Ghost introgression facilitates genomic divergence of a sympatric cryptic lineage in Cycas revoluta.

A cryptic lineage is a genetically diverged but morphologically unrecognized variant of a known species. Clarifying cryptic lineage evolution is essential for quantifying species diversity. In sympatric cryptic lineage divergence compared with allopatric divergence, the forces of divergent selection and mating patterns override geographical isolation. Introgression, by supplying preadapted or neutral standing genetic variations, can promote sympatric cryptic lineage divergence via selection. However, most studies concentrated on extant species introgression, ignoring the genetic legacy of introgression from extinct or unsampled lineages ("ghost introgression"). Cycads are an ideal plant for studying the influence of ghost introgression because of their common interspecific gene flow and past high extinction rate. Here, we utilized reference-based ddRADseq to clarify the role of ghost introgression in the evolution of a previously identified sympatric cryptic lineage in Cycas revoluta. After re-evaluating the evolutionary independency of cryptic lineages, the group-wise diverged single-nucleotide polymorphisms among sympatric and allopatric lineages were compared and functionally annotated. Next, we employed an approximate Bayesian computation method for hypothesis testing to clarify the cryptic lineage evolution and ghost introgression effect. SNPs with the genomic signatures of ghost introgression were further annotated. Our results reconfirmed the evolutionary independency of cryptic lineage among C. revoluta and demonstrated that ghost introgression to the noncryptic lineage facilitated their divergence. Gene function related to heat stress and disease resistance implied ecological adaptation of the main extant populations of C. revoluta.

Divergence With Gene Flow and Contrasting Population Size Blur the Species Boundary in Cycas Sect. Asiorientales, as Inferred From Morphology and RAD-Seq Data.

The divergence process of incipient species is fascinating but elusive by incomplete lineage sorting or gene flow. Species delimitation is also challenging among those morphologically similar allopatric species, especially when lacking comprehensive data. Cycas sect. Asiorientales, comprised of C. taitungensis and C. revoluta in the Ryukyu Archipelago and Taiwan, diverged recently with continuous gene flow, resulting in a reciprocal paraphyletic relationship. Their previous evolutionary inferences are questioned from few genetic markers, incomplete sampling, and incomprehensive morphological comparison by a long-term taxonomic misconception. By whole range sampling, this study tests the geographic mode of speciation in the two species of Asiorientales by approximate Bayesian computation (ABC) using genome-wide single nucleotide polymorphisms (SNPs). The individual tree was reconstructed to delimit the species and track the gene-flow trajectory. With the comparison of diagnostic morphological traits and genetic data, the allopatric speciation was rejected. Alternatively, continuous but spatially heterogeneous gene flow driven by transoceanic vegetative dispersal and pollen flow with contrasting population sizes blurred their species boundary. On the basis of morphological, genetic, and evolutionary evidence, we synonymized these two Cycas species. This study highlights not only the importance of the Kuroshio Current to species evolution but also the disadvantage of using species with geographically structured genealogies as conservation units.

Corrigendum: Divergence with gene flow and contrasting population size blur the species boundary in Cycas Sect. Asiorientales, as inferred from morphology and RAD-seq data.

[This corrects the article DOI: 10.3389/fpls.2022.824158.].

Merger of Betula tatewakiana (Betulaceae) from northern Japan with northeast Asian B. ovalifolia based on ploidy level.

It has been controversial whether Betula tatewakiana, a dwarf birch distributed in Hokkaido of northern Japan, is an endemic species or a synonym of B. ovalifolia broadly distributed in northeast Asia. The endemic hypothesis is based on the idea that B. tatewakiana is diploid while B. ovalifolia is tetraploid and that they are separated based on the ploidy level; however, no chromosome data have actually been published before. Resolving the taxonomic problem is crucial also in judging the conservation priority of B. tatewakiana in a global perspective. Our chromosome observation revealed that B. tatewakiana is tetraploid as well as B. ovalifolia. We also conducted morphological observations and clarified that B. tatewakiana is morphologically identical to B. ovalifolia in white hairs and dense resinous glands respectively on adaxial and abaxial leaf surfaces, in which they differ from closely related species in the same section Fruticosae. We conclude that the hypothesis that B. tatewakiana is a Hokkaido endemic based on the ploidy level is not supported and that B. tatewakiana should be merged with B. ovalifolia.

