Mitochondrial phylogeography of the long-eared bats (Plecotus) in the Mediterranean Palaearctic and Atlantic Islands

Long-eared bats of the genus Plecotus are widespread and common over most of the western Palaearctic. Based on recent molecular evidence, they proved to represent a complex of several cryptic species, with three new species being described from Europe in 2002. Evolutionary relationships among the different lineages are still fragmentary because of the limited geographic coverage of previous studies. Here we analyze Plecotus mitochondrial DNA sequences from the entire Mediterranean region and Atlantic Islands. Phylogenetic reconstructions group these western Palaearctic Plecotus into two major clades which split at least 5 Myr ago and that are each subdivided into further subgroups. An 'auritus group' includes the traditional P. auritus species and its sister taxon P. macrobullaris (=P. alpinus) plus related specimens from the Middle East. P. auritus and P. macrobullaris have broadly overlapping distributions in Europe, although the latter is apparently more restricted to mountain ranges. The other major clade, the 'austriacus group,' includes the European species P. austriacus and at least two other related taxa from North Africa (including P. teneriffae from the Canary Islands), the Balkans and Anatolia (P. kolombatovici). The sister species of this 'austriacus group' is P. balensis, an Ethiopian endemic. Phylogenetic reconstructions further suggest that P. austriacus reached Madeira during its relatively recent westward expansion through Europe, while the Canary Islands were colonized by a North African ancestor. Although colonization of the two groups of Atlantic Islands by Plecotus bats followed very distinct routes, neither involved lineages from the 'auritus group.' Furthermore, the Strait of Gibraltar perfectly segregates the distinct lineages, which confirms its key role as a geographic barrier. This study also stresses the biogeographical importance of the Mediterranean region, and particularly of North Africa, in understanding the evolution of the western Palaearctic biotas.     

Conflicting molecular phylogenies of European long-eared bats (Plecotus) can be explained by cryptic diversity

Conflicting phylogenetic signals of two data sets that analyse different portions of the same molecule are unexpected and require an explanation. In the present paper we test whether (i) differential evolution of two mitochondrial genes or (ii) cryptic diversity can better explain conflicting results of two recently published molecular phylogenies on the same set of species of long-eared bats (genus Plecotus). We sequenced 1714bp of three mitochondrial regions (16S, ND1, and D-loop) of 35 Plecotus populations from 10 European countries. A likelihood ratio test revealed congruent phylogenetic signals of the three data partitions. Our phylogenetic analyses demonstrated that the existence of a previously undetected Plecotus lineage caused the incongruities of previous studies. This lineage is differentiated on the species level and lives in sympatry with its sister lineage, Plecotus auritus, in Switzerland and Northern Italy. A molecular clock indicates that all European Plecotus species are of mid or late Pliocene origin. Plecotus indet. was previously described as an intergrade between P. auritus and Plecotus austriacus since it shares morphological characters with both. It is currently known from elevations above 800 m a.s.l. in the Alps, the Dinarian Alps and the Pindos mountains in Greece. Since we could demonstrate that incongruities of two molecular analyses simply arose from the mis-identification of one lineage, we conclude that molecular phylogenetic analyses do not free systematists from a thorough inclusion of morphological and ecological data.     

Molecular phylogeny of the Acre clade (Crassulaceae): Dealing with the lack of definitions for Echeveria and Sedum

The phylogenetic relationships within many clades of the Crassulaceae are still uncertain, therefore in this study attention was focused on the “Acre clade”, a group comprised of approximately 526 species in eight genera that include many Asian and Mediterranean species of Sedum and the majority of the American genera (Echeveria, Graptopetalum, Lenophyllum, Pachyphytum, Villadia, and Thompsonella). Parsimony and Bayesian analyses were conducted with 133 species based on nuclear (ETS, ITS) and chloroplast DNA regions (rpS16, matK). Our analyses retrieved four major clades within the Acre clade. Two of these were in a grade and corresponded to Asian species of Sedum, the rest corresponded to a European–Macaronesian group and to an American group. The American group included all taxa that were formerly placed in the Echeverioideae and the majority of the American Sedoideae. Our analyses support the monophyly of three genera – Lenophyllum, Thompsonella, and Pachyphytum; however, the relationships among Echeveria, Sedum and the various segregates of Sedum are largely unresolved. Our analyses represents the first broad phylogenetic framework for Acre clade, but further studies are necessary on the groups poorly represented here, such as the European and Asian species of Sedum and the Central and South American species of Echeveria.     

