Phylogeographical structure inferred from cpDNA sequence variation of <Emphasis Type="Italic">Zygophyllum xanthoxylon</Emphasis> across north-west China

Zygophyllum xanthoxylon, a desert species, displaying a broad east–west continuous distribution pattern in arid Northwestern China, can be considered as a model species to investigate the biogeographical history of this region. We sequenced two chloroplast DNA spacers (psbK-psbI and rpl32-trnL) in 226 individuals from 31 populations to explore the phylogeographical structure. Median-joining network was constructed and analysis of AMOVA, SMOVA, neutrality tests and distribution analysis were used to examine genetic structure and potential range expansion. Using species distribution modeling, the geographical distribution of Z. xanthoxylon was modeled during the present and at the Last Glacial Maximum (LGM). Among 26 haplotypes, one was widely distributed, but most was restricted to either the eastern or western region. The populations with the highest levels of haplotype diversity were found in the Tianshan Mountains and its surroundings in the west, and the Helan Mountains and Alxa Plateau in the east. AMOVA and SAMOVA showed that over all populations, the species lacks phylogeographical structure, which is speculated to be the result of its specific biology. Neutrality tests and mismatch distribution analysis support past range expansions of the species. Comparing the current distribution to those cold and dry conditions in LGM, Z. xanthoxylon had a shrunken and more fragmented range during LGM. Based on the evidences from phylogeographical patterns, distribution of genetic variability, and paleodistribution modeling, Z. xanthoxylon is speculated most likely to have originated from the east and migrated westward via the Hexi Corridor.     

Spatial patterns and intraspecific diversity of the glacial relict legume species <Emphasis Type="Italic">Vavilovia formosa</Emphasis> (Stev.) Fed. in Eurasia

Vavilovia formosa is one of five genera in tribe Fabeae, (Fabaceae, Leguminosae) with close phylogenetic relationships to Pisum. It grows in subalpine and alpine levels in Armenia, Azerbaijan, Georgia, Iran, Iraq, Lebanon, Russia and Turkey and is recognized as an endangered and protected plant. This study was conducted to reveal its intraspecific variability, as well as to predict the past, extant and future species distribution range. We analysed 51 accessions with common phylogenetic markers (trnF-trnL, trnS-trnG, matK, rbcL, psbA-trnH and ITS). These represent in total up to 2551 bp of chloroplast and 664 bp of nuclear sequences per sample. Two populations from Turkey and Armenia were analysed for genetic diversity by AFLP. Leaf morphometry was conducted on 1457 leaflets from 43 specimens. Extracted bioclimatic parameters were used for niche-modelling approach. Analysis of cpDNA revealed two haplotypes, 12 samples from Armenia, Daghestan, Nakhichevan and Iran belonged to H1 group, while 39 samples of all Turkish and part of Armenian were in H2 group. The mean intrapopulation diversity based on AFLP was low (H _E = 0.088) indicating limited outcrossing rate. A significantly positive correlation between geographical latitude and leaf area (\(\rho\) = 0.527, p < 0.05) was found. Niche modelling has shown temporal variation of predicted occurrence across the projected time periods. Vavilovia formosa has suffered a range reduction following climate warming after last glacial maximum, which classify this species as cold-adapted among the Fabeae species as well as a glacial relict.     

Molecular phylogenetic analysis of key <Emphasis Type="Italic">Jatropha</Emphasis> species inferred from nrDNA ITS and chloroplast (<Emphasis Type="Italic">trn</Emphasis>L-F and <Emphasis Type="Italic">rbc</Emphasis>L) sequences

The genus Jatropha (Euphorbiaceae) contains species that are of significant economic and ornamental value. However, Jatropha breeding material is rather limited due to incomplete information regarding phylogenetic relationships among germplasm resources. Phylogenetic analyses were performed based on the internal transcribed spacer of nuclear ribosomal DNA (nrDNA ITS), two chloroplast regions (trnL-F and rbcL), and the combined (ITS+trnL-F+rbcL) dataset among twenty-five specimens representing six key Jatropha species. Phylogenetic relationships of Jatropha were well resolved between subgenus Curcas and subgenus Jatropha, and demonstrated the intermediate position of section Polymorphae among sections of both subgenera. Jatropha curcas and J. integerrima demonstrated a close phylogenetic relationship. The molecular data agreed with the morphological classification that recognized J. multifida and J. podagrica in sec. Peltatae. The distinct intraspecific divergence that occurred in J. curcas could be attributed to restricted gene flow caused by geographical isolation and different ecological conditions. Phylograms produced with trnL-F and rbcL sequence data suggested slow rates of sequence divergence among Jatropha spp., while the ITS gene tree had good resolution suggesting high genetic variation of ITS among Jatropha species.     