Genetic diversity and population structure of Lychnis wilfordii (Caryophyllaceae) with newly developed 17 microsatellite markers.

Lychnis wilfordii (Regel) Maxim. is a perennial plant designated as an endangered species by the Korean government because of rapid reduction in its population size. Thus, a population genetic study of this species is needed to establish the strategy for management and conservation based on scientific evidences. The goals of this study were to develop useful microsatellite markers for L. wilfordii and to understand current genetic status of L. wilfordii in Korean peninsula. Seventeen microsatellite markers were identified using next-generation sequencing and bioinformatic analysis and then analyzed genetic diversity in one hundred forty-five individuals from Korea (KI1, KI2, and KP), China (CX, CF) and Russia (RP). Analysis of molecular variance (AMOVA), principal coordinates analysis (PCoA) and STRUCTURE results consistently showed discontinuity among L. wilfordii populations. AMOVA showed that the percentage of variation among populations was 53%, which was higher than the variation within populations (19%). PCoA showed that the populations were divided into three genetic clusters, (1) Chinese (CX, CF), (2) Russian (RP) populations and Korean populations (KI1, KI2) excluding KP, and (3) the KP population. In particular, KP, the most southern population on the Korean peninsula, showed significantly lower observed and expected heterozygosity, number of effective alleles, and Shannon index (I) than those of KI1 and KI2. L. wilfordii showed high differentiation between populations with low genetic diversity within populations. Among Korean populations, KP is likely to be affected by genetic drift due to small population size, low genetic diversity and limited gene flow.

Unique parallel radiations of high-mountainous species of the genus Sedum (Crassulaceae) on the continental island of Taiwan.

We explored the temporal and spatial diversification of the plant genus Sedum L. (Crassulaceae) in Taiwan based on molecular analysis of nrITS and cpDNA sequences from East Asian Sedum members. Our phylogenetic and ancestral area reconstruction analysis showed that Taiwanese Sedum comprised two lineages that independently migrated from Japan and Eastern China. Furthermore, the genetic distances among species in these two clades were smaller than those of other East Asian Sedum clades, and the Taiwanese members of each clade occupy extremely varied habitats with similar niches in high-mountain regions. These data indicate that species diversification occurred in parallel in the two Taiwanese Sedum lineages, and that these parallel radiations could have occurred within the small continental island of Taiwan. Moreover, the estimated time of divergence for Taiwanese Sedum indicates that the two radiations might have been correlated to the formation of mountains in Taiwan during the early Pleistocene. We suggest that these parallel radiations may be attributable to the geographical dynamics of Taiwan and specific biological features of Sedum that allow them to adapt to new ecological niches.

Distyly and floral morphology of Psychotria cephalophora (Rubiaceae) on the oceanic Lanyu (Orchid) Island, Taiwan.

BACKGROUND: Psychotria cephalophora Merr. (Rubiaceae), a shrub in oceanic islands of Taiwan and the Philippines, appears to be distylous, but distyly is usually rare on oceanic islands. To elucidate the functional breeding system of P. cephalophora can improve our understanding of plant reproductive ecology on oceanic islands. RESULTS: Field investigations on Lanyu (Orchid Island) off the coast of southeastern Taiwan revealed the flowers to be distylous with short (S)- and long (L)-styled morphs, with only one morph per individual. Laboratory observations revealed that both morphs had stainable pollen grains and indicated dimorphism in stigmatic papillae and pollen size. In hand pollination experiments, the pollen tubes reached the base of the style in intermorph crossing, whereas they rarely penetrated stylar tissue in intramorph crossing and selfing. Open pollinated S- and L-styled flowers produced fruit. CONCLUSIONS: The results indicate that the breeding system of P. cephalophora is morphologically and functionally distylous.

