Multiple nuclear genes stabilize the phylogenetic backbone of the genus Quercus

Phylogenetic relationships among 108 oak species (genus Quercus L.) were inferred using DNA sequences of six nuclear genes selected from the existing genomic resources of the genus. Previous phylogenetic reconstructions based on traditional molecular markers are inconclusive at the deeper nodes. Overall, weak phylogenetic signals were obtained for each individual gene analysis, but stronger signals were obtained when gene sequences were concatenated. Our data support the recognition of six major intrageneric groups Cyclobalanopsis, Cerris, Ilex, Quercus, Lobatae and Protobalanus. Our analyses provide resolution at deeper nodes but with moderate support and a more robust infrageneric classification within the two major clades, the ‘Old World Oaks’ (Cyclobalanopsis, Cerris, Ilex) and ‘New World Oaks’ (Quercus, Lobatae, Protobalanus). However, depending on outgroup choice, our analysis yielded two alternative placements of the Cyclobalanopsis clade within the genus Quercus. When Castanea Mill. was chosen as outgroup, our data suggested that the genus Quercus comprised two clades corresponding to two subgenera as traditionally recognized by Camus: subgenus Euquercus Hickel and Camus and subgenus Cyclobalanopsis Øersted (Schneider). However, when Notholithocarpus Manos, Cannon and S. Oh was chosen as an outgroup subgenus Cyclobalanopsis clustered with Cerris and Ilex groups to form the Old World clade. To assess the placement of the root, we complemented our dataset with published data of ITS and CRC sequences. Based on the concatenated eight gene sequences, the most likely root position is at the split between the ‘Old World Oaks’ and the ‘New World Oaks’, which is one of the alternative positions suggested by our six gene analysis. Using a dating approach, we inferred an Eocene age for the primary divergences in Quercus and a root age of about 50–55 Ma, which agrees with palaeobotanical evidence. Finally, irrespective of the outgroup choice, our data boost the topology within the New World clade, where (Protobalanus + Quercus) is a sister clade of Lobatae. Inferred divergence ages within this clade and the Cerris–Ilex clade are generally younger than could be expected from the fossil record, indicating that morphological differentiation pre-dates genetic isolation in this clade.     

壳斗科栎属青冈亚属的花粉形态及其系统学意义

对栎属青冈亚属17个种及炭栎花粉形态进行扫描电镜观察。结果表明:青冈亚属和炭栎花粉均为三拟孔沟,球形至长球形,(15～28.0)μm×(15.0～25.6)μm。花粉外壁纹饰可划分为3种:(1)蠕虫状(包括皱波状)、(2)聚合颗粒状、(3)棒状或小刺状。各纹饰在组间镶嵌分布。棒状及小刺状纹饰为栎属花粉较为原始式样,并为青冈亚属与高山栎组及部分巴东组(sect.Cerris)共有,推测其间具有较其它栎类更为接近的亲缘关系,可能为广义栎属中较为基础的类群,而与栎亚属sect.Quercuss.s.,Lobatae和Protobalanus关系较远。花粉形态对广义栎属系统分类价值仅适用于较高分类阶元。     

High variability of chloroplast DNA in three Mediterranean evergreen oaks indicates complex evolutionary history

Chloroplast DNA variation was studied in three evergreen Quercus species (Q. suber L., Q. ilex L. and Q. coccifera L.) from the Western Mediterranean Basin using PCR-RFLP. We studied five primer pair/enzyme combinations, four of them previously used in other European Quercus, obtaining a large number of haplotypes (81) grouped in three main types (suber type, ilex-coccifera I type and ilex-coccifera II type). Such level of haplotype diversity is higher than previously reported for the genus. Remarkable differences in haplotype richness between species have been found. Q. ilex and Q. coccifera usually share the same haplotypes, while a number of Q. suber populations possesses variants of the ilex-coccifera I type. This fact is interpreted as a result of genetic introgression between Q. suber and Q. ilex. Reproductive factors that could determine this exchange are discussed, as well as the influence of different species histories on the present structure of evergreen Quercus in the Western Mediterranean Basin.     