Ensifer mexicanus sp. nov. a new species nodulating Acacia angustissima (Mill.) Kuntze in Mexico

A new lineage of Ensifer nodulating the American legume Acacia angustissima in the tropical forest of Chiapas and Morelos, Mexico is described. Bacteria were identified as Ensifer with ssb or nolR specific primers. Phylogenetic analysis with partial sequences of the five chromosomal genes gyrA, nolR, recA, rpoB and rrs revealed that this new lineage is related to African Ensifer terangae. The results of total DNA-DNA hybridization and selected phenotypic tests among the A. angustissima strains and E. terangae indicated that they belong to different species. The phylogeny with the symbiotic nifH gene also separates this group as a different clade but with close affinities to bacteria belonging to the genus Ensifer isolated from American hosts. ITTG R7(T) (=CFN ER1001, HAMBI 2910, CIP 109033, ATCC BAA-1312, DSM18446) is the type strain of a new species for which the name Ensifer mexicanus sp. nov. is proposed.     

A molecular phylogeny of the nightjars (Aves: Caprimulgidae) suggests extensive conservation of primitive morphological traits across multiple lineages

We report a molecular re-assessment of the classification of the nightjars which draws conclusions that are strongly at odds with the traditional, morphology-based classifications. We used maximum likelihood and Bayesian methods to compare the cytochrome b gene for 14 species from seven of the 15 genera of the Caprimulgidae and partial cytochrome b sequence data was available for a further seven species including three further genera. We found that within the Caprimulgidae there were four geographically isolated clades with bootstrap support greater than 70%. One of these clades contained just Chordeiles species, the remaining three clades each contained a mixture of genera including Caprimulgus sp. A clade of exclusively South American nightjars included the genera Caprimulgus, Uropsalis, Eleopthreptus and Hydropsalis. A clade of African and Eurasian birds included Caprimulgus and Macrodipteryx. Phalaenoptilus nuttallii and Caprimulgus vociferous formed a clade of North American birds. Two ecological factors appear to make morphological classification potentially misleading: first, the apparent retention of primitive anti-predator and foraging-related traits across genetically divergent groups; second, rapid divergence in other traits, especially those related to mating, which generate high levels of morphological divergence between species that are genetically very similar. The cytochrome b data suggests that the genus Caprimulgus is not monophyletic and is restricted to Africa and Eurasia and that Caprimulgus species from outside this area have been misclassified as a consequence of retention of primitive adaptations for crepuscular/nocturnal living. Some other genera also appear to have little support from the cytochrome b data.     

Molecular systematics of Acarus siro s. lat., a complex of stored food pests

The astigmatid mite Acarus siro (Linnaeus 1758) is an important agricultural pest and environmental allergen. However, it is likely that many mites described in the literature as A. siro, collected from both outdoor and stored product habitats, may belong to one of its sibling species, A. farris [Ent. Ber. Amst. 2 (26) (1905) 20] or A. immobilis [Bull. Br. Mus. Nat. Hist. 11 (1964a) 413; Acarologia. 6 (Suppl) (1964) 101]. The three species are difficult to separate morphologically, gene exchange between some of them is possible and, although each species displays environmental preferences, they occur together in some environments. This raises a question about their separate species status. In a pilot study, we investigated whether genetic data supported the separate species status of these forms. Both nuclear (the second internal transcribed spacer region [ITS-2] of the ribosomal cistron) and mitochondrial (cytochrome oxidase subunit I, mtcoxI hereafter) loci were employed for this purpose. Mtcox1 data does not conflict the differentiation into three separate species and while the ITS2 data were problematic for this group of mites it suggested that a congener, Acarus gracilis [Ann. Mag. Nat. Hist. 10 (1957) 753], is basal to the A. siro species complex.     

