Yongpeng Ma

High Genetic Diversity and Low Differentiation of Michelia coriacea (Magnoliaceae), a Critically Endangered Endemic in Southeast Yunnan, China

Michelia coriacea, a critically endangered tree, has a restricted and fragmented distribution in Southeast Yunnan Province, China. The genetic diversity, genetic structure and gene flow in the three extant populations of this species were detected by 10 inter-simple sequence repeat (ISSR) markers and 11 simple sequence repeat (SSR) markers. Examination of genetic diversity revealed that the species maintained a relatively high level of genetic diversity at the species level (percentage of polymorphic bands) PPB = 96.36% from ISSRs; PPL (percentage of polymorphic loci) = 95.56% from SSRs, despite several fragmental populations. Low levels of genetic differentiation among the populations of M. coriacea were detected by Nei’s Gst = 0.187 for ISSR and Wright’s Fst = 0.090 for SSR markers, which is further confirmed by Bayesian model-based STRUCTURE and PCoA analysis that could not reveal a clear separation between populations, although YKP was differentiated to other two populations by ISSR markers. Meanwhile, AMOVA analysis also indicated that 22.84% and 13.90% of genetic variation existed among populations for ISSRs and SSRs, respectively. The high level of genetic diversity, low genetic differentiation, and the population, structure imply that the fragmented habitat and the isolated population of M. coriacea may be due to recent over-exploitation. Conservation and management of M. coriacea should concentrate on maintaining the high level of genetic variability through both in and ex-situ conservation actions.

Unidirectional hybridization and reproductive barriers between two heterostylous primrose species in north-west Yunnan, China

BACKGROUND AND AIMS Heteromorphy in flowers has a profound effect on breeding patterns within a species, but little is known about how it affects reproductive barriers between species. The heterostylous genus Primula is very diverse in the Himalaya region, but hybrids there have been little researched. This study examines in detail a natural hybrid zone between P. beesiana and P. bulleyana. METHODS Chloroplast sequencing, AFLP (amplified fragment length polymorphism) markers and morphological comparisons were employed to characterize putative hybrids in the field, using synthetic F1s from hand pollination as controls. Pollinator visits to parent species and hybrids were observed in the field. Hand pollinations were conducted to compare pollen tube growth, seed production and seed viability for crosses involving different morphs, species and directions of crossing. KEY RESULTS Molecular data revealed all hybrid derivatives examined to be backcrosses of first or later generations towards P. bulleyana: all had the chloroplast DNA (cpDNA) of this species. Some individuals had morphological traits suggesting they were hybrids, but they were genetically similar to P. bulleyana; they might have been advanced generation backcrosses. Viable F1s could not be produced with P. bulleyana pollen on P. beesiana females, irrespective of the flower morphs used. Within-morph crosses for each species had very low (<10 %) seed viability, whereas crosses between pin P. bulleyana (female) and pin P. beesiana had a higher seed viability of 30 %. Thus genetic incompatibility mechanisms back up mechanical barriers to within-morph crosses in each species, but are not the same between the two species. The two species share their main pollinators, and pollinators were observed to fly between P. bulleyana and hybrids, suggesting that pollinator behaviour may not be an important isolating factor. CONCLUSIONS Hybridization is strongly asymmetric, with P. bulleyana the only possible mother and all detected hybrids being backcrosses in this direction. Partial ecological isolation and inhibition of heterospecific pollen, and possibly complete barriers to F1 formation on P. beesiana, may be enough to make F1 formation very rare in these species. Therefore, with no F1 detected, this hybrid zone may have a finite life span as successive generations become more similar to P. bulleyana.

Rescuing Magnolia sinica (Magnoliaceae), a Critically Endangered species endemic to Yunnan, China

Magnolia sinica, a Critically Endangered tree endemic to Yunnan, China, is one of the 20 plant species with extremely small populations approved by the Yunnan government for urgent rescue action before 2015 Information on the geographical distribution and population size of this species had not previously been reported, hindering effective conservation. We therefore carried out a survey of the literature and of herbarium specimens, followed by a detailed field survey and morphological measurements and observations of surviving individuals. We located 52 individuals in the wild, in eight localities. Two distinguishing morphological characters (tepal colour and tepal number) were revised based on observations of all remaining wild individuals that produced flowers and on one 30-year-old flowering plant in Kunming Botanical Garden. The survival rate of individuals propagated from seed for ex situ conservation at the Garden was 100 % over. years; of 100 individuals transplanted to each of two reinforcement sites, 20 and 18, respectively, were alive after. years. We propose two groups of measures to protect M. sinica: (1) in situ conservation, population monitoring, and public engagement, and (2) ex situ conservation with reinforcement or reintroduction.