Molecular evidence for a natural diploid hybrid between <Emphasis Type="Italic">Miscanthus</Emphasis><Emphasis Type="Italic">sinensis</Emphasis> (Poaceae) and <Emphasis Type="Italic">M. sacchariflorus</Emphasis>

The hybrid origin of Miscanthus purpurascens has previously been proposed, primarily because of its intermediate morphology. In this study, phylogenies based on the DNA sequences from the internal transcribed spacer region of nuclear ribosomal DNA (nrDNA ITS), on the DNA sequences of the trnL intron and trnL-F intergenic spacer of chloroplast DNA, and on amplified fragment length polymorphism (AFLP) fingerprinting confirm that M. purpurascens originated through homoploid hybridization between M. sinensis and M. sacchariflorus. Two different types of ITS sequences were identified from almost all plants of M. purpurascens. One type was found to be closely related to M. sinensis and the other to M. sacchariflorus. Miscanthus purpurascens was found to possess many M. sinensis- and M. sacchariflorus-specific AFLP bands but no band specific to itself. Clustering with the Unweighted Pair Group Method with Arithmetic Mean and principal coordinate analysis based on the AFLP data also demonstrated that M. purpurascens is an approximate intermediate of the two species. In addition, M. purpurascens has the plastid genome of M. sinensis or M. sacchariflorus, suggesting that either species could be its maternal parent. All specimens of M. purpurascens and its coexisting parental species are identified as diploids (2n = 2x = 38). Possible mechanisms of natural hybridization, hybrid status, chloroplast DNA recombination, and evolutionary implications of this hybridization are also discussed.     

Differentiation of the endemic Greek genus <Emphasis Type="Italic">Hymenonema</Emphasis> and its relatives of subtribe Scolyminae (Compositae,Cichorieae) based on a multilocus species tree reconstruction

Hymenonema (Compositae, tribe Cichorieae) together with the genera Catananche, Gundelia, and Scolymus forms the subtribe Scolyminae. It is endemic to Greece and consists of two species, Hymenonema laconicum and Hymenonema graecum, which occur in the south Peloponnisos and central Aegean area, respectively. The present contribution aims at a phylogenetic reconstruction of evolutionary relationships among the 12 species of the subtribe, focusing on the temporal and spatial framework for its evolution. The phylogenetic relationships among the members of Scolyminae were inferred from molecular data based on the multi-copy region of the nrDNA internal transcribed spacers ITS1 and ITS2, two intergenic spacers of the cpDNA (trnL-trnF, rpl32-trnL), and one single-copy nuclear region (D10). The gene trees were reconstructed using Bayesian phylogenetic methods. All gene trees support the monophyly of Hymenonema and the sister-group relationship with the genus Scolymus. The further sister-group relationship of this group (Hymenonema–Scolymus) with Catananche is also supported by nrDNA and cpDNA analyses. Finally, a species tree (inferred in a Bayesian coalescent framework) was reconstructed and dates the divergence time between the two Hymenonema species to the Pleistocene (around 1.3 Ma ago). Maximum likelihood-based biogeographical reconstructions suggest a Miocene (pre-Messinian) differentiation of the subtribe on the northern Tethyan platform, followed by Miocene/Pliocene dispersal events to the western Mediterranean and North-African platforms and final, small-scale vicariance events within the genera in the Pleistocene.     