Coevolution of Cryptosporidium tyzzeri and the house mouse (Mus musculus)

Two house mouse subspecies occur in Europe, eastern and northern Mus musculus musculus (Mmm) and western and southern Mus musculus domesticus (Mmd). A secondary hybrid zone occurs where their ranges meet, running from Scandinavia to the Black Sea. In this paper, we tested a hypothesis that the apicomplexan protozoan species Cryptosporidium tyzzeri has coevolved with the house mouse. More specifically, we assessed to what extent the evolution of this parasite mirrors divergence of the two subspecies. In order to test this hypothesis, we analysed sequence variation at five genes (ssrRNA, Cryptosporidium oocyst wall protein (COWP), thrombospondin-related adhesive protein of Cryptosporidium 1 (TRAP-C1), actin and gp60) in C. tyzzeri isolates from Mmd and Mmm sampled along a transect across the hybrid zone from the Czech Republic to Germany. Mmd samples were supplemented with mice from New Zealand. We found two distinct isolates of C. tyzzeri, each occurring exclusively in one of the mouse subspecies (C. tyzzeri-Mmm and C. tyzzeri-Mmd). In addition to genetic differentiation, oocysts of the C. tyzzeri-Mmd subtype (mean: 4.24 × 3.69 μm) were significantly smaller than oocysts of C. tyzzeri-Mmm (mean: 4.49 × 3.90 μm). Mmm and Mmd were susceptible to experimental infection with both C. tyzzeri subtypes; however, the subtypes were not infective for the rodent species Meriones unguiculatus, Mastomys coucha, Apodemus flavicollis or Cavia porcellus. Overall, our results support the hypothesis that C. tyzzeri is coevolving with Mmm and Mmd.     

Phylogeography and species boundaries of the western North American Nightsnake (Hypsiglena torquata): revisiting the subspecies concept

The subspecies concept has received considerable debate throughout the past century. Subspecies were originally used to delineate potential incipient species, but were later employed to simply capture geographical variation. There is a recent trend to eliminate the trinomial in light of new evidence. Discrete, diagnosable lineages are elevated to specific status, while those that show clinal variation and/or appear to represent ecological pattern classes are placed in synonymy with the parent species and the subspecific epithets are disregarded. Here, I examine the species boundaries of nightsnakes (Hypsiglena torquata) using standard phylogeographic methods and mtDNA data from 178 individuals. Previously, seventeen subspecies of H. torquata were described. In this study, I recognize six species in what was previously considered H. torquata: one is novel, two were previously recognized subspecies, while the remaining three are wide-spread, polymorphic lineages, composed of multiple subspecies. I make the case to maintain the subspecific lineages in these wide-ranging species because they are geographically cohesive, morphologically discrete, and may represent incipient species within each complex, which have not yet achieved speciation. These subspecies are maintained, not only pending future investigations, but because they provide a useful identity for the taxonomy of this diverse lineage.     

星穗莎属，缅甸莎草科一新记录属

在近期缅甸植物考察中,发现1种莎草科(Cyperaceae)植物:云南星穗莎[Actinoschoenus yunnanensis (C. B. Clarke) Y. C.Tang]。该种植物代表缅甸莎草科1个新记录属:星穗莎属(Actinoschoenus Benth.),其主要特征为花序头状、小穗具1(2)花、花两性、雄蕊3、柱头3、小坚果倒卵形有3棱。     

