Ancient hybridization in Curcuma (Zingiberaceae)-Accelerator or brake in lineage diversifications?

Hybridization is a widespread phenomenon in the evolution of plants and exploring its role is crucial to understanding diversification processes of many taxonomic groups. Recently, more attention is focused on the role of ancient hybridization that has repeatedly been shown as triggers of evolutionary radiation, although in some cases, it can prevent further diversification. The causes, frequency, and consequences of ancient hybridization remain to be explored. Here, we present an account of several events of ancient hybridization in turmeric, the economically important plant genus Curcuma (Zingiberaceae), which harbors about 130 known species. We analyzed 1094 targeted low-copy genes and plastomes obtained by next-generation sequencing of 37 species of Curcuma, representing the known genetic diversity and spanning the geographical distribution of the genus. Using phylogenetic network analysis, we show that the entire genus Curcuma as well as its most speciose lineage arose via introgression from the genus Pyrgophyllum and one of the extinct lineages, respectively. We also document a single event of ancient hybridization, with C. vamana as a product, that represents an evolutionary dead end. We further discuss distinct circumstances of those hybridization events that deal mainly with (in)congruence in chromosome counts of the parental lineages.

Next-generation sequencing data show rapid radiation and several long-distance dispersal events in early Costaceae.

The monocot family Costaceae Nakai consists of seven genera but their mutual relationships have not been satisfactorily resolved in previous studies employing classical molecular markers. Phylogenomic analyses of 365 nuclear genes and nearly-complete plastome data provide almost fully resolved insights into their diversification. Paracostus is identified as sister to all other taxa, followed by several very short branches leading to discrete lineages, suggesting an ancient rapid radiation of these early lineages and leaving the exact relationships among them unresolved. Relationships among Chamaecostus, Dimerocostus and Monocostus confirmed earlier findings that these genera form a monophyletic group. The Afro-American Costus is also monophyletic. By contrast, Tapeinochilos appeared as a well-supported crown lineage of Cheilocostus rendering it paraphyletic. As these two genera differ morphologically from one another owing to a shift from insect- to bird-pollination, we propose to keep both names. The divergence time within Costaceae was estimated using penalized likelihood utilizing two fossils within Zingiberales, †Spirematospermum chandlerae and †Ensete oregonense, indicated a relatively recent diversification of Costaceae, between 18 and 9 Mya. Based on these data, the current pantropical distribution of the family is hypothesized to be the result of several long-distance intercontinental dispersal events, which do not correlate with global geoclimatic changes.

Phylogenomics and genome size evolution in Amomum s. s. (Zingiberaceae): Comparison of traditional and modern sequencing methods.

BACKGROUND AND AIMS: A targeted enrichment NGS approach was used to construct the phylogeny of Amomum Roxb. (Zingiberaceae). Phylogenies based on hundreds of nuclear genes, the whole plastome and the rDNA cistron were compared with an ITS-based phylogeny. Trends in genome size (GS) evolution were examined, chromosomes were counted and the geographical distribution of phylogenetic lineages was evaluated. METHODS: In total, 92 accessions of 54 species were analysed. ITS was obtained for 79 accessions, 37 accessions were processed with Hyb-Seq and sequences from 449 nuclear genes, the whole cpDNA, and the rDNA cistron were analysed using concatenation, coalescence and supertree approaches. The evolution of absolute GS was analysed in a phylogenetic and geographical context. The chromosome numbers of 12 accessions were counted. KEY RESULTS: Four groups were recognised in all datasets though their mutual relationships differ among datasets. While group A (A. subulatum and A. petaloideum) is basal to the remaining groups in the nuclear gene phylogeny, in the cpDNA topology it is sister to group B (A. repoeense and related species) and, in the ITS topology, it is sister to group D (the Elettariopsis lineage). The former Elettariopsis makes a monophyletic group. There is an increasing trend in GS during evolution. The largest GS values were found in group D in two tetraploid taxa, A. cinnamomeum and A. aff. biphyllum (both 2n = 96 chromosomes). The rest varied in GS (2C = 3.54-8.78 pg) with a constant chromosome number 2n = 48. There is a weak connection between phylogeny, GS and geography in Amomum. CONCLUSIONS: Amomum consists of four groups, and the former Elettariopsis is monophyletic. Species in this group have the largest GS. Two polyploids were found and GS greatly varied in the rest of Amomum.

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.

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.

Resolving the rapid plant radiation of early diverging lineages in the tropical Zingiberales: Pushing the limits of genomic data.

