Natural Hybridization and Introgression between Ligularia cymbulifera and L. tongolensis (Asteraceae,Senecioneae) in Four Different Locations

Natural hybridization has been considered to represent an important factor influencing the high diversity of the genus Ligularia Cass. in the Hengduan Mountains, China. Natural hybridization has been confirmed to occur frequently in Ligularia. To date, however, it has been demonstrated only within a single population. In this paper, we present evidence of natural hybridization in Ligularia from four different locations. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA and three chloroplast intergenic spacers (trnK-rps16, trnL-rpl32 and trnQ-5''rps16) of 149 accessions of putative hybrids and their putative parents (L. cymbulifera and L. tongolensis) were analyzed for evidence of hybridization. The ITS data clearly distinguished two putative parental species and sympatric L. vellerea and supported the hypothesis that those morphological intermediates were products of natural hybridization between L. cymbulifera and L. tongolensis. Moreover, several identified morphological parents were actual introgressed products. Because of hybridization and introgression, chloroplast DNA sequences generated a poorly resolved network. The present results indicate that varying degrees of hybridization and introgression occur differently depending on the habitat context. We conclude that gene flow caused by natural hybridization in Ligularia indeed plays an important role in the species diversity.     

Diversification and vicariance of desert plants: Evidence inferred from chloroplast DNA sequence variation of Lagochilus ilicifolius (Lamiaceae)

To document arid Northern China as a diversification center for desert plants, and to better understand the mechanisms of desert taxa diversification, we used five cpDNA spacers (trnL-trnF, rps16, psbA-trnH, psbK-psbI and trnS-trnG) to investigate Lagochilus ilicifolius in all distribution areas, including Northern China, Mongolia and Russia. Phylogenetic analyses showed that L. ilicifolius comprises two distinctive lineages, one distributed in China, and another in Mongolia–Russia. Our data confirmed that arid Northern China, is a distinctive area with many endemic genera. Biogeographic inferences, based on a Bayesian uncorrelated lognormal model together with molecular dating, suggested that the main diversification within the species occurred in the Pleistocene (ca. 1.38–0.3 Ma), resulting from the transition of the climate of Eurasia to a dry-cold pattern as well as the desertification caused the vicariance of desert plants.     

Molecular phylogeny of the endemic fern genera Cyrtomidictyum and Cyrtogonellum (Dryopteridaceae) from East Asia

Cyrtomidictyum Ching and Cyrtogonellum Ching are two eastern Asian endemic genera whose taxonomic affinities and phylogenetic relationships have long been controversial. The main uncertainty surrounds the separation of the two genera from the species-rich genus Polystichum. Here we present a phylogenetic study focusing on the phylogenetic relationships of these polystichoid ferns. We reconstructed the relationships based on DNA sequence variation in four chloroplast genome regions, rbcL, atpB, and the intergenic spacers (IGS) rps4-trnS and trnL-trnF. Maximum likelihood and Bayesian inference analyses confirm earlier results that were based on less comprehensive taxon sampling and either only a single gene (rbcL) or two IGS (rps4-trnS and trnL-trnF). Cyrtomidictyum is the sister of the clade of polystichoid ferns that includes Cyrtogonellum, Cyrtomium subser. Balansana and three sections of Polystichum. Cyrtogonellum groups with several species of Polystichum, and constitutes the sister taxon to Polystichum sect. Sphaenopolystichum. We support the recognition of Cyrtomidictyum as circumscribed initially, rather than expansion of the genus to include either several Polystichum species or Cyrtogonellum, some Polystichum and Cyrtomium species. The monophyly of Cyrtomidictyum is supported by morphological characters such as once-pinnate leaves, free venation, prolongated leaf apices, and exindusiate sori. Two synapomorphic indels in the chloroplast genome, one 15-bp deletion in rps4-trnS, and one 3-bp insertion in trnL-trnF further differentiate Cyrtomidictyum from other polystichoid ferns. The close affinity of Cyrtogonellum to section Sphaenopolystichum of Polystichum s.s. is highly supported by molecular data. However, no shared morphological characters or molecular indels have been detected, although the distinctness of Cyrtogonellum is shown by a 13-bp insertion in the rps4-trnS alignment.     

