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Variable yet vague: Questioning the utility of PHYB for barcoding in Potamogeton
Abstract
Potamogeton is a cosmopolitan genus of aquatic plants comprising ca. 72 species and 99 hybrids. Hybridization and polyploidization events are characteristic of its evolutionary history, thus molecular approach is desirable for confirmation of species and hybrids determinations. In this study, we tested the suitability of low-copy nuclear gene PHYB as a barcoding marker, as this gene was shown to have sufficient level of variability in Potamogeton in preceding studies. We cloned and sequenced part of gene PHYB exon 1 for 19 samples of 8 Potamogeton species. Our sampling took place in East Europe, Siberia, Russian Far East and NE North America. The obtained sequences were used to construct a genetic network. We report some features of PHYB which hamper its application as a barcode in Potamogeton. Most of the tetraploid species provided two sets of dissimilar PHYB sequences, which probably stand for two paralogs. PCR-derived errors, such as recombination and random substitutions, shifted the position of some sequences in the network. In addition, one haplotype was shared between two closely related species, breaking the species-specificity of PHYB barcode. Even though it may provide higher resolution for hybrid identification than ITS or 5S-NTS, using it as a marker for standard barcoding in this genus is not the best option.
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... Interspecific hybridization plays an important role in formation of plant diversity in general [e.g., [17][18][19][20][21] ]. In many groups of aquatic plants hybridization is one of the main sources of new, stable, and ecologically important taxa [e.g., [22][23][24][25][26][27][28][29] ]. ...
We demonstrate a wide distribution and abundance of hybrids between the river species Ranunculus aquatilis, R. fluitans and R. kauffmannii with the still water species R. circinatus (Batrachium, Ranunculaceae) in rivers of two postglacial landscapes of East Europe, i.e., Lithuania and Central European Russia. The Batrachium species and hybrid diversity is higher in the rivers of Lithuania (4 species and 3 hybrids vs. 2 and 1) and represented mainly by western R. aquatilis, R. fluitans and their hybrids whereas in Central European Russia, the East European species R. kauffmannii and its hybrid are the only dominant forms. Hybrids make up about 3/4 of the studied individuals found in 3/4 of the studied river localities in Lithuania and 1/3 of the individuals found in 1/3 of the localities in Central European Russia. Such extensive hybridization in river Batrachium may have arisen due to the specificity of rivers as open-type ecosystems. It may have been intensified by the transformation of river ecosystems by human activities and the postglacial character of the studied landscapes combined with ongoing climate change. Almost all hybrids of R. aquatilis, R. fluitans and R. kauffmannii originated from unidirectional crossings in which R. circinatus acted as a pollen donor. Such crossings could be driven by higher frequency and abundance of R. circinatus populations as well as by some biological mechanisms. Two hybrids, R. circinatus × R. fluitans and R. circinatus × R. kauffmannii, were formally described as R. × redundans and R. × absconditus. We found a hybrid which most likely originated from additional crossing between R. aquatilis and R. circinatus × R. fluitans.
The interspecific hybrid Potamogeton ×salicifolius (= P. lucens × P. perfoliatus), so far known only from several countries of Europe and a few countries of Asia, was discovered in Western Australia. Morphology of the Australian specimens fits the range of variation observed in Eurasian specimens. DNA sequencing confirmed the morphological identification as a hybrid P. lucens × P. perfoliatus, and identified P. lucens as the maternal parent. This identity is surprising because neither of the parental species are known to occur in Western Australia and P. lucens currently does not even occur in the continent. Three possible explanations for the occurrence of P. ×salicifolius in Western Australia are discussed: its relictual occurrence from a time when both the parental species occurred there, long-distance dispersal of a hybrid seed from Eurasia and the remnant of local cultivation or intentional planting.
Potamogeton is one of the most difficult groups to clarify in aquatic plants, which has an extensive range of interspecific morphological and ecological diversity. Internal transcribed spacer (ITS) is prevalent for phylogenetic analysis in plants. However, most researches demonstrate that ITS has a high percentage of homoplasy in phylogenetic datasets. In this study, eighteen materials were collected in Potamogeton from China and incongruence was shown between the rbcL and ITS phylogenies. To solve the discrepancy, we employed a novel barcode PHYB to improve resolution and accuracy of the phylogenetic relationships. The PHYB phylogeny successfully resolved the incongruence between the rbcL and ITS phylogenies. In addition, six hybrids were confirmed using PHYB, including P. compressus × P. pusillus, P. octandrus × P. oxyphyllus, P. gramineus × P. lucens, P. distinctus × P. natans, P. distinctus × P. wrightii, and S. pectinata × S. amblyophylla. Whereas, only one hybrid was identified (P. compressus × P. pusillus) by ITS, indicating that ITS homoplasy was present in Potamogeton and ITS was completely homogenized to one parental lineage. Thus, ITS might have limited utility for phylogenetic relationships in Potamogeton. It is recommended that a three-locus combination of chloroplast DNA gene, ITS and PHYB is potential to effectively reveal more robust phylogenetic relationships and species identification.
Single and low copy nuclear genes offer a larger number of, and more rapidly evolving, characters than the chloroplast and nuclear ribosomal gene sequences that have dominated plant phylogenetic studies to date. Until recently, only one or a few low copy nuclear gene markers were included in such studies. Now, the rapid adoption of “next generation sequencing” (NGS) techniques offers simpler and cheaper access to hundreds of, and not just tens of, coding and noncoding DNA regions. In this review, we describe the most commonly-used NGS methods available for accessing nuclear genes and discuss many NGS case studies that have been published in the last two to three years. These approaches include whole genome sequencing to target microsatellites, transcriptome sequencing, Exon-Primed Intron-Crossing sequencing (EPIC), targeted enrichment (or sequence capture), RAD sequencing (RAD-Seq, including genotyping-by-sequencing or GBS), and genome skimming. We also discuss some of the challenges to, and posed by,the NGS approaches.
Abstract Despite the potential power and extensive use of DNA variation for phylogenctic reconstruction, it has become increasingly apparent that DNA phylogenies are often discordant with organismal phylogenies, Factors responsible for discordance include ...