Seasonal changes of root hydraulic conductance (KRL) in four forest trees: an ecological interpretation

This paper deals with the possibility of relating root hydraulic parameters to an ecological index describing the continentality/oceanicity of four forest trees. Root hydraulic conductance ($K_R$) of seedlings of Fagus sylvatica L., Quercus ilex L., Quercus suber L. and Quercus pubescens Willd. was measured in May, August and November 1996. $K_R$ was calculated in terms of the relation of the water flow through intact root systems in situ measured with the pressure chamber, and the pressure driving the flow. The sufficiency of the root system to supply the foliage was estimated by dividing $K_R$ by the seedlings leaf surface area ($A_L$) thus obtaining $K_RL$. In the spring, $K_RL$ was largest in F. sylvatica and smallest in Q. pubescens with intermediate values recorded in Q. ilex and Q. suber. All the species studied showed a large decline in $K_RL$ just prior to the winter rest except for Q. suber which mantained $K_RL$ approximately constant through the period of study. In most cases, $K_RL$ changed in accordance with analogous changes in the flow. When the total seedlings' leaf surface area ($A_L$) was plotted versus $K_RL$, it appeared that $K_RL$ of Q. pubescens increased with $A_L$, proportionally, while $K_RL$ of F. sylvatica was inversely related to $A_L$. This, together with the largest $K_RL$ recorded in the summer in Q. pubescens, was interpreted as advantageous to this species (which is adapted to semi-arid environments) in that: (a) roots could supply water to foliage efficiently even during the adverse season and (b) the foliage growth could be sustained even in summer.No statistically significant relation of $K_RL$ to the continentality index calculated for the four species studied on the basis of their European distribution, was found to exist. Nonetheless, our data appear to be encouraging for future research aimed at better interpreting the typical distribution areas of plant species.     

Molecular and palaeoecological evidence for multiple glacial refugia for evergreen oaks on the Iberian Peninsula

Aim  A multiple glacial refugia hypothesis for Mediterranean plant species was tested with the evergreen Quercus complex ( Quercus suber L., Quercus ilex L. and Quercus coccifera L.) from the Iberian Peninsula, using molecular and palaeobotanical data.
Location  The Iberian Peninsula, which is an ecologically and physiographically complex area located on the western edge of the Mediterranean Basin.
Methods  We sampled 1522 individuals from 164 populations of Q. suber , Q. ilex and Q. coccifera . A review of the recent literature on fossil pollen and charcoal records and a nested clade analysis on chloroplast DNA polymerase chain reaction-restriction fragment length polymorphism was carried out to infer demographic and historical processes.
Results  The analysis indicates at least one glacial refugium for Q. suber in south-western Iberia. Extensive introgression of Q. suber with Q. ilex indicates several potential refugia in eastern Iberia. Past fragmentation was followed by a restricted range flow/range expansion, suggesting multiple refugia for Q. ilex–Q. coccifera elsewhere in central and northern Iberia and multiple areas of secondary contact. This finding is consistent with fossil records.
Main conclusions  The predicted multiple refugia during glacial periods indicates the existence of secondary post-glaciation contact areas. These areas contained complex diversity patterns resulting mainly from range expansions followed by isolation by distance. To a lesser degree, traces of restricted and long-distance dispersal were also found.     

Past distribution and ecology of the cork oak (Quercus suber) in the Iberian Peninsula: a pollen-analytical approach

Abstract.  This study presents pollen-analytical data from continental and offshore Iberian Peninsula sites that include pollen curves of Quercus suber , to provide information on the past distribution and ecology of the cork oak ( Q. suber ). Results centre on a new pollen record of Navarrés (Valencia, eastern Spain), which shows that the cork oak survived regionally during the Upper Pleistocene and was important during a mid-Holocene replacement of a local pine forest by Quercus -dominated communities. This phenomenon appears linked to the recurrence of fire and reinforces the value of the cork oak for reforestation programmes in fire-prone areas. In addition to Navarrés, other Late Quaternary pollen sequences (Sobrestany, Casablanca-Almenara, Padul, SU 8103, SU8113, 8057B) suggest last glacial survival of the cork oak in southern and coastal areas of the Peninsula and North Africa. Important developments also occur from the Late Glacial to the middle Holocene, not only in the west but also in the eastern Peninsula. It is suggested that, in the absence of human influence, Q. suber would develop in non-monospecific forests, sharing the arboreal stratum both with other sclerophyllous and deciduous Quercus and Pinus species.     

