A chloroplast genealogy of hordeum (poaceae): Long-term persisting haplotypes, incomplete lineage sorting, regional extinction, and the consequences for phylogenetic inference

To analyze reasons for inconclusive results of earlier chloroplast phylogenies in the grass genus Hordeum, we established a genealogy of chloroplast haplotypes by sequencing the trnL-trnF region in 875 individuals, covering all 31 species of the genus. Although the outcomes of phenetic and parsimony analyses of 88 haplotypes were ambiguous, a network approach showed that in Hordeum ancient chloroplast types co-occur with their descendants. Moreover, we found up to 18 different chloroplast haplotypes within a single species and up to 6 species sharing single haplotypes. Persisting polymorphisms together with incomplete lineage sorting occurred preferentially in the rapidly speciating New World taxa of the genus, where ancient chloroplast types have survived for at least 4 Myr. Lineages-through-time plots and a high number of missing chloroplast haplotypes indicated far-reaching extinction of chloroplast lineages in Europe and particularly the Mediterranean. Survival of these lineages in East Asia and North America resulted in chloroplast relationships that markedly differed from nuclear estimations of species relationships. Thus, even for the deepest splits in the genus, reaching back more than 9 Myr, no safe phylogenetic inference from chloroplast data is possible in Hordeum. The chloroplast genealogy, however, revealed biogeographic patterns and indicated processes involved in speciation in Hordeum. We conclude that the described phenomena are not restricted to Hordeum and that the knowledge of the chloroplast relationships within a genus is indispensable to prevent misinterpretation of phylogeographic data within single species.     

Isolation and characterization of 11 new microsatellite markers for Macaranga (Euphorbiaceae)

We provide primer sequences for 11 new polymorphic microsatellite markers developed in the tropical ant-plant genus Macaranga (Euphorbiaceae), after enrichment cloning of Macaranga tanarius and Macaranga hypoleuca. Allele numbers per locus ranged from two to 16 among 20 accessions of M. tanarius, and from three to 10 among 22 accessions of M. hypoleuca. Observed and expected heterozygosities ranged from 0.150 to 0.900 and from 0.375 to 0.894 in M. tanarius, and from 0.545 to 1.000 and from 0.434 to 0.870 in M. hypoleuca, respectively. Six of the 11 primer pairs successfully cross-amplified polymorphic polymerase chain reaction products in Macaranga winkleri.     

Production of food bodies on the reproductive organs of myrmecophytic Macaranga species (Euphorbiaceae): effects on interactions with herbivores and pollinators

In protective ant–plant mutualisms, plants offer ants food (such as extrafloral nectar and/or food bodies) and ants protect plants from herbivores. However, ants often negatively affect plant reproduction by deterring pollinators. The aggressive protection that mutualistic ants provide to some myrmecophytes may enhance this negative effect in comparison to plant species that are facultatively protected by ants. Because little is known about the processes by which myrmecophytes are pollinated in the presence of ant guards, we examined ant interactions with herbivores and pollinators on plant reproductive organs. We examined eight myrmecophytic and three nonmyrmecophytic Macaranga species in Borneo. Most of the species studied are pollinated by thrips breeding in the inflorescences. Seven of eight myrmecophytic species produced food bodies on young inflorescences and/or immature fruits. Food body production was associated with increased ant abundance on inflorescences of the three species observed. The exclusion of ants from inflorescences of one species without food rewards resulted in increased herbivory damage. In contrast, ant exclusion had no effect on the number of pollinator thrips. The absence of thrips pollinator deterrence by ants may be due to the presence of protective bracteoles that limit ants, but not pollinators, from accessing flowers. This unique mechanism may account for simultaneous thrips pollination and ant defense of inflorescences.     

A taxonomic reevaluation of freshwater ostracods (Crustacea,Ostracoda) referred to the genus Cypridopsis Brady, 1868 from Southeast Asia

A taxonomic reevaluation of five species of Southeast Asian freshwater ostracods formerly assigned to the genus Cypridopsis Brady, 1868 has resulted in the retention of Cypridopsis adusta Sars, 1903, Cypridopsis exigua Sars, 1903 and Cypridopsis dubia Sars, 1903 in Cypridopsis and transfer of Cypridopsis albida (Vavra) 1898 and Cypridopsis arsenia Tressler, 1937 to the genus Plesiocypridopsis Rome, 1965.     

