe Abrotrichini (Cricetidae,Sigmodontinae): integrating morphological and molecular evidence into a new classification

The tribe Abrotrichini (five genera and 14 living species) is a small clade within the speciose subfamily Sigmodontinae (Rodentia, Cricetidae), representing one of the extant successful radiations of mammals at southern high latitudes of the Neotropics. Its distribution is mostly Andean, reaching its greatest diversity in southern Argentina and Chile. We evaluate the phylogenetic relationships within this tribe through parsimony and Bayesian approaches based on 99 morphological characters (including 19 integumental characters, 38 skull characters, 31 dental characters, three postcranial skeletal characters, seven from the male accessory glands and phallus and one from the digestive system) and six molecular markers (one mitochondrial and five nuclear). We include representatives of all, except one, of the currently recognized species of living Abrotrichini plus one fossil form. Based on total evidence, we recovered a primary division between the genus Abrothrix and a group including the long‐clawed Abrotrichini, Chelemys, Geoxus, Notiomys and Pearsonomys. Both clades are recognized and named here as subtribes. The large degree of morphological variation observed within Abrothrix suggests that species in the genus fall into four groups, which we recognize as subgenera. In addition, the two known species of Chelemys do not form a monophyletic group, and Geoxus was recovered as paraphyletic with respect to Pearsonomys. To reconcile classification and phylogenetics, we describe a new genus for Chelemys macronyx and include Pearsonomys as a junior synonym of Geoxus. Our results highlight the importance of both morphology and molecules in resolving the phylogenetic relationships within this tribe. Based on biogeographical analyses, we hypothesize that Abrotrichini originated in south‐western South America by vicariance and then diversified mostly by successive dispersal events.     

Patterns of cryptic host specificity in duck lice based on molecular data

Documenting patterns of host specificity in parasites relies on the adequate definition of parasite species. In many cases, parasites have simplified morphology, making species delimitation based on traditional morphological characters difficult. Molecular data can help in assessing whether widespread parasites harbour cryptic species and, alternatively, in guiding further taxonomic revision in cases in which there is morphological variation. The duck louse genus Anaticola (Phthiraptera: Philopteridae), based on current taxonomy, contains both host‐specific and widespread species. Mitochondrial and nuclear DNA sequences of samples from this genus were used to document patterns of host specificity. The comparison of these patterns with morphological variations in Anaticola revealed a general correspondence between the groups identified by DNA sequences and morphology, respectively. These results suggest that a more thorough taxonomic review of this genus is needed. In general, the groups identified on the basis of molecular data were associated with particular groups of waterfowl (e.g. dabbling ducks, sea ducks, geese) or specific biogeographic regions (e.g. North America, South America, Australia, Eurasia).     

Accounting for molecular stochasticity in systematic revisions: Species limits and phylogeny of Paroaria

Different frameworks have been proposed for using molecular data in systematic revisions, but there is ongoing debate on their applicability, merits and shortcomings. In this paper we examine the fit between morphological and molecular data in the systematic revision of Paroaria, a group of conspicuous songbirds endemic to South America. We delimited species based on examination of >600 specimens, and developed distance-gap, and distance- and character-based coalescent simulations to test species limits with molecular data. The morphological and molecular data collected were then analyzed using parsimony, maximum likelihood, and Bayesian phylogenetics. The simulations were better at evaluating the new species limits than using genetic distances. Species diversity within Paroaria had been underestimated by 60%, and the revised genus comprises eight species. Phylogenetic analyses consistently recovered a congruent topology for the most recently derived species in the genus, but the most basal divergences were not resolved with these data. The systematic and phylogenetic hypotheses developed here are relevant to both setting conservation priorities and understanding the biogeography of South America.     

An interplay between forest and non‐forest South American avifaunas suggested by a phylogeny of lepidocolaptes woodcreepers (dendrocolaptinae)

Very few South American avian superspecies or species groups are composed of both forest and non‐forest taxa. The genus Lepidocolaptes comprises 8–9 species of woodcreepers, most of which are forest birds, but two species, L. angustirostris and L. souleyetii, inhabit open vegetations. Therefore, this genus should play an important role in the discussion about the relationships between forest and non‐forest South American avifaunas. Nucleotide sequences from two mitochondrial genes, cytochrome b and ND2, suggest that: (i) L. fuscus should be removed from the genus since its association with other members of this genus is poorly supported. This view has been pointed out also by morphological and behavioural data; (ii) the phylogenetic position of the open‐vegetation species within the Lepidocolaptes radiation indicate that the split between forest and non‐forest elements within this genus took place as recently as two million years ago. This result suggests that the evolutionary relationships between forest and non‐forest biotas in South America may have been more dynamic than previously thought.     