Study on chloroplast DNA diversity of cultivated and wild pears (<Emphasis Type="Italic">Pyrus</Emphasis> L.) in Northern China

Eight pairs of chloroplast DNA (cpDNA) universal primers selected from 34 pairs were used to assess the genetic diversity of 132 pear accessions in Northern China. Among them, six amplified cpDNA fragments showed genetic diversity. A total of 24 variable sites, including 1 singleton variable site and 23 parsimony informative sites, as well as 21 insertion-deletion fragments, were obtained from the combined cpDNA sequences (5309–5535 bp). Two trnL-trnF-487 haplotypes, five trnL-trnF-413 haplotypes, five rbcL haplotypes, six trnS-psbC haplotypes, eight accD-psaI haplotypes and 12 rps16-trnQ haplotypes were identified among the individuals. Twenty-one haplotypes were identified based on the combined fragments. The values of nucleotide diversity (P_i), average number of nucleotide differences (k) and haplotype diversity (H_d) were 0.00070, 3.56408 and 0.7960, respectively. No statistical significance was detected in Tajima’s D test. Remarkably, the important cpDNA haplotypes and their representing accessions were identified clearly in this study. H_19 was considered as one of the ancient haplotypes and was a divergent centre. H_16 was the most common haplotype of the wild accessions. H_2 was the haplotype representing the most pear germplasm resources (46 cultivars and two wild Ussurian Pear accessions), followed by haplotype H_5 (30 cultivars, two wild Ussurian Pear accessions and four sand pears in outgroups) representing the cultivars ‘Dangshan Suli’ and ‘Yali’, which harbour the largest and the second largest cultivation areas in China. More importantly, this study demonstrated, for the first time, the supposed evolution routes of Pyrus based on cpDNA divergence in the background of pear phylogeny in Northern China.     

Multilocus phylogenetic reconstruction informing polyploid relationships of <Emphasis Type="Italic">Aconitum</Emphasis> subgenus <Emphasis Type="Italic">Lycoctonum</Emphasis> (Ranunculaceae) in China

Polyploidization has long been recognized as one of the most important driving forces of plant evolution. Aconitum subgenus Lycoctonum (Ranunculaceae) has a wide distribution range and well-known background of polyploidy, thereby providing a potentially valuable model to explore polyploid origin and evolutionary history. However, the phylogeny of subg. Lycoctonum has not yet been completely resolved. In the current study, 29 species including diploid, tetraploid and hexaploid species were sampled in subg. Lycoctonum. Using four cpDNA regions (ndhF-trnL, psbA-trnH, psbD-trnT and trnT-L) and two nrDNA regions (internal transcribed spacer, ITS, and external transcribed spacer, ETS), phylogenetic relationship was first reconstructed for the polyploid species within subg. Lycoctonum. In combination with nuclear diversification rate estimation, cpDNA haplotype network, ancestral area reconstruction as well as morphological and karyotypic evidence, potential origin and divergence time were further assessed among the polyploid species. Hybridization was inferred for A. angustius and A. finetianum was suggested to be the potential maternal progenitor, due to their close phylogenetic relationship, highly similar morphologies and overlapping distribution range. Local origin was inferred for tetraploids in the Hengduan Mountains (HDM) with eight groups of chromosomes of four homeologous, which diverged approximately 3.00 Ma in the same period of the orogeny of the HDM. The hexaploid A. apetalum was inferred to suffer from geographical isolation due to the formation of the Qinghai–Tibetan Plateau (QTP) and the HDM. Hybridization and heterogeneous habitats in the HDM were suggested to play an important role in the polyploidization in subg. Lycoctonum.     

Geographic patterns of genetic variation in nuclear and chloroplast genomes of two related oaks (<Emphasis Type="Italic">Quercus aliena</Emphasis> and <Emphasis Type="Italic">Q. serrata</Emphasis>) in Japan: implications for seed and seedling transfer

In this study, we assessed geographic patterns of genetic variations in nuclear and chloroplast genomes of two related native oaks in Japan, Quercus aliena and Q. serrata, in order to facilitate development of genetic guidelines for transfer of planting stocks for each species. A total of 12 populations of Q. aliena and 44 populations of Q. serrata were analyzed in this study. Genotyping of nuclear microsatellites in Q. aliena was done with only nine populations (n = 212) due to limited numbers of individuals in two populations, while all 12 populations (n = 89) were used in sequencing chloroplast DNA (cpDNA). In Q. serrata, 43 populations (n = 1032) were genotyped by nuclear microsatellite markers, while cpDNA of 44 populations (n = 350) was sequenced. As anticipated, geographic patterns detected in the variations of Q. aliena’s nuclear genome and its chloroplast haplotype distribution clearly distinguished northern and southern groups of populations. However, those of Q. serrata were inconsistent. The geographic distribution of its chloroplast haplotypes tends to show the predicted differentiation between northern and southern lineages, but geographic signals in the genetic structure of its nuclear microsatellites are weak. Therefore, treating northern and southern regions of Japan as genetically distinct transferrable zones for planting stocks is highly warranted for Q. aliena. For Q. serrata, the strong NE-SW geographic structure of cpDNA should be considered.     