Phylogenetic analysis of Phlebotomus species belonging to the subgenus Larroussius (Diptera, psychodidae) by ITS2 rDNA sequences

In the genealogy of Phlebotomus (Diptera: Psychodidae), morphological analyses have indicated that the subgenus Larroussius is a monophyletic group which is most closely related to the subgenera Transphlebotomus and Adlerius. We conducted a phylogenetic analysis of the relationships among six representative species of the subgenus Larroussius and one species representatitive of the Phlebotomus subgenus, assessing sequences of the Second Internal Transcribed Spacer (ITS2) of the ribosomal RNA (rRNA). Three of the species (P. perniciosus, P. ariasi and P. perfiliewi perfiliewi) were collected in different parts of the Mediterranean area. The trees estimated from parsimony and neighbour-joining analyses supported the monophyly of the Larroussius subgenus inferred from the morphological analysis. According to our data, P. ariasi may be a sister group to the rest of the Larroussius subgenus, although additional sequence data are needed to confirm this observation. Our results suggest that P. perniciosus and P. longicuspis are distinct species, in spite of the fact that there are only slight morphological differences. The strict congruence between the phylogeny of the Larroussius subgenus inferred from the ITS2 sequences and that based on morphological studies further confirmed the ability of the spacer sequence to identify recently-derived affiliations.     

Frequent cytoplasmic exchanges between oak species that are not closely related: Quercus suber and Q. ilex in Morocco

Chloroplast (cp) and mitochondrial (mt) DNA variation were studied in 97 populations of cork oak (Quercus suber) in Morocco; in 31 of these populations, holm oak (Quercus ilex), a clearly distinct species, also occurred and was compared with Q. suber. Three cpDNA and one mtDNA primer pairs were used in the survey, each in combination with one restriction enzyme. Six haplotypes belonging to two very divergent lineages were detected; one lineage predominates in each species, and is probably ancestral, as inferred from comparisons with other oak species. In the mixed-species populations, cytoplasmic genomes were frequently shared across species, as indicated by an introgression ratio of 0.63. This index is a new measure of the propensity of species to share locally genetic markers, varying from zero (complete differentiation) to one (no differentiation). By contrast, more closely related deciduous oak species (Q. robur, Q. petraea and Q. pubescens) have introgression ratios varying from 0.82 to 0.97. The introgression events appear to have been more frequent in the direction Q. ilex (female) x Q. suber (male), a finding which seems attributable to the flowering phenology of these two species. This asymmetry may have favoured immigration of Q. suber beyond its main range, in regions already colonized by Q. ilex. There, rare hybridization and further introgression through long distance pollen flow have established populations that are morphologically indistinguishable from Q. suber but that have cytoplasmic genomes originating from the local Q. ilex populations.     

Evolution of Caulanthus amplexicaulis var. barbarae (Brassicaceae), a rare serpentine endemic plant: a molecular phylogenetic perspective

Intra- and interspecific phylogenetic relationships of the rare serpentine endemic taxon Caulanthus amplexicaulus var. barbarae and related taxa in the "Streptanthoid Complex" of genera (Streptanthus, Caulanthus, Guillenia) were examined using nuclear ribosomal internal transcribed spacer (ITS) and chloroplast trnL intron sequences. Phylogenetic hypotheses generated from 81 variable ITS nucleotide sites and six variable trnL nucleotide sites indicate that Streptanthus and Caulanthus are nonmonophyletic groups. Caulanthus amplexicaulis var. barbarae and its more widespread nonserpentine sister taxon Caulanthus amplexicaulis var. amplexicaulis formed a distinct monophyletic group. Among the taxa in our study, C. amplexicaulis was most closely related to Streptanthus tortuosus. The ITS sequences supported monophyly of subgenus Euclesia, which includes the bulk of the serpentine endemics in the Streptanthoid Complex. The serpentine taxa were nonmonophyletic, occurring in at least three distinct clades, suggesting that tolerance to serpentine may be gained or lost through relatively few genetic changes. Intraspecific ITS1 and ITS2 sequence divergence within C. amplexicaulis (1.3-1.8%) was higher than in comparable species (0.0-0.3%); implications of this genetic differentiation for the conservation status of C. amplexicaulis var. barbarae are discussed. Evidence is presented that supports a "biotype depletion" model for the origin of this rare endemic taxon.     