History, ocean channels, and distance determine phylogeographic patterns in three widespread Philippine fruit bats (Pteropodidae)

The comparative phylogeography of widespread, codistributed species provides unique insights into regional biodiversity and diversification patterns. I used partial DNA sequences of the mitochondrial genes ND2 and cyt b to investigate phylogeographic structure in three widespread Philippine fruit bats. Ptenochirus jagori is endemic to the oceanic region of the Philippines and is most abundant in lowland primary forest. Macroglossus minimus and Cynopterus brachyotis are most common in disturbed and open habitats and are not endemic. In all three, genetic differentiation is present at multiple spatial scales and is associated to some degree with Pleistocene landbridge island groups. In P. jagori and C. brachyotis, genetic distance is correlated with geographic distance; in C. brachyotis and M. minimus, it is correlated with the sea-crossing distance between islands. P. jagori has the least overall genetic structure of these three species, whereas C. brachyotis and M. minimus have more geographic association among haplotypes, suggesting that phylogeographic patterns are linked to ecology and habitat preference. However, contrary to expectation, the two widespread, disturbed habitat species have more structure than the endemic species. Mismatch distributions suggest rapid changes in effective population size in C. brachyotis and P. jagori, whereas M. minimus appears to be demographically more stable. Geologic and geographic history are important in structuring variation, and phylogeographic patterns are the result of dynamic long-term processes rather than simply reflecting current conditions.     

Phylogeny of Cerberus (Serpentes: Homalopsinae) and phylogeography of Cerberus rynchops: diversification of a coastal marine snake in Southeast Asia

Aim The biogeography of Southeast Asia has been greatly affected by plate tectonic events over the last 10 Myr and changing sea levels during the Quaternary. We investigated how these events may have influenced the evolution of Cerberus Cuvier, a marine coastal snake belonging to the Homalopsinae (Oriental‐Australian Rear‐fanged Water Snakes). This study is an expansion of a previous study on the biogeography and systematics of Cerberus. Location We obtained species from localities across the range of the widely distributed Cerberus: India, Sri Lanka, the Andaman islands, Myanmar, the Philippines, Borneo, Suluwesi, Sumatra, Vietnam, Thailand, Singapore and Australia. Methods We analysed mtDNA sequences (12S, ND3, ATPase, 2338 nucleotide characters) from 21 localities. The sample consisted of 65 Cerberus rynchops (Schneider), three Cerberus australis (Gray) and four Cerberus microlepis Boulenger. One Homalopsis buccata (Linnaeus), one Bitia hydroides Gray, one Enhydris enhydris (Schneider), and two Enhydris plumbea (Boie) were used as outgroups. Results We produced phylogenetic trees based on parsimony, maximum likelihood and Bayesian analysis. We did not find unambiguous support for the monophly of Cerberus. Cerberus austalis, H. buccata and all other Cerberus populations formed a three‐way basal polytomy under parsimony and C. australis formed the sister group to a clade consisting of H. buccata and all other Cerberus in likelihood and Bayesian analysis. The non‐Australian Cerberus were monophyletic and consisted of four primary biogeographical clades: Indian and Mayanmar, Philippines, Greater Sunda Islands and Suluwesi, and the Thai‐Malay peninsula and Gulf of Thailand. The range of genetic divergence between these clades and Australian Cerberus was 0.06–0.12. Genetic divergence among clades to the west of Australia was less pronounced (Thai‐Malay peninsula and Gulf of Thailand = 0.02–0.05; Sunda Islands and Suluwesi = 0.02–0.05; Philippines = 0.02–0.06; India and Myanmar = 0.04–0.06, Philippines = 0.02–0.5). Main conclusions Gyi [University of Kansas Publications, Museum of Natural History 20 (1970), 47] recognized three species of Cerberus: C. australis (from Australia), C. microlepis (known only from Lake Buhi in the Philippines), and the widely distributed C. rynchops (India to Wallacea). We did not find strong support for the monophyly of the genus. Cerberus australis is highly divergent from all other Cerberus lineages sampled from this region. The geographically widespread C. rynchops is resolved into four biogeographical clades (Indian and Myanmar, Philippines, Greater Sunda Islands and Suluwesi, and the Thai‐Malay Peninsula and Gulf of Thailand). We discuss how the dispersal biology of a salt‐water tolerant, coastal marine taxon and the complex geological history of the region (Tertiary plate tectonic movements and Quaternary sea‐level changes) could produce the observed patterns of diversification.     