DNA barcoding is currently a widely used and effective tool that enables rapid and accurate identification of plant species; however, none of the available loci work across all species. Because single-locus DNA barcodes lack adequate variations in closely related taxa, recent barcoding studies have placed high emphasis on the use of whole-chloroplast genome sequences which are now more readily available as a consequence of improving sequencing technologies. While chloroplast genome sequencing can already deliver a reliable barcode for accurate plant identification it is not yet resource-effective and does not yet offer the speed of analysis provided by single-locus barcodes to unspecialized laboratory facilities. Here, we review the development of candidate barcodes and discuss the feasibility of using the chloroplast genome as a super-barcode. We advocate a new approach for DNA barcoding that, for selected groups of taxa, combines the best use of single-locus barcodes and super-barcodes for efficient plant identification. Specific barcodes might enhance our ability to distinguish closely related plants at the species and population levels.
In order to establish a sound basis for systematic and evolutionary research, we determined the chromosome numbers of 181 samples of 47 species and 32 hybrids of Potamogetonaceae from 27 countries and areas, ranging from Greenland in the north to New Zealand in the south and reevaluated previously published counts. The first counts are reported here for 10 species and 25 hybrids of Potamogeton and for 1 species and 3 hybrids of Stuckenia. Both homoploid and heteroploid hybrids were identified, as well as hybrids resulting from the fusion of reduced and unreduced gametes. Three previously undetected hybrids of Potamogeton are described and validated as P. ×drepanoides, P. ×luxurians and P. ×serrulifer. The extensive within-species variation in chromosome numbers sometimes reported in the literature was not confirmed. Chromosome numbers appeared to be generally species-specific in Potamogetonaceae; the only exceptions were two sterile autotriploid plants detected within two otherwise fertile diploid Potamogeton species. Furthermore, chromosome numbers were often uniform even within species groups or genera and to some degree also clade-specific in phylogenetic trees based on nuclear ribosomal markers (ITS and 5S-NTS regions). In the largest genus, Potamogeton, there are two base numbers for diploids (x = 13 and x = 14) and three ploidy levels in species (diploids, tetraploids and octoploids; all polyploids were based on x = 13), in Stuckenia only hexaploids (also based on x = 13) occur and Groenlandia is monotypic with x = 15. A critical evaluation of the published counts revealed three major sources of error: (i) methodological problems due to difficult karyotypes, (ii) approximations based on wrong preconceptions and (iii) poor taxonomic treatments, misidentified species or unrecognized hybrids. We estimate that about 24% of the counts in original publications and up to 41% in chromosome atlases and indices are doubtful or demonstrably erroneous. Most of these were from a relatively few dubious sources whereas the majority of counts reported in the literature correspond to our findings. Two alternative scenarios for the evolution of chromosome numbers in this family are discussed in a phylogenetic context, with either x = 13 or x = 14 as the base chromosome number in the family; the base number of x = 7 suggested by some authors is refuted. In both scenarios, several aneuploid transitions between these karyotypes and a single change towards x = 15 have to be assumed. Polyploidizations are rare in this family and mostly associated with major evolutionary events. A single or a very few events led to a large species group of tetraploids in Potamogeton, and two subsequent rounds of polyploidization can explain the cytotype of Stuckenia, in which speciation took place entirely at the hexaploid level. Three octoploid species of Potamogeton had allopolyploid origins. This study gives an example of how the careful re-examination of chromosome numbers can substantially ameliorate interpretations of systematic and phylogenetic patterns.
In this paper Potamogeton ×clandestinus (Potamogetonaceae), a hybrid between P. crispus and P. natans, is described and illustrated. Hybrid populations were discovered in the rivers Koloshma, Nozhema and Suda in the northern part of European Russia (Vologda reg., Babaevo distr.). Confirmation of the hybrid origin is based on morphological and anatomical data and independently was tested by a direct sequencing of the nuclear ribosomal ITS region and rpl32-trnL intergenic spacer of chloroplast DNA. The additive ITS sequence pattern confirmed that P. ×clandestinus is a hybrid between P. crispus and P. natans. The rpl32-trnL intergenic spacer data revealed that P. crispus was the maternal parent of the hybrid. A detailed morphological and ecological description of P. ×clandestinus is provided and the taxonomic differences between the new hybrid and similar taxa are outlined.
DNA barcoding is the process of identification of species
based on nucleotide diversity of short DNA segments.
It is well established in animals with the
introduction of cytochrome c oxidase subunit 1 (COI)
as a standard barcode. In plants, however, due to the
difficulty in finding a universally acceptable barcode,
it is yet to be well established. Based on the relative
efficacy testing, the Consortium for the Barcode of
Life–Plant Working Group has recently identified a
few loci as potential barcode candidates and from
them a two-locus standard barcode (rbcL + matK) has
been recommended for initiating the barcoding process
of plant species. With 70% species discriminatory
power, this two-locus barcode is capable of serving
many projects, but for better resolution additional loci
need to be used. This article provides an overview of
the technical details, and merits and demerits of these
loci as plant barcodes.
Hybridization is a common phenomenon in many plant genera, and among them is the world’s largest aquatic genus, Potamogeton (Potamogetonaceae) with about 69 species and 50 hybrids reported. Here we provide genetic insights into Potamogeton hybrids from China, where ten or more putative hybrids exist, of which eight have been genetically confirmed. In the study presented here, we verified the hybrid status and inferred the origins of three Potamogeton hybrids using molecular phylogenetic analyses of plastid (chloroplast) trnT–trnF and nuclear ITS sequence data sets that include previously published and newly generated data. The hybrids identified were: 1) P. ×inbaensis, a known hybrid from Japan; 2) a hybrid between P. perfoliatus s.l. and P. wrightii, and 3) a hybrid between tetraploid maternal P. distinctus and diploid paternal P. octandrus. Potamogeton ×inbaensis is reported from China for the first time, whereas the latter two hybrids are new to science.
The occurrence and diversity of Potamogeton hybrids was investigated in eastern North America, the region of the highest diversity of Potamogeton species in the world. For various reasons, however, the existence of hybrids in this area has been largely overlooked. ITS direct sequencing and RFLPs revealed four previously unknown hybrids, which are described as Potamogeton × aemulans ( P. bicupulatus × P. epihydrus ), P. × mirabilis ( P. gramineus × P. oakesianus ), P. × versicolor ( P. epihydrus × P. perfoliatus ), and P. × absconditus ( P. perfoliatus × P. richardsonii ). These are the first confirmed hybrids involving P. bicupulatus , P. epihydrus , P. oakesianus , and P. richardsonii . Another hybrid, P. × nitens ( P. gramineus × P. perfoliatus ), is for the first time confirmed for North America with molecular markers. The hybrids’ maternal parents were revealed by cpDNA sequencing. Phenotypically, two of the hybrids more closely resemble other taxa than their parents. Hybrid diversity and recognition in North America and in Europe are compared. Morphological versus molecular identification of hybrids, occurrence of hybrids in the absence of their parents, parental species relationships, long-term persistence of hybrid clones, vegetative dispersal, frequency of hybridization events, and hybrid distribution patterns are discussed.