Ixeridium calcicola (Compositae), a new limestone endemic from Taiwan, with notes on its atypical basic chromosome number, phylogenetic affinities, and a limestone refugium hypothesis.

A new species Ixeridium calcicola (Compositae) endemic to middle altitude (ca 1,000-2,000 m asl) limestone mountains of eastcentral Taiwan is described based on morphological and chromosome cytological observations and molecular phylogenetic analyses. Ixeridium calcicola resembles Ixeridium transnokoense, endemic to upper montane and alpine ranges (2,600-3,500 m asl) of Taiwan, in the dwarf habit, but differs in the oblong to lanceolate leaf blades (vs. linear to linear-lanceolate), the presence of mucronulate teeth on the leaf margin and petiole (vs. smooth to very sparse), the dark purple lower leaf surface (vs. greenish), the capitulum with 10 to 12 florets (vs. 5 to 7) and 8 to 10 inner phyllaries (vs. 5, rarely to 7). The basic chromosome number in Ixeridium was known as X = 7. However, the new species has a basic chromosome number of X = 8, as recorded also in the closely related Ixeris. Molecular phylogenetic analyses with the expanded sampling of Ixeridium and Ixeris including both type species supported the monophyly of each of the genera and the placement of the new species in Ixeridium. The result of the phylogenetic analyses and detailed observation of the chromosome morphology revealed that X = 8 in Ixeridium calcicola is derived from centric fission in an ancestral karyomorphotype with X = 7 in Ixeridium. Ixeridium calcicola and Ixeridium transnokoense formed a Taiwan endemic lineage and their estimated divergence time was in the middle Pleistocene. Their common ancestral lineage may have experienced altitudinal distribution shifts in response to glacial-interglacial temperature fluctuation, and a lineage which had not retreated to alpine ranges in an interglacial period likely survived in a limestone refugium, where ordinary plant species did not grow, leading to allopatric speciation.

Begonia jinyunensis (Begoniaceae, section Platycentrum), a new palmately compound leaved species from Chongqing, China.

BACKGROUND: Continental China is the center of Begonia species diversity in Asia and contains more than 60 species out of about 110 named species of section Platycentrum. Mt. Jinyun, located in Chongqing City at the upper reaches of the Yangtze River, harbors a subtropical broadleaved forest with high species diversity. During a botanical survey in Mt. Jinyun, an unknown Begonia species of sect. Platycentrum with palmately compound leaves was collected and studied based on detailed morphological observations and cytological and molecular phylogenetic analyses. RESULTS: The unknown Begonia bears a superficial resemblance to B. hemsleyana in having palmately compound leaves, a feature unseen in other species of sect. Platycentrum in China. It is however sharply distinct from the latter in the acaulous habit with aerial stems seen only at anthesis and long rhizomes (vs. erect stems to 70 cm or taller with short rhizomes), 4-6 pinnatilobed leaflets with indistinct, decurrent petiolules (vs. 7-10 serrate leaflets with distinct petiolules), and white (vs. pink) tepals. Molecular phylogenetic analyses based on nuclear ribosomal DNA and chloroplast DNA sequences indicated that this species was allied to Platycentrum species occurring in Southwest and South-central China and Vietnam, including B. hemsleyana, and clearly separable from these species. Somatic chromosome number of 2n = 22 was reported for this unknown species. The diploid chromosome number is agreeable with those published for Begonia sect. Platycentrum. CONCLUSIONS: The unknown Begonia is confirmed to be a new species of sect. Platycentrum and hereto described as Begonia jinyunensis C.-I Peng, B. Ding & Q. Wang.