Strong reproductive isolation despite occasional hybridization between a widely distributed and a narrow endemic Rhododendron species

Reproductive isolation (RI) plays an important role for speciation, but assessing reproductive barriers at all life-cycle stages remains challenging. In plants, most studies addressing the topic have been focusing on herbs with short generation times. The present study attempted to quantify several reproductive barriers between a hybridizing species pair of long-lived woody rhododendrons. Consistent with findings of previous studies, pre-zygotic reproductive barriers contributed more to total RI than post-zygotic reproductive barriers. Especially in the more widespread species geographic isolation was an important barrier, and pollinator constancy contributed exceptionally to RI in both species. Additionally to strong pre-zygotic reproductive barriers, post-zygotic reproductive barriers were considerable, and had asymmetric tendencies favoring one of the species as maternal parent. Overall, despite occasional hybridization, the present study provides evidence for strong RI between R. cyanocarpum and R. delavayi.

Growth discrepancy between filament and style facilitates self‐fertilization in Brandisia hancei (Paulowniaceae)

Self-pollination has been hypothesized to be beneficial in environments where pollinators are rare as it can provide reproductive assurance. This study presents evidence for an autonomous self-fertilization mechanism in the winter flowering plant, Brandisia hancei. To determine changes in the spatial separation of stigma and anthers, the length of style and stamens was recorded. Additionally, pollination treatments were carried out to test fruit-set and seed production. Brandisia hancei is herkogamic in the early flowering stages. However, different growth rates of the filament and style lead to contact of stigma and anthers in the later stages, thereby facilitating self-pollination. The highest seeds number is produced under an out-crossing scenario but plants produce a considerable number of seeds even when purely selfed. Although pollinators are scarce, autonomous selfing alleviates the pollen limitation in B. hancei. Self-fertilization in B. hancei seems to be an adaptive strategy to ensure reproduction when pollinators are scarce.

Why is population information crucial for taxonomy? A case study involving a hybrid swarm and related varieties

Despite acceptance in the scientific community that population information and suites of characters are crucial for circumscription of taxonomic groups, new taxa continue to be published on the basis of few herbarium specimens. Given that there is increasing evidence that hybridisation plays an integral part in evolution, it is desirable to identify groups in which it occurs. In this study, we showcase how population variation can be used to identify distinguishing characters and why closely related species that are growing in sympatry should be considered when describing new taxonomic entities.

Pollinator-Mediated Isolation May Be an Underestimated Factor in Promoting Homoploid Hybrid Speciation