Molecular evidence for hybridization between invasive <Emphasis Type="Italic">Solidago canadensis</Emphasis> and native <Emphasis Type="Italic">S</Emphasis>. <Emphasis Type="Italic">virgaurea</Emphasis>

Hybridization between alien and native species is biologically very important and could lead to genetic erosion of native taxa. Solidago × niederederi was discovered over a century ago in Austria and described by Khek as a natural hybrid between the alien (nowadays regarded also as invasive) S. canadensis and native S. virgaurea. Although interspecific hybridization in the genus Solidago is considered to be relatively common, hybrid nature of S. × niederederi has not been independently proven using molecular tools, to date. Because proper identification of the parentage for the hybrid Solidago individuals solely based on morphological features can be misleading, in this paper we report an additive polymorphism pattern expressed in the ITS sequences obtained from individuals representing S. × niederederi, and confirm the previous hypothesis that the parental species of this hybrid are S. canadensis and S. virgaurea. Additionally, based on variability at the cpDNA rpl32-trnL locus, we showed that in natural populations hybridization occurs in both directions.     

Phylogenetic analysis of <Emphasis Type="Italic">IRIS</Emphasis> L. from China on chloroplast <Emphasis Type="Italic">TRN</Emphasis>L-F sequences

Phylogenetic relationships among the six Iris subgenera were reconstructed by chloroplast trnL-F sequences data using maximum likelihood. The entire matrix of aligned bases analyzed includes 1043 characters and the length of all sequences varied from 747 bp to 893 bp, and mutation sites accounted for 18.79% of the total length. The cluster analysis results accorded well with the subgeneric classification of Chinese Iris species. Results suggested rhizomes and sepals lacking ornament are ancestral characters, and subg. Xyridion and subg. Limniris are more primordial than another four subgenera. Subgenus Nepalensis and subg. Xyridion were resolved as monophyletic.     

Genetic divergence within the monotypic tree genus <Emphasis Type="Italic">Platycarya</Emphasis> (Juglandaceae) and its implications for species’ past dynamics in subtropical China

Subtropical East Asia harbours a large plant diversity that is often attributed to allopatric speciation in this topographically complex region characterized by a relative climate stability. Here, we use observations of Platycarya, a widespread subtropical Asian tree genus, to explore the consequences of past climate stability on species’ evolutionary history in subtropical China. This genus has a controversial taxonomy: while it is now prevailingly treated as monotypic, two species have been originally described, Platycarya strobilacea and P. longipes. Previous information from species distribution models, fossil pollen data and genetic data based on chloroplast DNA (cpDNA) were integrated with newly obtained genetic data from the two putative species. We used both cpDNA (psbA-trnH and trnL-F intergenic spacers, including a partial trnL gene sequence) and nuclear markers. The latter included sequences of the internal transcribed spacer region (ITS1–5.8S–ITS2) of the nuclear ribosomal DNA and random genomic single nucleotide polymorphisms. Using these nuclear genetic markers, we found interspecific genetic divergence fitting with the ‘two species’ scenario and geographically structured intraspecific variation. Using cpDNA markers, we also found geographically structured intraspecific variation. Despite deep inter- and intraspecific genetic divergence, we detected genetic admixture in southwest China. Overall, our findings of genetic divergence within Platycarya support the hypothesis of allopatric speciation. However, episodes of population interconnection were identified, at least in southwest China, suggesting that the genus has had a dynamic population history.     