Molecular phylogenetics and morphological evolution of St. John’s wort (Hypericum; Hypericaceae)

Phylogenetic hypotheses for the large cosmopolitan genus Hypericum (St. John’s wort) have previously been based on morphology, and molecular studies have thus far included only a few species. In this study, we used 360 sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) for 206 species representing Hypericum (incl. Triadenum and Thornea) and three other genera of Hypericaceae to generate an explicit phylogenetic hypothesis for the genus using parsimony and model-based methods. The results indicate that the small genus Triadenum is nested in a clade within Hypericum containing most of the New World species. Sister to Hypericum is Thornea from Central America. Within Hypericum, three large clades and two smaller grades were found; these are based on their general morphology, especially characters used previously in taxonomy of the genus. Relative to the most recent classification, around 60% of the sections of Hypericum were monophyletic. We used a Bayesian approach to reconstruct ancestral states of selected morphological characters, which resulted in recognition of characters that support major clades within the genus and a revised interpretation of morphological evolution in Hypericum. The shrubby habit represents the plesiomorphic state from which herbs evolved several times. Arborescent species have radiated convergently in high-elevation habitats in tropical Africa and South America.     

Phylogeny and biogeography of the staghorn fern genus Platycerium (Polypodiaceae, Polypodiidae)

The genus Platycerium is one of the few pantropical epiphytic fern genera with six species in Afro-Madagascar, 8-11 Australasian species, and a single species in tropical South America. Nucleotide sequences of four chloroplast DNA markers are employed to reconstruct the phylogeny of these ferns and to explore their historical biogeography. The data set was designed to resolve conflicting hypotheses on the relationships within the genus that were based on previous phylogenetic studies exploring morphological evidence. Our results suggest a basal split of Platycerium into two well-supported clades. One clade comprises species occurring in Africa, Madagascar, and South America, whereas the second clade contains exclusively Australasian species. The latter clade is further divided into a clade corresponding to P. bifurcatum and its putative segregates and a clade of seven species occurring from Indochina throughout the Malesian region to New Guinea and Australia. The Afro-Madagascan clade includes a clade of two species found in tropical Africa and a clade of four species that includes three species endemic to Madagascar. The single neotropical species of this genus, P. andinum, is nested within the Afro-Madagascan clade but is not closely related to any extant species.     

The genus Phymatolithon (Hapalidiaceae,Corallinales, Rhodophyta) in South Africa,including species previously ascribed to Leptophytum

Of the genera within the coralline algal subfamily Melobesioideae, the genera Leptophytum Adey and Phymatolithon Foslie have probably been the most contentious in recent years. In recent publications, the name Leptophytum was used in quotation marks because South African taxa ascribed to this genus had not been formally transferred to another genus or reduced to synonymy. The status and generic disposition of those species (L. acervatum, L. ferox, L. foveatum) have remained unresolved ever since Düwel and Wegeberg (1996) determined from a study of relevant types and other specimens that Leptophytum Adey was a heterotypic synonym of Phymatolithon Foslie. Based on our study of numerous recently collected specimens and of published data on the relevant types, we have concluded that each of the above species previously ascribed to Leptophytum represents a distinct species of Phymatolithon, and that four species (incl. P. repandum) of Phymatolithon are currently known to occur in South Africa.Here we present detailed illustrated accounts of each of the four species, including: new data on male and female/carposporangial conceptacles; ecological and morphological/anatomical comparisons; and a review of the information on the various features used previously to separate Leptophytum and Phymatolithon. Southern African species ascribed to the genus Phymatolithon may be separated from one another in the field by their growth forms, the substrata on which they are generally found, and the colour of living thalli. A key for identifying southern African specimens in the field is included. Our data support the conclusion that the characters upon which Leptophytum is based are unreliable for generic delimitation from Phymatolithon.     