Extreme habitats that emerged in the Pleistocene triggered divergence of weedy Youngia (Asteraceae) in Taiwan.

Weeds with broad distributions and large morphological variation are challenging for systematists and evolutionarily intriguing because their intensive dispersal would likely prevent local morphological differentiation. Study on weeds will help to understand divergence in plants unlikely to be affected by geographical and ecological barriers. We studied Youngia japonica based on nrDNA and cpDNA sequences. This is a widespread native in Asia and invasive worldwide; nevertheless, three subspecies (japonica, longiflora, and formosana) and an undescribed variant occur in Taiwan. Bayesian and the most parsimonious phylogenies revealed that subspecies longiflora is a different linage and independently arrived in Taiwan during the Pleistocene via land connection to the Asian Continent. Bayesian time estimation suggested that Youngia in Taiwan diverged in the lower Pleistocene or more recently. Extreme habitats that emerged in the Pleistocene, i.e., cold mountain ranges for subspecies formosana and xeric, raised coral reefs for the undescribed Youngia variant probably had triggered the divergence. Components of Youngia in Taiwan are not monophyletic; a coalescent-based test suggested incomplete lineage sorting. Nevertheless, the samples within each taxon share unique morphological features suggesting a common gene pool and each taxon has different dominant ITS and/or cpDNA types; these conditions suggest ongoing process toward monophyly via coalescent processes and support the delimitation of intraspecific taxa.

Development of microsatellite markers in heterostylous Hedyotis chrysotricha (Rubiaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed for a heterostylous herb, Hedyotis chrysotricha to investigate the effect of habitat fragmentation on its genetic diversity and population structure. METHODS AND RESULTS: Twelve primer sets were developed and their polymorphisms were tested on 47 individuals from two island populations of H. chrysotricha in Thousand Island Lake of China. The number of alleles per locus ranged from five to 10, with an average of seven alleles. Expected heterozygosity per locus ranged from 0.284 to 0.821 and observed heterozygosity ranged from 0.191 to 0.851. CONCLUSIONS: We showed that all of the 12 microsatellite markers developed for H. chrysotricha are polymorphic within populations, which should provide a powerful tool for assessing population structure and genetic diversity across fragmented and continuous populations, and for studying the genetic effects of habitat fragmentation on this species.

Isolation of compound microsatellite markers in Begonia fenicis (Begoniaceae) endemic to East and Southeast Asian islands.

PREMISE OF THE STUDY: We developed compound microsatellite markers for Begonia fenicis, a species endemic to eastern and southeastern Asian islands, to investigate geographical genetic structure. METHODS AND RESULTS: Using the compound microsatellite marker technique, 21 markers were developed and six polymorphic markers were characterized for samples from four islands in Taiwan and southern Japan. The number of alleles per locus ranged from two to six (mean = 4.33). Observed and expected heterozygosities were 0.125-0.725 (0.383) and 0.498-0.719 (0.641), and polymorphic information content was 0.371-0.664 (0.567). The interspecific transferability of the 21 markers was evaluated for eight species of the section Diploclinium from the Philippines; 15 markers were successfully amplified in one to eight species. CONCLUSIONS: These results indicate the utility of the six microsatellite markers in B. fenicis to investigate geographical genetic structure. The transferable markers are potentially useful for other species of the section.

Isolation of compound microsatellite markers for the endangered plant Neolitsea sericea (Lauraceae).

PREMISE OF THE STUDY: The development of compound microsatellite markers was conducted in Neolitsea sericea to investigate genetic diversity and population genetic structure of this endangered insular species. • METHODS AND RESULTS: Using the compound microsatellite marker technique, 10 compound microsatellite markers that were successfully amplified showed polymorphism when assessed in 55 individuals from two populations in East China and Japan. Overall, the number of alleles ranged from 3 to 17, with an average of 7.9 alleles per locus. In addition, these primers could be easily amplified in Neolitsea aurata var. paraciculata and N. aurata var. chekiangensis. • CONCLUSIONS: The highly polymorphic markers developed and characterized in this study will be useful for population genetic studies of N. sericea.