Estimating genealogical relationships among genes at the population level presents a number of difficulties to traditional methods of phylogeny reconstruction. These traditional methods such as parsimony, neighbour-joining, and maximum-likelihood make assumptions that are invalid at the population level. In this note, we announce the availability of a new software package, TCS, to estimate genealogical relationships among sequences using the method of Templeton et al. (1992) .
An account of the taxonomy of the genusPotamogeton L. with special reference to species description and delimitation is presented. A key to the species is given, based as far
as possible on vegetative characters. Detailed descriptions are provided for a total of 69 species which are regarded as sufficiently
well known. Special emphasis is laid both on a complete list of relevant characters as well as on the judgement of their respective
diagnostic values. All important synonyms are listed allowing direct access to most of the relevant taxonomic and floristicPotamogeton literature. 50 confirmed hybrids are listed and assigned to their putative parent species. Questions with respect to the
taxa listed are formulated in notes on each of the species. A more general view and questions on futurePotamogeton research are summarized in the conclusions.
Low-copy nuclear genes have been suggested as a promising source of independent phylogeographic markers in plants. However, the available studies at the intraspecific level have revealed that extracting information from them is frequently hampered by paralogy and lack of coalescence of alleles. It is thus relevant to test their utility with plants for which solid data from other markers are available. The aims of this study are to retrieve phylogeographic useful information in a low-copy nuclear gene by examining the congruence of the genetic variation with the geography, as well as with previous nuclear ribosomal, plastid, and amplified fragment length polymorphism (AFLP) markers. Seven combinations of primers have been assayed to characterize the structure of GapC (cytosolic glyceraldehyde 3-phosphate dehydrogenase) in Armeria pungens (Link) Hoffmanns. & Link, a linearly distributed AtlanticMediterranean disjunct sand-dune species. A matrix of 101 direct sequences from 71 individuals was analysed with statistical parsimony. To check the reliability of direct sequencing, 216 cloned sequences were also generated. Tests of recombination have also been attempted. By comparing nucleotide and amino acid sequences, three different paralogs (1, 2, 3) were identified of which paralog 2 was sampled for phylogeographic inference. Within this paralog, 13 alleles belonging in three different sequence types (I, II, III) were detected. These types are shown to correspond with lineages from the same locus whose splitting predates the origin of A.pungens, although type III could be a recent paralog. Allelic variation within types I and II followed a clear geographic trend supporting the two main genetic lineages detected in A.pungens with previous markers. This study suggests that information on the population history of a species can be retrieved, even if some uncertainty remains on the source of variation of low-copy nuclear gene sequences, either alleles from the same locus or paralogs.
Hagström's concepts of relationships in Potamogeton, as reflected by the subgeneric, sectional, and subsectional divisions in his classification of the genus are investigated by reconstruction of an evolutionary "tree" deduced from phylogenetic inferences stated in his monograph. These relationships are explored in the context of results from phenetic and cladistic numerical analyses performed using characters taken from the Hagström monograph that are scored for 26 subsections. The Hagström classification represents an amalgam of phenetic and cladistic interpretations and the hierarchy does not depict accurately fundamental evolutionary relationships indicated by numerical analyses. Recommendations are provided for developing and testing evolutionary hypotheses that are independent of relationships inferred by this long-standing but problematic classification.
The recognition of hybrids of linear-leaved taxa of Potamogeton (sect. Graminifolii) based on morphology is difficult and often debatable. As a consequence, currently only a few hybrid taxa are considered valid and many linear-leaved hybrids described in the past are not now recognized. On the other hand, the use of molecular tools has recently allowed more efficient tests of the origin of morphological forms and the tracking of hybridization events in Potamogeton systematics. In this paper, Potamogeton × maëmetsiae Zalewska-Gałosz et M. Ronikier nothosp. nov. (Potamogetonaceae), a hybrid between two linear-leaved species, P. friesii and P. rutilus, is described and illustrated. Hybrid plants were collected from two Central-European populations growing in Lake Skaidrys (Lithuania) and Soitsjärv (Estonia). The hybrid origin of the new entity was identified based on a morphological survey and independently confirmed using nuclear (ITS, 5S-NTS) and chloroplast (rpl32-trnL intergenic spacer) DNA sequence data and AFLP analysis of genetic structure. Differences between P. × maëmetsiae and similar taxa are outlined and other relevant details of the new hybrid discussed.
Hybridization is one of the main sources of taxonomic complexity in Potamogeton. In spite of long and intensive research, the total hybrid diversity and distribution of many hybrids are still insufficiently known. Identities of two currently recognized hybrids were tested using molecular analyses of plants from the original clones growing at the type localities of their correct names. Additive characters in direct sequencing of the ITS region and the placement of cloned sequences in a Bayesian consensus tree were used to identify the exact identity of these hybrids. Potamogeton ×lanceolatifolius is widely recognized as a hybrid P. gramineus × P. polygonifolius. However, the molecular analyses and cytological investigation identified this Swedish hybrid as P. gramineus × P. nodosus although P. nodosus currently does not occur in the whole of Scandinavia. This hybrid provides some insight into the former distribution area of P. nodosus as affected by past climatic changes. The recently detected P. ×vepsicus was originally described as P. alpinus × P. natans but the molecular investigation revealed that its correct identity is P. natans × P. praelongus. In both cases, the actual identities could hardly have been unequivocally deduced from the morphology of the hybrids. The existence of these hybrid combinations is here confirmed for the first time. The maternally inherited chloroplast rpl20-5'rps12 sequences were used to identify the female parental species. The name “P. ×argutulus, previously, but erroneously, used for a hybrid P. gramineus × P. nodosus, is typified and reduced to the synonymy of P. gramineus.