Many cases of rapid evolutionary radiations in plant and animal lineages are known; however phylogenetic relationships among these lineages have been difficult to resolve by systematists. Increasing amounts of genomic data have been sequentially applied in an attempt to resolve these radiations, dissecting their evolutionary patterns into a series of bifurcating events. Here we explore one such rapid radiation in the tropical plant order Zingiberales (the bananas and relatives) which includes eight families, approximately 110 genera, and more than 2600 species. One clade, the "Ginger families", including (Costaceae + Zingiberaceae) (Marantaceae + Cannaceae), has been well-resolved and well-supported in all previous studies. However, well-supported reconstructions among the "Banana families" (Musaceae, Heliconiaceae, Lowiaceae, Strelitziaceae), which most likely diverged about 90 Mya, have been difficult to confirm. Supported with anatomical, morphological, single locus, and genome-wide data, nearly every possible phylogenetic placement has been proposed for these families. In an attempt to resolve this complex evolutionary event, hybridization-based target enrichment was used to obtain sequences from up to 378 putatively orthologous low-copy nuclear genes (all ≥ 960 bp). Individual gene trees recovered multiple topologies among the early divergent lineages, with varying levels of support for these relationships. One topology of the "Banana families" (Musaceae (Heliconiaceae (Lowiaceae + Strelitziaceae))), which has not been suggested until now, was almost consistently recovered in all multilocus analyses of the nuclear dataset (concatenated - ExaML, coalescent - ASTRAL and ASTRID, supertree - MRL, and Bayesian concordance - BUCKy). Nevertheless, the multiple topologies recovered among these lineages suggest that even large amounts of genomic data might not be able to fully resolve relationships at this phylogenetic depth. This lack of well-supported resolution could suggest methodological problems (i.e., violation of model assumptions in both concatenated and coalescent analyses) or more likely reflect an evolutionary history shaped by an explosive, rapid, and nearly simultaneous polychotomous radiation in this group of plants towards the end of the Cretaceous, perhaps driven by vertebrate pollinator selection.

Very small relict populations suggest high extinction debt of gingers in primary forest fragments of a tropical city.

PREMISE OF THE STUDY: Tropical plant communities in fragmented forests are likely to experience an extinction debt, i.e., the habitat cannot support as many species as are present due to reduced habitat size and connectivity. There are few estimates of the number of species that represent extinction debt, and the number of extinctions over time has rarely been recorded. We recorded population sizes to assess threats and extinctions in gingers (sensu Zingiberales) in fragmented rainforest in Singapore, ca. 200 yr after fragmentation began. METHODS: We surveyed extant diversity and population sizes of gingers and used the results to estimate species survival. We critically assessed historic specimens to estimate initial extinctions and extinctions realized in present habitats. KEY RESULTS: We recorded 23 species, including five species previously presumed nationally extinct and four species omitted from the national checklist. The revised extinction rate is much lower than previously reported (12 vs. 37%). Most gingers have very small populations or miniscule ranges, implying that extinction debt has not been paid off. CONCLUSIONS: Ginger diversity remains high, but the number of species at immediate risk of extinction outnumber recorded extinctions. Although tropical forest fragments remain arks of plant diversity for a long time, extinction debt may be prevalent in all plant groups in Singapore. Slow relaxation of extinction debt should be explicitly identified as a conservation challenge and opportunity. For conserving plant diversity in tropical fragments, relaxation must be reversed through restoration of degraded landscapes and, where feasible, targeted ex situ conservation and planting.

Hybridization among distantly related species: Examples from the polyploid genus Curcuma (Zingiberaceae).

Discerning relationships among species evolved by reticulate and/or polyploid evolution is not an easy task, although it is widely discussed. The economically important genus Curcuma (ca. 120 spp.; Zingiberaceae), broadly distributed in tropical SE Asia, is a particularly interesting example of a group of palaeopolyploid origin whose evolution is driven mainly by hybridization and polyploidization. Although a phylogeny and a new infrageneric classification of Curcuma, based on commonly used molecular markers (ITS and cpDNA), have recently been proposed, significant evolutionary questions remain unresolved. We applied a multilocus approach and a combination of modern analytical methods to this genus to distinguish causes of gene tree incongruence and to identify hybrids and their parental species. Five independent regions of nuclear DNA (DCS, GAPDH, GLOBOSA3, LEAFY, ITS) and four non-coding cpDNA regions (trnL-trnF, trnT-trnL, psbA-trnH and matK), analysed as a single locus, were employed to construct a species tree and hybrid species trees using (*)BEAST and STEM-hy. Detection of hybridogenous species in the dataset was also conducted using the posterior predictive checking approach as implemented in JML. The resulting species tree outlines the relationships among major evolutionary lineages within Curcuma, which were previously unresolved or which conflicted depending upon whether they were based on ITS or cpDNA markers. Moreover, by using the additional markers in tests of plausible topologies of hybrid species trees for C. vamana, C. candida, C. roscoeana and C. myanmarensis suggested by previous molecular and morphological evidence, we found strong evidence that all the species except C. candida are of subgeneric hybrid origin.