A tale of two clades: Comparative study of Glyptodon Owen and Glyptotherium Osborn (Xenarthra,Cingulata, Glyptodontidae)

Glyptodon and Glyptotherium represent the most conspicuous taxa of late Neogene and Pleistocene glyptodonts in South America and North America, respectively. The earliest records of Glyptodon in South America are 1.07 Ma (late early Pleistocene, Calabrian), although the possibility that “Paraglyptodon uquiensis” represents a Pliocene specimen of Glyptodon cannot be rejected. Glyptotherium originated from South American ancestry in northern South America or Central America about 3.9 Ma (early late Pliocene, Zanclean) or earlier. The diversity of South American Glyptodon is currently under study, but preliminary evidence would indicate that no more than three species (G. munizi, G. elongatus and G. reticulatus) are valid, plus a possible new Andean species. In turn, according to the updated taxonomy proposed herein, Glyptotherium includes two chronospecies. The earliest species, Gl. texanum, differs only slightly from the latest species, Gl. cylindricum. The relationship of Glyptodon and Glyptotherium has been problematical since the discovery of the North American lineage, at first identified as various species of Glyptodon and later considered a separate genus. Glyptodon is recognized as a natural group and recent taxonomic and phylogenetic revisions place all North American glyptodontines into Glyptotherium. In this paper, we propose a detailed morphological comparison between the southern South American species of Glyptodon and Glyptotherium in order to identify diagnostic differences and potential synapomorphies. Both genera can be distinguished mainly by differences in the skull, mandible, dentition, dorsal carapace, and caudal armor, Glyptodon being somewhat larger than Glyptotherium. Both clades show a highly conservative evolution, which could be interpreted as an anagenesis. The scarce records of glyptodonts in Central America show more morphological affinity with Glyptotherium than with Glyptodon.     

A New Fishfly Species (Megaloptera: Corydalidae: Neohermes Banks) Discovered from North America by a Systematic Revision,with Phylogenetic and Biogeographic Implications

The taxonomy of Megaloptera from the Nearctic region is fairly well known and their faunal diversity has been largely surveyed, even in relatively remote regions. However, the evolutionary history of Nearctic Megaloptera is still poorly known with phylogenetic and biogeographic studies lacking. In this paper, we report a new fishfly species of the endemic North American genus Neohermes Banks, 1908, increasing the total number known of species to six. This new species (Neohermes inexpectatus sp. nov.) is currently known to occur only in California (USA) and is apparently confined to the Northern Coastal Range. The new species resembles the three Neohermes species from eastern North America based on the relatively small body size and the presence of female gonostyli 9. However, our phylogenetic analysis using adult morphological data recovered the new species as the sister species to the remaining Neohermes, which includes two species from western North America and three from eastern North America. According to the present interspecific phylogeny of Neohermes, with reconstructed ancestral areas, the initial divergence within the genus was found to take place in western North America, with a subsequent eastward dispersal. This likely lead to the modern distribution of Neohermes in eastern North America with the closure of the Mid-Continental Seaway, which separated western and eastern North America in the Mid-Late Cretaceous (100–80 MYA) and finally disappeared at the end of the Cretaceous (70 MYA). The uplift of the Cordilleran System probably accounted for the divergence between the eastern and two western Neohermes species.     

Phylogenetic position of a remarkable new fideliine bee from northern Chile (Hymenoptera: Megachilidae)

Fideliine bees are an archaic group with a disjunct distribution mostly restricted to deserts of South America and South Africa. This group was previously thought to be more diverse in Africa than in South America, where only one genus (Neofidelia) comprising five species is known. Here we describe a species belonging to a second South American genus: Xenofidelia colorada Packer gen. et sp.n. , from northern Chile. The species is illustrated and its phylogenetic position within Megachilidae is assessed using morphological, molecular and combined data. The 214 character morphological matrix includes 55 new characters with an additional 16 hitherto unexplored for megachilid phylogeny. The molecular dataset is based upon seven nuclear gene sequences, totalling 6439 bp, many of which are published for the first time for particular megachilid taxa. In all analyses, Xenofidelia was found as sister to Neofidelia (endemic to Chile and Peru). It differs from that genus most notably in its short mouthparts, absence of a glossal rod, unmodified female metabasitarsus and an elongate and horizontal dorsal surface of the metapostnotum. Morphological and combined data support a monophyletic Fideliinae (excluding Pararhophites), while molecular data alone failed to recover fideliine monophyly. Dating analyses suggest that Xenofidelia and Neofidelia diverged 34.3–40.6 Ma, indicating that New World fideliines were probably present in arid habitats of South America during the Eocene. This divergence time predates both the main orogenic events that resulted in the formation of the Andean mountains and the origin of hyperarid conditions in the Atacama Desert; it also corresponds to a period prior to the origin of the summer rainfall area in the far north of Chile where the new genus is found. These results support the view that arid habitats have been present continuously in South America since the Eocene. This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:EA69BB4A‐6F59‐4A15‐AB44‐2A8949E3CF8F .     