Potamogeton is a taxonomically problematic genus of aquatic monocotyledons, which has received limited phylogenetic study using molecular data. The group is known for extensive vegetative plasticity, confusing patterns of morphological variation and propensity for hybridization. Potamogeton gemmiparus and P. vaseyi are of conservation concern in North America where both are listed as imperiled. These vegetatively similar species are particularly difficult to distinguish in the absence of floating leaves. We studied both species and putatively related taxa in subsection Pusilli (e.g. P. clystocarpus, P. foliosus, and P. pusillus) to elucidate relationships and to develop an identification method using molecular markers. Phylogenetic analyses of nrITS and trnK 5′ intron sequence data clearly endorse the recognition of P. gemmiparus and P. vaseyi as distinct species but call into question the subspecific circumscription of P. pusillus currently followed in North America. Our data resolved P. pusillus in a clade with P. foliosus, separated substantially from P. berchtoldii (= P. pusillus subsp. tenuissimus), thus supporting the recognition of P. berchtoldii as a distinct species. Using molecular cloning techniques, we documented three clear examples of interspecific hybridization (P. foliosus × P. pusillus, P. berchtoldii × P. vaseyi, and P. gemmiparus × P. vaseyi). Simple DNA polymorphisms also indicated several P. berchtoldii × P. gemmiparus hybrids. The narrowly distributed P. gemmiparus and P. clystocarpus are similar morphologically and genetically to the wide-ranging P. berchtoldii, with which they both hybridize. We recommend either the recognition of P. gemmiparus and P. clystocarpus as distinct species, or more suitably as subspecies of P. berchtoldii, for which two new combinations are provided: P. berchtoldii subsp. gemmiparus and P. berchtoldii subsp. clystocarpus.
Andropogoneae is a monophyletic tribe of 85 genera that includes Zea and Sorghum. All members exhibit C 4 photosynthesis and have inflorescences of paired spikelets. Previous studies of the chloroplast gene ndhF and the nuclear gene GBSSI identified numerous mutations that distinguish genera of the tribe but do not indicate relationships among them; the deep branches of the trees are quite short. Here we add newly collected data from phytochrome B to the data from the other two genes. The same pattern holds, with very short branches along the backbone of the tree indicating that the tribe resulted from rapid radiation. The phylogeny shows a single origin of a disarticulating rachis, which is a synapomorphy for the tribe. We find strong support for a core Andropogoneae that includes Andropogon, Bothriochloa, Capillipedium, Cymbopogon, Dichan-thium, Heteropogon, Hyparrhenia, and Schizachyrium and support for its relationship with an expanded Saccharinae that includes Microstegium. The combined data reject the monophyly of subtribes Andropogoninae and Anthistiriinae and provide evidence that subtribes Sorghinae, Saccharinae, and Rottboelliinae are para-or polyphyletic, as is the traditional Maydeae. A relationship with Zea and Tripsacum is indicated for Elionurus, while Chionachne and Phacelurus are shown to diverge early in the history of the tribe. Arundinella hirta and Arundinella nepalensis can be included in an expanded Andropogoneae.
The tropical seagrass genus, Halodule, is distributed in warm to tropical areas throughout the world. We performed separate molecular phylogenetic analyses of Halodule based on both plastid and nuclear DNA sequences, followed by haplotype analysis, focusing on plants in the western Pacific area. One western tropical Atlantic species, H. wrightii s.l., and two western Pacific species, H. pinifolia and H. uninervis, were recognised and characterised by leaf morphology. Because samples from the western tropical Atlantic and the western Pacific were nested with each other, allopatric differentiation into both areas may have occurred during the early stages of the evolution of the genus. A hybrid of Halodule was detected at two nearby localities in Okinawa, Japan, together with its co-occurring maternal and paternal species, H. uninervis and H. pinifolia, respectively. Haplotype analysis provided evidence that the hybrid resulted from a single hybridisation event, followed by clonal dispersal to its present localities.
Unlabelled:
The 32-bit Windows application START is implemented using Visual Basic and C(++) and performs analyses to aid in the investigation of bacterial population structure using multilocus sequence data. These analyses include data summary, lineage assignment, and tests for recombination and selection.
Availability:
START is available at http://outbreak.ceid.ox.ac.uk/software.htm.
Contact:
keith.jolley@ceid.ox.ac.uk
A two-marker combination of plastid rbcL and matK has previously been recommended as the core plant barcode, to be supplemented with additional markers such as plastid trnH-psbA and nuclear ribosomal internal transcribed spacer (ITS). To assess the effectiveness and universality of these barcode markers in seed plants, we sampled 6,286 individuals representing 1,757 species in 141 genera of 75 families (42 orders) by using four different methods of data analysis. These analyses indicate that (i) the three plastid markers showed high levels of universality (87.1-92.7%), whereas ITS performed relatively well (79%) in angiosperms but not so well in gymnosperms; (ii) in taxonomic groups for which direct sequencing of the marker is possible, ITS showed the highest discriminatory power of the four markers, and a combination of ITS and any plastid DNA marker was able to discriminate 69.9-79.1% of species, compared with only 49.7% with rbcL + matK; and (iii) where multiple individuals of a single species were tested, ascriptions based on ITS and plastid DNA barcodes were incongruent in some samples for 45.2% of the sampled genera (for genera with more than one species sampled). This finding highlights the importance of both sampling multiple individuals and using markers with different modes of inheritance. In cases where it is difficult to amplify and directly sequence ITS in its entirety, just using ITS2 is a useful backup because it is easier to amplify and sequence this subset of the marker. We therefore propose that ITS/ITS2 should be incorporated into the core barcode for seed plants.
Molecular phylogenetic studies of palms (Arecaceae) have not yet provided a fully resolved phylogeny of the family. There is a need to increase the current set of markers to resolve difficult groups such as the Neotropical subtribe Bactridinae (Arecoideae: Cocoseae). We propose the use of two single-copy nuclear genes as valuable tools for palm phylogenetics.
New primers were developed for the amplification of the AGAMOUS 1 (AG1) and PHYTOCHROME B (PHYB) genes. For the AGAMOUS gene, the paralogue 1 of Elaeis guineensis (EgAG1) was targeted. The region amplified contained coding sequences between the MIKC K and C MADS-box domains. For the PHYB gene, exon 1 (partial sequence) was first amplified in palm species using published degenerate primers for Poaceae, and then specific palm primers were designed. The two gene portions were sequenced in 22 species of palms representing all genera of Bactridinae, with emphasis on Astrocaryum and Hexopetion, the status of the latter genus still being debated.
The new primers designed allow consistent amplification and high-quality sequencing within the palm family. The two loci studied produced more variability than chloroplast loci and equally or less variability than PRK, RPBII and ITS nuclear markers. The phylogenetic structure obtained with AG1 and PHYB genes provides new insights into intergeneric relationships within the Bactridinae and the intrageneric structure of Astrocaryum. The Hexopetion clade was recovered as monophyletic with both markers and was weakly supported as sister to Astrocaryum sensu stricto in the combined analysis. The rare Astrocaryum minus formed a species complex with Astrocaryum gynacanthum. Moreover, both AG1 and PHYB contain a microsatellite that could have further uses in species delimitation and population genetics.
