Comparative Phylogeography in Rainforest Trees from Lower Guinea,Africa

Comparative phylogeography is an effective approach to assess the evolutionary history of biological communities. We used comparative phylogeography in fourteen tree taxa from Lower Guinea (Atlantic Equatorial Africa) to test for congruence with two simple evolutionary scenarios based on physio-climatic features 1) the W-E environmental gradient and 2) the N-S seasonal inversion, which determine climatic and seasonality differences in the region. We sequenced the trnC-ycf6 plastid DNA region using a dual sampling strategy: fourteen taxa with small sample sizes (dataset 1, mean n = 16/taxon), to assess whether a strong general pattern of allele endemism and genetic differentiation emerged; and four taxonomically well-studied species with larger sample sizes (dataset 2, mean n = 109/species) to detect the presence of particular shared phylogeographic patterns. When grouping the samples into two alternative sets of two populations, W and E, vs. N and S, neither dataset exhibited a strong pattern of allelic endemism, suggesting that none of the considered regions consistently harboured older populations. Differentiation in dataset 1 was similarly strong between W and E as between N and S, with 3–5 significant F _ST tests out of 14 tests in each scenario. Coalescent simulations indicated that, given the power of the data, this result probably reflects idiosyncratic histories of the taxa, or a weak common differentiation pattern (possibly with population substructure) undetectable across taxa in dataset 1. Dataset 2 identified a common genetic break separating the northern and southern populations of Greenwayodendron suaveolens subsp. suaveolens var. suaveolens, Milicia excelsa, Symphonia globulifera and Trichoscypha acuminata in Lower Guinea, in agreement with differentiation across the N–S seasonal inversion. Our work suggests that currently recognized tree taxa or suspected species complexes can contain strongly differentiated genetic lineages, which could lead to misinterpretation of phylogeographic patterns. Therefore the evolutionary processes of such taxa require further study in African tropical rainforests.     

Comparative chloroplast and nuclear DNA analysis of Castanea species in the southern region of the USA

Boundaries between American Castanea species (Castanea dentata, the American chestnut and C. pumila var. pumila, the Allegheny chinkapin, and var. ozarkensis, the Ozark chinkapin) have been difficult to establish because of intraspecific variation, interspecific similarities and the incidence of chestnut blight, which has prevented trees from maturing. In this study, informative chloroplast (cp) DNA and nuclear sequences from Castanea taxa were analyzed to gain a better understanding of their phylogeography in North America. Our emphasis has been on the most southern Castanea population in the Appalachian region, known for its morphological diversity. This Ruffner Mountain (Alabama) population shows a high number of unique haplotypes, which can be divided into two main groups. One group shares homology with the widespread and evolutionarily recent C. dentata haplotype. The other group shares homology with American chestnuts and Allegheny chinkapin taxa from southern states. This group has been the result of recent and more ancient cp capture and hybridization, indicative of hybrid zone clustering and glacial refugial origin. The range of C. pumila must have been more extensive along the Coastal Plains region, since only a few mutations separate the Ozark chinkapin from the main Allegheny chinkapin haplotype. The geographic origin of the American Castanea species complex appears to be in the Gulf Coast region.     

Intraspecific classification of Ficus deltoidea Jack subsp. deltoidea (Moraceae) in Peninsular Malaysia based on morphological and molecular variations

Taxonomic classification of the important medicinal plant, Ficus deltoidea (Moraceae), is challenging because of the variability of its leaves and fig forms that occur within the species. We developed 16 nuclear Simple Sequence Repeat (nSSR) markers, and characterized them using 24 individuals from a natural population. We then studied the intraspecific variation of F. deltoidea subsp. deltoidea in Peninsular Malaysia using morphological and molecular approaches. Based on the morphological variations, we further determined the varieties that occur regionally under the above subspecies based on the leaf characteristics. As for molecular data sets, we used both chloroplast DNA (cpDNA) and nSSR markers to elucidate the phylogenetic relationship among the varieties. The cpDNA dendrogram yielded poorer resolution where most of the clades were forming paraphyletic complex. The cluster analysis based on nSSR is largely congruent with the morphological classification, with F. deltoidea subsp. deltoidea classified into four main varieties, namely var. deltoidea, var. angustifolia, var. kunstleri and var. lutescens. Our study demonstrates the applicability of molecular approach in complementing the conventional taxonomic classification.     