It is widely acknowledged that allopolyploidy via hybridization and genome doubling can easily lead to speciation, as polyploid hybrids can be immediately isolated from diploid parental taxa due to high levels of sterility caused by uneven numbers of chromosome complements in their progeny (Soltis and Soltis, 2009; Abbott et al., 2013). However, homoploid hybrid speciation (HHS), in which a hybrid lineage becomes genetically isolated from their parents and functions as a distinct evolutionary unit, has been thought to be fairly uncommon (Chase et al., 2010; Servedio et al., 2013). Schumer et al. (2014) proposed three criteria for proving HHS: evidence of hybridization in the genome, reproductive isolation (RI) of hybrid lineages from parental species, and evidence that this reproductive isolation is a consequence of hybridization. According to these criteria, only one case of HHS in sunflowers was strictly documented in plants till now (Schumer et al., 2014). These criteria highlight two reasons why HHS is harder to detect and prove than allopolyploid speciation: the first is the lack of chromosome number change, whereas the second is that molecular evidence for hybridization becomes harder to detect as the parent species become more closely related, and it is known that HHS tends to involve more closely related parent species than allopolyploidy events (Abbott et al., 2013). Inconsistent with the low rate of documented HHS events in plants, a recent simulation study published in PLOS Genetics by Schumer et al. (2015) predicts that homoploid hybrids could rapidly become isolated from parental species by fixing combinations of genes that hinder successful reproduction with parental species, even in the presence of substantial ongoing immigration from parental species to the new homoploid hybrid species. The main reason for the discrepancy might lie in the fact that most population genetics studies are merely focused on evidence of hybridization in the genome, without examining the strength of RI which can be assessed by other integrative methods. Pollination biology approach can address the extent of RI between hybrids and parental species. For example, the detailed examination of flowering time difference and the pattern of pollen flow mediated by pollinator behavior quantified by pollen DNA barcoding method could provide the strength of pre-pollination RI between parental species and hybrids whereas post-pollination RI could be assessed by comparing pollen tube growth and reproductive fitness between parental species and hybrids (e.g., fruit set, seed set, seed viability) using artificial pollination (e.g., Ma et al., 2014; Bell et al., 2016; Ma et al., 2016). After the confirmation of certain traits that originate from hybridization and are responsible for RI, QTL can be then applied to link the variation of traits to specific genetic loci. It should be pointed out that abiotic ecological factors could promote RI by long-term geographic isolation between hybrids and parents. In this commentary, however, we focus on flowering plants with an entomoplily system that could sometimes build up RI between hybrids and parents by biotic factors with a relative short time. It is a common phenomenon in entomophilous plants that post-zygotic RI like hybrid inviability and/or sterility is not strong enough to select against all the hybrid progeny. Early-generation hybrids with morphological intermediacy are likely to be less effective in attracting the pollinator(s) of either parent, given long term interaction between and adaptive evolution of flower characters of parental species and pollinators (Chase et al., 2010). Thus, strong selection against hybrids with intermediate morphologies would be expected to occur frequently. In addition, even where certain pollinators are shared between hybrids and parental species, theory predicts that once F1s are casually formed, backcrosses to either or both parental species should frequently occur, therefore hybrids can be easily re-absorbed by parental species (Arnold, 1997). In rare cases where F1s dominate hybrid zones, selection against second generation hybrids appears to prevent both interspecific gene flow and the possible creation of homoploid hybrid species (Milne et al., 2003; Milne and Abbott, 2008). However, entomoplily in some flowering plants seems much more complicated than theoretical predictions suggest, because of highly specific interactions between plants and pollinators. Interestingly, three recent studies on pollination biology have indicated that HHS in entomophilous plants should not be so uncommon. The first case involves pollinator mediated RI between the homoploid hybrid species Penstemon clevelandii and its parental species, by flower color shift due to hybridization (Wolfe et al., 1998). The magenta flower color of P. clevelandii has established RI by selection for a distinct bee and hummingbird pollination syndrome, whereas one of its parental species has red flower and is pollinated by hummingbirds, and the other has lavender flower and is pollinated by wasps (Wolfe et al., 1998). The second well documented case is Iris nelsonii, a diploid species that originated from hybridization between I. fulva, I. hexagona, and I. brevicaulis. It is isolated from one of its progenitors (I. hexagona) by attracting hummingbirds due to large red flowers, while I. hexagona is mainly pollinated by bumblebees due to blue flowers, although other isolation barriers such as habitat and environmental isolation are also present (Taylor et al., 2013). The two study cases mentioned above show a partial shift of pollinator preferences creating pre-zygotic RI in the pollination process, due to flower color differentiation as a result of hybridization. The latest case involves Narcissus hybrids which recruit novel pollinators under natural conditions (Marques et al., 2016). Their results showed that the hybrids not only emitted new floral volatiles, but also recruited new pollinators of ants, which were never witnessed in their parental species. This provides strong evidence for pollinator mediated RI between hybrids and parental species (Marques et al., 2016). Overall, after molecular confirmation of hybridization, work remains to be done for examining RI between hybrids and their parents and further testing if the characters responsible for RI originate from hybridization. Recent studies on entomophlilous plants, albeit only three cases, have proved the power of homoploid hybridization in creating new traits responsible for RI. We therefore emphasize the need for field investigation of pollinator mediated RI after hybridization is confirmed by molecular data for entomophlilous plants. We believe that HHS may not be uncommon in flowering plants, especially in many genera in which hybridization is thought to play an important role in their evolution and speciation; the rarity of proven examples likely reflects difficulty of detection. Integrative approaches to investigating potential cases should examine the possibility of pollinator-mediated isolation from parental species, and how this might have been generated during hybridization. The Sino-Himalaya region, where many congeneric species occur sympatrically and are interfertile, is an ideal place for conducting such studies.