AG1 and PHYB provide additional phylogenetic information within the palm family, and should prove useful in combination with other genes to improve the resolution of palm phylogenies.
Isoëtes, a heterosporous lycopod with a fossil record dating back to the Paleozoic, has numerous putative allopolyploids (resulting from hybridization events coupled with doubling of chromosome number). By using the highly variable nucleotide sequences from the second intron of a LFY homologue in Isoëtes, species could be delimited and hybrid origins determined. The data suggest that reticulate evolution is both common and complex within a more derived species complex of Isoëtes. Sequences of identifiable parentage and sequences that are unlike any diploid species known were recovered, leading to the conclusion that one or both of the putative parents have not yet been discovered or are extinct. A range of observations concerning allopolyploid speciation were categorized as follows: (1) verification of previous hypotheses regarding parentage (e.g., I. riparia, I. appalachiana), (2) determination that two morphologically distinct allotetraploid species can share the same parentage (I. azorica and I. acadiensis), (3) recognition of a cryptic allotetraploid species, indicated by the presence of different parental genomes (I. "appalachiana" from Florida), and (4) identification of allotetraploid species with one or two unknown parents (e.g., I. tuckermanii, I. acadiensis, I. azorica, and I. hyemalis). Some sequences from diploid species are remarkably uniform among populations (e.g., I. echinospora from various locations in North America, Iceland, and Wales), while others are variable at the subspecies level (e.g., northern and southern populations within I. engelmannii).
Cryptic species are morphologically indistinguishable, yet reproductively isolated. Morphological boundaries between species can also be obscured by hybridization and clonality. Determining the roles of reproductive isolation, hybridization, and clonality in morphologically indistinguishable taxa is essential to determining appropriate species-level taxonomic rankings for conservation purposes. The taxonomic status of the endangered Little Aguja pondweed of west Texas, Potamogeton clystocarpus, is uncertain due to a lack of fixed morphological differences between it and two sympatric congeners. Morphology, amplified fragment length polymorphisms (AFLPs), and sequences of the internal transcribed spacer (ITS) region and trnL-F intron and spacer were used to determine the degree of genetic distinctiveness, hybridization and clonality for this rare species. AFLPs indicate that P. clystocarpus is a genetically distinct lineage compared to P. pusillus and P. foliosus. No hybrids involving P. clystocarpus were detected, but two putative hybrids involving P. pusillus and P. foliosus were identified. Clonal growth was only detected in P. pusillus. A combination of morphological and molecular markers was successful in determining the genetic distinctiveness of an endangered cryptic species, Potamogeton clystocarpus. Further sampling in this and adjacent drainages is necessary to assess the degree of endemism of P. clystocarpus and confidently rule out hybridization and clonality in this taxon.
Using a low-copy nuclear gene region (LEAFY second intron) we show multiple instances of allopolyploid speciation in Persicaria (Polygonaceae), which includes many important weeds. Fifteen species seem to be allopolyploids, which is higher than the number found in previous comparisons of chloroplast DNA and nuclear ribosomal internal transcribed spacer (nrITS) phylogenies. This underestimation of the extent of allopolyploidy is due in at least three cases to homogenization of nrITS toward the maternal lineage. One of the diploid species, P. lapathifolia, has been involved in at least six cases of allopolyploid speciation. Of the diploids, this species is the most widespread geographically and ecologically and also bears more numerous and conspicuous flowers, illustrating ecologic factors that may influence hybridization frequency. With a few exceptions, especially the narrowly endemic hexaploid, P. puritanorum, the allopolyploid species also are widespread, plastic, ecological generalists. Hybridization events fostered by human introductions may be fueling the production of new species that have the potential to become aggressive weeds.
• hybridization
• LEAFY intron
• phylogeny
• Polygonum
• invasive species
We sought to test the utility of two single-copy nuclear genes for resolving phylogenetic relationships within the woody plant tribe, Acereae (Sapindaceae). Acereae comprises Acer (125+ spp.) and Dipteronia (2 spp.), two genera that possess schizocarpic fruits, which split into two winged mericarps. In Acer, the mericarps are elongated with a basally arranged locule and a dorsal or distal wing. In Dipteronia, the mericarp is obovate, and the locule is located centrally and surrounded by the wing. We analyzed 35 species of Acer representing 12 of 16 taxonomic sections plus Dipteronia sinensis to elucidate the phylogeny of Acereae using the single-copy nuclear genes AT103 and SQD1. Both genes exhibited limited variation in Acereae and, therefore, provided limited support for phylogenetic relationships. The phylogeny of concatenated AT103 and SQD1 showed Dipteronia sinensis within Acer with negligible support (0.14 posterior probability, < 50% maximum parsimony bootstrap, MP-BS), a position that is congruent with results from prior studies using chloroplast DNA and internal transcribed spacer (ITS). Based on our results and results from prior studies, we discuss implications for leaf and fruit evolution in Acereae.
Potamogetonaceae is one of the most difficult groups to clarify in aquatic plants. The current studies are mostly concentrated on interspecific identification in Potamogeton. Promising barcoding markers are poor to discriminate intraspecific genetic variety in Potamogeton. In this present study, we tested eight barcoding markers for intraspecific divergence in Potamogeton, including seven cpDNA barcodes (trnL-trnL-trnF, rpl20-rps12, trnG intron, trnH-psbA, matK and rbcL) and two nuclear markers (ITS and PHYB). The results demonstrated that PHYB showed a significantly higher degree of intraspecific divergence in Potamogeton than the others. Five barcodes including trnL-trnL-trnF, trnG intron, matK，rbcL and ITS exhibited a low level of intraspecific variability in Potamogeton. In addition, rpl20-rps12 and trnH-psbA did not show an intraspecific variability in this genus. Thus, PHYB is the most efficient candidate for intraspecific divergence in Potamogeton. This study provides a solid foundation for Potamogeton to explore evolutionary process, and contributes to investigating the relationships between species distribution and geographic arrangement in Potamogeton.