Dispersal across southern Iberian refugia? integrating RAPDs, sequence data and morphometrics inArmeria (plumbaginaceae)

A southern Spanish massif (Tejeda/Almijara range, Málaga province, SE Spain) has been previously identified as a contact zone for genotypes of a rare taxon,Armeria villosa subsp.bernisii, and a frequent one,A. filicaulis, based on (1) the discovery of a species-independent geographically structured pattern of variation for nuclear ribosomal ITS sequence data, and (2) the sharing of chloroplast haplotypes, which reveal horizontal transfer between the species. This study uses RAPD data, as a total DNA marker, and morphometrics, as potentially revealing hybridization and introgression, to throw further light on the origin of the above mentioned contact zone. Individuals of the two taxa sampled from the range do not show a F1 hybrid profile for RAPD or for morphometrics. To integrate these results with the previously published sequence data (ITS and chloroplast spacertrnL-F) it is proposed that introgressive hybridization has occurred inA. villosa subsp.bernisii, whereas forA. filicaulis the contact zone occurs at the intraspecific level. With the available data, the contact between individuals ofArmeria with different genotypes in the two taxa may have implied westward migration from a biodiverse massif like Sierra Nevada, and this may apply to other organisms although further data are needed to confirm it.     

Limited structure and widespread diversity suggest potential buffers to genetic erosion in a threatened grassland shrub Pimelea spinescens (Thymelaeaceae)

Pimelea spinescens is a critically endangered species of the temperate grasslands of southeastern Australia. Two subspecies are recognised. Subspecies, P. spinescens subsp. spinescens, formerly common and widespread, is found in isolated remnants of previously extensive grasslands. The second subspecies, subsp. pubiflora, is thought to have been historically rare with only two geographically-isolated extant populations. The grassland communities exist now as fragmented remnants representing <1 % of their extent prior to European settlement in the early 1800s. Conservation management strategies for species in these critically endangered ecosystems rely on an understanding of genetic diversity and population structure to ensure long-term evolutionary potential. We used chloroplast DNA (cpDNA) and microsatellite markers to examine the population genetic structure of both subspecies. Analysis of cpDNA revealed 14 haplotypes with high divergence between the single haplotype found in subsp. pubiflora and most remaining haplotypes restricted to subsp. spinescens. Microsatellites also indicated high genetic differentiation between subspecies but little evidence of sub-structuring within either subspecies. Results suggest that seed dispersal has not been as limited as previously thought. In this fragmented habitat, a lack of genetic structure suggests buffers to genetic erosion, with current patterns reflecting genetic diversity prior to fragmentation. Plant longevity and the presence of seed banks may contribute to the maintenance of these patterns, resulting in a lag between fragmentation and genetic erosion. Whilst factors such as longevity and seed banks may be preserving historic genetic diversity, management is required to ensure the maintenance of this diversity into the future.     

Genetic variation in Mediterranean Helichrysum italicum (Asteraceae; Gnaphalieae): do disjunct populations of subsp. microphyllum have a common origin?

The yellow‐flowered everlasting daisy Helichrysum italicum (Asteraceae, Gnaphalieae) is widely distributed in the Mediterranean basin, where it grows in continuous and widespread populations in diverse open habitats. Helichrysum italicum subsp. microphyllum has a disjunct distribution in the Balearic Islands (Majorca and Dragonera), Corsica, Sardinia, Crete and Cyprus. Numerous morphological intermediates between subsp. italicum and subsp. microphyllum are known from Corsica, where the two subspecies co‐occur. The aims of the study were to investigate if subsp. microphyllum has a common origin, constituting an independent gene pool from subsp. italicum, or if the morphological differences between subsp. microphyllum and subsp. italicum have arisen independently in different locations from a common wider gene pool. Our analyses of AFLP, cpDNA sequences and morphological characters show that there is geographic structure to the genetic variation within H. italicum, with eastern and western Mediterranean groups, which do not correspond with the division into subsp. microphyllum and subsp. italicum as currently circumscribed. Local selection on quantitative trait loci provides sufficient explanation for the morphological divergence observed and is consistent with genetic data. Within the western Mediterranean group of the species we found considerable polymorphism in chloroplast DNA sequences among and within some populations. Comparison with chloroplast DNA sequences from other Helichrysum species showed that some chloroplast haplotypes are shared across species.     