Cytosolic ascorbate peroxidase in some species of the Quercus genus

ABSTRACT

Six species of the Quercus genus (Quercus ilex L., Q. coccifera L., Q. suber L., Q. trojana Webb, Q. macrolepis Kotschy, Q. cerris L.) have been screened for cytosolic ascorbate peroxidase (APX) by means of native polyacrylamide gel electrophoresis (PAGE). A single isozyme was found in five species (Q. trojana, Q. suber, Q. cerris, Q. macrolepis and Q. coccifera), while Q. ilex shows two different APX proteins. The data showed marked similarities among Q. trojana, Q. suber, Q. cerris and Q. macrolepis with respect to the electrophoretic mobility. The validity of APX electrophoretic patterns in systematic studies is discussed.     

Molecular systematics of Trilliaceae II. Phylogenetic analyses of Trillium and its allies using sequences of rbcL and matK genes of cpDNA and internal transcribed spacers of 18S–26S nrDNA

Coding regions of the rbcL and matK genes of cp DNA and internal transcribed spacers (ITS) of nuclear ribosomal DNA were sequenced to study phylogenetic relationships within and among all four genera of Trilliaceae: Trillium, Paris, Daiswa and Kinugasa . The rbcL gene has evolved much slower than matK and in particular ITS; hence the phylogenetic trees based on the rbcL gene show a much lower resolution than trees based on either matK or ITS. The general topology of phylogenetic trees resulting from separate parsimony analyses of the matK and ITS sequences are relatively congruent, with the exception of the placement of T. pusillum . Both matK and ITS phylogenies reveal that T. rivale diverges at the base of the trees. In both trees, Paris, Daiswa and Kinugasa form a relatively weakly supported group. Within this group, the allo-octaploid Kinugasa japonica is the sister group of Daiswa species. The Paris–Daiswa – Kinugasa group, the major Trillium group, and T. undulatum and T. govanianum showed a loosely related topology, but their affinities are not evident according to these two molecular markers. However, phylogenetic analysis of amino acid sequences derived from matK shows that T. rivale together with clades T. undulatum–T. govanianum, Daiswa–Kinugasa and Paris is basally diverged as a sister group to the remainder of Trillium .     

Are the Platyhelminthes a monophyletic primitive group? An assessment using 18S rDNA sequences

In most zoological textbooks, Platyhelminthes are depicted as an early- emerging clade forming the likely sister group of all the other Bilateria. Other phylogenetic proposals see them either as the sister group of most of the Protostomia or as a group derived from protostome coelomate ancestors by progenesis. The main difficulty in their correct phylogenetic placing is the lack of convincing synapomorphies for all Platyhelminthes, which may indicate that they are polyphyletic. Moreover, their internal phylogenetic relationships are still uncertain. To test these hypotheses, new complete 18S rDNA sequences from 13 species of "Turbellaria" have been obtained and compared to published sequences of 2 other "Turbellaria," 3 species of parasitic Platyhelminthes, and several diploblastic and deuterostome and protostome triploblastics. Maximum-parsimony, maximum-likelihood, and neighbor-joining methods were used to infer their phylogeny. The results show the order Catenulida to form an independent early- branching clade and emerge as a potential sister group of the rest of the Bilateria, while the rest of Platyhelminthes (Rhabditophora), which includes the parasites, form a clear monophyletic group closely related to the protostomes. The order Acoela, morphologically considered as candidates to be ancestral, are shown to be fast-clock organisms for the 18S rDNA gene. Hence, long-branching of acoels and insufficient sampling of catenulids and acoels leave their position still unresolved and call for further studies. Within the Rhabditophora, our analyses suggest (1) a close relationship between orders Macrostomida and Polycladida, forming a clear sister group to the rest of orders; (2) that parasitic platyhelminthes appeared early in the evolution of the group and form a sister group to a still-unresolved clade made by Nemertodermatida, Lecithoepitheliata, Prolecithophora, Proseriata, Tricladida, and Rhabdocoela; and (3) that Seriata is paraphyletic.      