使用 RASP 软件预测中国柴胡属的起源和扩散简

采用系统发育的祖先性状重构软件RASP（Reconstruct Ancestral State in Phylogenies）,首次使用扩散隔离分析的统计学方法（Statistical Dispersal-Vicariance Analysis,S-DIVA）和Binary Bayesian MCMC（BBM）方法,选择粗糙西风芹（Seseli squarrulosum）和竹叶西风芹（S.mairei）为外类群,对来自中国的26个柴胡属植物的核糖体内转录间隔区（Internal Transcribed Spacer,ITS）和叶绿体rps16序列进行分子系统分析。结果表明,ITS和rps16序列集所构建的重要节点中,祖先分布区概率占绝对优势的是中国南方地区,推测中国南方是中国柴胡属的起源中心,且时间—事件（Time Event,TE）曲线结果表明距今20和2.5百万年（20和2.5 Ma Bp）出现扩散峰值,在15 Ma Bp出现谷值,推测其20 Ma Bp（庐山亚冰期）,南方和北方种类交流隔离,中国北方分布类型为主体的类群,以散点式的分布,南方分布类型为主体的类群,形成次生分化中心,并按照一定的路径向外扩散,形成对外扩散的高峰期;15 Ma Bp时,以南方为冰期的避难所,出现扩散的低谷期;2.5 Ma Bp（大理亚冰期）左右,因为青藏高原的隆起,中国柴胡属种类再一次发生物种多样化和形成向外扩散的高峰期。     

DNA barcoding approach fails to discriminate Central European bladderworts (Utricularia,Lentibulariaceae), but provides insights concerning their evolution

Abstract

The main features to distinguish the seven native Utricularia species occurring in central Europe are found in flower shape, but being rarely flowering identification is often doubtful and uncertain. A recent morphometric work highlighted that there are no univocal reliable extra-floral morphological features allowing a safe identification at species level. Therefore, DNA barcoding approach is attempted here. Molecular analyses were performed to search for DNA barcodes using nuclear ITS (rDNA), plastid (cpDNA) trnL-trnF IGS and rps16 intron sequences. Generally, the barcoding approach failed to discriminate Utricularia species, although it could be of some help in the U. minor aggregate. With few exceptions, U. bremii shows peculiar DNA regions different from U. minor for both plastid markers investigated. However, interesting hypotheses could be derived from the obtained networks, including hybridization events to explain the rise of mostly sterile species, such as U. stygia. This species clusters with the other species of the U. intermedia aggregate in plastid phylogenetic graphs, while it is closely related to species of the U. minor aggregate in ITS phylogenetic graphs. Additionally to U. stygia, U. ochroleuca also shows some incongruences in the different markers, at least for some accessions, pointing to the possible occurrence of hybrids.     

Phylogenetic split of Larix: evidence from paternally inherited cpDNA trnT-trnF region

A molecular phylogeny of Larix comprising 12 species was constructed from the sequence analysis of the paternally inherited cpDNA trnT-trnF region of 46 individuals. The most parsimonious tree split Larix into three sister clades: one clade was composed of two North American species, the other two were short-bracted and long-bracted species of Eurasia respectively except that L. sibirica was clustered in the long-bracted clade. The difference between the present cpDNA phylogeny and previous nrDNA ITS phylogeny in the position of L. sibirica seems to suggest that ancient cytoplasmic gene flow might exist between sections Larix and Multiserialis. The short-bracted L. laricina and long-bracted L. occidentalis have an identical sequence of the trnT-trnF region, which implies that the bract length divergence among North American larches might have occurred recently or chloroplast capture happened during the early differentiation of the two species. The cpDNA results also shed some light on the biogeography of Larix.     

Climatic Factors Drive Population Divergence and Demography: Insights Based on the Phylogeography of a Riparian Plant Species Endemic to the Hengduan Mountains and Adjacent Regions