Plio-Pleistocene history and phylogeography of Acacia senegal in dry woodlands and savannahs of sub-Saharan tropical Africa: evidence of early colonisation and recent range expansion

Drylands are extensive across sub-Saharan Africa, socio-economically and ecologically important yet highly sensitive to environmental changes. Evolutionary history, as revealed by contemporary intraspecific genetic variation, can provide valuable insight into how species have responded to past environmental and population changes and guide strategies to promote resilience to future changes. The gum arabic tree (Acacia senegal) is an arid-adapted, morphologically diverse species native to the sub-Saharan drylands. We used variation in nuclear sequences (internal transcribed spacer (ITS)) and two types of chloroplast DNA (cpDNA) markers (PCR-RFLP, cpSSR) to study the phylogeography of the species with 293 individuals from 66 populations sampled across its natural range. cpDNA data showed high regional and rangewide haplotypic diversity (h_T(cpSSR)=0.903–0.948) and population differentiation (G_ST(RFLP)=0.700–0.782) with a phylogeographic pattern that indicated extensive historical gene flow via seed dispersal. Haplotypes were not restricted to any of the four varieties, but showed significant geographic structure (G_ST(cpSSR)=0.392; R_ST=0.673; R_ST>R_ST (permuted)), with the major division separating East and Southern Africa populations from those in West and Central Africa. Phylogenetic analysis of ITS data indicated a more recent origin for the clade including West and Central African haplotypes, suggesting range expansion in this region, possibly during the Holocene humid period. In conjunction with paleobotanical evidence, our data suggest dispersal to West Africa, and across to the Arabian Peninsula and Indian subcontinent, from source populations located in the East African region during climate oscillations of the Plio-Pleistocene.     

Multiple lines of evidence supports the two varieties of Halenia elliptica (Gentianaceae) as two species

Delimiting species requires multiple sources of evidence. Here, we delimited two varieties of Halenia elliptica (Gentianaceae) using several lines of evidence, including morphological traits and mating system in a sympatric population, phylogenetic relationships based on nrITS and cpDNA (rpl16) data, and complete chloroplast genome sequences. Comparative analysis of 21 morphological traits clearly separates the two varieties of H. elliptica. Examination of the flowering process and pollination treatments indicate that H. elliptica var. grandiflora produces seeds via outcrossing, whereas H. elliptica var. elliptica produces seeds via mixed mating. Furthermore, hand-pollinated hybridization of the two varieties produced no seeds. Observations of pollinators showed that when bees began a pollination bout on H. elliptica var. grandiflora they preferred to continue pollinating this variety; however, when they began a pollination bout on H. elliptica var. elliptica, they showed no preference for either variety. Phylogenetic analysis confirmed the monophyly of H. elliptica, which was further divided into two monophyletic clades corresponding to the two varieties. A large number of variants from the chloroplast genomes reflected remarkable genetic dissimilarities between the two varieties of H. elliptica. We recommend that the two varieties of H. elliptica should be revised as two species (H. elliptica and H. grandiflora). Our findings indicate that H. elliptica varieties may have split into two separate species due to a shift in mating system, changes in flowering phenology and/or post-pollination reproductive isolation.     

Mitochondrial DNA variation and reticulate evolution of the genus Abies

The sequences of three regions of mitochondrial DNA (mtDNA) of a total length of 5226 bp were used to study the phylogeography of the genus Abies. The mtDNA haplotype network, comprising 36 studied Abies taxa, consisted of two branches; the first represented all American species plus two Asian, and the second included the remaining Eurasian species. Within these clusters, the haplotypes formed nine major groups, generally corresponding to the clades of the previously obtained phylogeny based on chloroplast DNA (cpDNA), but the relationships of these groups were significantly different; species assignment to the particular mtDNA haplotype group was more in line with its geographical distribution. In addition, the mtDNA haplotype network contains cycles indicating the recombination. It is assumed that the incongruence of cpDNA and mtDNA phylogenies is caused by the introgression capture of alien mtDNA during species hybridization and thus contains information about past migrations. The cases of incongruence of mitochondrial and chloroplast DNA suggesting a migration of Abies between Asia and North America are discussed.     