Extreme long-distance dispersal of the lowland tropical rainforest tree Ceiba pentandra L. (Malvaceae) in Africa and the Neotropics

Many tropical tree species occupy continental expanses of rainforest and flank dispersal barriers such as oceans and mountains. The role of long-distance dispersal in establishing the range of such species is poorly understood. In this study, we test vicariance hypotheses for range disjunctions in the rainforest tree Ceiba pentandra, which is naturally widespread across equatorial Africa and the Neotropics. Approximate molecular clocks were applied to nuclear ribosomal [ITS (internal transcribed spacer)] and chloroplast (psbB-psbF) spacer DNA sampled from 12 Neotropical and five West African populations. The ITS (N=5) and psbB-psbF (N=2) haplotypes exhibited few nucleotide differences, and ITS and psbB-psbF haplotypes were shared by populations on both continents. The low levels of nucleotide divergence falsify vicariance explanations for transatlantic and cross-Andean range disjunctions. The study shows how extreme long-distance dispersal, via wind or marine currents, creates taxonomic similarities in the plant communities of Africa and the Neotropics.     

Natural hybridization and introgression in sympatric Ligularia species (Asteraceae, Senecioneae)

The difficulty in clarifying species of genus Ligularia Cass.has been attributed to rapid and continuous allopatric speciation in small and isolated populations,combined with interspecific diploid hybridization in the Qinghai-Tibetan Plateau and adjacent areas.However,no concrete example has been reported to prove this hypothesis.We studied a natural mixed population of six species of Ligularia in which some individuals were morphologically intermediate between L.subspicata and L.nelumbifolia.Based on DNA sequences (trnC-E trnL-rpL32,trnQ5'rpsl6,trnK-rpsl6,and internal transcribed spacer) and inter-simple sequence repeat data,we concluded that putative hybrids are primarily products of hybridization between L.nelumbifolia and L.subspicata.The other four species or additional,unknown species may also be involved in hybridization.This hybridization is bidirectional but asymmetrical.Hybrid individuals were mostly the first generation,but F2 and later-generation hybrids were also present.Moreover,the backcrossed individuals detected indicate that natural gene flow occurs among at least three Ligularia species.Hybrids may become stabilized to form new species or may function as intermediates in evolutionary diversification.     

Chloroplast DNA phylogeography of European ashes, Fraxinus sp. (Oleaceae): roles of hybridization and life history traits

We investigated range-wide phylogeographic variation in three European ash species (Fraxinus sp., Oleaceae). Chloroplast DNA (cpDNA) microsatellites were typed in the thermophilous Fraxinus angustifolia and Fraxinus ornus and the observed haplotypes and the geographic distribution of diversity were compared to cpDNA data previously obtained in the more cold-tolerant Fraxinus excelsior. We found wide-ranging haplotype sharing between the phylogenetically close F. angustifolia and F. excelsior, suggesting hybridization (i) in common glacial refuges in the Iberian Peninsula, northern Italy, the eastern and/or Dinaric Alps and the Balkan Peninsula, and/or (ii) during postglacial recolonization. The data allowed us to propose additional glacial refuges for F. angustifolia in southern Italy and in Turkey, and populations from the latter region were particularly polymorphic. There was evidence for refuge areas in Italy, the Balkan Peninsula and Turkey for F. ornus, which did not share any single chloroplast haplotype with the other species. In both F. angustifolia and F. ornus, cpDNA diversity (h(S) = 0.027 and h(S) = 0.009, respectively) was lower and fixation levels (G(ST) = 0.964 and G(ST) = 0.983, respectively) higher than in sympatric F. excelsior (h(S) = 0.096, G(ST) = 0.870). These diversity patterns could be due to temperature tolerance or the demographic history.     

