Evolutionary patterns of variations in chromosome counts and genome sizes show positive correlations with taxonomic diversity in tropical gingers.

PREMISE: Cytogenetic traits such as an organism's chromosome number and genome size are taxonomically critical as they are instrumental in defining angiosperm diversity. Variations in these traits can be traced to evolutionary processes such as polyploidization, although geographic variations across cytogenetic traits remain underexplored. In the pantropical monocot family Zingiberaceae (~1500 species), cytogenetic traits have been well documented; however, the role of these traits in shaping taxonomic diversity and biogeographic patterns of gingers is not known. METHODS: A time-calibrated Bayesian phylogenetic tree was constructed for 290 taxa covering three of the four subfamilies in Zingiberaceae. We tested models of chromosome number and genome size evolution within the family and whether lineage age, taxonomic diversity, and distributional range explain the variations in the cytogenetic traits. Tests were carried out at two taxonomic ranks: within Zingiberaceae and within genus Hedychium using correlations, generalized linear models and phylogenetic least square models. RESULTS: The most frequent changes in chromosome number within Zingiberaceae were noted to be demi-polyploidization and polyploidization (~57% of the time), followed by ascending dysploidy (~27%). The subfamily Zingiberoideae showed descending dysploidy at its base, while Alpinioideae showed polyploidization at its internal nodes. Although chromosome counts and genome sizes did not corroborate with each other, suggesting that they are not equivalent; higher chromosome number variations and higher genome size variations were associated with higher taxonomic diversity and wider biogeographic distribution. CONCLUSIONS: Within Zingiberaceae, multiple incidences of polyploidization were discovered, and cytogenetic events appear to have reduced the genome sizes and increased taxonomic diversity, distributional ranges and invasiveness.

Floral evolution and pollinator diversification in Hedychium: Revisiting Darwin's predictions using an integrative taxonomic approach.

PREMISE: Hedychium J. Koenig (Zingiberaceae) is endemic to the Indo-Malayan Realm and is known for its colorful and fragrant flowers. Historically, two different pollination syndromes characterize Hedychium: diurnal or bird pollination, and nocturnal or moth pollination. In this study, we aim to understand the evolution of nocturnal and diurnal flowers, and to test its putative association with lineage diversification in Hedychium. METHODS: A molecular tree of Hedychium was used as a scaffold upon which we estimated ancestral character states, phylogenetic signals, and correlations for certain categorical and continuous floral traits. Furthermore, we used phylomorphospace and trait-dependent diversification rate estimation analyses to understand phenotypic evolution and associated lineage diversification in Hedychium. RESULTS: Although floral color and size lacked any association with specific pollinators, white or pale flowers were most common in the early branching clades when compared to bright-colored flowers, which were more widely represented in the most-derived clade IV. Five categorical and two continuous characters were identified to have informative evolutionary patterns, which also emphasized that ecology may have played a critical role in the diversification of Hedychium. CONCLUSIONS: From our phylogenetic analyses and ecological observations, we conclude that specializations in pollinator interactions are rare in the hyperdiverse clade IV, thus challenging the role of both moth-specialization and bird-specialization as central factors in the diversification of Hedychium. However, our results also suggest that clade III (predominantly island clade) may show specializations, and future studies should investigate ecological and pollinator interactions, along with inclusion of new traits such as floral fragrance and anthesis time.

Himalayan orogeny and monsoon intensification explain species diversification in an endemic ginger (Hedychium: Zingiberaceae) from the Indo-Malayan Realm.

The Indo-Malayan Realm is a biogeographic realm that extends from the Indian Subcontinent to the islands of Southeast Asia (Malay Archipelago). Despite being megadiverse, evolutionary hypotheses explaining taxonomic diversity in this region have been rare. Here, we investigate the role of geoclimatic events such as Himalayan orogeny and monsoon intensification in the diversification of the ginger-lilies (Hedychium J.Koenig: Zingiberaceae). We first built a comprehensive, time-calibrated phylogeny of Hedychium with 75% taxonomic and geographic sampling. We found that Hedychium is a very young lineage that originated in Northern Indo-Burma, in the Late Miocene (c. 10.6 Ma). This was followed by a late Neogene and early Quaternary diversification, with multiple dispersal events to Southern Indo-Burma, Himalayas, Peninsular India, and the Malay Archipelago. The most speciose clade IV i.e., the predominantly Indo-Burmese clade also showed a higher diversification rate, suggesting its recent rapid radiation. Our divergence dating and GeoHiSSE results demonstrate that the diversification of Hedychium was shaped by both the intensifications in the Himalayan uplift as well as the Asian monsoon. Ancestral character-state reconstructions identified the occurrence of vegetative dormancy in both clades I and II, whereas the strictly epiphytic growth behavior, island dwarfism, lack of dormancy, and a distinct environmental niche were observed only in the predominantly island clade i.e., clade III. Finally, we show that the occurrence of epiphytism in clade III corresponds with submergence due to sea-level changes, suggesting it to be an adaptive trait. Our study highlights the role of recent geoclimatic events and environmental factors in the diversification of plants within the Indo-Malayan Realm and the need for collaborative work to understand biogeographic patterns within this understudied region. This study opens new perspectives for future biogeographic studies in this region and provides a framework to explain the taxonomic hyperdiversity of the Indo-Malayan Realm.