Evidence of three parallel evolutions of leaf dwarfism and phytogeography in Lysimachia sect. Nummularia in Japan and Taiwan.

Phylogenetic analyses were performed using nrITS variations of eight species including one variety of the genus Lysimachia sect. Nummularia in Japan and Taiwan, with another 34 Lysimachia species and Glaux maritime obtained from a DNA database. Among the four dwarf-leaf taxa in Japan and Taiwan, L. japonica var. minutissima of Japan and two Taiwanese species were placed into two different clades; and L. liukiuensis of Japan, was positioned outside of these two clades. These results suggest that three independent dwarfism events generated the four dwarf-leaf taxa; and that there were likely multiple migration events from China to Japan and Taiwan.

Cryptochrome and Period Proteins Are Regulated by the CLOCK/BMAL1 Gene: Crosstalk between the PPARs/RXRalpha-Regulated and CLOCK/BMAL1-Regulated Systems.

Feeding and the circadian system regulate lipid absorption and metabolism, and the expression of enzymes involved in lipid metabolism is believed to be directly controlled by the clock system. To investigate the interaction between the lipid metabolism system and the circadian system, we analyzed the effect of a CLOCK/BMAL1 heterodimer on the transcriptional regulation of PPAR-controlled genes through PPAR response elements (PPREs). Transcription of acyl-CoA oxidase, cellular retinol binding protein II (CRBPII), and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase was altered by CLOCK/BMAL1, and transcriptional activity via PPRE by PPARs/RXRalpha was enhanced by CLOCK/BMAL1 and/or by PPARs ligand/activators. We also found that CLOCK/BMAL1-mediated transcription of period (PER) and cryptochrome (CRY) was modulated by PPARalpha/RXRalpha. These results suggest that there may be crosstalk between the PPARs/RXRalpha-regulated system and the CLOCK/BMAL1-regulated system.

Breakdown of distyly in a tetraploid variety of Ophiorrhiza japonica (Rubiaceae) and its phylogenetic analysis.

We examined the floral morph of tetraploid Ophiorrhiza japonica Blume var. amamiana Hatus. and diploid O. japonica var. japonica to elucidate the association of distyly and ploidy levels. Chloroplast DNA phylogeny was reconstructed to determine the number of tetraploidization events and floral morph shifts in O. japonica. All individuals of O. japonica var. amamiana proved to be long-homostylous, whereas O. japonica var. japonica was distylous with typical long- and short-styled flowers. Distyly is related to the ploidy level. The bagging treatment of flowers indicated that O. japonica var. amamiana is self-compatible and potentially automatically self-pollinating. In cpDNA sequencing analysis, no haplotype was shared between the two varieties. The cpDNA haplotype network displayed the monophyly of O. japonica var. amamiana, suggesting a single origin of this variety. Hence, both tetraploidization and the breakdown of distyly to homostyly in O. japonica var. amamiana likely occurred just once. Because O. japonica var. amamiana having the morphological and cytological entity is recognized as a single lineage and clearly separated from O. japonica var. japonica, this variety can be considered to be a distinct species. We therefore propose to raise O. japonica var. amamiana to the rank of species.

Disruption of the murine intestinal alkaline phosphatase gene Akp3 impairs lipid transcytosis and induces visceral fat accumulation and hepatic steatosis.