Quaternary climatic factors have played a significant role in population divergence and demography. Here we investigated the phylogeography of Osteomeles schwerinae, a dominant riparian plant species of the hot/warm-dry river valleys of the Hengduan Mountains (HDM), Qinling Mountains (QLM) and Yunnan-Guizhou Plateau (YGP). Three chloroplast DNA (cpDNA) regions (trnD-trnT, psbD-trnT, petL-psbE), one single copy nuclear gene (glyceraldehyde 3-phosphate dehydrogenase; G3pdh), and climatic data during the Last Interglacial (LIG; c. 120–140 ka), Last Glacial Maximum (LGM; c. 21 ka), and Current (c. 1950–2000) periods were used in this study. Six cpDNA haplotypes and 15 nuclear DNA (nDNA) haplotypes were identified in the 40 populations of O. schwerinae. Spatial Analysis of Molecular Variance, median-joining networks, and Bayesian phylogenetic trees based on the cpDNA and nDNA datasets, all suggested population divergence between the QLM and HDM-YGP regions. Our climatic analysis identified significant heterogeneity of the climatic factors in the QLM and HDM-YGP regions during the aforementioned three periods. The divergence times based on cpDNA and nDNA haplotypes were estimated to be 466.4–159.4 ka and 315.8–160.3 ka, respectively, which coincide with the time of the weakening of the Asian monsoons in these regions. In addition, unimodal pairwise mismatch distribution curves, expansion times, and Ecological Niche Modeling suggested a history of population expansion (rather than contraction) during the last glaciation. Interestingly, the expansion times were found being well consistent with the intensification of the Asian monsoons during this period. We inferred that the divergence between the two main lineages is probably caused by disruption of more continuous distribution because of weakening of monsoons/less precipitation, whilst subsequent intensification of the Asian monsoons during the last glaciation facilitated the expansion of O. schwerinae populations.     

Multi-gene region phylogenetic analyses suggest reticulate evolution and a clade of Australian origin among paleotropical woody bamboos (Poaceae: Bambusoideae: Bambuseae)

The paleotropical woody bamboo subtribe Bambusinae has not been satisfactorily circumscribed and remains a major taxonomic problem. Genera such as Holttumochloa, Mullerochloa and Temburongia have not been confidently assigned to this subtribe. The phylogenetic relationships among genera hitherto placed in or near the Bambusinae, together with the related subtribes Racemobambosinae and Melocanninae, were investigated using three chloroplast DNA markers (rps16-trnQ, trnC-rpoB and trnD-T intergenic spacers) and a nuclear DNA marker (granule-bound starch synthase I, GBSSI) for a sample of 51 ingroup species and 2 outgroup species. Phylogenetic analyses revealed four distinct lineages among the members of the currently recognized Bambusinae: (1) the Bambusa-Dendrocalamus-Gigantochloa (BDG) complex, (2) the Holttumochloa-Kinabaluchloa clade, (3) the Dinochloa-Mullerochloa-Neololeba-Sphaerobambos (DMNS) clade and (4) Temburongia simplex. The BDG complex, which comprises the core of the Bambusinae, appears to have a complex evolutionary history as indicated by incongruence between the cpDNA and the nuclear gene topologies. Introgressive hybridization and incomplete lineage sorting are possible underlying causes for this complexity. The distinction of the climbing-scrambling bamboo lineages from the core Bambusinae and Racemobambosinae suggests directions for investigating the possible existence of further subtribes with increased taxon and geographical sampling. Possible biogeographic scenarios associated with the Holttumochloa-Kinabaluchloa clade and the DMNS clade are discussed.     

Phylogeny of Androcymbium (Colchicaceae) based on morphology and DNA sequences

The inclusion of species of Colchicum within Androcymbium in a previous cpDNA phylogeny of the Colchicaceae, questioned the monophyly of Androcymbium, and it was proposed to unite the two genera in Colchicum. Here we expand the previous phylogenetic analyses of Androcymbium by increasing the taxon sampling and adding more data. The analysis include 29 of the 57 species of Androcymbium, more cpDNA (trnL intron, trnL-trnF IGS, trnY-trnD IGS, and trnH-psbA IGS), and nDNA (RNApol2 intron 23) regions, and morphological and life-history traits data. Both parsimony and Bayesian inference were used. According to our data there is no reason to expand Colchicum to include Androcymbium, but we support the inclusion of Bulbocodium and Merendera within Colchicum. Morphology and life history traits are the main arguments in favor of recognizing Androcymbium as a well-circumscribed genus. In the phylogeny two Androcymbium groups are clearly differentiated: (1) one including species from Western and Eastern South Africa, Namibia, and North Africa, and (2) one including species from the north west of South Africa and south of Namibia.     

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.     