Using patterns of genetic structure based on microsatellite loci to test hypotheses of current hybridization,ancient hybridization and incomplete lineage sorting in Conradina (Lamiaceae)

Hybridization and/or incomplete sorting of ancestral polymorphism are commonly implicated to explain discordant phylogenetic analyses of closely related species complexes. One genus in which these phenomena have been suggested to have played major roles based on phylogenetic data is Conradina, a genus of mints (Lamiaceae) endemic to the southeastern USA containing several endangered species. The goals of this study were to use microsatellite data to better understand patterns of genetic structure in Conradina, to test hypotheses of recent or ancient hybridization and incomplete lineage sorting, and to clarify species boundaries. Individuals from 55 populations representing all Conradina species were genotyped using 10 microsatellite loci. Analyses of the patterns of genetic structure in Conradina revealed a clear differentiation of populations following recognized species boundaries, indicating that species have diverged from one another genetically and interspecific hybridization has not occurred recently. Neither ancient hybridization nor incomplete lineage sorting is supported as the sole cause of species nonmonophyly, suggesting that both may have contributed to patterns found in phylogenetic trees; however, analyses of other types of data may be more appropriate to distinguish between these two hypotheses. Because all described species appear to be valid entities, the current listing status of most endangered species of Conradina is appropriate; however, populations of Conradina canescens are genetically differentiated into three groups, each of which may merit species status, and several recently discovered populations of Conradina in Dunn's Creek State Park in Florida are highly differentiated genetically and also appear to represent a new species.     

Biogeographical implications of mitochondrial DNA variation in the Bockadam snake (Cerberus rynchops, Serpentes: Homalopsinae) in Southeast Asia

Aim The biogeography of Southeast Asia has been greatly affected by Pleistocene sea‐level changes and the consequent alteration of coastline and land mass configurations. We investigated the effect of these Pleistocene events on genetic divergence in Cerberus rynchops, the Bockdam, an Asian water snake (Homalopsinae) associated with tidal mudflats and coastal mangrove forests in Southeast Asia. Location Localities for the Cerberus included the Andaman sea coast of Thailand, Sumatra, and Borneo (Sunda Shelf localities), the Philippines and Sulawesi, and from northern Australia (Sahul Shelf). Methods We analysed mtDNA sequences (12 s, 16 s, and cyt b) from seven C. rynchops populations (twenty‐six specimens), from two specimens of Cerberus microlepis (a freshwater species known only from Lake Buhi in the Philippines), and from one Enhydris enhyris (Schneider), another homalopsine used as an outgroup. Results We found a strong correlation between genetic divergence and geographical distance (r = 0.922, P < 0.001) and the biogeographic history of the region. Cerberus rynchops populations from the Sunda Shelf localities, the Philippines, Sulawesi and C. microlepis from the Philippines were genetically similar (mean divergence = 2.1%, range = 0.7–2.8%) compared to the C. rynchops population from northern Australia (mean divergence from all other Cerberus populations = 6.6%, range = 6.0–7.3%). This divergence was comparable to that observed between the E. enhydris outgroup and all Cerberus populations (mean distance = 7.3%, range = 6.8–8.6%). Main conclusions These findings suggest a relatively high degree of movement and gene flow among Sunda Shelf localities, the Philippines, and Sulawesi (biogeographic region west of Weber’s Line) and isolation of the northern Australian (Sunda Shelf) Cerberus. Taxa will be differentially affected by these Pleistocene sea level changes dependent on their physiology and ecology. We discuss how the dispersal of the coastal, saltwater tolerant C. rynchops would have been affected by changing configurations of Pleistocene coastlines and the implications of these results for the systematics of Cerberus.     