Intestinal alkaline phosphatase (IAP) is involved in the process of fat absorption, a conclusion confirmed by an altered lipid transport and a faster body weight gain from 10 to 30 wk in both male and female mice with a homozygous null mutation of the IAP coding gene (Akp3(-/-) mice). This study was aimed to delineate morphologically and quantitatively the accelerated lipid absorption in male Akp3(-/-) mice. Feeding a corn oil bolus produced an earlier peak of triacylglycerol in serum (2 vs. 4 h for Akp3(-/-) and wild-type mice, respectively) and an approximately twofold increase in serum triacylglycerol concentration in Akp3(-/-) mice injected with a lipolysis inhibitor, Triton WR-1339. A corn oil load induced the threefold enlargement of the Golgi vacuoles in male wild-type mice but not in Akp3(-/-) mice, indicating that absorbed lipids rarely reached the Golgi complex and that the transcytosis of lipid droplets does not follow the normal pathway in male Akp3(-/-) mice. Force feeding an exaggerated fat intake by a 30% fat chow for 10 wk induced obesity in both male Akp3(-/-) and wild-type mice, and therefore no phenotypic difference was observed between the two. On the other hand, the forced high-fat chow induced an 18% greater body weight gain, hepatic steatosis, and visceral fat accumulation in female Akp3(-/-) mice but not in female wild-type controls. These results provide further evidence that IAP is involved in the regulation of the lipid absorption process and that its absence leads to progressive metabolic abnormalities in certain fat-forced conditions.

Phylogenetic systematics of the monotypic genus Hayataella (Rubiaceae) endemic to Taiwan.

Hayataella (Rubiaceae) is a monotypic genus endemic to Taiwan that comprises H. michelloides. In recent years, Hayataella was considered to be synonymous with Ophiorrhiza; however, no specific data have been reported, and the systematic treatment of Hayataella has been unclear. To elucidate the systematic treatment of Hayataella, molecular phylogenetic analyses based on ITS of nrDNA, atpB-rbcL, and trnK/matK of cpDNA were conducted with four Ophiorrhiza species. In the Bayesian and the most parsimonious trees, H. michelloides was included in the Ophiorrhiza clade. The monotypic status of Hayataella is, therefore, not considered appropriate, and the combination Ophiorrhiza michelloides (Masam.) H. S. Lo is supported.

Apoptosis of endothelial cells may be mediated by genes of peroxisome proliferator-activated receptor gamma 1 (PPARgamma 1) and PPARalpha genes.

Apoptosis in human umbilical vein endothelial cells (HUVECs) was prevented by transfection with the gene for the human full-length peroxisome proliferator-activated receptor alpha (PPARalpha), or acyl-coenzyme A synthetase (AcylCS) into HUVECs. In contrast, ligands/activators of PPARgamma 1 induced apoptosis by a cytochrome c-dependent mechanism in HUVECs transfected with human full-length PPARgamma 1, but not in hepatocytes. Co-transfection of PPARgamma 1 and PPARalpha protected the HUVEC apoptosis. The results suggest that the apoptosis of endothelial cells may be mediated by genes of PPARgamma1 and PPARalpha.

Electrophoretically unique amylases in rat livers: phylogenic and ontogenic study on the mammalian liver.

Liver amylase activity in rodents was assayed with Blue Starch as substrate, and found to be higher than in humans or pigs. Based on the result of concanavalin A affinity chromatography, we found that the sugar moieties of amylase molecules increased in parallel with amylase activity in the tested mammals. However, the amounts of amylase proteins determined by Western bloting with anti-human salivary-type antibody as the probe, were similar to the levels in mammalian livers. Moreover, a similar expression of amylase mRNA was also detected in the mammalian livers by a reverse transcriptional-polymerase chain reaction using primers specific for the human salivary and/or pancreatic amylase complementary DNA (cDNA) sequences. The amylase was detected at the catalytic activity, protein molecule and mRNA levels in rat liver at all ages from fetus to adult. Salivary-type liver amylase activity increased up to one week after birth, and was maintained at the adult level thereafter. However, based on the results of the electrophoretic mobility test, livers with accelerated amylase activity, e.g., at 2-4 weeks after birth or during liver regeneration after partial hepatectomy, were also found to express an amylase electrophoretical identical to pancreatic-type amylase in addition to salivary-type activity. These results suggest that the liver may express an etopic amylase in a certain condition.