Cryptic diversity in European bats

Different species of bat can be morphologically very similar. In order to estimate the amount of cryptic diversity among European bats we screened the intra- and interspecific genetic variation in 26 European vespertilionid bat species. We sequenced the DNA of subunit 1 of the mitochondrial protein NADH dehydrogenase (ND1) from several individuals of a species, which were sampled in a variety of geographical regions. A phylogeny based on the mitochondrial (mt) DNA data is in good agreement with the current classification in the family. Highly divergent mitochondrial lineages were found in two taxa, which differed in at least 11% of their ND1 sequence. The two mtDNA lineages in Plecotus austriacus correlated with the two subspecies Plecotus austriacus austriacus and Plecotus austriacus kolombatovici. The two mtDNA lineages in Myotis mystacinus were partitioned among two morphotypes. The evidence for two new bat species within Europe is discussed. Convergent adaptive evolution might have contributed to the morphological similarity among distantly related species if they occupy similar ecological niches. Closely related species may differ in their ecology but not necessarily in their morphology. On the other hand, two morphologically clearly different species (Eptesicus serotinus and Eptesicus nilssonii) were found to be genetically very similar. Neither morphological nor mitochondrial DNA sequence analysis alone can be guaranteed to identify species.     

吉林省发现长尾鼠耳蝠

在吉林省通化市采集到长尾鼠耳蝠(Myotis frater)样本9只,为吉林省蝙蝠科新纪录,鉴定为长尾亚种M.f.longicaudatus.本文给出了该蝙蝠的特征描述和相关测量数据,并与文献记录进行了比较.     

Molecular genetics of <Emphasis Type="Italic">Cicindela (Cylindera) terricola</Emphasis> and elevation of <Emphasis Type="Italic">C. lunalonga</Emphasis> to species level,with comments on its conservation status

Cicindela (Cylindera) terricola Say is one of the most widespread and variable species of Nearctic Cicindelidae with six recognized subspecies. Cicindela t. lunalonga Schaupp (1884) is known from few museum specimens collected prior to 1979. The goal of this study was to resolve the uncertain taxonomic status of C. t. lunalonga using mitochondrial DNA analysis of cytochrome b and cytochrome oxidase subunit I and determine its conservation status. In phylogenetic reconstruction using distance and parsimony methods, all members of the terrricola group were recovered as monophyletic and embedded within outgroup species of the subgenus Cylindera, while C. lunalonga was recovered as sister to all other members of the C. terricola clade. Cicindela lunalonga exhibited an exceptionally high (mean of 6.36%) pairwise sequence divergence for both genes against all C. terricola surveyed. For the cytochrome oxidase subunit I alone the pairwise divergence was 3.9–4.8% (4.58% avg.). The lowest divergences were between C. lunalonga and C. terricola subspecies of the American southwest (C. t. cinctipennis and C. t. kaibabensis), rather than with the closest geographic neighbors (C. t. imperfecta). We conclude that based on strict monophyly and pairwise sequence divergence, C. lunalonga is a distinct species. Our study of museum specimens and extensive field surveys suggest this species has been extirpated from all sites in the San Joaquin Valley and perhaps all but one of the historic sites throughout its range. Thus, it should be considered for Federal listing as an endangered species.     

疏花长柄山蚂蝗复合体(豆科)的修订

通过文献、标本研究,结合野外考察及数据统计分析,确认疏花长柄山蚂蝗复合体包括4种1亚种,讨论了各分类群的重要性状,并给出了新的检索表。将湘西长柄山蚂蝗(Hylodesmum laxum subsp.falfolium)处理为原亚种的新异名;订正了《中国植物志》和《Flora of China》等著作中该类群的几个错误的地理分布,记载了我国几个省区的分布新记录,并给出了凭证标本。     

Cenozoic biogeography and evolution in direct-developing frogs of Central America (Leptodactylidae: Eleutherodactylus) as inferred from a phylogenetic analysis of nuclear and mitochondrial genes