Taxonomic revision of the Chilean Puya species (Puyoideae, Bromeliaceae), with special notes on the Puya alpestris-Puya berteroniana species complex

The Chilean Puya species (Puyoideae, Bromeliaceae) constitute an early diverging group within the genus and thus are key taxa for the understanding of the early evolution and biogeography of Puya. However, a modern taxonomic treatment including information from molecular phylogenetic studies is still lacking. Here, a taxonomic revision of the Chilean species of Puya is presented based on morphological, molecular, and biogeographic data. A re-evaluation of the widely applied concept of P. berteroniana led us to the conclusion that the type of P. berteroniana is of hybrid origin and is maintained as Puya × berteroniana. Our studies revealed that the name P. berteroniana has been widely misapplied to what in fact is the northern metapopulation of P. alpestris, which is here described as a new subspecies, Puya alpestris subsp. zoellneri, a member of subgenus Puya. A lectotype is designated for Puya copiapina and a neotype for P. chilensis. Altogether, six Puya species, two subspecies, four varieties and one hybrid taxon are recognized for the Chilean flora in this revision: P. alpestris subsp. alpestris and subsp. zoellneri; P. boliviensis; P. chilensis; P. gilmartiniae; P. coerulea var. coerulea, var. intermedia, var. monteroana, and var. violacea; P. venusta; and P. × berteroniana. A key is provided for their identification.     

The Phenotype of a Virescent Chloroplast Mutation in Tobacco Is Associated with the Absence of a 37.5 kD Thylakoid Polypeptide

The Vir-c mutation is a virescent chloroplast mutation found in a line of plants derived from protoplast fusions between a Nicotina tabacum line and a line containing N. tabacum nuclei with Nicotiana suaveolens cytoplasm. Vir-c displays a lag period in chlorophyll accumulation and granal stack formation in young leaves. We examined total chloroplast protein in young leaves and showed the mutant contains 1.3 to 2.1 times less stromal protein, and 2.9 to 4.3 times less thylakoid protein when compared to the N. tabacum var “Turkish Samsun” control. Electrophoretic patterns of total thylakoid proteins indicated three polypeptides were specifically decreased in amount within the context of the overall reduction in thylakoid protein. Electrophoresis of thylakoid proteins synthesized by chloroplasts isolated from half-expanded leaves demonstrated that mutant chloroplasts did not synthesize a 37.5 kilodalton polypeptide which was synthesized by “Samsun” chloroplasts. A polypeptide of this molecular weight was synthesized by Vir-c chloroplasts isolated from mature leaves which had recovered the normal phenotype. Restriction digestion and electrophoresis of the mutant's chloroplast DNA produced a pattern of restriction fragments different from either N. tabacum or N. suaveolens chloroplast DNA.     

Acacia gum exudates from species of the series gummiferae

An analytical study of the gum exudates from the African species Acacia ehrenbergiana (three specimens), A.xanthophloea (two specimens), A.hockii and A.sieberana var.villosa, and of the Australian species A.calcigera, has been made. There are now 19 species within the series Gummiferae Benth. for which gum parameters are available; of these, only A. ehrenbergiana gum displays a slightly negative optical rotation. The data for the three gum specimens from A. ehrenbergiana give a further example of the extent to which the gum from different trees of one particular species can vary in composition. The data for A.sieberana var. villosa gum are compared with the values established previously for subsp. sieberana; the differences between varieties of one species are similar in extent to those established for some subspecies. Although A. xanthophloea, A. hockii, A. ehrenbergiana, A. seyal and A. karroo are regarded as being very closely related botanically, the values for some of their analytical parameters differ considerably and strongly support the view that it is correct to retain them as distinct species.     