A Draft Genome of the Ginger Species Alpinia nigra and New Insights into the Genetic Basis of Flexistyly.

Angiosperms possess various strategies to ensure reproductive success, such as stylar polymorphisms that encourage outcrossing. Here, we investigate the genetic basis of one such dimorphism that combines both temporal and spatial separation of sexual function, termed flexistyly. It is a floral strategy characterised by the presence of two morphs that differ in the timing of stylar movement. We performed a de novo assembly of the genome of Alpinia nigra using high-depth genomic sequencing. We then used Pool-seq to identify candidate regions for flexistyly based on allele frequency or coverage differences between pools of anaflexistylous and cataflexistylous morphs. The final genome assembly size was 2 Gb, and showed no evidence of recent polyploidy. The Pool-seq did not reveal large regions with high FST values, suggesting large structural chromosomal polymorphisms are unlikely to underlie differences between morphs. Similarly, no region had a 1:2 mapping depth ratio which would be indicative of hemizygosity. We propose that flexistyly is governed by a small genomic region that might be difficult to detect with Pool-seq, or a complex genomic region that proved difficult to assemble. Our genome will be a valuable resource for future studies of gingers, and provides the first steps towards characterising this complex floral phenotype.

Species complex delimitations in the genus Hedychium: A machine learning approach for cluster discovery.

PREMISE: Statistical methods used by most morphologists to validate species boundaries (such as principal component analysis [PCA] and non-metric multidimensional scaling [nMDS]) are limiting because these methods are mostly used as visualization methods, and because the groups are identified by taxonomists (i.e., supervised), adding human bias. Here, we use a spectral clustering algorithm for the unsupervised discovery of species boundaries followed by the analysis of the cluster-defining characters. METHODS: We used spectral clustering, nMDS, and PCA on 16 morphological characters within the genus Hedychium to group 93 individuals from 10 taxa. A radial basis function kernel was used for the spectral clustering with user-specified tuning values (gamma). The goodness of the discovered clusters using each gamma value was quantified using eigengap, a normalized mutual information score, and the Rand index. Finally, mutual information-based character selection and a t-test were used to identify cluster-defining characters. RESULTS: Spectral clustering revealed five, nine, and 12 clusters of taxa in the species complexes examined here. Character selection identified at least four characters that defined these clusters. DISCUSSION: Together with our proposed character analysis methods, spectral clustering enabled the unsupervised discovery of species boundaries along with an explanation of their biological significance. Our results suggest that spectral clustering combined with a character selection analysis can enhance morphometric analyses and is superior to current clustering methods for species delimitation.

Rediscovery of four narrow endemic Didymocarpus species (Gesneriaceae) from Mizoram, India, with revised species descriptions and lectotypifications.

Here we report the rediscovery of four endemic gesneriads from the state of Mizoram, India, after a span of 86 to 90 years since their last collection. The four species belong to the genus Didymocarpus Wall. and they are: D. adenocarpus, D. lineicapsa, D. parryorum, and D. wengeri. We present revised morphological descriptions, photographs, and designate lectotypes for D. parryorum and D. wengeri. During our study we came across several discrepancies between morphological characters assigned to these four species in the protologue and morphological characters present (or absent) in the type specimens and in plants recollected by us. We list these discrepancies in a section titled 'amendments to protologue'. Based on the high endemicity and critical conservation status of all the four rediscovered species, we suggest that floristic studies along with large-scale biogeographic studies should be prioritized in the Indo-Burmese region.

Low-cost FloPump for regulated air sampling of volatile organic compounds.

PREMISE: We present a low-cost, battery-operated, portable pump, "FloPump," which allows regulated air sampling for the study of volatile organic compounds (VOCs). VOCs are routinely investigated in applications such as atmospheric chemistry, agriculture, and fragrance biology. METHODS AND RESULTS: We compared the performance of FloPump with the Supelco pump in collecting VOCs using two test samples: guava fruit (Psidium guajava) and a perfume. The sampling and identification of volatiles was carried out using a dynamic headspace sampling method followed by gas chromatography-mass spectrometry. We show that the sampling efficiency of FloPump is comparable to the commercial pump, and at an affordable cost of ~US$115 (~86% cheaper), it provides a viable option for researchers interested in sampling volatiles on a constrained budget. CONCLUSIONS: Accurate air sampling is critical for the study of VOCs. We propose that FloPump will make air sampling more affordable, thus encouraging studies of VOCs.