Secondary pollen presentation on anther hairs enhances reproductive fitness in Brandisia hancei, a protogynous perennial with autonomous selfing

ABSTRACT Background: Secondary pollen presentation (SPP) is a phenomenon where pollen is relocated from within the anthers to other floral structures for presentation. Aims: We report a new mechanism of androecial SPP on anther hairs, which is involved in increasing reproductive fitness in Brandisia hancei. Methods: The morphological change of anther hairs during floral ageing was recorded, and pollen grains were quantified after anther hair removal to clarify the roles played by hairs in pollen retention. Additionally, fruit set and seed set were assessed to investigate the efficiency of anther hairs to reproductive success. Results: Young flowers of B. hancei showed approach herkogamy, while autonomous selfing was achieved by changes in relative positions of the stigma and anthers during floral ageing. In B. hancei, anthers developed characteristic hairs of epidermal origin, and hairs originated along the furrow between the paired pollen sacs and were used for SPP after anther dehiscence. Anther hairs increased pollen retention by over 70% in later floral-age stages, and seed set decreased by about 30% after anther hairs had been removed. Conclusions: In the process of autonomous selfing, SPP on anther hairs enhances both the male and female functions of the flowers of B. hancei.

The draft genome assembly of Rhododendron delavayi Franch. var. delavayi

Abstract Rhododendron delavayi Franch. is globally famous as an ornamental plant. Its distribution in southwest China covers several different habitats and environments. However, not much research had been conducted on Rhododendron spp. at the molecular level, which hinders understanding of its evolution, speciation, and synthesis of secondary metabolites, as well as its wide adaptability to different environments. Here, we report the genome assembly and gene annotation of R. delavayi var. delavayi (the second genome sequenced in the Ericaceae), which will facilitate the study of the family. The genome assembly will have further applications in genome-assisted cultivar breeding. The final size of the assembled R. delavayi var. delavayi genome (695.09 Mb) was close to the 697.94 Mb, estimated by k-mer analysis. A total of 336.83 gigabases (Gb) of raw Illumina HiSeq 2000 reads were generated from 9 libraries (with insert sizes ranging from 170 bp to 40 kb), achieving a raw sequencing depth of ×482.6. After quality filtering, 246.06 Gb of clean reads were obtained, giving ×352.55 coverage depth. Assembly using Platanus gave a total scaffold length of 695.09 Mb, with a contig N50 of 61.8 kb and a scaffold N50 of 637.83 kb. Gene prediction resulted in the annotation of 32 938 protein-coding genes. The genome completeness was evaluated by CEGMA and BUSCO and reached 95.97% and 92.8%, respectively. The gene annotation completeness was also evaluated by CEGMA and BUSCO and reached 97.01% and 87.4%, respectively. Genome annotation revealed that 51.77% of the R. delavayi genome is composed of transposable elements, and 37.48% of long terminal repeat elements (LTRs). The de novo assembled genome of R. delavayi var. delavayi (hereinafter referred to as R. delavayi) is the second genomic resource of the family Ericaceae and will provide a valuable resource for research on future comparative genomic studies in Rhododendron species. The availability of the R. delavayi genome sequence will hopefully provide a tool for scientists to tackle open questions regarding molecular mechanisms underlying environmental interactions in the genus Rhododendron, more accurately understand the evolutionary processes and systematics of the genus, facilitate the identification of genes encoding pharmaceutically important compounds, and accelerate molecular breeding to release elite varieties.

Evaluation of RFID Tags to Permanently Mark Trees in Natural Populations

Long-term ecological and genetic studies in natural populations of tree species require marking techniques so that individuals can be re-visited over time, even in difficult terrain. Both GPS coordinates and physical labels have disadvantages that can make re-finding trees difficult. We tested passive and semi-active radio frequency identification (RFID) tags and readers as a means to relocate individual trees. Passive RFID tags do not provide a good solution because of low transmission power of hand-held readers and strong directionality. Semi-active RFID tags provide detection over longer distances, but also suffer from strong directionality. Active RFID tags promise an improvement over semi-passive tags, and could be evaluated in a future study. We conclude that RFID technology has the potential to improve the ability of researchers to locate individual trees repeatedly under natural conditions, and can be used in conjunction with other marking techniques such as physical tags and GPS coordinates.