A phylogenetic and geotectonic scenario to explain the biogeography of the Lophopidae (Hemiptera, Fulgoromorpha)

The Lophopidae are found in South America, Africa, Australia, India and Southeast Asia. This distribution appears to be typically Gondwanan, triggered by tectonic events beginning over 100 Ma. However, within the Fulgoromorpha, the lophopids are considered to be relatively recently. In this study, biological, geological and phylogenetic information is evaluated to provide a parsimonious explanation for the distribution of the group and its geographic region of ancestral origin. The Lophopidae can be divided into four monophyletic groups. The ancestors of two groups appear to have originated somewhere along the western Pacific island arc system. Another group appears to have an origin in Southeast Asia. A reliable explanation for the ancestral origin of the fourth group was not possible because it consists of only one genus present in Central and South America. A biogeographic map of the two groups of lophids of the western Pacific island arc is concordant with their phylogeny based on biological and morphological data. Based on this finding, the best explanation for the origin and evolution of the Lophopidae and their current distribution of these lophopids is through vicariance. Similar types of eco-evolutionary events explain radiation and distribution of the Lophopidae, in general.     

Onset of cryptic vicariance in the Japanese dormouse Glirulus japonicus (Mammalia, Rodentia) in the Late Tertiary, inferred from mitochondrial and nuclear DNA analysis

The sequences of the mitochondrial cytochrome b gene and restriction site variation in the spacer region of the nuclear ribosomal RNA gene [rDNA-restriction fragment length polymorphism (RFLP)] were analysed to determine the phylogeographic structure of the Japanese dormouse ( Glirulus japonicus ), which is threatened by deforestation and has been designated an endangered species in Japan. The phylogenetic tree of cytochrome b grouped G. japonicus into six geographical populations: north-eastern Honshu (I), central Honshu (II), west-central Honshu/Kii Peninsula (III), western Honshu (IV), Shikoku (V), and westernmost Honshu/Kyushu (VI); the genetic distances among these groups suggest divergence in the Late Tertiary. The lineage of group VI was located at the basal position in the phylogenetic tree, followed by the radiation of the other lineages. An rDNA-RFLP analysis of 15 restriction sites roughly supported such genetic isolation; groups I, II, III, IV, V and VI have five, two, one, one, one and four unique restriction sites, respectively, revealing four geographic groups as cryptic species: I, II, III + IV + V and VI. Our results reveal the ancient divergences of the local population, which has a complicated evolutionary history, and should be useful in developing a framework for the conservation of this species.     

Taxonomic Notes on Nasutitermes and Bulbitermes (Termitidae, Nasutitermitinae) from the Sunda region of Southeast Asia based on morphological and molecular characters

The Sunda region of Southeastern Asia is rich in termite fauna, but termites from this region have been poorly described. In this study, we described eight species from two diverse genera from this region, and from the family Termitidae. We describe Bulbitermes 4 spp. and Nasutitermes 4 spp. from new field collections. Where possible we examine original holotype specimens, and describe the essential morphological characters for soldier and worker castes. We devise two new bifurcating keys to guide the field identification of each species. In addition, we develop a nucleotide sequence profile for the COI gene. From this molecular character matrix, we use Neighbour-Joining analysis to test the monophyly of each morphospecies and genus. We find that the morphological and molecular characters are highly concordant, whereby all taxa appear to represent distinct molecular clades. For termites, there is therefore agreement between the morphological taxonomic characters used to sort species from a bifurcating key and the molecular taxonomic characters used to sort species on a bifurcating tree. This joint analysis suggests that DNA barcoding holds considerable promise for termite taxonomy, especially for diverse clades like Bulbitermes and Nasutitermes for which a global morphological key would be intractable.     

Phylogeography of the mountain chickadee (Poecile gambeli): diversification, introgression, and expansion in response to Quaternary climate change

Since the late 1990s, molecular techniques have fuelled debate about the role of Pleistocene glacial cycles in structuring contemporary avian diversity in North America. The debate is still heated; however, there is widespread agreement that the Pleistocene glacial cycles forced the repeated contraction, fragmentation, and expansion of the North American biota. These demographic processes should leave genetic 'footprints' in modern descendants, suggesting that detailed population genetic studies of contemporary species provide the key to elucidating the impact of the late Quaternary (late Pleistocene-Holocene). We present an analysis of mitochondrial DNA (mtDNA) variation in the mountain chickadee (Poecile gambeli) in an attempt to examine the genetic evidence of the impact of the late Quaternary glacial cycles. Phylogenetic analyses reveal two strongly supported clades of P. gambeli: an Eastern Clade (Rocky Mountains and Great Basin) and a Western Clade (Sierra Nevada and Cascades). Post-glacial introgression is apparent between these two clades in the Mono Lake region of Central California. Within the Eastern Clade there is evidence of isolation-by-distance in the Rocky Mountain populations, and of limited gene flow into and around the Great Basin. Coalescent analysis of genetic variation in the Western Clade indicates that northern (Sierra Nevada/Cascades) and southern (Transverse/Peninsular Ranges) populations have been isolated and evolving independently for nearly 60,000 years.     