Phylogenetic utility of structural alterations found in the chloroplast genome of pear: hypervariable regions in a highly conserved genome

The genome structure of pear chloroplast DNA (cpDNA) is extremely highly conserved in comparison with that of other angiosperms, and therefore, relatively few phylogenetic analyses for pear (Pyrus spp.) have been carried out using cpDNA as a marker. In this study, we identified two hypervariable regions in intergenic spacers of cpDNA from 21 species in Pyrus. One of these regions is 857 bp in length and lies between the accD-psaI genes, and the other is a 904-bp region between the rps16-trnQ genes. The mutation rate of gaps for the two regions was 10 and 26 times higher, respectively, than the base change rate. Twenty-five haplotypes were revealed among 21 species in Pyrus by 36 mutations found in the two regions. These included 27 gaps and 9 base changes but excluded cpSSRs. Phylogenetic relationships between the 25 haplotypes were generated by haplotype network analysis. The 25 haplotypes represented three groups (types A–C) with two large deletions, one 228 bp in length between the accD-psaI genes and the other 141 bp between the rps16-trnQ genes. Types A and B consisted mostly of pears native to East and South Asia. Type C contained mainly Pyrus communis and wild relatives native to Europe, West and Central Asia, Russia, and Africa. Type B might have diverged from primitives such as pea pears in type A. Phylogenetic utility of structural alterations (gaps) occurring in the hypervariable regions of Pyrus cpDNA is discussed.     

Karyotype and cytogeography of the genus Heracleum (Apiaceae) in the Hengduan Mountains

Abstract In the present study, the karyotypes of 34 populations belonging to 11 species and one variety of Heracleum from the Hengduan Mountains in China were examined. Chromosome numbers and the karyotypes of three species (H. souliei, H. kingdoni, and H. wenchuanense) are reported for the first time, as are the karyotypes of H. moellendorffii and H. henryi (tetraploid). Populations of H. candicans, H. franchetii, and H. kingdoni in the Hengduan Mountains were found to consist of a mixture of diploid and tetraploid plants. Except for four species of Heracleum, namely H. candicans, H. franchetii, H. henryi, and H. kingdoni, which have both diploid and tetraploid karyotypes, all other species of Heracleum are were found to be diploid. All karyotypes were found to belong to the 2A type of Stebbins, with the exception of H. candicans var. obtusifolium, which belongs to 2B, and H. hemsleyanum and H. franchetii (Mt. Dujuan, Daocheng, Sichuan, China), which belong to 1A. There was only a slight difference in the karyotype asymmetry index, which suggests a close kinship for species of Heracleum and that the entire phylogenetic development of Heracleum is relatively primitive. Species that exhibited advanced morphological features were also more advanced in karyotype structure, with the order of karyotype evolution being 1A→2A→2B. This phenomenon indicates that the species distributed in the Hengduan Mountains have not diverged completely and that the Hengduan Mountains are a relatively young and active area for the evolution of Heracleum. Polyploidization in Heracleum may be an important evolutionary mechanisms for some species, generating diversity. The biological attributes, distribution range, and the geological history of the genus have all played a part in accelerating the evolution through polyploidization or aneuploidization. It is known that as the distribution latitude of Heracleum decreases from north to south, the chromosome number, ploidy level, and asymmetry structure appear to increase. In the Hengduan Mountains, these tendencies are also evident. Finally, based on all the available cytogeographic data, we speculate that the more advanced tetraplont or aneuploid species of Heracleum in India may be derived from early diplont species that were distributed in the Caucasus region and Hengduan Mountains. The dispersal of Heracleum was from Eurasia to India, because this correlates with the emergence of the Himalayan Mountains through tectonic movement. Thus, the Hengduan Mountains are not only a center of diversity for Heracleum, but also a center of active speciation in modern times.     

Molecular identification and phylogeny of an aquatic moss species in Antarctic lakes

Due to the morphological variability, the identification of moss species can be difficult when the plant grows in submerged environments. The taxonomic status of an aquatic moss found in lakes of the Sôya Coast region, East Antarctica, had been controversial, and then, it was investigated by molecular phylogenetic and haplotype network analysis of two chloroplast regions (rps4 and trnL-F) and/or the nuclear ribosomal ITS region. Based on the results of the analyses, the moss was assigned to the genus Leptobryum and determined to be conspecific with Leptobryum wilsonii (Mitt.) Broth. described from South America. Almost no genetic variation was observed between all samples from Antarctic lakes and some samples of L. wilsonii from Chile. Molecular and geohistorical evidence suggests that immigration of L. wilsonii into Antarctic lakes took place during the Holocene via long-distance dispersal from South America. This study gives a clear example of the widespread assumption that most of the Antarctic moss species are post-glacial immigrants.     