Potamogeton is a cosmopolitan genus of 90–95 species in which numerous hybridization events have occurred worldwide. A plant recently collected from Argentina exhibited ambiguous morphology that does not match any species of the genus. We aimed to assess if the plant coexisting with another Potamogeton species is a product of reticulate evolution. A concatenated plastid DNA (psbA-trnH, trnL intron, and trnL-trnF) and nuclear ribosomal DNA (5S-NTS) data sets, primarily based on previous studies sample set mainly consisting of American and Asian species, were analyzed using Bayesian inference. Nuclear ribosomal ITS sequences were also obtained from five Argentina materials. We recovered similar topologies from both the plastid DNA and nuclear ribosomal 5S-NTS analyses in which most specimens are consistently placed. The specimen of primal interest from Argentina strongly clustered with co-occurring linear-leaved species in the 5S-NTS tree, but was genetically identical to broad-leaved ones in the plastid DNA analysis. The ITS sequence of the specimen was the same as that of the linear-leaved species and no polymorphisms were observed. Considering the discrepant phylogenetic positions between the trees and lack of ITS infra-individual variations, the origin of the specimen from Argentina is better explained by hybridization and subsequent introgression than other possibilities, such as extensive morphological variation.
Premise of the study:
Noncoding chloroplast DNA (NC-cpDNA) sequences are the staple data source of low-level phylogeographic and phylogenetic studies of angiosperms. We followed up on previous papers (tortoise and hare II and III) that sought to identify the most consistently variable regions of NC-cpDNA. We used an exhaustive literature review and newly available whole plastome data to assess applicability of previous conclusions at low taxonomic levels.
Methods:
We aligned complete plastomes of 25 species pairs from across angiosperms, comparing the number of genetic differences found in 107 NC-cpDNA regions and matK. We surveyed Web of Science for the plant phylogeographic literature between 2007 and 2013 to assess how NC-cpDNA has been used at the intraspecific level.
Key results:
Several regions are consistently the most variable across angiosperm lineages: ndhF-rpl32, rpl32-trnL((UAG)), ndhC-trnV((UAC)), 5'rps16-trnQ((UUG)), psbE-petL, trnT((GGU))-psbD, petA-psbJ, and rpl16 intron. However, there is no universally best region. The average number of regions applied to low-level studies is ∼2.5, which may be too little to access the full discriminating power of this genome.
Conclusions:
Plastome sequences have been used successfully at lower and lower taxonomic levels. Our findings corroborate earlier works, suggesting that there are regions that are most likely to be the most variable. However, while NC-cpDNA sequences are commonly used in plant phylogeographic studies, few of the most variable regions are applied in that context. Furthermore, it appears that in most studies too few NC-cpDNAs are used to access the discriminating power of the cpDNA genome.
Numerous studies have identified low copy nuclear genes (LCNG) with phylogenetic potential throughout angiosperms, several specifically focused on Leguminosae. However, phylogenetic resolution at the species- to subspecies-level is often inferred based only on a small subset of taxa scattered throughout the higher level study group. This study aims to reassess the phylogenetic resolution of 19 previously published nuclear regions in Leguminosae using 18 species from two clades of the Caesalpinieae representing both distantly related genera and closely related species. Nuclear regions were amplified and aligned throughout the sampled taxa. Sequences were cloned when polymorphism was noted. The plastid loci matK, rps16, trnL, trnD–trnT and the nuclear ribosomal ITS regions were also analyzed for comparison. Phylogenetic analyses using parsimony were performed on individual matrices. Three nuclear regions were eliminated due to the non-specificity of the primers (RNAH, PTSB, GI). Four regions showed lower resolution than predicted from previous studies (MMK1, CYB6, RBPCO, EFGC), while three revealed greater resolution than anticipated (SQD1, AT103, EIF3E). Three other markers indicated previously unidentified duplication events for the genus Caesalpinia s.l. (ATCP and AROB) and at the base of the Caesalpinia and Peltophorum clades (CALTL). Phylogenies reconstructed from the intron-spanning regions AIGP, SHMT, AT103 and EIF3E are congruent with ITS and plastid data and show the best phylogenetic potential for studies of closely related species of caesalpinioid legumes. We present a screening strategy for the evaluation of LCNG for phylogenetic studies.
Due to insufficient morphological differentiation, hybridization among linear-leaved species of Potamogeton is rarely observed and recognized. Here, we applied molecular tools (sequencing of nrITS and rpl32-trnL cpDNA intergenic spacer) to study several natural, central European populations of morphologically intermediate forms between two closely related species, P. acutifolius and P. compressus, to examine their taxonomical status and test whether they represented the variation range of the species or are results of interspecific hybridization. Both DNA regions provided distinguishing characters between putative parental species. The ITS sequences from all the morphologically intermediate specimens displayed an additive pattern combining the variation of both parental species, providing evidence for the hybrid origin of all these plants. This case study suggests that hybrids of linear-leaved Potamogeton species are more common than usually believed, but have been difficult to identify without molecular tools.
The paper provides a novel molecular demonstration of the hybrid origin of two rare Potamogeton natans hybrids, namely P. x gessnacensis (=P. natans x P. polygonifolius) and P. x vepsicus (=P. natans x P. praelongus), based on nuclear and plastid DNA sequence data. The results of the DNA-based survey were somehow unexpected due to incongruence with the primary background of a preliminary morphological identification of the putative hybrid collections. Our results further demonstrate the incertainty in treatment of hybrids in several taxa of Potamogeton based on morphological characteristics. In this context, we suggest that morphological characters and chorological data of at least a part of P. natans hybrids, particularly P. x sparganiifolius (=P. gramineus x P. natans)and P. x vepsicus, should be verified. Taking into account the new data, other recently published records and rectification of erroneous reports, we also present a reappraisal of the current knowledge on Potamogeton hybrid diversity in Poland as compared with selected other European areas well studied in this respect (British Isles, Czech Republic, Germany), with a notion on general rules shaping the distribution pattern of these taxa in Europe. According to the current data, 12 Potamogeton hybrids have been recorded so far in Poland. Among 32 Potamogeton hybrids reported at least in one of the aforementioned areas, Potamogeton x angustifolius, P. x nitens and P. x salicifolius are the most common taxa, while 18 hybrids are based on records from a single country and predominantly from few localities only. Sparse records of at least part of the rare hybrid taxa reflect a still insufficient state of knowledge on the regional hybrid diversity. However, both at the scale of Poland and Europe, the likely general pattern of the occurrence of Potamogeton hybrids in Europe may indicate that their diversity and frequency of occurrence increase with latitude.