Review of the Southeast Asian species of the Aenictus javanus and Aenictus philippinensis species groups (Hymenoptera, Formicidae, Aenictinae)

The Southeast Asian species of the Aenictus javanus and Aenictus philippinensis groups are revised. Six species (four named and two new species) of the Aenictus javanus group occurring in this area are: Aenictus doydeei Jaitrong & Yamane, 2011, Aenictus duengkaei Jaitrong & Yamane, sp. n., Aenictus javanus Emery, 1896, Aenictus longinodus Jaitrong & Yamane, sp. n., Aenictus nishimurai Terayama & Kubota, 1993, and Aenictus piercei Wheeler & Chapman, 1930. Four species (three named and one new species) are recognized in the Aenictus philippinensis group: Aenictus pangantihoni Zettel & Sorger, 2010, Aenictus philippinensis Chapman, 1963, Aenictus punctatus Jaitrong & Yamane, sp. n., and Aenictus rabori Chapman, 1963. Aenictus piercei is removed from the members of the Aenictus piercei group sensu Jaitrong and Yamane (2011) and transferred to the Aenictus javanus group. Lectotypes and paralectotypes are designated for Aenictus piercei and Aenictus rabori. Size variation occurs among individuals from single colonies of the Aenictus javanus group, while the workers in the Aenictus philippinensis group are clearly monomorphic.     

Phylogeny, divergence times and species limits of spiny lizards (Sceloporus magister species group) in western North American deserts and Baja California

The broad distribution of the Sceloporus magister species group (squamata: phrynosomatidae) throughout western North America provides an appropriate model for testing biogeographical hypotheses explaining the timing and origins of diversity across mainland deserts and the Baja California Peninsula. We inferred concordant phylogenetic trees describing the higher-level relationships within the magister group using 1.6 kb of mitochondrial DNA (mtDNA) and 1.7 kb of nuclear DNA data. These data provide strong support for the parallel divergence of lineages endemic to the Baja California Peninsula (S. zosteromus and the orcutti complex) in the form of two sequential divergence events at the base of the magister group phylogeny. A relaxed phylogenetic analysis of the mtDNA data using one fossil and one biogeographical constraint provides a chronology of these divergence events and evidence that further diversification within the Baja California clades occurred simultaneously, although patterns of geographical variation and speciation between clades differ. We resolved four major phylogeographical clades within S. magister that (i) provide a novel phylogenetic placement of the Chihuahuan Desert populations sister to the Mojave Desert; (ii) illustrate a mixed history for the Colorado Plateau that includes Mojave and Sonoran Desert components; and (iii) identify an area of overlap between the Mojave and Sonoran Desert clades near Yuma, Arizona. Estimates of bidirectional migration rates among populations of S. magister using four nuclear loci support strong asymmetries in gene flow among the major mtDNA clades. Based on the nonexclusivity of mtDNA haplotypes, nuclear gene flow among populations and wide zones of phenotypic intergradation, S. magister appears to represent a single geographically variable and widespread species.     

A morphological study and hybridization analysis of Caloglossa leprieurii (Ceramiales, Rhodophyta) from Japan, Singapore and Australia

Morphological and hybridization experiments were performed on Caloglossa leprieurii (Montagne) J. Agardh collected from Japan, Singapore and Australia in order to evaluate taxonomic characters of this species. Within C. leprieurii at least four mating groups were recognized from the Indo-Pacific region. These mating groups could be characterized by the blade width at the inter-node and the cell-row numbers on the opposite side derived from the first axial cell at the main axis, though these properties showed a certain variability even in the same plant under both field and culture conditions. The phylogenetic relationship and geographic distribution of the four mating groups are discussed.