We report the first phylogenetic analysis of DNA sequence data for the Central American component of the genus Eleutherodactylus (Anura: Leptodactylidae: Eleutherodactylinae), one of the most ubiquitous, diverse, and abundant components of the Neotropical amphibian fauna. We obtained DNA sequence data from 55 specimens representing 45 species. Sampling was focused on Central America, but also included Bolivia, Brazil, Jamaica, and the USA. We sequenced 1460 contiguous base pairs (bp) of the mitochondrial genome containing ND2 and five neighboring tRNA genes, plus 1300 bp of the c-myc nuclear gene. The resulting phylogenetic inferences were broadly concordant between data sets and among analytical methods. The subgenus Craugastor is monophyletic and its initial radiation was potentially rapid and adaptive. Within Craugastor, the earliest splits separate three northern Central American species groups, milesi, augusti, and alfredi, from a clade comprising the rest of Craugastor. Within the latter clade, the rhodopis group as formerly recognized comprises three deeply divergent clades that do not form a monophyletic group; we therefore restrict the content of the rhodopis group to one of two northern clades, and use new names for the other northern (mexicanus group) and one southern clade (bransfordii group). The new rhodopis and bransfordii groups together form the sister taxon to a clade comprising the biporcatus, fitzingeri, mexicanus, and rugulosus groups. We used a Bayesian MCMC approach together with geological and biogeographic assumptions to estimate divergence times from the combined DNA sequence data. Our results corroborated three independent dispersal events for the origins of Central American Eleutherodactylus: (1) an ancestor of Craugastor entered northern Central America from South American in the early Paleocene, (2) an ancestor of the subgenus Syrrhophus entered northern Central America from the Caribbean at the end of the Eocene, and (3) a wave of independent dispersal events from South America coincided with formation of the Isthmus of Panama during the Pliocene. We elevate the subgenus Craugastor to the genus rank.     

A new record and an evaluation of the phylogenetic relationships of Abrothrix olivaceus markhami (Rodentia: Sigmodontinae)

Recent phylogenetic studies of the subfamily of cricetid rodents (Sigmodntinae) have validated the taxonomic classification at the tribal level of the Andean Clade. It is possible that some endemic species from Patagonian South America are part of this new tribe, but previous studies have not evaluated this hypothesis due to the difficulty of obtaining samples. In this study, we evaluate the phylogenetic relationships of Akodon markhami (Pine, 1973), a species endemic to the Chilean Patagonia, using individuals recently captured at the type locality of this taxon. Our results indicate that this form of Akodon corresponds to a subspecies of Abrothrix olivaceus, and should be incorporated into the Andean Clade as a geographic race of this widely distributed species on the South American continent. Based on a molecular clock calibration, we dated the origin of this geographic race during the last glacial cycles of the Quaternary, as the result of a vicariant process.     

Molecular systematics and phylogeography of the gigantic earthworms of the Metaphire formosae species group (Clitellata, Megascolecidae)

The earthworms of the Metaphire formosae species group distributed in Taiwan are members of the Pheretima complex within the Megascolecidae. In this study, the systematics and phylogeography of this species group were investigated using DNA sequences of mitochondrial cytochrome c oxidase subunit I (COI), 16S ribosomal (r)RNA, and NADH dehydrogenase subunit 1 (ND1). The results indicated that the 13 taxa of the M. formosae species group form a clade, including a cryptic species discovered in this study. In addition, Metaphire hengchunensis (James, S.W., Shih, H.-T., Chang, H.-W., 2005. Seven new species of Amynthas (Clitellata: Megascolecidae) and new earthworm records from Taiwan. J. Nat. Hist. 39, 1007-1028) should be regarded as a subspecies of Metaphire paiwanna Tsai, C.-F., Shen, H.-P., Tsai, S.-C., 2000a. Native and exotic species of terrestrial earthworm (Oligochaeta) in Taiwan with reference to Northeast Asia. Zool. Stud. 39, 285-294, Tsai, C.-F., Tsai, S.-C., Liaw, G.-J., 2000b. Two new species of pontandric pheretimoid earthworms belonging to the genus Metaphire (Megascolecidae: Oligochaeta) from Taiwan. J. Nat. Hist. 34, 1731-1741, and Metaphire bununa glareosa Tsai et al. 2000 should be elevated to specific status. Phylogeographical inferences showed that allopatric speciation occurred in this species group during the rapid uplift of the main island of Taiwan between 5.0 and 2.5 million years ago. Our analysis exposes non-monophyly within each of the genera Amynthas and Metaphire, and more generally within the Pheretima complex. Further revisions of this speciose complex are urgently needed.     