Seed morphology and anatomy and its utility in recognizing subfamilies and tribes of Zingiberaceae.

PREMISE OF THE STUDY: Recent phylogenetic analyses based on molecular data suggested that the monocot family Zingiberaceae be separated into four subfamilies and four tribes. Robust morphological characters to support these clades are lacking. Seeds were analyzed in a phylogenetic context to test independently the circumscription of clades and to better understand evolution of seed characters within Zingiberaceae. METHODS: Seventy-five species from three of the four subfamilies were analyzed using synchrotron based x-ray tomographic microscopy (SRXTM) and scored for 39 morphoanatomical characters. KEY RESULTS: Zingiberaceae seeds are some of the most structurally complex seeds in angiosperms. No single seed character was found to distinguish each subfamily, but combinations of characters were found to differentiate between the subfamilies. Recognition of the tribes based on seeds was possible for Globbeae, but not for Alpinieae, Riedelieae, or Zingibereae, due to considerable variation. CONCLUSIONS: SRXTM is an excellent, nondestructive tool to capture morphoanatomical variation of seeds and allows for the study of taxa with limited material available. Alpinioideae, Siphonochiloideae, Tamijioideae, and Zingiberoideae are well supported based on both molecular and morphological data, including multiple seed characters. Globbeae are well supported as a distinctive tribe within the Zingiberoideae, but no other tribe could be differentiated using seeds due to considerable homoplasy when compared with currently accepted relationships based on molecular data. Novel seed characters suggest tribal affinities for two currently unplaced Zingiberaceae taxa: Siliquamomum may be related to Riedelieae and Monolophus to Zingibereae, but further work is needed before formal revision of the family.

Ancient hybridization and repetitive element proliferation in the evolutionary history of the monocot genus Amomum (Zingiberaceae).

Genome size variation is a crucial aspect of plant evolution, influenced by a complex interplay of factors. Repetitive elements, which are fundamental components of genomic architecture, often play a role in genome expansion by selectively amplifying specific repeat motifs. This study focuses on Amomum, a genus in the ginger family (Zingiberaceae), known for its 4.4-fold variation in genome size. Using a robust methodology involving PhyloNet reconstruction, RepeatExplorer clustering, and repeat similarity-based phylogenetic network construction, we investigated the repeatome composition, analyzed repeat dynamics, and identified potential hybridization events within the genus. Our analysis confirmed the presence of four major infrageneric clades (A-D) within Amomum, with clades A-C exclusively comprising diploid species (2n = 48) and clade D encompassing both diploid and tetraploid species (2n = 48 and 96). We observed an increase in the repeat content within the genus, ranging from 84% to 89%, compared to outgroup species with 75% of the repeatome. The SIRE lineage of the Ty1-Copia repeat superfamily was prevalent in most analyzed ingroup genomes. We identified significant difference in repeatome structure between the basal Amomum clades (A, B, C) and the most diverged clade D. Our investigation revealed evidence of ancient hybridization events within Amomum, coinciding with a substantial proliferation of multiple repeat groups. This finding supports the hypothesis that ancient hybridization is a driving force in the genomic evolution of Amomum. Furthermore, we contextualize our findings within the broader context of genome size variations and repeatome dynamics observed across major monocot lineages. This study enhances our understanding of evolutionary processes within monocots by highlighting the crucial roles of repetitive elements in shaping genome size and suggesting the mechanisms that drive these changes.

Curcumamaxwellii and C.rubroaurantiaca (Zingiberaceae, Zingiberoideae), two new species from Thailand.

Curcumamaxwelliisp. nov. and Curcumarubroaurantiacasp. nov. (Zingiberaceae, Zingiberoideae, Zingibereae), two new red-orange-flowered species from Thailand, are described. They are compared to the morphologically closest species from the Curcumasubgen.Ecomatae and detailed descriptions, colour plates and information on their distribution, ecology, phenology and uses are provided. Preliminary IUCN conservation assessments for both of these species are proposed as Least Concern.

A new species and new combination in basally flowering Vietnam Costaceae.

Cheilocostuscandidus sp. nov. (Costaceae), a basally flowering spiral ginger with cream white flowers from southern Vietnam, is described and illustrated here. A new combination, Cheilocostustonkinensis (Gagnep.) Skornick., is proposed here and the lectotype is designated. A key to Cheilocostus in Vietnam is included.

Hanguanadeflexa (Hanguanaceae), a new forest species from Sarawak, Borneo.