Like most aquatic plants, the pondweeds (Potamogetonaceae) are among the most phenotypically reduced and plastic of all angiosperms. As such, hypotheses of structural homology present difficulties for morphological phylogenetic reconstruction. We used non‐coding nuclear and plastid DNA data to address Potamogetonaceae relationships and accompanying issues in character evolution and biogeography. Genera currently assigned to Potamogetonaceae, plus Zannichellia , formed a strongly supported monophyletic group. Potamogeton and Stuckenia ( Potamogeton subg. Coleogeton ) were both resolved as monophyletic. Within Potamogeton proper, two major clades followed the traditional split between broad‐ and narrow‐leaved species, with the latter condition optimized as basal. Heterophylly (submerged plus floating leaves) has evolved several times, and the ancestral distribution for Potamogeton appears to be Northern Hemispheric. Our phylogenetic results have provided a useful genetic framework from which to interpret morphological, cytological and biogeographical evolution.
© The Willi Hennig Society 2006.
In the genus Potamogeton, high morphologic and ecologic diversification and difficulty in evaluating systematic usefulness of various characteristics have led to the definition of many small groups of species corresponding to sections or subsections. The 18 species of Japanese Potamogeton that have high variation represent 11 of the 14 species groups proposed in a recent treatment of the genus. We performed phylogenetic analyses of these 18 species of Japanese Potamogeton with three allied genera by using the noncoding region of chloroplast DNA between trnT (UGU) and trnL (UAA). Our phylogenetic tree revealed that Japanese Potamogeton were divided into two main groups (I and II). In general, each group was characterized by the shape of the submerged leaves and anatomic features of the stem. Group I comprised eight species with broad submerged leaves, and these species lacked sub-epidermal bundles in the stem. Group II comprisesd 10 species characterized by linear submerged leaves and the presence of sub-epidermal bundles. Group II was further divided into two subgroups (IIa and IIb). Subgroup IIa taxa did not have a dormant period or physiologically specialized turions, and the stele was of the trio or proto-type. In comparison, subgroup IIb members have dormant period in the form of axillary or apical turions, and stele types were oblong or circular. Potamogeton crispus and P. maackianus, each of which has distinct morphology and ecology, are included in group I but differed from the other Potamogeton in having unique length mutations and substitutions in the trnT–trnL sequence.
In a review of chromosome numbers in the genus Potamogeton, we highlight numerous errors that have crept into the literature. These have resulted chiefly from reliance on abstracts in chromosome number indices and compilations, rather than on the original publications, but partly also because of misleading summaries even in the primary literature. We present a list of counts that we believe are original and genuine, and a list of those that were never made but which nevertheless appear in the literature. Scrutiny of the list of accepted counts indicates that aneuploidy is widespread in the genus and that transition between the two common chromosome numbers (2n=26 and 2n=28) has occurred several times. Currently available data are insufficient to resolve the question of the ancestral base number. We also present details of the first chromosome counts from English populations of five taxa: P. polygonifolius Pourr. (2n=28), P. pectinatus L. (2n=ca. 78), P. perfoliatus L. (2n=ca. 52), P.×nitens Weber (P. gramineus×P. perfoliatus) (2n=ca. 52) and P.×salicifolius Wolfg. (P. lucens×P. perfoliatus) (2n=ca. 52).
Sequences of the chloroplast trnT-trnF region were analyzed for species of the genus Potamogeton distributed in China to reconstruct phylogenetic relationships. The phylogenetic analyses showed that the genus Potamogeton could be divided into two clades. Eighteen species formed a monophyletic clade while the remaining four formed a second, distinguishable one, supporting the conventional treatment that the genus Potamogeton contains the submerged linear-leaved group and the submerged broad-leaved group. The first clade, which represented the subgenus Potamogeton, could be further divided into two subclades. The second clade, which represented the subgenus Coleogeton, displayed a close phylogenetic relationship with the subgenus Potamogeton and occupied a unique position within the genus Potamogeton. This finding suggested that the treatment of the subgenus Coleogeton, which was once regarded as the genus Stuckenia Börner, may need to be reconsidered. Furthermore, identification of maternal donors of some hybrids was successfully applied based on sequence of maternally inherited chloroplast genome. The female parents of three putative hybrids, P. × malainoides, P. × anguillanus, and P. × orientalis, were proved to be accordant with previous morphological conclusions.
Organismal phylogeny provides a crucial evolutionary framework for many studies and the angiosperm phylogeny has been greatly improved recently, largely using organellar and rDNA genes. However, low-copy protein-coding nuclear genes have not been widely used on a large scale in spite of the advantages of their biparental inheritance and vast number of choices. Here, we identified 1083 highly conserved low-copy nuclear genes by genome comparison. Furthermore, we demonstrated the use of five nuclear genes in 91 angiosperms representing 46 orders (73% of orders) and three gymnosperms as outgroups for a highly resolved phylogeny. These nuclear genes are easy to clone and align, and more phylogenetically informative than widely used organellar genes. The angiosperm phylogeny reconstructed using these genes was largely congruent with previous ones mainly inferred from organellar genes. Intriguingly, several new placements were uncovered for some groups, including those among the rosids, the asterids, and between the eudicots and several basal angiosperm groups. These conserved universal nuclear genes have several inherent qualities enabling them to be good markers for reconstructing angiosperm phylogeny, even eukaryotic relationships, further providing new insights into the evolutionary history of angiosperms.
Hybridization and polyploidization are important evolutionary processes in higher plants and have greatly enriched the diversity
of the genus Potamogeton (Potamogetonaceae). To study the phylogenetic relationships and hybrid origin of Potamogeton species, 35 accessions representing 20 species, including diploids, tetraploids and hexaploids, and three hybrids were collected
in China and their ribosomal internal transcribed spacers (ITS) were cloned, sequenced and statistically analyzed. The data
showed that ITS sequences were informative to analyze the phylogeny of Potamogeton, and the phylogenetic tree revealed that Potamogeton species examined could be mainly divided into two groups (Group I and II), corresponding to subgenus Potamogeton and subgenus Coleogeton, respectively. Then, the evolutionary mechanism on the polyploidy of Potamogeton species was discussed. P. natans probably was an allotetraploid and one of its parent might result from aneuploidy change of species with 2n=28. P. hubeiensis might be derived from the hybridization between P. octandrus and P. cristatus. We suggested that both P. lucens and P. maackianus probably were allotetraploids, and P. obtusifolius might be a diploid hybrid between P. compressus and P. pusillus. Moreover, P. malainoides might have undergone biased concerted evolution toward one of its parent P. wrightii, and P. intortusifolius might be a synonymy of P. × anguillanus.