Hedychiumziroense (Zingiberaceae), a new species of ginger lily from Northeast India.

We describe Hedychiumziroense sp. nov. from Northeast India (NE India) which was discovered during one of our recent botanical explorations in Arunachal Pradesh. We provide detailed morphological comparison of this species with four other Hedychium species (H.griersonianum R.M.Sm., H.ellipticum Buch.-Ham. ex Sm., H.gomezianum Wall. and H.yunnanense Gagnep.), with which it shares some morphological similarities. The new species is characterised by a dense cylindrical spike, pubescent rachis, folded bracts, 2-3 flowers per cincinnus, deeply cleft labellum and a distinctive late monsoonal flowering phenology from August to September.

Describing terminologies and discussing records: More discoveries of facultative vivipary in the genus Hedychium J.Koenig (Zingiberaceae) from Northeast India.

The authors introduce the term facultative vivipary for the first time in gingers and elaborate on this reproductive strategy. Four new observations of facultative vivipary are reported in the genus Hedychium which were discovered during botanical explorations by the authors in Northeast India (NE India) over the past three years. The viviparous taxa are H. marginatum C.B.Clarke, H. speciosum var. gardnerianum (Ker Gawl.) Sanoj & M.Sabu (previously, H. gardnerianum Sheppard ex Ker Gawl.), H. thyrsiforme Buch.-Ham. ex Sm. and H. urophyllum G.Lodd. The authors also attempt to summarise the occurrence of vivipary in the family Zingiberaceae from published reports and to clarify a taxonomic misidentification in a previously known report of vivipary in Hedychium elatum.

miRNAome expression profiles in the gonads of adult Melopsittacus undulatus.

The budgerigar (Melopsittacus undulatus) is one of the most widely studied parrot species, serving as an excellent animal model for behavior and neuroscience research. Until recently, it was unknown how sexual differences in the behavior, physiology, and development of organisms are regulated by differential gene expression. MicroRNAs (miRNAs) are endogenous short non-coding RNA molecules that can post-transcriptionally regulate gene expression and play a critical role in gonadal differentiation as well as early development of animals. However, very little is known about the role gonadal miRNAs play in the early development of birds. Research on the sex-biased expression of miRNAs in avian gonads are limited, and little is known about M. undulatus. In the current study, we sequenced two small non-coding RNA libraries made from the gonads of adult male and female budgerigars using Illumina paired-end sequencing technology. We obtained 254 known and 141 novel miRNAs, and randomly validated five miRNAs. Of these, three miRNAs were differentially expressed miRNAs and 18 miRNAs involved in sexual differentiation as determined by functional analysis with GO annotation and KEGG pathway analysis. In conclusion, this work is the first report of sex-biased miRNAs expression in the budgerigar, and provides additional sequences to the avian miRNAome database which will foster further functional genomic research.

Using an integrated approach to identify cryptic species, divergence patterns and hybrid species in Asian ladies' tresses orchids (Spiranthes, Orchidaceae).

Spiranthes (∼36 species, Orchidaceae) is a small genus with a global distribution. It has a center of diversity in North America with only a few species occurring in Asia. This study focuses on the Asian Spiranthes with an emphasis on understanding their biogeographic relationships and species delimitations using molecular markers. Our phylogenetic trees based on nuclear (ITS) and chloroplast (trnL-trnLF, matK and trnS-G) sequences from samples across their range in Asia revealed the Asian Spiranthes are monophyletic. Ancestral area optimization suggested that North America forms the ancestral region for the Asian Spiranthes rather than Europe suggesting that they originated from a single long-distance dispersal event. Our study also revealed the presence of a cryptic species S. himalayensis, which was discovered based on molecular data thus emphasizing the importance of wide geographical sampling in phylogenetic studies. Sequences of cloned ITS provided support for the hypothesis that natural hybridization between S. sinensis and the newly described S. himalayensis resulted in the allotetraploid S. hongkongensis, with S. himalayensis as the paternal parent. One of the species complexes known in Asia is the S. sinensis complex, which shows a wide occurrence and is known for local geographical variants. Some of these variants have been described as new species in Australia and New Zealand. Our studies show that all the sampled variants including the Australian and New Zealand species show monophyly despite having long branches. This suggests that there may be high rates of gene flow between the geographically distinct forms resulting in lack of species resolution within the S. sinensis complex.