Global chloroplast phylogeny and biogeography of bracken (Pteridium; Dennstaedtiaceae)

Bracken ferns (genus Pteridium) represent an ancient species complex with a natural worldwide distribution. Pteridium has historically been treated as comprising a single species, but recent treatments have recognized several related species. Phenotypic plasticity, geographically structured morphological variation, and geographically biased sampling have all contributed to taxonomic confusion in the genus. We sampled bracken specimens worldwide and used variable regions of the chloroplast genome to investigate phylogeography and reticulate evolution within the genus. Our results distinguish two major clades within Pteridium, a primarily northern hemisphere Laurasian/African clade, which includes all taxa currently assigned to P. aquilinum, and a primarily southern hemisphere Austral/South American clade, which includes P. esculentum and P. arachnoideum. All European accessions of P. aquilinum subsp. aquilinum appear in a monophyletic group and are nested within a clade containing the African P. aquilinum taxa (P. aquilinum subsp. capense and P. aquilinum subsp. centrali-africanum). Our results allow us to hypothesize the maternal progenitors of two allotetraploid bracken species, P. caudatum and P. semihastatum. We also discuss the biogeography of bracken in the context of the chloroplast phylogeny. Our study is one of the first to take a worldwide perspective in addressing variation in a broadly distributed species complex.     

Chloroplast evidence for geographic stasis of the Australian bird‐dispersed shrub Tasmannia lanceolata (Winteraceae)

Few chloroplast‐based genetic studies have been undertaken for plants of mesic temperate forests in the southern hemisphere and fossil‐based models have provided evidence of vegetation history only at the broadest scales in this region. This study investigates the chloroplast DNA phylogeography of Tasmannia lanceolata (Winteraceae), a fleshy‐fruited, bird‐dispersed shrub that is widespread in the mountains of southeastern Australia and Tasmania. Thirty haplotypes were identified after sequencing 3206 bp of chloroplast DNA in each of 244 individuals collected across the species’ range. These haplotypes showed unexpectedly strong phylogeographic structuring, including a phylogeographic break within a continuous part of the species’ range, with the distribution of four major clades mostly not overlapping, and geographic structuring of haplotypes within these clades. This strong geographic patterning of chloroplast DNA provided evidence for the survival of T. lanceolata in multiple putative wet forest refugia as well as evidence for additional wet forest species refugia in southeastern Australia. In western Tasmania lower haplotype diversity below the LGM tree line compared to above the LGM tree line suggests that glacial refugia at high altitudes may have been important for T. lanceolata. The level of geographic structuring in T. lanceolata is similar to gravity dispersed southern hemisphere plants such as Nothofagus and Eucalyptus. Behavioural traits of the birds transporting seed may have had a strong bearing on the limited transport of T. lanceolata seed, although factors limiting establishment, possibly including selection, may also have been important.     

Genomic fingerprinting versus nuclear gene sequences: A comparative approach for studying the Lupinus montanus (Fabaceae) species complex

The taxonomy and systematics of Mexican Lupinus are lacking in resolution, because the taxa are distinguished using a few minor and inconsistent morphological characters. The use of molecular markers can contribute to resolving such issues. In this study, we focused on two varieties of the Lupinus montanus complex (Fabaceae) in Mexico, L. montanus subsp. montanus var. montanus and L. montanus subsp. montanus var. nelsonii, and aimed to determine the most suitable genetic markers for clarifying taxon delimitation based on morphology. We compared hypervariable Inter Simple Sequence Repeats (ISSR) to gene sequences (External Transcribed Spacer (ETS) and Conserved Orthologous Sets (COS)).Distance analysis (ISSR) and maximum likelihood (ETS + COS) generated congruent results but with much more variability and resolution for ISSR. These data confirm the potential of ISSR for working at low taxonomic level and their reliability compared to that of gene sequences.     

Shared phylogeographic patterns and widespread chloroplast haplotype sharing in Eucalyptus species with different ecological tolerances

We examined the phylogeography of three south-east Australian trees (Eucalyptus delegatensis, Eucalyptus obliqua, and Eucalyptus regnans) with different tolerances, in terms of cold, drought, fire and soil to explore whether species with different ecologies share major phylogeographic patterns. A second aim of this study was to examine geographic patterns of chloroplast DNA (cpDNA) haplotype sharing among the three study species. Trees of E. delegatensis (n?=?120), E. obliqua (n?=?265) and E. regnans (n?=?270) were genotyped with five cpDNA microsatellite markers. The species shared major phylogeographic disjunctions, and common patterns at proposed glacial refugia (generally high haplotype diversity) and areas thought to have been treeless during the Last Glacial Maximum (LGM) (low diversity). Inter-specific sharing of haplotypes was extensive, and fixation of shared, regional haplotypes was more frequent in areas postulated as having been treeless at the LGM. Despite ecological differences, chloroplast microsatellite data suggest the three species have responded to past climatic changes in a similar way, by persisting in multiple, generally common refugia. We suggest that the natural ability of eucalypt species to hybridise with others with quite different or broader ecological tolerances may provide an “insurance policy” for response to rapidly changing abiotic conditions.     