Competitive strategies for water availability in two Mediterranean Quercus species

Competition for water availability was studied in a mixed natural stand of Quercus suber L. and Quercus cerris L. growing in Sicily by measuring diurnal changes of leaf conductance to water vapour ( g _L), water potential ( Ψ _L) and relative water content ( RWC ) in April, July and October 1997 as well as the seasonal changes in root hydraulic conductance per unit leaf surface area ( K _RL). Quercus cerris behaved as a drought-tolerant species, with strong reductions of K _RL, Ψ _L, and RWC in the summer. By contrast, Q. suber appeared to withstand summer drought by an avoidance strategy based on reducing g _L, maintaining Ψ _L and RWC high and K _RL at the same level as that measured in the spring. A 'conductance ratio' ( CR ) was calculated in terms of the ratio of g _L to K _RL. Seasonal changes of this ratio contrasted in the two species, thus suggesting that Q. suber and Q. cerris did not really compete for available water. In the summer, when Q. suber was extracting water from the soil to maintain high leaf hydration, Q. cerris had restricted water absorption, thus suffering drought but tolerating its effects. The possibility that cohabitation of drought-tolerant with drought-avoiding species can be generalized is also discussed.     

Secondary Structure of the ITS1 Transcript and its Application in a Reconstruction of the Phylogeny of Boraginales

Abstract: In this study, we present the possibilities for calculating systematic relationships on higher taxonomical levels based exclusively on ITS1. This is demonstrated for Boraginales (Boraginaceae s.str., Cordiaceae, Ehretiaceae, Heliotropiaceae, Hydrophyllaceae and Lennoaceae). Secondary structure of the ITS1 region is more conserved than the primary structure (i.e., sequence itself) and is therefore a tool for optimizing alignments. It increases the number of structural characters. Information inferred from the secondary structure enables us to construct well-resolved phylogenetic trees at higher taxonomic levels. A general secondary structure of ITS1 for Boraginales, with four major helices, is proposed here. In each subtaxon, derivations from this structure are found. The paraphyly of Boraginaceae s.l. is evident from both a comparison of secondary structures and from bootstrap analysis. Boraginaceae s.str. are the sister group of a clade formed by Hydrophyllaceae, Cordiaceae, Ehretiaceae, Heliotropiaceae and Lennoaceae. The last four taxa constitute a monophyletic group which is the sister group of Hydrophyllaceae. Lennoaceae are closely related to Ehretiaceae, and these two taxa in turn are the sister group of Cordiaceae.     

Molecular analyses of the genus Ilex (Aquifoliaceae) in southern South America, evidence from AFLP and ITS sequence data

In order to clarify the relationships among southern South American (sSA) representatives of the genus Ilex, an amplified fragment length polymorphism (AFLP) analysis was accomplished. In addition, the phylogenetic relationships of the species were studied using ribosomal internal transcribed spacer (ITS) sequence data alone and in combination with AFLP data, taking into account the possible existence of paralogous sequences and the influence of alignment parameters. To explore stability of phylogenetic hypotheses, a sensitivity analysis was performed using 15 indel-substitution models. Within each species assayed, the AFLPs allowed the recognition of several diagnostic bands. Furthermore, the AFLP analysis revealed that individuals belonging to the same morpho-species formed coherent clades. In addition, some cases of geographical association were noted. Studies on ITS sequences revealed divergence between data obtained herein and sequence data downloaded from GenBank. The sensitivity analyses yielded different interspecific hypotheses of relationships. Notwithstanding, analyses of the ITS data alone and in combination with AFLPs, rendered clades stable to variation in the analytical parameters. Topologies obtained for the AFLPs, the ITS data alone and the combined analyses, demonstrated the existence of a group formed by I. argentina, I. brasiliensis, I. brevicuspis, I. integerrima, and I. theezans, and that I. dumosa and I. paraguariensis were distantly related to the former. Incongruence with traditional taxonomical treatments was found.     