Phylogeny and taxonomy of the damselfly genus Enallagma and related taxa (Odonata: Zygoptera: Coenagrionidae)

Abstract The zygopteran genus Enallagma has been the subject of numerous behavioural and ecological studies, but phylogenetic relationships among species have been examined only within eastern North America, and even the composition and diagnosis of the genus are unclear on a world-wide basis. Most authorities currently recognize about seventy species within Enallagma , comprising two major radiations, in North America and Africa. This study, using morphological data, demonstrates that the North American and a few related Palaearctic species form a monophyletic group that is quite distinct from the African species. The latter are themselves divided into at least three, and probably four, separate clades, one of which may be related to E. parvum of India. Consequently, three of Kennedy's long disused genera, Africallagma , Amphiallagma and Proischnura ( Kennedy, 1920 ) are resurrected and two new genera, Azuragrion gen.n. and Pinheyagrion gen.n. are established for the remaining African taxa. Finally, Enallagma is divided into two subgenera, Enallagma s.s ., the typical 'bluets', including many North American, Holarctic and Palaearctic species, and Chromatallagma subgen.n., comprising a group of species of more variable colour that is confined to North America, the Caribbean and northernmost South America.     

Phylogeny of Dendroctonus bark beetles (Coleoptera: Curculionidae: Scolytinae) inferred from morphological and molecular data

Bark beetles in the genus Dendroctonus may attack and kill several species of coniferous trees, some of them causing major economic losses in temperate forests throughout North and Central America. For this reason, they have been widely studied. However, various aspects of the taxonomy and evolutionary history of the group remain contentious. The genus has been subdivided in species groups according to morphological, biological, karyological or molecular attributes, but the evolutionary affinities among species and species groups within the genus remain uncertain. In this study, phylogenetic relationships among Dendroctonus species were reassessed through parsimony‐based cladistic analysis of morphological and DNA sequence data. Phylogenetic inference was based on 36 morphological characters and on mitochondrial DNA sequences of the cytochrome oxidase I (COI) gene. Analyses were carried out for each dataset, as well as for the combined data analysed simultaneously, under equal and implied weights. According to the combined analysis, the genus Dendroctonus is a monophyletic group defined by at least three synapomorphic characters and there are four main lineages of varied composition and diversity within the genus. Within these lineages, several monophyletic groups match, to some extent, species groups defined by previous authors, but certain groups proposed by those authors are polyphyletic or paraphyletic.     

Phylogenetic relationships in Ephedra (Ephedraceae) inferred from chloroplast and nuclear DNA sequences

Sequences of the nuclear ribosomal DNA internal transcribed spacer region 1 and the chloroplast-encoded genes maturase K and ribulose-1,5 biphosphate carboxylase large subunit were obtained from species of Ephedra (Ephedraceae) representing the geographic range and morphological diversity of the genus. Phylogenetic analyses of the DNA data indicate that relationships within the genus are better predicted by geographic region of origin than by ovulate cone characters. The sampled species with dry, winged (versus fleshy) ovulate cone bracts or single-seeded cones do not form monophyletic groups and therefore the previous classification systems of Ephedra based on these aspects of bract morphology appear to be largely unnatural. Three groups were identified among the Old World species studied, one comprising European and Mediterranean species and two including only Asian species. The sequence data suggest a possible early divergence of a New World clade of Ephedra from among the Old World groups. The South American species form a distinct clade apparently related to one of two groups of North American species, which accords with a frequent floristic pattern of close relationships between species groups in western South America and southwestern North America.