High genetic diversity in an endangered medicinal plant, <Emphasis Type="Italic">Saussurea involucrata</Emphasis> (<Emphasis Type="Italic">Saussurea</Emphasis>, Asteraceae), in western Tianshan Mountains,China

Saussurea involucrata (Asteraceae) is a medicinal and second-degree national priority endangered plant that is mainly distributed in the high latitude region of the western Tianshan Mountains. The population is fragmented and isolated, and extensive human impact merits a suitable and specific conservation strategy, which can be compiled based on the genetic diversity, population structure, and demographic history. Phylogeographic studies were conducted on a total of five natural populations and 150 individuals were sampled. Data from three cpDNA intergenic spacer regions (trnL-F, matK, and ndhF-rpl32) and nrDNA ITS sequences showed that twelve haplotypes in cpDNA and five haplotypes in nrDNA indicated high genetic diversity among populations sampled (H _T?=?0.820 and 0.756) and within populations sampled (H _S?=?0.792 and 0.721). Additionally, the high genetic diversity did not mirror genetic structure in either cpDNA (F _ST?=?0.03153, G _ST?>?N _ST, p?<?0.05) or nrDNA (F _ST?=?0.03666, meaningless G _ST and N _ST). Two groups (north and south) were determined for a SAMOVA analysis. Based on this analysis, the demographic history was conducted with a Bayesian Skyline Plot and Isolation with Migration analysis, which showed sustainable and stable extension without a marked bottleneck. Divergence time was indicated at c. 6.25 Mya (90%HPD: 15.30–0.22 Mya) in the Miocene, which is consistent with the formation of the Kelasu section of Tianshan. The southern populations in the Bayanbulak and Gonglu regions require additional attention and transplanting would be an effective way to restore rare cpDNA haplotypes, increase effective population size, and migration rate. Our results suggested that in situ conservation of S. involucrata in western Tianshan should be the main strategy for protection and recovery of the species.     

Phylogeography of East Asian <Emphasis Type="Italic">Lespedeza buergeri</Emphasis> (Fabaceae) based on chloroplast and nuclear ribosomal DNA sequence variations

The dynamic changes in land configuration during the Quaternary that were accompanied by climatic oscillations have significantly influenced the current distribution and genetic structure of warm-temperate forests in East Asia. Although recent surveys have been conducted, the historical migration of forest species via land bridges and, especially, the origins of Korean populations remains conjectural. Here, we reveal the genetic structure of Lespedeza buergeri, a warm-temperate shrub that is disjunctively distributed around the East China Sea (ECS) at China, Korea, and Japan. Two non-coding regions (rpl32-trnL, psbA-trnH) of chloroplast DNA (cpDNA) and the internal transcribed spacer of nuclear ribosomal DNA (nrITS) were analyzed for 188 individuals from 16 populations, which covered almost all of its distribution. The nrITS data demonstrated a genetic structure that followed geographic boundaries. This examination utilized AMOVA, comparisons of genetic differentiation based on haplotype frequency/genetic mutations among haplotypes, and Mantel tests. However, the cpDNA data showed contrasting genetic pattern, implying that this difference was due to a slower mutation rate in cpDNA than in nrITS. These results indicated frequent migration by this species via an ECS land bridge during the early Pleistocene that then tapered gradually toward the late Pleistocene. A genetic isolation between western and eastern Japan coincided with broad consensus that was suggested by the presence of other warm-temperate plants in that country. For Korean populations, high genetic diversity indicated the existence of refugia during the Last Glacial Maximum on the Korean Peninsula. However, their closeness with western Japanese populations at the level of haplotype clade implied that gene flow from western Japanese refugia was possible until post-glacial processing occurred through the Korea/Tsushima Strait land bridge.     

Pleistocene shaping of genetic diversity in a monsoon-affected environment: the case of <Emphasis Type="Italic">Gymnosporia</Emphasis> (Celastraceae) in the southern Arabian Peninsula