Molecular systematics of Apiaceae subfamily Apioideae: phylogenetic analyses of nuclear ribosomal DNA internal transcribed spacer and plastid RPO C1 intron sequences

Evolutionary relationships among representatives of Apiaceae (Umbelliferae) subfamily Apioideae have been inferred from phylogenetic analyses of nuclear ribosomal DNA internal transcribed spacer (ITS 1 and ITS 2) and plastid rpoC1 intron sequences. High levels of nucleotide sequence variation preclude the use of the ITS region for examining relationships across subfamilial boundaries in Apiaceae, whereas the rpoC1 intron is more suitably conserved for family-wide phylogenetic study but is too conserved for examining relationships among closely related taxa. In total, 126 ITS sequences from subfamily Apioideae and 100 rpoC1 intron sequences from Apiaceae (all three subfamilies) and outgroups Araliaceae and Pittosporaceae were examined. Phylogenies estimated using parsimony, neighbor-joining, and maximum likelihood methods reveal that: (1) Apiaceae subfamily Apioideae is monophyletic and is sister group to Apiaceae subfamily Saniculoideae; (2) Apiaceae subfamily Hydrocotyloideae is not monophyletic, with some members strongly allied to Araliaceae and others to Apioideae + Saniculoideae; and (3) Apiaceae subfamily Apioideae comprises several well-supported subclades, but none of these coincide with previously recognized tribal divisions based largely on morphological and anatomical characters of the fruit. Four major clades in Apioideae are provisionally recognized and provide the framework for future lower level phylogenetic analyses. A putative secondary structure model of the Daucus carota (carrot) rpoC1 group II intron is presented. Of its six major structural domains, domains II and III are the most, and domains V and VI the least, variable.     

Phylogenetic relationships within Pappophoreae s.l. (Poaceae: Chloridoideae): Additional evidence based on ITS and trnL-F sequence data

Historically, Pappophoreae included the genera Cottea, Enneapogon, Kaokochloa, Pappophorum and Schmidtia. Some authors consider this tribe as a well-supported monophyletic group; while other evidences reveals Pappophoreae as polyphyletic, with Pappophorum separated from the rest of the tribe. When the latter happens, it can form a clade with Tridens flavus. Molecular phylogenetic analyses of the subfamily Chloridoideae have included few species of Pappophoreae; therefore, further research involving more representatives of this tribe is needed. With the aim of providing new evidence to help clarify the phylogenetic position of Pappophorum and its relationships with other genera of the tribe and the subfamily Chloridoideae, eight new sequences of ITS and trnL-F regions of Pappophoreae species were generated. These sequences were analyzed together with other available sequence data obtained from GenBank, using maximum parsimony and Bayesian inference, for individual (trnL-F or ITS) or combined trnL-F/ITS data sets. All analyses reveal that Pappophoreae is polyphyletic, with Pappophorum separated from the rest of the tribe forming a well-supported clade sister to Tridens flavus.     

Contrasting nuclear and cytoplasmic exchanges between phylogenetically distant oak species (Quercus suber L. and Q. ilex L.) in Southern France: inferring crosses and dynamics

Gene flow is particularly frequent in the genus Quercus (oaks), especially between closely related species. We focus here on Quercus ilex and the cork-producing Quercus suber , which occasionally hybridize although they are phylogenetically markedly separated. Morphological observations were combined with both allozymic and chloroplastic diagnostic markers to characterize hybridization and introgression and to infer their dynamics in two French regions (French Catalonia and Provence), which are separated by several hundred kilometres. Some hybrids were found in both regions, indicating recent hybridization events. As expected from previous studies, most hybrids resulted from ♀ Q. ilex  × ♂ Q. suber crosses, but our data showed that the reciprocal cross is also possible. Partial independence between nuclear and chloroplastic introgression was observed in the two species. Nuclear introgression was limited in both species and both regions, with no preferred direction. In Provence, chloroplastic introgression was very rare in both species. Conversely, all Q. suber individuals from French Catalonia were introgressed by Q. ilex chlorotypes. This might be explained by introgression in the Iberian Peninsula antedating the first occurrence of the two species in French Catalonia. We also observed a new chlorotype that was created locally, and was exchanged between the two species. However, the two species still remain genetically differentiated. The dynamics and complexity of exchanges and the factors determining them (including human management of Q. suber ) are discussed.