Evolutionary Conservation and Emerging Functional Diversity of the Cytosolic Hsp70:J Protein Chaperone Network of Arabidopsis thaliana.

Heat shock proteins of 70 kDa (Hsp70s) partner with structurally diverse Hsp40s (J proteins), generating distinct chaperone networks in various cellular compartments that perform myriad housekeeping and stress-associated functions in all organisms. Plants, being sessile, need to constantly maintain their cellular proteostasis in response to external environmental cues. In these situations, the Hsp70:J protein machines may play an important role in fine-tuning cellular protein quality control. Although ubiquitous, the functional specificity and complexity of the plant Hsp70:J protein network has not been studied. Here, we analyzed the J protein network in the cytosol of Arabidopsis thaliana and, using yeast genetics, show that the functional specificities of most plant J proteins in fundamental chaperone functions are conserved across long evolutionary timescales. Detailed phylogenetic and functional analysis revealed that increased number, regulatory differences, and neofunctionalization in J proteins together contribute to the emerging functional diversity and complexity in the Hsp70:J protein network in higher plants. Based on the data presented, we propose that higher plants have orchestrated their "chaperome," especially their J protein complement, according to their specialized cellular and physiological stipulations.

Two new species of Gingers (Zingiberaceae) from Myanmar.

Two new species of gingers (Zingiberaceae), Globba sherwoodiana W.J. Kress & V. Gowda sp. nov., and Curcuma arracanensis W.J. Kress & V. Gowda sp. nov., from Myanmar are described. The new species of Globba is currently only known in cultivation and is commonly grown and sold in markets in Myanmar. In contrast Curcuma arracanensis has been collected from a single restricted region in the cloud forests of the Rakhine Yoma above the Bay of Bengal in western Myanmar. Three-locus DNA barcodes were generated as aids for the identification of the two new species.

The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes: Ardeidae): sequence, structure and phylogenetic analysis.

Ixobrychus cinnamomeus is a member of the large wading bird family, known as Ardeidae. In the present study, we determined the complete mitochondrial genome of I. cinnamomeus for use in future phylogenetic analysis. This circular mitochondrial genome is 17,180 bp in length and composed of 13 protein-coding genes, 22 tRNA genes, two rRNA genes and one putative control region. Three conserved domains and a minisatellite of 17 nucleotides with 22 tandem repeats were detected at the end of the control region. Phylogenetic relationships were reconstructed using the nucleotide and corresponding amino acid datasets of 12 concatenated protein-coding genes from the mitochondrial genome. Using maximum likelihood, maximum parsimony and Bayesian inference methods, the monophyly of Ciconiidae, Ardeidae and Threskiornithidae were confirmed; however, the monophyly of traditional Ciconiiformes and Pelecaniformes failed to be recovered. Although further studies are recommended to clarify relationships among and within the orders of Ciconiiformes, Pelecaniformes, Suliformes and Phaethontiformes, our results provide preliminary exploratory results that can be useful in the current understanding of avian phylogenetics.

Development and characterization of microsatellite loci for two Caribbean Heliconia (Heliconiaceae: H. bihai and H. caribaea).

PREMISE OF THE STUDY: Microsatellite loci were developed to characterize genetic variation and population subdivision in Heliconia bihai and H. caribaea from the Caribbean Islands. METHODS AND RESULTS: A total of 13 new microsatellite markers were developed and characterized in the two Caribbean heliconias. Di-, tri-, and tetranucleotide repeats were identified with one to 17 alleles per locus, and the observed heterozygosity ranged from 0.13 to 0.87. Additionally, cross-species amplification was successful in eight out of 13 loci. CONCLUSIONS: The microsatellite loci developed have discriminatory potential to be used in genetic characterizations of Caribbean Heliconia. Both H. bihai and H. caribaea are known to have adaptive interactions with their hummingbird pollinators, and the characterized microsatellite markers will be used to study mating system, genetic structure, and phylogeographic patterns in Caribbean Heliconia.

Butterfly pollination and high-contrast visual signals in a low-density distylous plant.

In low-density butterfly-pollinated Mussaenda frondosa (Rubiaceae), flowers attract pollinators at short distances while conspicuous, non-rewarding accessory bracts are detectable at long distances by long-ranging pollinators such as the birdwing butterfly Troides minos that did not detect flower-bearing plants in the absence of these bracts. However, even in the absence of flowers, the white, ultraviolet-absorbing bracts attracted butterflies that visited flowerless plants. Although flower visits by short-ranging territorial butterflies declined significantly on removal of bracts, they did not cease completely. Nectar-robbing carpenter bees and birds did not change their behaviour following bract removal. Bract removal caused a significant decline in fruit set, indicating their importance as visual signals to pollinators.