The Gymnosporia dhofarensis complex (Celastraceae) consists of the closely related taxa G. dhofarensis (Sebsebe) Jordaan and G. parviflora (Vahl) Chiov. and occurs in the fragmented and isolated monsoon-affected refugia of the coastal mountains in a vegetation of relics of (semi-)evergreen Afro-montane woodland and drier habitats (deciduous woodland, open dry scrub) of the southern Arabian Peninsula. To study the phylogeography of the complex, 536 individuals from 31 populations were analysed using cpDNA-RFLP, while a subset of 320 individuals from 20 populations was analysed using AFLP fingerprinting. Additionally, for 46 accessions from populations throughout the distribution range of the complex and 10 individuals of G. senegalensis (Lam.) Exell. s.l. sequences of nrDNA ITS were established to infer ribotype variation and allow dating of diversification events. Phylogeographical reconstructions based on six detected chloroplast haplotypes show a distinct haplotype distribution pattern with a main split between western (Northern Yemen/Jabal Eraf/Jabal Al Aghbar/Jabal Urays; G. parviflora) and eastern populations (Ras Fartak/Hawf Mts/Dhofar Mts; G. dhofarensis). The results of AFLP fingerprinting are congruent with chloroplast data but offer an even more detailed view into the intra-regional population differentiation. The analysis of nrDNA ITS variants shows that G. dhofarensis s.l. is well separated from other Gymnosporia representatives in the study (i.e. G. senegalensis s.l. and G. aff. divaricata). Based on nrDNA ITS sequence variation, the main inter-regional split between western (G. parviflora) and eastern populations (G. dhofarensis) is dated to 0.9–2.2 Ma before present.     

Molecular phylogenetic and biogeographical analysis of <Emphasis Type="Italic">Nitraria</Emphasis> based on nuclear and chloroplast DNA sequences

Based upon DNA sequences from six plastid regions (rbcL, psbB-psbH, trnL-trnF, rpS16, psbA-trnH, rpS16-trnK) and the internal transcribed spacer (ITS) region of nuclear ribosomal DNA, the phylogenetic relationships in the genus Nitraria and family Nitrariaceae are investigated by using methods of maximum parsimony, maximum likelihood, and Bayesian inference. Our study strongly supports the monophyly of Nitraria. Nitraria can be divided into four parts, namely, the N. sphaerocarpa group, N. retusa group, the N. roborowskii and N. tangutorum group, and a group consisting of N. schoberi, N. komarovii, N. sibirica, and N. billardieri. Ancestral area reconstruction using S-Diva shows that eastern Central Asia is most likely the place of origin, and then dispersals occurred to western Central Asia, Africa, and Australia.     

Complete chloroplast genome sequence of <Emphasis Type="Italic">Capsicum</Emphasis><Emphasis Type="Italic">baccatum</Emphasis> var. <Emphasis Type="Italic">baccatum</Emphasis>

Molecular markers derived from the complete chloroplast genome can provide effective tools for species identification and phylogenetic resolution. Complete chloroplast (cp) genome sequences of Capsicum species have been reported. We herein report the complete chloroplast genome sequence of Capsicum baccatum var. baccatum, a wild Capsicum species. The total length of the chloroplast genome is 157,145 bp with 37.7 % overall GC content. One pair of inverted repeats, 25,910 bp in length, was separated by a small single-copy region (17,974 bp) and large single-copy region (87,351 bp). This region contains 86 protein-coding genes, 30 tRNA genes, 4 rRNA genes, and 11 genes contain one or two introns. Pair-wise alignments of chloroplast genome were performed for genome-wide comparison. Analysis revealed a total of 134 simple sequence repeat (SSR) motifs and 282 insertions or deletions variants in the C. baccatum var. baccatum cp genome. The types and abundances of repeat units in Capsicum species were relatively conserved, and these loci could be used in future studies to investigate and conserve the genetic diversity of the Capsicum species.     

<Emphasis Type="Italic">Viola woosanensis</Emphasis>, a recurrent spontaneous hybrid between <Emphasis Type="Italic">V. ulleungdoensis</Emphasis> and <Emphasis Type="Italic">V. chaerophylloides</Emphasis> (Violaceae) endemic to Ulleung Island,Korea