Genetic nature of eastern wolves: Past, present and future

Eastern North American wolves have long been recognized as morphologically distinct from both coyotes and gray wolves. This has led to questions regarding their origins and taxonomic status. Eastern wolves are mainly viewed as: (1) a smaller subspecies of gray wolf (Canis lupus lycaon), potentially the result of historical hybridization between gray wolves (C. lupus) and red wolves (C. rufus), (2) a hybrid, the result of gray wolf (C. lupus) and coyote (C. latrans) interbreeding, or (3) a distinct species, C. lycaon, closely related to the red wolf (C. rufus). Although debate persists, recent molecular studies suggest that the eastern wolf is not a gray wolf subspecies, nor the result of gray wolf/coyote hybridization. Eastern wolves were more likely a distinct species, C. lycaon, prior to the eastward spread of coyotes in the late 1800s. However, contemporary interbreeding exits between C. lycaon to both C. lupus and C. latrans over much of its present range complicating its present taxonomic characterization. While hybridization may be reducing the taxonomic distinctiveness of C. lycaon, it should not necessarily be viewed as negative influence. Hybridization may be enhancing the adaptive potential of eastern wolves, allowing them to more effectively exploit available resources in rapidly changing environments.     

Cophylogeny and disparate rates of evolution in sympatric lineages of chewing lice on pocket gophers

Although molecular-based phylogenetic studies of hosts and parasites are increasingly common in the literature, no study to date has examined two congeneric lineages of parasites that live in sympatry on the same lineage of hosts. This study examines phylogenetic relationships among chewing lice (Phthiraptera: Trichodectidae) of the Geomydoecus coronadoi and Geomydoecus mexicanus species complexes and compares these to phylogenetic patterns in their hosts (pocket gophers of the rodent family Geomyidae). Sympatry of congeneric lice provides a natural experiment to test the hypothesis that closely related lineages of parasites will respond similarly to the same host. Sequence data from the mitochondrial COI and the nuclear EF-1alpha genes confirm that the two louse complexes are reciprocally monophyletic and that individual clades within each species complex parasitize a different species of pocket gopher. Phylogenetic comparisons reveal that both louse complexes show a significant pattern of cophylogeny with their hosts. Comparisons of rates of nucleotide substitution at 4-fold degenerate sites in the COI gene indicate that both groups of lice have significantly higher basal mutation rates than their hosts. The two groups of lice have similar basal rates of mutation, but lice of the G. coronadoi complex show significantly elevated rates of nucleotide substitution at all sites. These rate differences are hypothesized to result from population-level phenomena, such as effective population size, founder effects, and drift, that influence rates of nucleotide substitution.     

GyrB sequence analysis and MALDI-TOF MS as identification tools for plant pathogenic Clavibacter

The bacterial genus Clavibacter has only one species, Clavibacter michiganensis, containing five subspecies. All five are plant pathogens, among which three are recognized as quarantine pests (mentioned on the EPPO A2 list). Prevention of their introduction and epidemic outbreaks requires a reliable and accurate identification. Currently, identification of these bacteria is time consuming and often problematic, mainly because of cross-reactions with other plant-associated bacteria in immunological tests and false-negative results in PCR detection methods. Furthermore, distinguishing closely related subspecies is not straightforward. This study aimed at evaluating the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and a fragment of the gyrB sequence for the reliable and fast identification of the Clavibacter subspecies. Amplification and sequencing of gyrB using a single primer set had sufficient resolution and specificity to identify each subspecies based on both sequence similarities in cluster analyses and specific signatures within the sequences. All five subspecies also generated distinct and reproducible MALDI-TOF MS profiles, with unique and specific ion peaks for each subspecies, which could be used as biomarkers for identification. Results from both methods were in agreement and were able to distinguish the five Clavibacter subspecies from each other and from representatives of closely related Rathayibacter, Leifsonia or Curtobacterium species. Our study suggests that proteomic analysis using MALDI-TOF MS and gyrB sequence are powerful diagnostic tools for the accurate identification of Clavibacter plant pathogens.