Colour polymorphism in common primrose (Primula vulgaris Huds.): many colours–many species?

Primula vulgaris exhibits flower colour polymorphism in the eastern part of its range, especially pronounced on the NE coast of the Black Sea. This polymorphism in the Caucasian populations has been taxonomically described and some segregated species are listed as rare and endangered. We used sequence variation in two chloroplast noncoding regions (trnL–trnF and rpll32–trnL) and the complete nuclear internal transcribed spacer (ITS) of ribosomal DNA region to investigate correspondence between flower colour and geographical distribution of both nuclear and chloroplast haplotypes. It appears that variability in these DNA regions does not correlate with flower colour, being, however, clearly structured geographically. We used nested clade analysis to explore this geographical structure. It seems that the territory of the Colchis refugium on the E coast of the Black Sea contains both the highest flower colour and haplotype diversities. The results suggest that common primroses colonized the NE coast of the Black Sea from this refugium, spreading along the coast westward. At the same time, the analysis of ITS haplotypes indicates that P. vulgaris colonized the Crimea from NW Anatolia. This makes it clear that no segregated species can be recognized within flower colour polymorphic P. vulgaris in the Caucasus region. However, its phylogeography needs further detailed study on a broader scale.     

Towards a legal framework for systematic conservation: identification and development of allele-specific PCR markers for conspecific varieties of an endangered perennial herb <Emphasis Type="Italic">Primula kisoana</Emphasis> Miquel based on sequence variation of chloroplast DNA

Primula kisoana var. kisoana (Primulaceae) is a narrow endemic found only in extremely restricted areas of central Honshu Island, Japan. Although the species is included as ‘Critically Endangered (CR)’ in the latest Japanese Red List, it is not covered by legislation such as the ‘Law for the Conservation of Endangered Species of Wild Fauna and Flora, Japan’. This poor conservation status is due, largely, to the presence of another conspecific variety, var. shikokiana, which is regarded as less threatened than var. kisoana. In this study, we investigated the genetic dissimilarity between the two varieties by examining sequence variation in three noncoding regions of chloroplast DNA. Ten distinct haplotypes were detected, none of which were distributed across populations and varieties. The genetic differentiation between the two varieties revealed by an AMOVA and a minimum spanning network suggests that var. kisoana and var. shikokiana should be conserved and managed as separate units. Using the allele-specific PCR method, four primer pairs were developed at the point mutations and insertion/deletion loci that exhibited only inter-variety variation. These markers will facilitate reliable identification of var. kisoana, assisting in the implementation of restoration programs including the introduction of seedlings and construction of a legal framework for conservation.     

Essential oil constituents and RAPD markers to establish species relationship in Cymbopogon Spreng. (Poaceae)

Nineteen Cymbopogon taxa belonging to 11 species, two varieties, one hybrid taxon and four unidentified species were analysed for their essential oil constituents and RAPD profiles to determine the extent of genetic similarity and thereby the phylogenetic relationships among them. Remarkable variation was observed in the essential oil yield ranging from 0.3% in Cymbopogon travancorensis Bor. to 1.2% in Cymbopogon martinii (Roxb.) Wats var. motia. Citral, a major essential oil constituent, was employed as the base marker for chemotypic clustering. Based on genetic analysis, elevation of Cymbopogon flexuosus var. microstachys (Hook. F.) Soenarko to species status and separate species status for C. travancorensis Bor., which has been merged under C. flexuosus (Steud.) Wats were suggested towards resolving some of the taxonomic complexes in Cymbopogon. The separate species status for the earlier proposed varieties of C. martinii (motia and sofia) is further substantiated by these analyses. The unidentified species of Cymbopogon have been observed as intermediate forms in the development of new taxa.