Ulleung Island is an oceanic volcanic island in Korea, which has never been connected to the adjacent continent. Previous studies highlighted Ulleung Island as an excellent system to study the pattern and process of early stages of flowering plant evolutions on oceanic island. The predominant mode of speciation in flowering plants on Ulleung Island appears to be anagenesis. However, the potentially important role of hybrid speciation among incompletely reproductively isolated lineages cannot be ruled out. Viola woosanensis (Violaceae) is of purportedly hybrid origin between V. ulleungdoensis (i.e., formerly recognized as V. selkirkii in Ulleung Island) and V. chaerophylloides, based on morphology. To examine the origin of V. woosanensis, we sampled a total of 80 accessions, including V. woosanensis and its putative parental species and sequenced nrDNA ITS, and four highly variable chloroplast noncoding regions (trnL-trnF, rpl16 intron, atpF-atpH, and psbA-trnH). Representative species of Viola from Korea were also included in the phylogenetic analyses (maximum parsimony, maximum likelihood, and Bayesian inference). Additive polymorphic sites in the nrDNA ITS regions were confirmed by cloning amplicons from representative species. The molecular data strongly supported the hybrid origin of V. woosanensis, and the maternal and paternal parent were determined to be V. ulleungdoensis and V. chaerophylloides, respectively. The presence of two parental ribotypes in V. woosanensis (with the exception in one population) was confirmed by cloning, suggesting V. woosanensis is primarily the F₁ generation. No trace of backcrossing and introgression with its parents was detected due to low fertility of hybrid species. We found a multiple and unidirectional hybrid origin of V. woosanensis. Additional studies are required to determine which factors contribute to asymmetric gene flow of Viola species in Ulleung Island.     

Genetic Differentiation of Colombian Populations of <Emphasis Type="Italic">Anopheles darlingi</Emphasis> Root (Diptera: Culicidae)

Anopheles darlingi Root is a primary vector of malaria in the neotropic region, a species not just highly anthropophilic but very efficient in transmitting Plasmodium species and considered the most important vector in the Amazon region. The main goal of this study was to determine the genetic structure of the A. darlingi populations using microsatellites (STR) in western and eastern regions of Colombia. DNA extraction was done with the cited protocol of band using the Genomic Prep? cell and tissue isolation commercial kits. We used the STR reported by Conn et al (Mol Ecol Notes 1: 223-225, 2001). The analysis with STR proved there was a high genetic diversity and significant alterations of the Hardy-Weinberg equilibrium. The greatest genetic diversity was recorded in Mitu (Vaupes) (Na = 14, Ho = 0.520). The lowest was in Pueblo Nuevo (Cordoba) (Na = 12, Ho = 0.457). The eastern region and the Mitu (Vaupes) populations presented the highest number of primer alleles (Ap = 30; Ap = 13; Ap = 9), with variations between 0.010 and 0.097. The AMOVA revealed that the whole population underwent moderate genetic differentiation (F _ST = 0.063, p < 0.05). The same differentiation was noticed (0.06 < F _ST > 0.06, p < 0.05) with five of the six populations included in this job, and there was a low differentiation in the Las Margaritas (Santander) area (F _ST = 0.02s3, p < 0.05). Our results suggest a slight positive correlation, which does not show a statistical significance between the geographic and genetic distances, probably suggesting that the moderate genetic differentiation found between pairs of populations does not need to be explained for the hypothesis of separation by distance.     

Phylogeny of the Neotropical sages (<Emphasis Type="Italic">Salvia</Emphasis> subg. <Emphasis Type="Italic">Calosphace</Emphasis>; Lamiaceae) and insights into pollinator and area shifts

Salvia subg. Calosphace (Lamiaceae, Lamiales) is a highly diverse clade endemic to the New World. The phylogenetic relationships of Calosphace have been previously investigated using DNA sequences of nuclear ITS region and plastid psbA–trnH intergenic spacer, but the resulting trees lack resolution and support for many clades. The present paper reassesses the phylogenetic relationships of subgenus Calosphace, including a broader taxon sampling, with a special focus on representing previously unsampled sections, and using an additional plastid marker (trnL–trnF region). Our results show increased resolution and overall patterns of support, recovering ten main clades. Within core Calosphace, the most inclusive of these main clades, 17 new subclades were identified. Of the 42 sections for which more than one species was analysed, only 12 are monophyletic. Our biogeographical analysis identified at least twelve migrations to South America from Mexican and Central American lineages, in agreement with previous suggestions of multiple origins of South American Calosphace diversity. This analysis also confirmed a colonization of the Antilles by Andean lineages. The reconstruction of ancestral states of pollination syndromes showed multiple shifts to ornithophily from melittophily and one reversal to the latter.