Hanguanadeflexa sp. nov. (Hanguanaceae) from Lawas district, Sarawak, Malaysia (northern Borneo) is described and illustrated, bringing the total number of species in Borneo to eight. The new species differs from all other recognized Hanguana species by a combination of flat leaf blade, deflexed infructescences, one-seeded dull red fruits with centrally positioned stigma and globose seed with wedge-shaped ostiole. Revised key for Bornean Hanguana species is presented.

Population genomics reveal apomixis in a novel system: uniclonal female populations dominate the tropical forest herb family, Hanguanaceae (Commelinales).

The abundance of apomixis in tropical plant genera is poorly understood, and this affects the understanding of speciation and evolution. Hanguanaceae is a tropical monogeneric, dioecious plant family. All but two species are solitary herbs with no capability to spread vegetatively. Viable seeds are often produced when males have not been observed. Our aim was to investigate the presence of apomixis in Hanguana. We used reduced representation genomics to study phylogenetics and genetic variability in all populations of Hanguana in Singapore. We measured genome sizes and estimated ploidy levels in 10 species. Almost all taxa tested were genetically uniform (uniclonal) regardless of the extent of their distribution. The distribution of single clones over distinct localities supports our hypothesis of apomictic reproduction. Only one sexually reproducing native species was detected. Triploid and pentaploid states support our hypothesis that the type of apomixis in Hanguana is gametophytic. Population genomics tools offer a quick and cost-effective way of detecting excess clonality and thereby inferring apomixis. In the case of Hanguana, the presence of male plants is a strong indicator of sexual reproduction, whereas genome triplication is indicative of apomictic reproduction.

Species diversity driven by morphological and ecological disparity: a case study of comparative seed morphology and anatomy across a large monocot order.

Phenotypic variation can be attributed to genetic heritability as well as biotic and abiotic factors. Across Zingiberales, there is a high variation in the number of species per clade and in phenotypic diversity. Factors contributing to this phenotypic variation have never been studied in a phylogenetic or ecological context. Seeds of 166 species from all eight families in Zingiberales were analyzed for 51 characters using synchrotron based 3D X-ray tomographic microscopy to determine phylogenetically informative characters and to understand the distribution of morphological disparity within the order. All families are distinguishable based on seed characters. Non-metric multidimensional scaling analyses show Zingiberaceae occupy the largest seed morphospace relative to the other families, and environmental analyses demonstrate that Zingiberaceae inhabit both temperate and tropical regions, while other Zingiberales are almost exclusively tropical. Temperate species do not cluster in morphospace nor do they share a common suite of character states. This suggests that the diversity seen is not driven by adaptation to temperate niches; rather, the morphological disparity seen likely reflects an underlying genetic plasticity that allowed Zingiberaceae to repeatedly colonize temperate environments. The notable morphoanatomical variety in Zingiberaceae seeds may account for their extraordinary ecological success and high species diversity as compared to other Zingiberales.

Chromosome numbers and genome size variation in Indian species of Curcuma (Zingiberaceae).

BACKGROUND AND AIMS: Genome size and chromosome numbers are important cytological characters that significantly influence various organismal traits. However, geographical representation of these data is seriously unbalanced, with tropical and subtropical regions being largely neglected. In the present study, an investigation was made of chromosomal and genome size variation in the majority of Curcuma species from the Indian subcontinent, and an assessment was made of the value of these data for taxonomic purposes. METHODS: Genome size of 161 homogeneously cultivated plant samples classified into 51 taxonomic entities was determined by propidium iodide flow cytometry. Chromosome numbers were counted in actively growing root tips using conventional rapid squash techniques. KEY RESULTS: Six different chromosome counts (2n = 22, 42, 63, >70, 77 and 105) were found, the last two representing new generic records. The 2C-values varied from 1.66 pg in C. vamana to 4.76 pg in C. oligantha, representing a 2.87-fold range. Three groups of taxa with significantly different homoploid genome sizes (Cx-values) and distinct geographical distribution were identified. Five species exhibited intraspecific variation in nuclear DNA content, reaching up to 15.1 % in cultivated C. longa. Chromosome counts and genome sizes of three Curcuma-like species (Hitchenia caulina, Kaempferia scaposa and Paracautleya bhatii) corresponded well with typical hexaploid (2n = 6x = 42) Curcuma spp. CONCLUSIONS: The basic chromosome number in the majority of Indian taxa (belonging to subgenus Curcuma) is x = 7; published counts correspond to 6x, 9x, 11x, 12x and 15x ploidy levels. Only a few species-specific C-values were found, but karyological and/or flow cytometric data may support taxonomic decisions in some species alliances with morphological similarities. Close evolutionary relationships among some cytotypes are suggested based on the similarity in homoploid genome sizes and geographical grouping. A new species combination, Curcuma scaposa (Nimmo) Skornick. & M. Sabu, comb. nov., is proposed.