Aquatic angiosperms are widely recognized as a biological group sharing attributes associated with adaptations to the aquatic condition. Clonal growth, high vagility of vegetative propagules, and rare to sporadic sexual reproduction are common convergences among aquatic plants, and play central, interacting roles in various evolutionary factors. In this review, two important evolutionary factors, hybridization and chromosome number variation, are discussed with respect to interactions involving clonal growth, vagility, and asexuality. Asexual reproduction emerges as a significant evolutionary catalyst allowing for the perpetuation of hybrid offspring and anomalous cytotypic variants. Inherent phenotypic plasticity in aquatic plants is difficult to discern from both hybrid individuals and cytotypic variants. Detailed studies of putative ‘hybrids’ in some groups may reveal a higher incidence of cytotypic variants at the basis of morphological differences previously attributed to hybridization.
This is a time of unprecedented transition in DNA sequencing technologies. Next-generation sequencing (NGS) clearly holds promise for fast and cost-effective generation of multilocus sequence data for phylogeography and phylogenetics. However, the focus on non-model organisms, in addition to uncertainty about which sample preparation methods and analyses are appropriate for different research questions and evolutionary timescales, have contributed to a lag in the application of NGS to these fields. Here, we outline some of the major obstacles specific to the application of NGS to phylogeography and phylogenetics, including the focus on non-model organisms, the necessity of obtaining orthologous loci in a cost-effective manner, and the predominate use of gene trees in these fields. We describe the most promising methods of sample preparation that address these challenges. Methods that reduce the genome by restriction digest and manual size selection are most appropriate for studies at the intraspecific level, whereas methods that target specific genomic regions (i.e., target enrichment or sequence capture) have wider applicability from the population level to deep-level phylogenomics. Additionally, we give an overview of how to analyze NGS data to arrive at data sets applicable to the standard toolkit of phylogeography and phylogenetics, including initial data processing to alignment and genotype calling (both SNPs and loci involving many SNPs). Even though whole-genome sequencing is likely to become affordable rather soon, because phylogeography and phylogenetics rely on analysis of hundreds of individuals in many cases, methods that reduce the genome to a subset of loci should remain more cost-effective for some time to come.
The phytochrome nuclear gene family encodes photoreceptor proteins that mediate developmental responses to red and far red light throughout the life of the plant. From studies of the dicot flowering plant Arabidopsis, the family has been modeled as comprising five loci, PHYA-PHYE. However, it has been shown recently that the Arabidopsis model may not completely represent some flowering plant groups because additional PHY loci related to PHYA and PHYB of Arabidopsis apparently have evolved independently several times in dicots, and monocot flowering plants may lack orthologs of PHYD and PHYE of Arabidopsis. Nonetheless, the phytochrome nucleotide data were informative in a study of organismal evolution because the loci occur as single copy sequences and appear to be evolving independently. We have continued our investigation of the phytochrome gene family in flowering plants by sampling extensively in the grass family. The phytochrome nuclear DNA data were cladistically analyzed to address the following questions: (1) Are the data consistent with a pattern of differential distribution of phytochrome genes among monocots and higher dicots, with homologs of PHYA, B, C, D, and E present in higher dicots, but of just PHYA, B, and C in monocots, and (2) what phylogenetic pattern within Poaceae do they reveal? Results of these analyses, and of Southern blot experiments, are consistent with the observation that the phytochrome gene family in grasses comprises the same subset of loci detected in other monocots. Furthermore, for studies of organismal phylogeny in the grass family, the data are shown to provide significant support for relationships that are just weakly resolved by other data sets.
Phylogenetic analyses of partial phytochrome B (PHYB) nuclear DNA sequences provide unambiguous resolution of evolutionary relationships within Poaceae. Analysis of PHYB nucleotides from 51 taxa representing seven traditionally recognized subfamilies clearly distinguishes three early-diverging herbaceous "bambusoid" lineages. First and most basal are Anomochloa and Streptochaeta, second is Pharus, and third is Puelia. The remaining grasses occur in two principal, highly supported clades. The first comprises bambusoid, oryzoid, and pooid genera (the BOP clade); the second comprises panicoid, arundinoid, chloridoid, and centothecoid genera (the PACC clade). The PHYB phylogeny is the first nuclear gene tree to address comprehensively phylogenetic relationships among grasses. It corroborates several inferences made from chloroplast gene trees, including the PACC clade, and the basal position of the herbaceous bamboos Anomochloa, Streptochaeta, and Pharus. However, the clear resolution of the sister group relationship among bambusoids, oryzoids, and pooids in the PHYB tree is novel; the relationship is only weakly supported in ndhF trees and is nonexistent in rbcL and plastid restriction site trees. Nuclear PHYB data support Anomochlooideae, Pharoideae, Pooideae sensu lato, Oryzoideae, Panicoideae, and Chloridoideae, and concur in the polyphyly of both Arundinoideae and Bambusoideae.
Intraspecific gene evolution cannot always be represented by a bifurcating tree. Rather, population genealogies are often multifurcated, descendant genes coexist with persistent ancestors and recombination events produce reticulate relationships. Whereas traditional phylogenetic methods assume bifurcating trees, several networking approaches have recently been developed to estimate intraspecific genealogies that take into account these population-level phenomena.
Low-copy nuclear genes in plants are a rich source of phylogenetic information. They hold a great potential to improve the robustness of phylogenetic reconstruction at all taxonomic levels, especially where universal markers such as cpDNA and nrDNA are unable to generate strong phylogenetic hypotheses. Low-copy nuclear genes, however, remain underused in plant phylogenetic studies due to practical and theoretical complications in unraveling the evolutionary dynamics of nuclear gene families. The lack of the universal markers or universal PCR primers of low-copy nuclear genes has also hampered their phylogenetic utility. It has recently become clear that low-copy nuclear genes are particularly helpful in resolving close interspecific relationships and in reconstructing allopolyploidization in plants. Gene markers that are widely, if not universally, useful have begun to emerge. Although utilizing low-copy nuclear genes usually requires extra lab work such as designing PCR primers, PCR-cloning, and/or Southern blotting, rapid accumulation of gene sequences in the databases and advances in cloning techniques have continued to make such studies more feasible. With the growing number of theoretical studies devoted to the gene tree and species tree problem, a solid foundation for reconstructing complex plant phylogenies based on multiple gene trees began to build. It is also realized increasingly that fast evolving introns of the low-copy nuclear genes will provide much needed phylogenetic information around the species boundary and allow us to address fundamental questions concerning processes of plant speciation. Phylogenetic and molecular evolutionary analyses of developmentally important genes will add a new dimension to systematic and evolutionary studies of plant diversity.