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.     

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.     

'Missing link' species Capsella orientalis and Capsella thracica elucidate evolution of model plant genus Capsella (Brassicaceae)

To elucidate the evolutionary history of the genus Capsella, we included the hitherto poorly known species C. orientalis and C. thracica into our studies together with C. grandiflora, C. rubella and C. bursa‐pastoris. We sequenced the ITS and four loci of noncoding cpDNA regions (trnL – F, rps16, trnH –psbA and trnQ –rps16). Sequence data were evaluated with parsimony and Bayesian analyses. Divergence time estimates were carried out with the software package BEAST. We also performed isozyme, cytological, morphological and biogeographic studies. Capsella orientalis (self‐compatible, SC; 2n = 16) forms a clade (eastern lineage) with C. bursa‐pastoris (SC; 2n = 32), which is a sister clade (western lineage) to C. grandiflora (self‐incompatible, SI; 2n = 16) and C. rubella (SC; 2n = 16). Capsella bursa‐pastoris is an autopolyploid species of multiple origin, whereas the Bulgarian endemic C. thracica (SC; 2n = 32) is allopolyploid and emerged from interspecific hybridization between C. bursa‐pastoris and C. grandiflora. The common ancestor of the two lineages was diploid and SI, and its distribution ranged from eastern Europe to central Asia, predominantly confined to steppe‐like habitats. Biogeographic dynamics during the Pleistocene caused geographic and genetic subdivisions within the common ancestor giving rise to the two extant lineages.     

Genetic differentiation and delimitation of Pugionium dolabratum and Pugionium cornutum (Brassicaceae)

Species delimitation has been a major research focus in evolutionary biology. However, the genetic delimitation of recently diverged species varies depending on the markers examined. In this study, we aimed to examine genetic differentiation and delimitations between only two species of Pugionium Gaertn (Brassicaceae)—Pugionium cornutum (L.) Gaertn and Pugionium dolabratum Maxim—that occur in the desert habitats of central Asia and have parapatric distributions. We genotyped 169 individuals from 25 populations, using two chloroplast (cp) DNA fragments (trnV-trnM and trnS-trnfM), seven simple repeated sequence (SSR) loci and the nuclear ribosomal internal transcribed spacer (ITS). Four cp haplotypes were identified, three of which commonly occur in the two species, suggesting incomplete species-specific lineage sorting. Between-species cpDNA differentiation (F _CT) was low, even lower than among populations of the same species. However, we found higher than average SSR F _CT values, and both Bayesian clustering of SSR variables and maximum-likelihood genetic analyses divided all sampled individuals into two groups, agreeing well with morphological separation, although gene flow between species was obvious according to the SSR loci data. However, two types of ITS sequences were highly consistent with the morphological delimitation of the two species in all sampled individuals. These results together suggest that these two species shared numerous ancestral cpDNA polymorphisms and point to the importance of nuclear DNA (ITS or genetic accumulation at multiple loci) in delimiting recently diverged species.     

Molecular evidence confirms the parasite Congracilaria babae (Gracilariaceae,Rhodophyta) from Malaysia

A phylogenetic study of two parasites found on the common host species Gracilaria salicornia from Japan and Malaysia based on nuclear, plastid, and mitochondrial molecular markers was conducted. The Japanese and Malaysian parasites were placed in the same cluster in the nuclear phylogenies inferred from the LSU rRNA gene and ITS region, supporting the monophyly of these parasites despite the slight anatomical variations observed. In addition to the molecular analyses, shared morphological and reproductive features including the lack of rhizoids, similar coloration to hosts, and the presence of deep spermatangial conceptacles supported the inclusion of both the Japanese and Malaysian parasites in Congracilaria babae. Phylogenetic analyses based on ITS sequences indicated that C. babae most likely evolved directly from its current host species, G. salicornia. C. babae was shown to be a member of Gracilaria sensu stricto along with its host. C. babae growing on G. salicornia is characterized by having plastid rbcL and mitochondrial cox1 gene sequences identical to those of its host, whereas the nuclear LSU rRNA gene and ITS region sequences are different from those of its host.